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A Whole-Genome, Radiation Hybrid Mapping Resource of Hexaploid Wheat
Abstract
Generating a contiguous, ordered reference sequence of a complex genome such as hexaploid wheat (2n=6x=42; ~17GB) is a challenging task due to its large, highly repetitive, and allopolyploid genome. In wheat, ordering of whole-genome or hierarchical shotgun sequencing contigs is primarily based on recombination and comparative genomics-based approaches. However, comparative genomics approaches are limited to syntenic inference and recombination is suppressed within the peri-centromeric regions of wheat chromosomes, thus, precise ordering of physical maps and sequenced contigs across the whole genome using these approaches is nearly impossible. We developed a whole-genome, radiation-hybrid (RH) resource and tested it by genotyping a set of 115 randomly selected lines on a high density SNP array. At the whole-genome level, 26,299 SNP markers were mapped on the RH panel and provided an average mapping resolution of ~248Kb/cR1500 with a total map length of 6,866 cR1500 . The 7,296 unique mapping bins provided a 5-8 fold higher resolution than genetic maps used in similar studies. Most strikingly, the RH map had uniform bin resolution across the entire chromosome(s), including peri-centromeric regions. Our research provides a valuable and low-cost resource for anchoring and ordering sequenced BAC and NGS contigs. The WGRH developed for reference wheat line Chinese Spring (CS-WGRH), will be useful for anchoring and ordering sequenced BAC and NGS based contigs for assembling a high-quality, reference sequence of hexaploid wheat. Additionally, this study provides an excellent model for developing similar resources for other polyploid species. This article is protected by copyright. All rights reserved.
... This method uses gamma irradiationinduced chromosomal breaks to order marker scaffolds in a given RH panel Harris 1975, 1977;Riera-Lizarazu et al. 2000Wardrop et al. 2002). In plants, RH mapping was first explored to map maize chromosome 9 (Riera-Lizarazu et al. 2000) and was subsequently applied in barley (Wardrop et al. 2002) and wheat (Kalavacharla et al. 2006;Riera-Lizarazu et al. 2010;Tiwari et al. 2012aTiwari et al. , 2016. ...
... Wheat has a completely sequenced genome and very often its telomeric regions are referred to as distal parts while the centromeric and pericentromeric regions are referred to as the proximal parts of the genome Walkowiak et al. 2020). The proximal part of the wheat chromosomes shows highly reduced recombination events and much lower gene density with more than 30% of wheat genes residing in the proximal or pericentromeric regions (Saintenac et al. 2009;Tiwari et al. 2016;IWGSC 2018). It is well documented that precise mapping of wheat genes in low-recombination region (2/3 of a given chromosome) of wheat chromosomes poses a big challenge (Johnson et al. 2008;Saintenac et al. 2009;Luo et al. 2009;Tiwari et al. 2016). ...
... The proximal part of the wheat chromosomes shows highly reduced recombination events and much lower gene density with more than 30% of wheat genes residing in the proximal or pericentromeric regions (Saintenac et al. 2009;Tiwari et al. 2016;IWGSC 2018). It is well documented that precise mapping of wheat genes in low-recombination region (2/3 of a given chromosome) of wheat chromosomes poses a big challenge (Johnson et al. 2008;Saintenac et al. 2009;Luo et al. 2009;Tiwari et al. 2016). RH mapping is a recombination-independent approach that provides an alternative to genetic mapping approaches (Tiwari et al. 2012bBassi et al. 2013;Mahlandt et al. 2021). ...
Key Message
This work reports the physical mapping of an important gene affecting spike compactness located in a low-recombination region of hexaploid wheat. This work paves the way for the eventual isolation and characterization of the factor involved but also opens up possibilities to use this approach to precisely map other wheat genes located on proximal parts of wheat chromosomes that show highly reduced recombination.
Abstract
Mapping wheat genes, in the centromeric and pericentromeric regions (~ 2/3rd of a given chromosome), poses a formidable challenge due to highly suppressed recombination. Using an example of compact spike locus (C-locus), this study provides an approach to precisely map wheat genes in the pericentromeric and centromeric regions that house ~ 30% of wheat genes. In club-wheat, spike compactness is controlled by the dominant C-locus, but previous efforts have failed to localize it, on a particular arm of chromosome 2D. We integrated radiation hybrid (RH) and high-resolution genetic mapping to locate C-locus on the short arm of chromosome 2D. Flanking markers of the C-locus span a physical distance of 11.0 Mb (231.0–242 Mb interval) and contain only 11 high-confidence annotated genes. This work demonstrates the value of this integrated strategy in mapping dominant genes in the low-recombination regions of the wheat genome. A comparison of the mapping resolutions of the RH and genetic maps using common anchored markers indicated that the RH map provides ~ 9 times better resolution that the genetic map even with much smaller population size. This study provides a broadly applicable approach to fine map wheat genes in regions of suppressed recombination.
... However, RH mapping does not depend on allelic polymorphism and uses presence (retention) versus absence (deletion) marker assay. This characteristic of RH mapping allows the mapping of even monomorphic markers, thus dramatically increasing the number of markers that can be mapped Tiwari et al. 2016). This also means that RH mapping can be an excellent tool to assign genes or gene based markers, which are mostly conserved and show very low genetic polymorphism among species (Kumar et al. 2012b). ...
... This also means that RH mapping can be an excellent tool to assign genes or gene based markers, which are mostly conserved and show very low genetic polymorphism among species (Kumar et al. 2012b). A simple example for this could be the mapping of D-genome markers in wheat Tiwari et al. 2016). ...
... Individually, RH mapping studies were able to map 580 and 671 markers on D-genome of wheat Tiwari et al. 2012), whereas, genetic populations were able to map a total of 41-219 markers using the same DArT platform of wheat (Akbari et al. 2006;Singh et al. 2010;Sorrells et al. 2011;Wang et al. 2011). Similar results were also observed for SNP markers (Tiwari et al. 2016). This clearly shows that RH mapping approach could map many folds more markers than the traditionally used genetic or recombination mapping approach. ...
Identification of the QTL/genes associated with traits of interest determines the successful application of those genes for crop improvement. After identification of QTL, fine mapping and cloning of important QTL can also enhance our understanding of the genetic structure of the underlying genes responsible for the phenotypic variation. Genetic or recombination mapping is the most common approach used to identify, map and clone QTL or genes in plants. However, genetic mapping approach for fine mapping or map-based cloning is associated with many drawbacks including the need of developing a homogenous and large population, the availability of polymorphic markers, and the poor recombination in certain chromosomal regions. In this article, we describe an alternative approach, called radiation hybrid (RH) mapping, for forward genetic studies in plants. This approach has been extensively used in animal system and offer greater prospects for forward genetic studies in plants as well. The RH mapping uses radiation induced chromosomal breaks to map markers, and thus offers many advantages compared to traditionally used genetic mapping approach, particularly for loci located in recombination cold spots and for the traits which lack genetic diversity. Here, we reviewed the progress made in application of RH approach for forward genetics in plants.
... The third approach -in silico anchoring -was used to anchor physical map contigs to Ae. tauschii linkage map, Chinese Spring x Renan F 6 RIL map (2413 7D SNP markers [37]), Chinese Spring consensus map v.3 (2323 7D DArTseq markers, Suppl. Table B) and Chinese Spring radiation hybrid map (485 7DS SNP markers from iSelect 90 K SNP array, [38,39]). Sequences of all markers were analyzed for homology with 7DS MTP sequences using BLASTn. ...
... Ordering physical map and sequence contigs in pericentromeric regions poses a major challenge in genomic projects due to suppressed recombination. Here we applied an integrated multiple-map [38]. ...
... Table C). The wheat radiation hybrid map, not relying on genetic recombination and showing a high resolution in the pericentromeric region of 7D chromosome [38], appeared a promising genomic resource. Unfortunately, we failed to allocate SNP markers from the RH map to the 100 7DS centromeric contigs. ...
Bread wheat (Triticum aestivum L.) is a staple food for a significant part of the world's population. The growing demand on its production can be satisfied by improving yield and resistance to biotic and abiotic stress. Knowledge of the genome sequence would aid in discovering genes and QTLs underlying these traits and provide a basis for genomics-assisted breeding. Physical maps and BAC clones associated with them have been valuable resources from which to generate a reference genome of bread wheat and to assist map-based gene cloning. As a part of a joint effort coordinated by the International Wheat Genome Sequencing Consortium, we have constructed a BAC-based physical map of bread wheat chromosome arm 7DS consisting of 895 contigs and covering 94% of its estimated length. By anchoring BAC contigs to one radiation hybrid map and three high resolution genetic maps, we assigned 73% of the assembly to a distinct genomic position. This map integration, interconnecting a total of 1,713 markers with ordered and sequenced BAC clones from a minimal tiling path, provides a tool to speed up gene cloning in wheat. The process of physical map assembly included the integration of the 7DS physical map with a whole-genome physical map of Aegilops tauschii and a 7DS Bionano genome map, which together enabled efficient scaffolding of physical-map contigs, even in the non-recombining region of the genetic centromere. Moreover, this approach facilitated a comparison of bread wheat and its ancestor at BAC-contig level and revealed a reconstructed region in the 7DS pericentromere.
... This repair pathway is error prone and can cause various kinds of genomic rearrangements such as interstitial deletions, insertions, inversions and translocations (Pipiras et al., 1998;Puchta, 2005). Ionizing radiation-induced chromosomal breaks occur randomly and are evenly distributed across the entire chromosomes, including pericentromeric regions (Kumar et al., 2012;Tiwari et al., 2016). ...
... RH mapping played a major role in whole genome sequencing and assembly of human (Lander et al.2001) and animal genomes (Faraut et al., 2009). Moreover, RH mapping has successfully supported high-resolution mapping of individual wheat chromosomes 1D (Kalavacharla et al. 2006), 3B (Kumar et al. 2012;Paux et al. 2008), 6B (Kobayashi et al. 2015) and 4A (Balcárková et al., 2017), the D-sub-genome (Riera-Lizarazu et al. 2010) and the whole genome of the hexaploid wheat (Tiwari et al. 2016). ...
... By contrast, radiation induced deletion mapping was able to unequivocally resolve order for most markers. Gamma radiation affects the entire genome causing homogeneous marker loss regardless of positions within the chromosomes (Bassi et al., 2013;Riera-Lizarazu et al., 2010;Tiwari et al., 2016). Accordingly, radiation induced breaks were found to be evenly distributed across the 5AS chromosome, agreeing with previous RH mapping results (Balcárková et al., 2017;Bassi et al., 2016;Bassi et al., 2013;Kalavacharla et al., 2006;Kumar et al., 2012Kumar et al., , 2105Mazaheri et al., 2015;Tiwari et al., 2016;Tiwari et al., 2012). ...
The Qfhs.ifa-5A allele, contributing to enhanced Fusarium head blight resistance in wheat, resides in a low recombinogenic region of chromosome 5A close to the centromere. A near isogenic RIL population segregating for the Qfhs.ifa-5A resistance allele was developed and among 3650 lines as few as four recombined within the pericentromeric C-5AS1-0.40 bin, yielding only a single recombination point. Genetic mapping of the pericentromeric region using a recombination dependent approach was thus not successful. To facilitate fine-mapping the physically large Qfhs.ifa-5A interval, two gamma-irradiated deletion panels were generated: (1) Seeds of line NIL3 carrying the Qfhs.ifa-5A resistance allele in an otherwise susceptible background were irradiated and plants thereof were selfed to obtain deletions in homozygous state. (2) A radiation hybrid panel was produced using irradiated pollen of the wheat line Chinese Spring (CS) for pollinating the CS-nullisomic5Atetrasomic5B. In total 5157 radiation selfing and 276 radiation hybrid plants were screened for deletions on 5AS and plants containing deletions were analysed using 102 5AS specific markers. Combining genotypic information of both panels yielded an 817 fold map improvement (cR/cM) for the centromeric bin and was 389 fold increased across the Qfhs.ifa-5A interval compared to the genetic map, with an average map resolution of 0.77 Mb/cR. We successfully proved that the RH mapping technique can effectively resolve marker order in low-recombining regions, including pericentromeric intervals, and simultaneously allow developing an in vivo panel of sister lines differing for induced deletions across the Qfhs.ifa-5A interval that can be used for phenotyping.
... and centromeric and pericentromeric regions are referred to as the proximal part of the genomeWalkowiak et al. 2020). The proximal part of the wheat chromosomes shows highly reduced recombination events and much lower gene density with more than 30% of wheat genes residing in the proximal or pericentromeric regions(Saintenac et al. 2009;Tiwari et al., 2016, IWGSC, 2018. It is well documented that precise mapping of wheat genes in low recombination region ( ...
Mapping wheat genes, in the centromeric and pericentromeric regions (~2/3 rd of a given chromosome), poses a formidable challenge due to highly suppressed recombination. Using an example of compact spike locus ( C-locus) , this study provides an approach to precisely map wheat genes in the pericentromeric and centromeric regions that house ~30% of wheat genes. In Club-wheat, spike compactness is controlled by the dominant C-locus, but previous efforts have failed to localize it , on a particular arm of chromosome 2D. We integrated radiation hybrid (RH) and high-resolution genetic mapping to locate C-locus on the short arm of chromosome 2D. Flanking markers of the C-locus span a physical distance of 11.0 Mb (231.0-242 Mb interval) and contain only 11 high-confidence annotated genes. This work demonstrates the value of this integrated strategy in mapping dominant genes in the low-recombination regions of the wheat genome. A comparison of the mapping resolutions of the RH and genetic maps using common anchored markers indicated that the RH map provides ~9 times better resolution that the genetic map even with much smaller population size. This study provides a broadly applicable approach to fine-map wheat genes in regions of suppressed recombination.
... These molecular factors have been employed in proposing the strategies to resist, tolerate and or adapt the plants to abiotic and biotic stresses, which is critical for sustainable agriculture. of tools, databases and diverse skills in computing, statistics and technologies such as cloud computing and high-performance computing. The rapid decrease in the cost of nextgeneration sequencing (NGS) technology helps to characterize genome, exome, transcriptome of plants in addition to understanding genes, proteins, non-coding molecules, genetic variants, quantitative trait loci (QTLs), genetic composition and molecular pathways (Scossa et al., 2016, Tiwari et al., 2016. For instance, high-throughput genomic studies coupled with rapid phenotyping systems would help to identifying associated genomic regions and candidate genes with differential expression for different biotic and abiotic stresses. ...
Natural and human activities have increased the greenhouse emissions and
st it will continue to boost global temperature in the 21 century. In this paper, we
discuss the profound impact of climate on plant diseases — if the climatic
conditions are not favourable to disease, a vulnerable host will not be infected by
a virulent pathogen. Variable concentrations of CO , temperature, and availability 2
of water may induce positive, neutral, or negative effects on disease development.
Nevertheless, the basic concept of interactions of host-pathogen-environment
may theoretically be applied to all pathosystems. Environmental factors also
in
... Nevertheless, technologies are now available to overcome this difficulty. Radiation hybrids have been employed as an alternative in high-resolution physical mapping (Kumar et al. 2012;Tiwari et al. 2016;Buerstmayr et al. 2018), although not as successful as expected, particularly for complex traits. Moreover, fine mapping coupled with RNA-seq or mutagenesis-combined MutChromSeq are among the alternatives for candidate gene identification in recombination-cold regions (Deng et al. 2019;Sanchez-Martin et al. 2016). ...
Key message
KT1 was validated as a novel thickness QTL with major effects on wheat kernel dimensions and weight and fine mapped to a 0.04 cM interval near the chromosome-5A centromere.
Abstract
Kernel size, the principal grain weight determining factor of wheat and a target trait for both domestication and artificial breeding, is mainly defined by kernel length (KL), kernel width (KW) and kernel thickness (KT), of which KW and KT have been shown to be positively related to grain weight (GW). Qkt.nau-5A, a major QTL for KT, was validated using the QTL near-isogenic lines (NILs) in three genetic backgrounds. Genetic analysis using two F2 populations derived from the NILs showed that Qkt.nau-5A was dominant for thicker kernel and inherited like a single gene and therefore was designated as Kernel Thickness 1 (KT1). With 77 recombinant lines identified from a total of 19,160 F2 plants from the two NIL-derived F2 populations, KT1 was mapped to the 0.04 cM Xwgrb1356-Xwgrb1619 interval, which was near the centromere and displayed strong recombination suppression. The KT1 interval showed positive correlation with KW and GW and negative correlation with KL and therefore could be used in breeding for cultivars with round-shaped kernels that are beneficial to higher flour yield. KT1 candidate identification could be achieved through combination of sequence variation analysis with expression profiling of the annotated genes in the interval.
... This technique may provide uniform mapping resolution across the genome (Tiwari et al. 2012. In the RH mapping approach, radiation-induced chromosomal breaks can be used to generate overlapping deletions, and these can be used to produce a high-resolution marker scaffold (Riera-Lizarazu et al. 2010;Kumar et al. 2015;Tiwari et al. 2016;IWGSC 2018). Deletion-based genotyping favors the mapping of dominant genes with large effect because the phenotypes are easily distinguishable when the causal genetic factors are deleted. ...
Key message
This work reports a quick method that integrates RH mapping and genetic mapping to map the dominant Mov-1 locus to a 1.1-Mb physical interval with a small number of candidate genes.
Abstract
Bread wheat is an important crop for global human population. Identification of genes and alleles controlling agronomic traits is essential toward sustainably increasing crop production. The unique multi-ovary (MOV) trait in wheat holds potential for improving yields and is characterized by the formation of 2–3 grains per spikelet. The genetic basis of the multi-ovary trait is known to be monogenic and dominant in nature. Its precise mapping and functional characterization is critical to utilizing this trait in a feasible manner. Previous mapping efforts of the locus controlling multiple ovary/pistil formation in the hexaploid wheat have failed to produce a consensus for a particular chromosome. We describe a mapping strategy integrating radiation hybrid mapping and high-resolution genetic mapping to locate the chromosomal position of the Mov-1 locus in hexaploid wheat. We used RH mapping approach using a panel of 188 lines to map the Mov-1 locus in the terminal part of long arm of wheat chromosome 2D with a map resolution of 1.67 Mb/cR1500. Then using a genetic population of MOV × Synthetic wheat of F2 lines, we delineated the Mov-1 locus to a 1.1-Mb physical region with a small number of candidate genes. This demonstrates the value of this integrated strategy to mapping dominant genes in wheat.
... This indicates the potential of both the oligos identified and induction of chromosomal mutations through irradiation in peanut chromosome engineering. The chromosomal variations could be used for translocation or deletion mapping, radiation hybrid mapping and gene mapping of peanut as has been reported previously [50,51]. ...
Background
Chromosomal variants play important roles in crop breeding and genetic research. The development of single-stranded oligonucleotide (oligo) probes simplifies the process of fluorescence in situ hybridization (FISH) and facilitates chromosomal identification in many species. Genome sequencing provides rich resources for the development of oligo probes. However, little progress has been made in peanut due to the lack of efficient chromosomal markers. Until now, the identification of chromosomal variants in peanut has remained a challenge.
Results
A total of 114 new oligo probes were developed based on the genome-wide tandem repeats (TRs) identified from the reference sequences of the peanut variety Tifrunner (AABB, 2n = 4x = 40) and the diploid species Arachis ipaensis (BB, 2n = 2x = 20). These oligo probes were classified into 28 types based on their positions and overlapping signals in chromosomes. For each type, a representative oligo was selected and modified with green fluorescein 6-carboxyfluorescein (FAM) or red fluorescein 6-carboxytetramethylrhodamine (TAMRA). Two cocktails, Multiplex #3 and Multiplex #4, were developed by pooling the fluorophore conjugated probes. Multiplex #3 included FAM-modified oligo TIF-439, oligo TIF-185-1, oligo TIF-134-3 and oligo TIF-165. Multiplex #4 included TAMRA-modified oligo Ipa-1162, oligo Ipa-1137, oligo DP-1 and oligo DP-5. Each cocktail enabled the establishment of a genome map-based karyotype after sequential FISH/genomic in situ hybridization (GISH) and in silico mapping. Furthermore, we identified 14 chromosomal variants of the peanut induced by radiation exposure. A total of 28 representative probes were further chromosomally mapped onto the new karyotype. Among the probes, eight were mapped in the secondary constrictions, intercalary and terminal regions; four were B genome-specific; one was chromosome-specific; and the remaining 15 were extensively mapped in the pericentric regions of the chromosomes.
Conclusions
The development of new oligo probes provides an effective set of tools which can be used to distinguish the various chromosomes of the peanut. Physical mapping by FISH reveals the genomic organization of repetitive oligos in peanut chromosomes. A genome map-based karyotype was established and used for the identification of chromosome variations in peanut following comparisons with their reference sequence positions.
... This indicates the potential of both the oligos identi ed and induction of chromosomal mutations through irradiation in peanut chromosome engineering. The chromosomal variations could be used for translocation or deletion mapping, radiation hybrid mapping, and gene mapping of peanut as has been reported previously [50,51]. ...
Background: Chromosomal variants play important roles in crop breeding and genetic research. The development of single-stranded oligonucleotide (oligo) probes simplifies the process of fluorescence in situ hybridization (FISH) and facilitates chromosomal identification in many species. Genome sequencing provides rich resources for the development of oligo probes. However, little progress has been made in peanut due to the lack of efficient chromosomal markers. Until now, the identification of chromosomal variants in peanut has remained a challenge.
Results: A total of 114 new oligo probes were developed based on the genome-wide tandem repeats (TRs) identified from the reference sequences of the peanut variety Tifrunner (AABB, 2n = 4x = 40) and the diploid species Arachis ipaensis (BB, 2n = 2x = 20). These oligos were classified into 28 types based on their positions and overlapping signals in chromosomes. For each type, a representative oligo was selected and modified with green fluorescein 6-carboxyfluorescein (FAM) or red fluorescein 6-carboxytetramethylrhodamine (TAMRA). Two cocktails, Multiplex #3 and Multiplex #4, were developed by pooling the fluorophore conjugated probes. Multiplex #3 included FAM-modified oligo TIF-439, oligo TIF-185-1, oligo TIF-134-3, and oligo TIF-165. Multiplex #4 included TAMRA-modified oligo Ipa-1162, oligo Ipa-1137, oligo DP-1, and oligo DP-5. Each cocktail enabled the establishment of a genome map-based karyotype after sequential FISH/genomic in situ hybridization (GISH) and in silico mapping. Furthermore, we identified 14 chromosomal variants of peanut induced by radiation exposure. A total of 28 representative probes were further chromosomally mapped onto the new karyotype. Among the probes, eight were mapped in the secondary constrictions, intercalary, and terminal regions; four were B genome-specific; one was chromosome-specific; and the remaining 15 were extensively mapped in the pericentric regions of chromosomes.
Conclusions: The development of new oligo probes provides an effective set of tools which can be used to distinguish the various chromosomes of the peanut. Physical mapping by FISH reveals the genomic organization of repetitive oligos in peanut chromosomes. A genome map-based karyotype was established and used for the identification of chromosome variations in peanut following comparisons with their reference sequence positions.
... This indicates the potential of both the oligos identi ed and radiation in peanut chromosome engineering. The chromosomal variations could be used for translocation or deletion mapping, radiation hybrid mapping, and gene mapping of peanut, as has been reported previously [50,51]. ...
Background: Chromosomal variants play important roles in crop breeding and genetic research. The development of single-stranded oligonucleotide (oligo) probes simplifies the process of fluorescence in situ hybridization (FISH) and facilitates chromosomal identification in many species. Genome sequencing provides rich resources for the development of oligo probes. However, little progress has been made in peanut. Thus, the identification of chromosomal variants in peanut remains a challenge, owing to a lack of efficient chromosomal markers.
Results: A total 114 new oligo probes were developed, based on the genome-wide tandem repeats (TRs) identified from the reference sequences of the peanut variety Tifrunner (AABB, 2n = 4x = 40) and the diploid species Arachis ipaensis (BB, 2n = 2x = 20). These oligos were classified into 28 types, based on their positions, and overlapping signals in chromosomes. For each oligo types, a single and representative oligos was selected and modified with 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA). Based on these 28 probes, a new multiplex #3 cocktail was developed with FAM-modified TIF-439, TIF-185-1, TIF-134-3, and TIF-165-3, and TAMRA-modified Ipa-1162, Ipa-1137, DP-1, and DP-5. This cocktail enabled the establishment of a genome map-based karyotype after sequential FISH/genomic in situ hybridization (GISH) and in silico mapping. Furthermore, we identified 14 chromosomal variants of peanut induced by radiation. A total of 28 representative probes were further chromosomally mapped onto the new karyotype. Among the probes, eight were mapped in the secondary constrictions, and intercalary and terminal regions; four were B genome-specific; one was chromosome-specific; and the other 15 were extensively mapped in the pericentric regions of chromosomes.
Conclusions: The development of new oligo probes provides effective tools, which can be used to distinguish various chromosomes of peanut. Physical mapping reveals the genomic organization of repetitive oligos in peanut chromosomes by FISH. Following comparisons with their positions in the reference sequences, a genome map-based karyotype was established and used for the identification of chromosome variations in peanut.
... Conventional breeding may help in wheat improvement, but it is rather slow, due to difficulties in crossing with wild relatives and limited hereditary base [1,2]. Transformation of wheat was the bottommost among monocots, due to its complicated genome (2n = 28 for Triticum durum and 2n = 42 for Triticum aestivum) as it is rich in repetitive sequences causing difficulties in regeneration and transformation [3][4][5]. ...
Wheat is a major cereal crop for humans but quite recalcitrant in transformation. Establishment
of regeneration system in wheat using immature embryos is not easy and time/cost-consuming.
Herein, we developed a regeneration and transformation system using mature seeds in four pasta
wheat cultivars. The MS medium with 2.0 mg/l 2,4-D and 2 mg/l BA was the optimum medium
for developing shoots from calli. Wheat cultivars showed different regeneration frequencies
response due to their genetic makeup. The cultivar Sohag-3 produced the highest regeneration
frequency (93.2%) among the tested cultivars. Developed cultivars Sohag-3 and ACSAD1105
mature embryos were co-cultivated with Agrobacterium tumefaciens strain GV3101 with the
binary vector pISV2678 harboring the bar gene and β-glucuronidase (gus) gene. The
transformation efficiencies were 12.3 and 9.1% for cultivars Sohag-3 and ACSAD1105,
respectively. The polymerase chain reaction (with specific primers for the transgenes) and the
dot blot hybridization were used to confirm the integration of the transgene in transformed
plants. The transformation percentages were reduced according to their expression and reached
5.6 and 4.6% for cultivars Sohag-3 and ACSAD1105, respectively. RT-PCR and northern blot
analysis confirmed the expression of the gus gene only in the transgenic plants. The procedures
developed in this study demonstrate the ability to produce transgenic wheat plants expressing the
gus gene; hence, this protocol could be used to regenerate transgenic wheat plants expressing
desirable and selective genes
... Rapidly declining cost of next-generation sequencing (NGS) technology to characterize genome, exome or transcriptome of plants leads to its broad adoption in the agricultural setting. The application of NGS technologies to understand plant molecular biology includes, but is not limited to aid, discovery research to understand genetic composition, genetic variants, quantitative trait loci (QTLs), genes, proteins, non-coding molecules and molecular pathways (Allwright and Taylor, 2016;Kage et al., 2016;Scossa et al., 2016;Tiwari et al., 2016;Huttenhofer and Vogel, 2006). In plant biology research, NGS technologies also help researchers to sequence and identify novel plant species that available in major plant databases. ...
Plants are essential facilitators of human life on planet earth. Plants play a critical functional role in mediating the quality of air, availability of food and the sustainability of agricultural resources. However, plants are in constant interaction with its environment and often hampered by various types of stresses like biotic and abiotic ones. Biotic stress is a significant reason for crop-loss and causes yield loss in the range of 31–42%, post-harvest loss due to biotic stress is in the range of 6–20%, and abiotic stress causes 6–20% of the crop damage. Recognizing the molecular factors driving plant stress-related events, and developing molecular strategies to aid plants to tolerate, resist or adapt to biotic and abiotic stress are critical for sustainable agriculture practice. In this review, we discuss how recent advances in bioinformatics, plant genomics, and data science could help to improve our understanding of plant stress biology and improve the scale of global food production. We present various areas of scientific and technological advances, such as increased availability of genomics data through whole genome sequencing that require attention. We also discuss emerging techniques including CRISPR-Cas9 based genome engineering systems to develop plant varieties that can handle combinatorial stress signals. Growing trend of converging multiple omics technologies and availability of accurate, multi-scale models of plant stress through the study of orthologs and synteny studies, would improve our knowledge of how plants perceive, respond, and manage stress to thrive as resilient crop species and thus help to reduce global food crisis.
... Using the same approach, construction of radiation hybrid maps for chromosome 4A, 5A and 3B were also prepared. Later, RH maps for the whole D sub-genome and the whole wheat genome involving all the three sub-genomes (A, B and D) were also produced [57,107]. ...
The chromosome number of hexaploid wheat (2n = 6x = 42) was first worked out in 1918 and was followed by research on intergeneric and interspecific hybridization, mainly in Japan under the leadership of Hitoshi Kihara. Starting in 1935, Ernie Sears in USA produced a large collection of aneuploids including nullisomics, monosomics, trisomics, tetrasomics, ditelocentrics and nullisomic–tetrasomic (NT) lines. In parallel, through a study of meiotic chromosome pairing in hybrids produced through interspecific and intergeneric crosses, progenitors of the A and D genomes of 6x wheat were determined with certainty; the progenitor of B genome was difficult and still not known with certainty. The diploidizing system and the Ph1 locus restricting meiotic chromosome pairing to only between homologous (but not between homoeologous) chromosomes was also initially discovered by Okamoto (Wheat Inf Serv 5:6, 1957), and was subsequently studied in more detail by Ralph Riley and his group at Cambridge, UK during late 1950s. With the establishment of International Triticeae Mapping Initiative (ITMI) and the development of ITMI mapping population in 1990, first genetic maps were produced for all the seven homoeologous groups involving 21 wheat chromosomes by 1995. High-density genetic maps were developed later through large-scale development of SNPs through next generation sequencing (NGS) technology. During 1990s, TR Endo and BS Gill at Kansas State University, USA also produced > 400 chromosome deletion stocks that were extensively used for the development of physical maps for all the 21 wheat chromosomes. More recently, during the last 12 years (2005–2017), cytogenomics research was conducted, which included separation of longest chromosome 3B and the 40 chromosome arms for the remaining 20 chromosomes through flow sorting and their utilization in the development of BAC libraries that were used for developing BAC-based physical maps. These maps were used in generating chromosome survey sequences, and assembly of the whole genome sequence of 6x wheat Chinese Spring. During 2017–2018, whole genome sequences for a wild emmer tetraploid wheat (T. turgidum) and those for the two diploid species (T. urartu, Ae. tauschii) representing the donors of A and D sub-genomes of 6x wheat, were also published. For hexaploid wheat, estimates of a pangenome with ~ 140,000 genes and that of the core genome with ~ 80,000 genes also became available. These resources are already being extensively utilized and will continue to be utilized throughout the world for several decades not only for basic research, but also for the improvement of wheat crop to feed the growing world population.
The book was inadvertently published with an incorrect name information for one of the Chapter author as Body Mori, instead it should be “Boyd A. Mori” in the front matter and Chapter 20.
In order to produce successful varieties, wheat breeding programs must develop several strategies that fall under one of the following topics: line development, population improvement, and selection methods. This chapter focuses on breeding activities related to population improvement and selection methods, while Chap. 10.1007/978-3-030-90673-3_5 discusses line development. The objective of population improvement is to enhance the entire genetic base of the breeding program, while selection methods aim to identify breeding lines with superior potential or performance. As with line development approaches, numerous population improvement and selection methods have been developed in order to enhance breeding program efficiency and achieve genetic improvement. This chapter will provide an overview of population improvement and selection methods in the context of wheat breeding, discuss their advantages and disadvantages, and summarize empirical studies that have evaluated them in order to inform breeding program design.
The main objective of a plant breeding program is to deliver superior germplasm for farmers in a defined set of environments, or a target population of environments (TPE). Historically, CIMMYT has characterized the environments in which the developed germplasm will be grown. The main factors that determine when and where a wheat variety can be grown are flowering time, water availability and the incidence of pests and diseases. A TPE consists of many (population) environments and future years or seasons, that share common variation in the farmers’ fields, it can also be seen as a variable group of future production environments. TPEs can be characterized by climatic, soil and hydrological features, as well as socioeconomic aspects. Whereas the selection environments (SE) are the environments where the breeder does the selection of the lines. The SE are identified for predicting the performance in the TPE, but the SE may not belong to the TPE. The utilization of advanced statistical methods allows the identification of GEI to obtain higher precision when estimating the genetic effects. Multi-environmental testing (MET) is a fundamental strategy for CIMMYT to develop stable high grain yielding germplasm in countries with developing economies. An adequate MET strategy allows the evaluation of germplasm in stress hotspots and the identification of representative and correlated sites; thus, breeders can make better and targeted decisions in terms of crossing, selection and logistic operations.
Increasing the genetic diversity of wheat is key to its future production in terms of increasing yields, resistance to diseases and adaptability to fluctuations in global climate. The use of the progenitor species of wheat and also its wild relatives uniquely provides a route to vastly increase the genetic variation available to wheat breeders for the development of new, superior wheat varieties. The introduction of genetic variation from the wild relatives of wheat in the form of introduced chromosome segments or introgressions, has taken place for hundreds of years, albeit largely unintentionally in farmers’ fields. However, the use of the wild relatives became more systematic from the 1950s onwards. The work has previously been hampered due to a lack of technology for the identification and characterisation of the introgressions and consequently the strategic use of the wild relatives. The advances in molecular biology over recent years now make it possible to generate wheat/wild relative introgressions on a scale not previously possible. In fact, the greatest threat to this area of work is now the lack of scientists/breeders with the understanding of chromosomes and their manipulation.
The chapter aims to provide guidance on how phenotyping may contribute to the genetic advance of wheat in terms of yield potential and resilience to adverse conditions. Emphasis will be given to field high throughput phenotyping, including affordable solutions, together with the need for environmental and spatial characterization. Different remote sensing techniques and platforms are presented, while concerning lab techniques only a well proven trait, such as carbon isotope composition, is included. Finally, data integration and its implementation in practice is discussed. In that sense and considering the physiological determinants of wheat yield that are amenable for indirect selection, we highlight stomatal conductance and stay green as key observations. This choice of traits and phenotyping techniques is based on results from a large set of retrospective and other physiological studies that have proven the value of these traits together with the highlighted phenotypical approaches.
Abiotic stresses, such as drought and high temperature, significantly limit wheat yield globally and the intensity and frequency of these stresses are projected to increase in most wheat growing areas. Wheat breeders have incrementally improved the tolerance of cultivars to these stresses through empirical selection in the environment, however new phenotyping and genetic technologies and strategies can significantly improve rates of genetic gain. The integration of new tools and knowledge in the plant breeding process, including better breeding targets, improved choice of genetic diversity, more efficient phenotyping methods and strategy and optimized integration of genetic technologies in the context of several commonly used wheat breeding strategies is discussed. New knowledge and tools that improve the efficiency and speed of wheat improvement can be integrated within the scaffold of most wheat breeding strategies without significant increase in cost.
New varieties of crops are developed to provide farmers seeds of cultivars that are acquainted with specific environmental or management conditions to realize best yield and quality. Seed is the carrier of genetic potential for the performance of a crop, hence is considered the most vital input in agriculture. Wheat being self-pollinated, it is not necessary to buy seed every year as in case of hybrids. Seeds are multiplied through an informal or formal approach. In most developing countries, informal wheat seed sector is dominant. Seed production follows well defined steps wherein a particular class of seed is grown to deliver another class of seed to the farmer. In general, there are four classes of seeds in wheat – nucleus, breeder, foundation and certified, although in some cases registered seed is also produced. The strength of the seed sector varies across countries – strong in developed countries but moderate to weak in the Global South. In most countries seed production and its marketing is regulated and both public and private sectors are involved. In counties with a not so strong seed sector, a fast track approach for varietal release and seed dissemination has been advocated to meet the challenges of climate change and transboundary diseases.
Without higher yielding and more climate resilient crop varieties, better agronomy and sustainable inputs, the world is on a course for catastrophes in food and nutritional security with all the associated social and political implications. Achieving food and nutritional security is one of the most important Grand Challenges of this century. These circumstances demand new systems for improving wheat to sustain current needs and future demands. This chapter presents some of the networks that have been developed over the years to help address these challenges. Networks help to: identify the most urgent problems based on consensus; identify and bridge knowledge silos; increase research efficacy and efficiency by studying state of the art germplasm and sharing common research environments/platforms so multiple strands of research can be cross-referenced; and creating communities of practice where the modus operandi becomes cooperation towards common goals rather than competition. Networks can also provide identity and visibility to research programs and their stakeholders, thereby lending credibility, increasing investment opportunities and accelerating outputs and dissemination of valuable new technologies.
Micronutrients are essential for plant growth although required in only very small amounts. There are eight micronutrients needed for healthy growth of wheat: chlorine, iron, boron, manganese, zinc, copper, nickel and molybdenum. Several factors will influence the availability of micronutrients, including levels in the soil, and mobility or availability. Zinc deficiency is the most significant problem globally followed by boron, molybdenum, copper, manganese and iron. Deficiency is usually addressed through application of nutrients to seeds, or through foliar spays when symptoms develop. There is considerable genetic variation in the efficiency of micronutrient uptake in wheat, but this is not a major selection target for breeding programs given the agronomic solutions. However, for some micronutrients, the concentrations in the soil can be very high and result in toxicity. Of the micronutrients, the narrowest range between deficiency and toxicity is for boron and toxicity is a significant problem in some regions. Although not a micronutrient, aluminium toxicity is also a major factor limiting yield in many areas, usually associated with a low soil pH. Agronomic solutions for boron and aluminium toxicity are difficult and expensive. Consequently, genetic approaches have dominated the strategies for addressing toxicity and good sources of tolerance are available.
Wheat is a staple for rich and poor alike. Its improvement as a discipline was boosted when statisticians first distinguished heritable variation from environment effects. Many twentieth century crop scientists contributed to the Green Revolution that tripled yield potential of staple crops but yield stagnation is now a concern, especially considering the multiple challenges facing food security. Investments in modern technologies – phenomics, genomics etc. – provide tools to take both translational research and crop breeding to the next level. Herein wheat experts address three main themes: “Delivering Improved Germplasm” outlining theory and practice of wheat breeding and the attendant disciplines; ‘Translational Research to Incorporate Novel Traits’ covers biotic and abiotic challenges and outlines links between more fundamental research and crop breeding. However, effective translational research takes time and can be off-putting to funders and scientists who feel pressure to deliver near-term impacts. The final section ‘Rapidly Evolving Technologies & Likely Potential’ outlines methods that can boost translational research and breeding. The volume by being open access aims to disseminate a comprehensive textbook on wheat improvement to public and private wheat breeders globally, while serving as a benchmark of the current status as we address the formidable challenges that agriculture faces for the foreseeable future.
Wheat plants are infected by diverse pathogens of economic significance. They include biotrophic pathogens like mildews and rusts that require living plant cells to proliferate. By contrast necrotrophic pathogens that cause diseases such as tan spot, Septoria nodurum blotch and spot blotch require dead or dying cells to acquire nutrients. Pioneering studies in the flax plant-flax rust pathosystem led to the ‘gene-for-gene’ hypothesis which posits that a resistance gene product in the host plant recognizes a corresponding pathogen gene product, resulting in disease resistance. In contrast, necrotrophic wheat pathosystems have an ‘inverse gene-for-gene’ system whereby recognition of a necrotrophic fungal product by a dominant host gene product causes disease susceptibility, and the lack of recognition of this pathogen molecule leads to resistance. More than 300 resistance/susceptibility genes have been identified genetically in wheat and of those cloned the majority encode nucleotide binding, leucine rich repeat immune receptors. Other resistance gene types are also present in wheat, in particular adult plant resistance genes. Advances in mutational genomics and the wheat pan-genome are accelerating causative disease resistance/susceptibility gene discovery. This has enabled multiple disease resistance genes to be engineered as a transgenic gene stack for developing more durable disease resistance in wheat.
For more than a century, breeding has delivered huge benefits as a major driver of increased wheat productivity and of stability in the face of inevitable disease threats. Thus, the real cost of this staple grain has been reduced for billions of consumers. Steady breeding progress has been seen across many important traits of wheat, currently for potential yield averaging about 0.6% p.a. This yield progress continues to rely of extensive multilocational yield testing but has, however, become more difficult, even as new breeding techniques have improved efficiency. Breeding will continue to evolve as new approaches, being proposed with increasing frequency, are tested and found useful or not. High throughput phenotyping (HTPP), applying modern crop physiology, and molecular markers and genomic selection (GS) are in this phase right now. Such new techniques, along with pre-breeding for new traits, will likely play a larger role in this future improvement of wheat. New tools will also include genetic engineering (GE), as society’s need for its benefits become more urgent. The steady privatization of breeding seems unlikely to cease in the developed world but will continue to struggle elsewhere. It would seem wise, however, that a significant portion of the world’s pre-breeding research remains in the public sector, while maintaining close and equitable contact with those delivering new varieties.
In order to produce successful varieties, wheat breeding programs must develop several strategies that fall under one of the following topics: line development, population improvement, and selection methods. Part I of this chapter focuses on breeding activities related to line development, while Part II discusses population improvement and selection methods. Line development refers to the process of obtaining homozygous inbreds derived from crosses between parental lines. A wide variety of line development methods have been proposed in pursuit of greater efficiency and effectiveness. This chapter aims to provide basic knowledge on line development methods in relation to wheat breeding, describe how and why they came about, and synthesize the results of empirical studies that have evaluated them in order to foster critical thinking and innovation in breeding strategy design.
Established breeding methods for wheat in dry environments continue to make gains. It will remain the cornerstone for wheat improvement. This Chapter discusses proven methods to make additional gains. It discusses a way to benchmark yield potential in dry environments and how this can be used to determine whether unexpected agronomic or genetic factors are limiting yields. It examines opportunities, advantages and disadvantages of trait-based selection methods for dry environments, and it presents a framework by which important traits can be selected. Both high throughput and marker-based methods of selection are examined for their success and feasibility of use in breeding. It also highlights the importance of agronomic approaches in combination with breeding to continue to improve yield potential in water limited environments. Finally, the elements of success of translation from research to the delivery of new varieties is examined.
Wheat improvement relies on genetic diversity associated with variation in target traits. While traditionally the main sources of novel genetic diversity for breeding are wheat varieties or various wild relatives of wheat, advances in gene mapping and genome editing technologies provide an opportunity for engineering new variants of genes that could have beneficial effect on agronomic traits. Here, we provide the overview of the genome editing technologies and their application to creating targeted variation in genes that could enhance wheat productivity. We discuss the potential utility of the genome editing technologies and CRISPR-Cas-induced variation incorporated into the pre-breeding pipelines for wheat improvement.
While the three rusts are the most predominant wheat diseases in the global scale, various other diseases dominate in different geographical regions. In this chapter, some major non-rust diseases of wheat with global and/or regional economic importance are addressed, including three spike diseases (Fusarium head blight, wheat blast, and Karnal bunt), four leaf spotting diseases (tan spot, Septoria nodorum blotch, spot blotch, and Septoria tritici blotch), and several root diseases.KeywordsHead blight diseasesLeaf spotting diseasesRoot diseases
Beginning in the first decade of 1900, pioneering research in disease resistance and seed color inheritance established the scientific basis of Mendelian inheritance in wheat breeding. A series of breakthroughs in chromosome and genome analysis beginning in the 1920s and continuing into the twenty-first century have impacted wheat improvement. The application of meiotic chromosome pairing in the 1920s and plasmon analysis in the 1950s elucidated phylogeny of the Triticum-Aegilops complex of species and defined the wheat gene pools. The aneuploid stocks in the 1950s opened floodgates for chromosome and arm mapping of first phenotypic and later protein and DNA probes. The aneuploid stocks, coupled with advances in chromosome banding and in situ hybridization in the 1970s, allowed precise chromosome engineering of traits in wide hybrids. The deletion stocks in the 1990s were pivotal in mapping expressed genes to specific chromosome bins revealing structural and functional differentiation of chromosomes along their length and facilitating map-based cloning of genes. Advances in whole-genome sequencing, chromosome genomics, RH mapping and functional tools led to the assembly of reference sequence of Chinese Spring and multiple wheat genomes. Chromosome and genomic analysis must be integrated into wheat breeding and wide-hybridizaton pipeline for sustainable crop improvement.
This chapter provides an analysis of the processes determining the yield potential of wheat crops. The structure and function of the wheat crop will be presented and the influence of the environment and genetics on crop growth and development will be examined. Plant breeding strategies for raising yield potential will be described, with particular emphasis on factors controlling photosynthetic capacity and grain sink strength.KeywordsYield potentialGrain sink strengthRadiation-use efficiencyTrait-based breeding
Sound experimental design underpins successful plant improvement research. Robust experimental designs respect fundamental principles including replication, randomization and blocking, and avoid bias and pseudo-replication. Classical experimental designs seek to mitigate the effects of spatial variability with resolvable block plot structures. Recent developments in experimental design theory and software enable optimal model-based designs tailored to the experimental purpose. Optimal model-based designs anticipate the analytical model and incorporate information previously used only in the analysis. New technologies, such as genomics, rapid cycle breeding and high-throughput phenotyping, require flexible designs solutions which optimize resources whilst upholding fundamental design principles. This chapter describes experimental design principles in the context of classical designs and introduces the burgeoning field of model-based design in the context of plant improvement science.
High temperature stress is a primary constraint to maximal yield in wheat, as in nearly all cultivated crops. High temperature stress occurs in varied ecoregions where wheat is cultivated, as either a daily chronic metabolic stress or as an acute episodic high heat shock during critical periods of reproductive development. This chapter focuses on defining the key biochemical processes regulating a plant’s response to heat stress while highlighting and defining strategies to mitigate stress and stabilize maximal yield during high temperature conditions. It will weigh the advantages and disadvantages of heat stress adaptive trait breeding strategies versus simpler integrated phenotypic selection strategies. Novel remote sensing and marker-assisted selection strategies that can be employed to combine multiple heat stress tolerant adaptive traits will be discussed in terms of their efficacy. In addition, this chapter will explore how wheat can be re-envisioned, not only as a staple food, but also as a critical opportunity to reverse climate change through unique subsurface roots and rhizomes that greatly increase wheat’s carbon sequestration.
The three rusts are the most damaging diseases of wheat worldwide and continue to pose a threat to global food security. In the recent decades, stem rust races belonging to the Ug99 (TTKSK) and Digalu (TKTTF) race group resurfaced as a major threat in Africa, the Middle East and Europe threatening global wheat production. In addition, the evolution and migration of new aggressive races of yellow rust adapted to warmer temperatures into Europe and Asia from Himalayan region are becoming a significant risk in several wheat production environments. Unique and complex virulence patterns, continuous evolution to overcome effective resistance genes in varieties, shifts in population dynamics, transboundary migration have resulted in localized/regional epidemics leading to food insecurity threats. This underscores the need to identify, characterize, and deploy effective rust resistant genes from diverse sources into pre-breeding lines and future wheat varieties. The use of genetic resistance and deployment of multiple race specific and pleiotropic adult plant resistance genes in wheat lines can enhance resistance durability. Recent advances in sequencing annotated wheat reference genome with a detailed analysis of gene content among sub-genomes will not only accelerate our understanding of the genetic basis of rust resistance bread wheat, at the same time wheat breeders can now use this information to identify genes conferring rust resistance. Progress in genetic mapping techniques, new cloning techniques and wheat transformation methods over the last two decades have not only resulted in characterizing new genes and loci but also facilitated rapid cloning and stacking multiple genes as gene cassettes which can be future solution for enhancing durable resistance.
Crop simulation models are robust tools that enable users to better understand crop growth and development in various agronomic systems for improved decision making regarding agricultural productivity, environmental sustainability, and breeding. Crop models can simulate many agronomic treatments across a wide range of spatial and temporal scales, allowing for improved agricultural management practices, climate change impact assessment, and development of breeding strategies. This chapter examines current applications of wheat crop models and explores the benefits from model improvement and future trends, such as integration of G × E × M and genotype-to-phenotype interactions into modeling processes, to improve wheat ( Triticum spp.) production and adaptation strategies for agronomists, breeders, farmers, and policymakers.
Since its domestication around 10,000 years ago, wheat has played a crucial role in global food security. Wheat now supplies a fifth of food calories and protein to the world’s population. It is the most widely cultivated crop in the world, cultivated on 217 million ha annually. This chapter assesses available data on wheat production, consumption, and international trade to examine the global supply and demand conditions for wheat over the past quarter century and future implications. There is continued urgency to enhance wheat productivity to ensure global food security given continued global population growth and growing popularity of wheat based processed foods in the Global South. To enhance productivity while staying within planetary boundaries, there is a need for substantive investments in research and development, particularly in support of wheat’s role in agri-food systems in the Global South.
In general terms, pre-breeding links needed traits to new varieties and encompasses activities from discovery research, exploration of gene banks, phenomics, genomics and breeding. How does pre-breeding given its importance differ from varietal-based breeding? Why is pre-breeding important? Pre-breeding identifies trait or trait combinations to help boost yield, protect it from biotic or abiotic stress, and enhance nutritional or quality characteristics of grain. Sources of new traits/alleles are typically found in germplasm banks, and include the following categories of ‘exotic’ material: obsolete varieties, landraces, products of interspecific hybridization within the Triticeae such as chromosome translocation lines, primary synthetic genotypes and their derivatives, and related species mainly from the primary or secondary gene pools (Genus: Triticum and Aegilops ). Genetic and/or phenotyping tools are used to incorporate novel alleles/traits into elite varieties. While pre-breeding is mainly associated with use of exotics, unconventional crosses or selection methodologies aimed to accumulate novel combinations of alleles or traits into good genetic backgrounds may also be considered pre-breeding. In the current chapter, we focus on pre-breeding involving research-based screening of genetic resources, strategic crossing to combine complementary traits/alleles and progeny selection using phenomic and genomic selection, aiming to bring new functional diversity into use for development of elite cultivars.
The plant net genetic merit is a linear combination of trait breeding values weighted by its respective economic weights whereas a linear selection index (LSI) is a linear combination of phenotypic or genomic estimated breeding values (GEBV) which is used to predict the net genetic merit of candidates for selection. Because economic values are difficult to assign, some authors developed economic weight-free LSI. The economic weights LSI are associated with linear regression theory, while the economic weight-free LSI is associated with canonical correlation theory. Both LSI can be unconstrained or constrained. Constrained LSI imposes restrictions on the expected genetic gain per trait to make some traits change their mean values based on a predetermined level, while the rest of the traits change their values without restriction. This work is geared towards plant breeders and researchers interested in LSI theory and practice in the context of wheat breeding. We provide the phenotypic and genomic unconstrained and constrained LSI, which together cover the theoretical and practical cornerstone of the single-stage LSI theory in plant breeding. Our main goal is to offer researchers a starting point for understanding the core tenets of LSI theory in plant selection.
The current and future trends in population growth and consumption patterns continue to increase the demand for wheat. Wheat is a major source and an ideal vehicle for delivering increased quantities of zinc (Zn), iron (Fe) and other valuable bioactive compounds to population groups who consume wheat as a staple food. To address nutritious traits in crop improvement, breeding feasibility must be assessed and nutrient targets defined based on their health impact. Novel alleles for grain Zn and Fe in competitive, profitable, Zn enriched wheat varieties have been accomplished using conventional breeding techniques and have been released in South Asia and Latin America, providing between 20% and 40% more Zn than local commercial varieties and benefitting more than four million consumers. Future challenges include accelerating and maintaining parallel rates of genetic gain for productivity and Zn traits and reversing the trend of declining nutrients in wheat that has been exacerbated by climate change. Application of modern empirical and analytical technologies and methods in wheat breeding will help to expedite genetic progress, shorten time-to-market, and achieve mainstreaming objectives. In exploiting synergies from genetic and agronomic options, agronomic biofortification can contribute to achieving higher Zn concentrations, stabilize Zn trait expression, and increase other grain minerals, such as selenium or iodine. Increasing Fe bioavailability in future breeding and research with other nutrients and bioactive compounds is warranted to further increase the nutritious value of wheat. Crop profiles must assure value propositions for all actors across the supply chain and consider processors requirements in product development.
Key message
Discovery and mapping of a susceptibility factor located on the short arm of wheat chromosome 7A whose deletion makes plants resistant to Fusarium head blight.
Abstract
Fusarium head blight (FHB) disease of wheat caused by Fusarium spp. deteriorates both quantity and quality of the crop. Manipulation of susceptibility factors, the plant genes facilitating disease development, offers a novel and alternative strategy for enhancing FHB resistance in plants. In this study, a major effect susceptibility gene for FHB was identified on the short arm of chromosome 7A (7AS). Nullisomic–tetrasomic lines for homoeologous group-7 of wheat revealed dosage effect of the gene, with tetrasomic 7A being more susceptible than control Chinese Spring wheat, qualifying it as a genuine susceptibility factor. Five chromosome 7A inter-varietal substitution lines and a tetraploid Triticum dicoccoides 7A substitution line showed similar susceptibility as that of Chinese Spring, indicating toward the commonality of the susceptibility factor among these diverse genotypes. The susceptibility factor was named as Sf-Fhb-7AS and mapped on chromosome 7AS to a 48.5–50.5 Mb peri-centromeric region between del7AS-3 and del7AS-8. Our results showed that deletion of Sf-Fhb-7AS imparts 50–60% type 2 FHB resistance and its manipulation can be used to enhance resistance against FHB in wheat.
Fusarium head blight (FHB) disease of wheat caused by Fusarium spp. deteriorates both quantity and quality of the crop. Manipulation of susceptibility factors, the genes facilitating disease development in plants, offers a novel and alternative strategy for enhancing FHB resistance in plants. In this study, a major effect susceptibility gene for FHB was identified on the short arm of chromosome 7A (7AS). Nullisomic-tetrasomic lines for homoeologous group-7 of wheat revealed dosage effect of the gene, with tetrasomic 7A being more susceptible than control Chinese Spring wheat, qualifying it as a bonafide susceptibility factor. The gene locus was conserved in six chromosome 7A inter-varietal wheat substitution lines of diverse origin and a tetraploid Triticum dicoccoides genotype. The susceptibility gene was named as SF 7AS FHB and mapped on chromosome 7AS to 48.5-50.5 Mb peri-centromeric region between del7AS-3 and del7AS-8. Our results showed that deletion of SF 7AS FHB imparts ~ 50-60% type 2 FHB resistance (against the spread of the fungal pathogen) and its manipulation may lead to enhanced resistance against FHB in wheat.
Highlight
Discovery and mapping of a conserved susceptibility factor located on the short arm of wheat chromosome 7A whose deletion makes plants resistant to Fusarium Head Blight.
The structure and function of every living organism in the biosphere are shaped and directed by its genome, respectively. The genome of an organism is an abode of all the genes and other DNA segments, known and yet to be known, responsible for its metabolism, survival, growth, development, interaction with the environment, defense, reproduction, senescence, etc. To exploit any species as a biological resource at molecular level to serve human kind in form of good(s) and/or service(s), primarily requires a thorough understanding of its genes and their locations in the genome. Genome mapping is a technique that aids in determining the location of genes, and/or its(or their) markers(s), of interest in relation to other gene(s), and/or its (or their) marker(s), within the genome. Since all the genes of commercial interest are nor present in the same individual/taxa, it is essential to move gene(s) of interest from available source(s) to the required target(s), which is made easy with the knowledge of genome map. Genome mapping has wider applications in modern molecular biology: from the applied areas, such as genetic improvement of organisms, to the fundamental research such as genome sequencing. It needs proper tools and techniques such as various kinds of markers and mapping populations. To choose an appropriate tool and method requires thorough knowledge of myriad basket of available genome mapping tools. This chapter brings a complete, comprehensive and updated information that is necessary to understand genome of interest and use appropriate mapping tools, such as markers and mapping populations, for mapping of genes of interest in the genome of an organism of interest, ultimately to help deriving useful product(s) and/or service(s) to meet various needs of humankind in the wake of everchanging environment and never-receding human populations. This chapter also offers future prospects of available genome mapping tools and opportunities to develop new tools in the light of advanced techniques available for DNA sequencing and rapid generation advancement of mapping populations.
Several molecular markers have been developed for breeding major crops owing to their significance, ease, and suitability. Out of these DNA markers are frequently used ones; therefore, in this chapter, we describe the DNA markers to map major genes with regard to their principle, applicability, and methods. The two major classes of DNA markers are based on (i) DNA hybridization, e.g., restriction fragment polymorphism, DNA chips, etc.,. and (ii) polymerase chain reaction (PCR), e.g., SSR, RAPD, AFLP, and SNP. Developing trait-linked markers involves the segregation of populations demonstrating target traits followed by reliable phenotyping methods. With the help of these techniques, trait-linked markers may be used in two situations: (i) in the absence of any biological information and (ii) with available information about the trait.
A reference sequence of the barley genome—some 12 years ago—this goal seemed unrealistic to achieve based on the available technology. Still, a group of international barley scientists developed a vision of how such a resource could be developed by joint efforts and by revisiting once established strategies against new possibilities facilitated through technological innovation. This chapter provides an overview of the main steps taken toward the publication of a first reference sequence of the barley genome in 2017 and how this represents the beginning rather than the end of genome research in barley.
Insights from the annotated wheat genome
Wheat is one of the major sources of food for much of the world. However, because bread wheat's genome is a large hybrid mix of three separate subgenomes, it has been difficult to produce a high-quality reference sequence. Using recent advances in sequencing, the International Wheat Genome Sequencing Consortium presents an annotated reference genome with a detailed analysis of gene content among subgenomes and the structural organization for all the chromosomes. Examples of quantitative trait mapping and CRISPR-based genome modification show the potential for using this genome in agricultural research and breeding. Ramírez-González et al. exploited the fruits of this endeavor to identify tissue-specific biased gene expression and coexpression networks during development and exposure to stress. These resources will accelerate our understanding of the genetic basis of bread wheat.
Science , this issue p. eaar7191 ; see also p. eaar6089
Polyploidy is the major mechanism of speciation in flowering plants. All genomes of ancient species that are the progenitors of extant plant species experienced polyploidization. Three consecutive stages of polyploidization, i.e., ancient polyploidization, tetra-, and hexaploidization, resulted in the emergence of modern allohexaploid bread wheat Triticum aestivum L. with the BBAADD genome. Polyploidization and subsequent stabilization of the polyploid genome of T. aestivum led, on one hand, to cytological diploidization and, on the other hand, to structural and functional asymmetry of its three subgenomes. In recent years, there has been a sharp increase in the data accumulation on the origin and structure of the bread wheat genomes a result of analysis of genomes and transcripomes of natural and synthetic wheats using modern mapping and sequencing methods. This review provides up-to-date information on the peculiarities of the T. aestivum genome reorganization, which affected its structure and functioning.
Various factors affecting in vitro regeneration like different carbon sources, different gelling agents, and growth additives were assessed comprehensively for callus induction and plant regeneration for five Indian wheat cultivars using mature embryos as the explants for the first time. The tissue culture responses of cultivars WH-1105, HD-2967, and PBW-343 have not been reported earlier. Besides, the effect of different concentrations of the cytokinin, zeatin has also been optimized. Using the optimized factors, the efficiency of five different varieties, i.e., HD 2967, C 306, RAJ 3765, WH 1105, and PBW 343 was evaluated for regeneration. Modified MS basal medium containing dicamba reduced precocious germination of the embryo and induced embryogenic callus more efficiently. Removal of embryogenic calli from non-regenerable structures during early callus phase improved plant regeneration. These calli on zeatin (1.0 mgl-1) and dicamba (0.1 mgl-1) containing medium showed the highest regeneration frequency (98%) with a maximum of 8-9 shoots per calli. Maltose had the maximum callusing and regeneration percentage than other carbon sources. Various gelling agents did not have any significant difference on the regeneration. Of all the varieties, C-306 and HD-2967 were found to be more regenerative and can be used in transformation experiments.
Background:
The large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high resolution genome maps with saturated marker scaffolds to anchor and orient BAC contigs/ sequence scaffolds for whole genome assembly. Radiation hybrid (RH) mapping has proven to be an excellent tool for the development of such maps for it offers much higher and more uniform marker resolution across the length of the chromosome compared to genetic mapping and does not require marker polymorphism per se, as it is based on presence (retention) vs. absence (deletion) marker assay.
Methods:
In this study, a 178 line RH panel was genotyped with SSRs and DArT markers to develop the first high resolution RH maps of the entire D-genome of Ae. tauschii accession AL8/78. To confirm map order accuracy, the AL8/78-RH maps were compared with:1) a DArT consensus genetic map constructed using more than 100 bi-parental populations, 2) a RH map of the D-genome of reference hexaploid wheat 'Chinese Spring', and 3) two SNP-based genetic maps, one with anchored D-genome BAC contigs and another with anchored D-genome sequence scaffolds. Using marker sequences, the RH maps were also anchored with a BAC contig based physical map and draft sequence of the D-genome of Ae. tauschii.
Results:
A total of 609 markers were mapped to 503 unique positions on the seven D-genome chromosomes, with a total map length of 14,706.7 cR. The average distance between any two marker loci was 29.2 cR which corresponds to 2.1 cM or 9.8 Mb. The average mapping resolution across the D-genome was estimated to be 0.34 Mb (Mb/cR) or 0.07 cM (cM/cR). The RH maps showed almost perfect agreement with several published maps with regard to chromosome assignments of markers. The mean rank correlations between the position of markers on AL8/78 maps and the four published maps, ranged from 0.75 to 0.92, suggesting a good agreement in marker order. With 609 mapped markers, a total of 2481 deletions for the whole D-genome were detected with an average deletion size of 42.0 Mb. A total of 520 markers were anchored to 216 Ae. tauschii sequence scaffolds, 116 of which were not anchored earlier to the D-genome.
Conclusion:
This study reports the development of first high resolution RH maps for the D-genome of Ae. tauschii accession AL8/78, which were then used for the anchoring of unassigned sequence scaffolds. This study demonstrates how RH mapping, which offered high and uniform resolution across the length of the chromosome, can facilitate the complete sequence assembly of the large and complex plant genomes.
Polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible to construct a mapping population.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0582-8) contains supplementary material, which is available to authorized users.
We produced a reference sequence of the 1-gigabase chromosome 3B of hexaploid
bread wheat. By sequencing 8452 bacterial artificial chromosomes in pools, we
assembled a sequence of 774 megabases carrying 5326 protein-coding genes,
1938 pseudogenes, and 85% of transposable elements. The distribution of structural
and functional features along the chromosome revealed partitioning correlated with
meiotic recombination. Comparative analyses indicated high wheat-specific inter- and
intrachromosomal gene duplication activities that are potential sources of variability
for adaption. In addition to providing a better understanding of the organization,
function, and evolution of a large and polyploid genome, the availability of a
high-quality sequence anchored to genetic maps will accelerate the identification
of genes underlying important agronomic traits.
An ordered draft sequence of the 17-gigabase hexaploid bread wheat (Triticum aestivum) genome has been produced by sequencing isolated chromosome arms. We have annotated 124,201 gene loci distributed nearly
evenly across the homeologous chromosomes and subgenomes. Comparative gene analysis of wheat subgenomes and extant diploid
and tetraploid wheat relatives showed that high sequence similarity and structural conservation are retained, with limited
gene loss, after polyploidization. However, across the genomes there was evidence of dynamic gene gain, loss, and duplication
since the divergence of the wheat lineages. A high degree of transcriptional autonomy and no global dominance was found for
the subgenomes. These insights into the genome biology of a polyploid crop provide a springboard for faster gene isolation,
rapid genetic marker development, and precise breeding to meet the needs of increasing food demand worldwide.
High-density single nucleotide polymorphism (SNP) genotyping arrays are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships between individuals in populations and studying marker-trait associations in mapping experiments. We developed a genotyping array including about 90 000 gene-associated SNPs and used it to characterize genetic variation in allohexaploid and allotetraploid wheat populations. The array includes a significant fraction of common genome-wide distributed SNPs that are represented in populations of diverse geographical origin. We used density-based spatial clustering algorithms to enable high-throughput genotype calling in complex data sets obtained for polyploid wheat. We show that these model-free clustering algorithms provide accurate genotype calling in the presence of multiple clusters including clusters with low signal intensity resulting from significant sequence divergence at the target SNP site or gene deletions. Assays that detect low-intensity clusters can provide insight into the distribution of presence-absence variation (PAV) in wheat populations. A total of 46 977 SNPs from the wheat 90K array were genetically mapped using a combination of eight mapping populations. The developed array and cluster identification algorithms provide an opportunity to infer detailed haplotype structure in polyploid wheat and will serve as an invaluable resource for diversity studies and investigating the genetic basis of trait variation in wheat.
Next-generation, whole genome shotgun (WGS) assemblies of complex genomes are highly enabling, but fail to link nearby sequence contigs with each other or provide a linear order of contigs along individual chromosomes. Here, we introduce a strategy based on sequencing progeny of a segregating population that allows the de novo production of a genetically anchored, linear assembly of the gene space of an organism. We demonstrate the power of the approach by reconstructing the chromosomal organization of the gene space of barley, a large, complex and highly repetitive 5.1-Gb genome. We evaluate the robustness of the new assembly by comparison to a recently released physical and genetic framework of the barley genome, and to different genetically ordered sequence-based genotypic datasets. The method is independent of the need for any prior sequence resources and will enable the rapid and cost efficient establishment of powerful genomic information for many species. This article is protected by copyright. All rights reserved.
As for other major crops, achieving a complete wheat genome sequence is essential for the application of genomics to breeding new and improved varieties. To overcome the complexities of the large, highly repetitive and hexaploid wheat genome, the International Wheat Genome Sequencing Consortium established a chromosome-based strategy that was validated by the construction of the physical map of chromosome 3B. Here, we present improved strategies for the construction of highly integrated and ordered wheat physical maps, using chromosome 1BL as a template, and illustrate their potential for evolutionary studies and map-based cloning.
Using a combination of novel high throughput marker assays and an assembly program, we developed a high quality physical map representing 93% of wheat chromosome 1BL, anchored and ordered with 5,489 markers including 1,161 genes. Analysis of the gene space organization and evolution revealed that gene distribution and conservation along the chromosome results from the superimposition of the ancestral grass and recent wheat evolutionary patterns leading to a peak of synteny in the central part of the chromosome arm and an increased density of non collinear genes towards the telomere. With a density of about 11 markers per Mb, the 1BL physical map provides 916 markers, including 193 genes, for fine mapping the 40 QTLs mapped on this chromosome.
Here, we demonstrate that high marker density physical maps can be developed in complex genomes such as wheat to accelerate map-based cloning, gain new insights into genome evolution, and provide a foundation for reference sequencing.
The current limitations in genome sequencing technology require the construction of physical maps for high-quality draft sequences of large plant genomes, such as that of Aegilops tauschii, the wheat D-genome progenitor. To construct a physical map of the Ae. tauschii genome, we fingerprinted 461,706 bacterial artificial chromosome clones, assembled contigs, designed a 10K Ae. tauschii Infinium SNP array, constructed a 7,185-marker genetic map, and anchored on the map contigs totaling 4.03 Gb. Using whole genome shotgun reads, we extended the SNP marker sequences and found 17,093 genes and gene fragments. We showed that collinearity of the Ae. tauschii genes with Brachypodium distachyon, rice, and sorghum decreased with phylogenetic distance and that structural genome evolution rates have been high across all investigated lineages in subfamily Pooideae, including that of Brachypodieae. We obtained additional information about the evolution of the seven Triticeae chromosomes from 12 ancestral chromosomes and uncovered a pattern of centromere inactivation accompanying nested chromosome insertions in grasses. We showed that the density of noncollinear genes along the Ae. tauschii chromosomes positively correlates with recombination rates, suggested a cause, and showed that new genes, exemplified by disease resistance genes, are preferentially located in high-recombination chromosome regions.
Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGA(m)A(m)), is central to wheat evolution, domestication and genetic improvement. The progenitor species of the A genome is the diploid wild einkorn wheat T. urartu, which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome) and Ae. tauschii (the donor of the D genome), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T. urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T. urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.
About 8,000 years ago in the Fertile Crescent, a spontaneous hybridization of the wild diploid grass Aegilops tauschii (2n = 14; DD) with the cultivated tetraploid wheat Triticum turgidum (2n = 4x = 28; AABB) resulted in hexaploid wheat (T. aestivum; 2n = 6x = 42; AABBDD). Wheat has since become a primary staple crop worldwide as a result of its enhanced adaptability to a wide range of climates and improved grain quality for the production of baker's flour. Here we describe sequencing the Ae. tauschii genome and obtaining a roughly 90-fold depth of short reads from libraries with various insert sizes, to gain a better understanding of this genetically complex plant. The assembled scaffolds represented 83.4% of the genome, of which 65.9% comprised transposable elements. We generated comprehensive RNA-Seq data and used it to identify 43,150 protein-coding genes, of which 30,697 (71.1%) were uniquely anchored to chromosomes with an integrated high-density genetic map. Whole-genome analysis revealed gene family expansion in Ae. tauschii of agronomically relevant gene families that were associated with disease resistance, abiotic stress tolerance and grain quality. This draft genome sequence provides insight into the environmental adaptation of bread wheat and can aid in defining the large and complicated genomes of wheat species.
Carh
ta Gene: is an integrated genetic and radiation hybrid (RH) mapping tool which can deal with multiple populations, including mixtures
of genetic and RH data. Carh
ta Gene: performs multipoint maximum likelihood estimations with accelerated expectation–maximization algorithms for some pedigrees
and has sophisticated algorithms for marker ordering. Dedicated heuristics for framework mapping are also included. Carh
ta Gene: can be used as a C++ library, through a shell command and a graphical interface. The XML output for companion tools is integrated.
Availability: The program is available free of charge from www.inra.fr/bia/T/CarthaGene for Linux, Windows and Solaris machines (with Open Source).
Contact: tschiex{at}toulouse.inra.fr
Bread wheat (Triticum aestivum) is a globally important crop, accounting for 20 per cent of the calories consumed by humans. Major efforts are underway worldwide to increase wheat production by extending genetic diversity and analysing key traits, and genomic resources can accelerate progress. But so far the very large size and polyploid complexity of the bread wheat genome have been substantial barriers to genome analysis. Here we report the sequencing of its large, 17-gigabase-pair, hexaploid genome using 454 pyrosequencing, and comparison of this with the sequences of diploid ancestral and progenitor genomes. We identified between 94,000 and 96,000 genes, and assigned two-thirds to the three component genomes (A, B and D) of hexaploid wheat. High-resolution synteny maps identified many small disruptions to conserved gene order. We show that the hexaploid genome is highly dynamic, with significant loss of gene family members on polyploidization and domestication, and an abundance of gene fragments. Several classes of genes involved in energy harvesting, metabolism and growth are among expanded gene families that could be associated with crop productivity. Our analyses, coupled with the identification of extensive genetic variation, provide a resource for accelerating gene discovery and improving this major crop.
Physical mapping and genome sequencing are underway for the ≈17 Gb wheat genome. Physical mapping methods independent of meiotic recombination, such as radiation hybrid (RH) mapping, will aid precise anchoring of BAC contigs in the large regions of suppressed recombination in Triticeae genomes. Reports of endosperm development following pollination with irradiated pollen at dosages that cause embryo abortion prompted us to investigate endosperm as a potential source of RH mapping germplasm. Here, we report a novel approach to construct RH based physical maps of all seven D-genome chromosomes of the hexaploid wheat 'Chinese Spring', simultaneously. An 81-member subset of endosperm samples derived from 20-Gy irradiated pollen was genotyped for deletions, and 737 markers were mapped on seven D-genome chromosomes. Analysis of well-defined regions of six chromosomes suggested a map resolution of ∼830 kb could be achieved; this estimate was validated with assays of markers from a sequenced contig. We estimate that the panel contains ∼6,000 deletion bins for D-genome chromosomes and will require ∼18,000 markers for high resolution mapping. Map-based deletion estimates revealed a majority of 1-20 Mb interstitial deletions suggesting mutagenic repair of double-strand breaks in pollen provides a useful resource for RH mapping and map based cloning studies.
Advancements in next-generation sequencing technology have enabled whole genome re-sequencing in many species providing unprecedented discovery and characterization of molecular polymorphisms. There are limitations, however, to next-generation sequencing approaches for species with large complex genomes such as barley and wheat. Genotyping-by-sequencing (GBS) has been developed as a tool for association studies and genomics-assisted breeding in a range of species including those with complex genomes. GBS uses restriction enzymes for targeted complexity reduction followed by multiplex sequencing to produce high-quality polymorphism data at a relatively low per sample cost. Here we present a GBS approach for species that currently lack a reference genome sequence. We developed a novel two-enzyme GBS protocol and genotyped bi-parental barley and wheat populations to develop a genetically anchored reference map of identified SNPs and tags. We were able to map over 34,000 SNPs and 240,000 tags onto the Oregon Wolfe Barley reference map, and 20,000 SNPs and 367,000 tags on the Synthetic W9784 × Opata85 (SynOpDH) wheat reference map. To further evaluate GBS in wheat, we also constructed a de novo genetic map using only SNP markers from the GBS data. The GBS approach presented here provides a powerful method of developing high-density markers in species without a sequenced genome while providing valuable tools for anchoring and ordering physical maps and whole-genome shotgun sequence. Development of the sequenced reference genome(s) will in turn increase the utility of GBS data enabling physical mapping of genes and haplotype imputation of missing data. Finally, as a result of low per-sample costs, GBS will have broad application in genomics-assisted plant breeding programs.
Reference populations are valuable resources in genetics studies for determining marker order, marker selection, trait mapping, construction of large-insert libraries, cross-referencing marker platforms, and genome sequencing. Reference populations can be propagated indefinitely, they are polymorphic and have normal segregation. Described are two new reference populations who share the same parents of the original wheat reference population Synthetic W7984 (Altar84/ Aegilops tauschii (219) CIGM86.940) x Opata M85, an F(1)-derived doubled haploid population (SynOpDH) of 215 inbred lines and a recombinant inbred population (SynOpRIL) of 2039 F(6) lines derived by single-plant self-pollinations. A linkage map was constructed for the SynOpDH population using 1446 markers. In addition, a core set of 42 SSR markers was genotyped on SynOpRIL. A new approach to identifying a core set of markers used a step-wise selection protocol based on polymorphism, uniform chromosome distribution, and reliability to create nested sets starting with one marker per chromosome, followed by two, four, and six. It is suggested that researchers use these markers as anchors for all future mapping projects to facilitate cross-referencing markers and chromosome locations. To enhance this public resource, researchers are strongly urged to validate line identities and deposit their data in GrainGenes so that others can benefit from the accumulated information.
Sponsored by the National Science Foundation and the U.S. Department of Agriculture, a wheat genome sequencing workshop was held November 10-11, 2003, in Washington, DC. It brought together 63 scientists of diverse research interests and institutions, including 45 from the United States and 18 from a dozen foreign countries (see list of participants at http://www.ksu.edu/igrow). The objectives of the workshop were to discuss the status of wheat genomics, obtain feedback from ongoing genome sequencing projects, and develop strategies for sequencing the wheat genome. The purpose of this report is to convey the information discussed at the workshop and provide the basis for an ongoing dialogue, bringing forth comments and suggestions from the genetics community.
Because of the huge size of the common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) genome of 17,300 Mb, sequencing and mapping of the expressed portion is a logical first step for gene discovery. Here we report mapping of 7104 expressed sequence tag (EST) unigenes by Southern hybridization into a chromosome bin map using a set of wheat aneuploids and deletion stocks. Each EST detected a mean of 4.8 restriction fragments and 2.8 loci. More loci were mapped in the B genome (5774) than in the A (5173) or D (5146) genomes. The EST density was significantly higher for the D genome than for the A or B. In general, EST density increased relative to the physical distance from the centromere. The majority of EST-dense regions are in the distal parts of chromosomes. Most of the agronomically important genes are located in EST-dense regions. The chromosome bin map of ESTs is a unique resource for SNP analysis, comparative mapping, structural and functional analysis, and polyploid evolution, as well as providing a framework for constructing a sequence-ready, BAC-contig map of the wheat genome.
This report describes the rationale, approaches, organization, and resource development leading to a large-scale deletion bin map of the hexaploid (2n = 6x = 42) wheat genome (Triticum aestivum L.). Accompanying reports in this issue detail results from chromosome bin-mapping of expressed sequence tags (ESTs) representing genes onto the seven homoeologous chromosome groups and a global analysis of the entire mapped wheat EST data set. Among the resources developed were the first extensive public wheat EST collection (113,220 ESTs). Described are protocols for sequencing, sequence processing, EST nomenclature, and the assembly of ESTs into contigs. These contigs plus singletons (unassembled ESTs) were used for selection of distinct sequence motif unigenes. Selected ESTs were rearrayed, validated by 5' and 3' sequencing, and amplified for probing a series of wheat aneuploid and deletion stocks. Images and data for all Southern hybridizations were deposited in databases and were used by the coordinators for each of the seven homoeologous chromosome groups to validate the mapping results. Results from this project have established the foundation for future developments in wheat genomics.
Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM) and barley (Oregon Wolfe Barley) recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.
To improve our understanding of the organization and evolution of the wheat (Triticum aestivum) genome, we sequenced and annotated 13-Mb contigs (18.2 Mb) originating from different regions of its largest chromosome, 3B (1 Gb), and produced a 2x chromosome survey by shotgun Illumina/Solexa sequencing. All regions carried genes irrespective of their chromosomal location. However, gene distribution was not random, with 75% of them clustered into small islands containing three genes on average. A twofold increase of gene density was observed toward the telomeres likely due to high tandem and interchromosomal duplication events. A total of 3222 transposable elements were identified, including 800 new families. Most of them are complete but showed a highly nested structure spread over distances as large as 200 kb. A succession of amplification waves involving different transposable element families led to contrasted sequence compositions between the proximal and distal regions. Finally, with an estimate of 50,000 genes per diploid genome, our data suggest that wheat may have a higher gene number than other cereals. Indeed, comparisons with rice (Oryza sativa) and Brachypodium revealed that a high number of additional noncollinear genes are interspersed within a highly conserved ancestral grass gene backbone, supporting the idea of an accelerated evolution in the Triticeae lineages.
Chromosomal breaks occurred in the progeny of a common wheat (Triticum aestivum L. em Thell; 2n = 6x = 42, genome formula AABBDD) cultivar Chinese Spring with a monosomic addition of an alien chromosome from Aegilops cylindrica Host (2n = 4x = 28, CCDD) or A. triuncialls L. (2n = 4x = 28, UUCC) or a chromosomal segment from A. speltoides Tausch (2n = 2x = 14, SS). We identified 436 deletions by C-banding. The deletion chromosomes were transmitted stably to the offspring. We
selected deletion homozygotes in the progeny of the deletion heterozygotes and established homozygous lines for about 80%
of the deletions. We falled to establish homozygous lines for most of the deletions in the short arm of chromosome 2A and
for all deletions in the short arm of chromosome 4B, because plants homozygous for these deletions were sterile. We could
not obtain any homozygotes for larger deletions in the long arms of chromosomes 4A, 5A, 5B, and 5D. The deletion stocks showed
variations in morphological, physiological, and bichemical traits, depending on the size of their chromosomal deficiency,
and are powerful tools for physical mapping of wheat chromosomes.
Chromosome 1H (approximately 622 Mb) of barley (Hordeum vulgare) was isolated by flow sorting and shotgun sequenced by GSFLX pyrosequencing to 1.3-fold coverage. Fluorescence in situ hybridization and stringent sequence comparison against genetically mapped barley genes revealed 95% purity of the sorted chromosome 1H fraction. Sequence comparison against the reference genomes of rice (Oryza sativa) and sorghum (Sorghum bicolor) and against wheat (Triticum aestivum) and barley expressed sequence tag datasets led to the estimation of 4,600 to 5,800 genes on chromosome 1H, and 38,000 to 48,000 genes in the whole barley genome. Conserved gene content between chromosome 1H and known syntenic regions of rice chromosomes 5 and 10, and of sorghum chromosomes 1 and 9 was detected on a per gene resolution. Informed by the syntenic relationships between the two reference genomes, genic barley sequence reads were integrated and ordered to deduce a virtual gene map of barley chromosome 1H. We demonstrate that synteny-based analysis of low-pass shotgun sequenced flow-sorted Triticeae chromosomes can deliver linearly ordered high-resolution gene inventories of individual chromosomes, which complement extensive Triticeae expressed sequence tag datasets. Thus, integration of genomic, transcriptomic, and synteny-derived information represents a major step toward developing reference sequences of chromosomes and complete genomes of the most important plant tribe for mankind.
High resolution physical maps of vertebrate species' chromosomes empower comparative genomics discovery and are indispensible for sequence assembly precision.
Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR amplification. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1% for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays were shown to be useful for genotyping wheat cultivars. This study demonstrated that the GoldenGate assay is a very efficient tool for high-throughput genotyping of polyploid wheat, opening new possibilities for the analysis of genetic variation in wheat and dissection of genetic basis of complex traits using association mapping approach.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-009-1059-5) contains supplementary material, which is available to authorized users.
In wheat (Triticum aestivum L.), the crossover (CO) frequency increases gradually from the centromeres to the telomeres. However, little is known about the factors affecting both the distribution and the intensity of recombination along this gradient. To investigate this, we studied in detail the pattern of CO along chromosome 3B of bread wheat. A dense reference genetic map comprising 102 markers homogeneously distributed along the chromosome was compared to a physical deletion map. Most of the COs (90%) occurred in the distal subtelomeric regions that represent 40% of the chromosome. About 27% of the proximal regions surrounding the centromere showed a very weak CO frequency with only three COs found in the 752 gametes studied. Moreover, we observed a clear decrease of CO frequency on the distal region of the short arm. Finally, the intensity of interference was assessed for the first time in wheat using a Gamma model. The results showed m values of 1.2 for male recombination and 3.5 for female recombination, suggesting positive interference along wheat chromosome 3B.
As the staple food for 35% of the world's population, wheat is one of the most important crop species. To date, sequence-based
tools to accelerate wheat improvement are lacking. As part of the international effort to sequence the 17–billion–base-pair
hexaploid bread wheat genome (2n = 6x = 42 chromosomes), we constructed a bacterial artificial chromosome (BAC)–based integrated physical map of the largest chromosome,
3B, that alone is 995 megabases. A chromosome-specific BAC library was used to assemble 82% of the chromosome into 1036 contigs
that were anchored with 1443 molecular markers, providing a major resource for genetic and genomic studies. This physical
map establishes a template for the remaining wheat chromosomes and demonstrates the feasibility of constructing physical maps
in large, complex, polyploid genomes with a chromosome-based approach.
Radiation hybrid (RH) mapping, a somatic cell genetic technique, was developed as a general approach for constructing long-range
maps of mammalian chromosomes. This statistical method depends on x-ray breakage of chromosomes to determine the distances
between DNA markers, as well as their order on the chromosome. In addition, the method allows the relative likelihoods of
alternative marker orders to be determined. The RH procedure was used to map 14 DNA probes from a region of human chromosome
21 spanning 20 megabase pairs. The map was confirmed by pulsed-field gel electrophoretic analysis. The results demonstrate
the effectiveness of RH mapping for constructing high-resolution, contiguous maps of mammalian chromosomes.
Physical mapping of wheat chromosomes has revealed small chromosome segments of high gene density and frequent recombination interspersed with relatively large regions of low gene density and infrequent recombination. We constructed a detailed genetic and physical map of one highly recombinant region on the long arm of chromosome 5B. This distally located region accounts for 4% of the physical size of the long arm and at least 30% of the recombination along the entire chromosome. Multiple crossovers occurred within this region, and the degree of recombination is at least 11-fold greater than the genomic average. Characteristics of the region such as gene order and frequency of recombination appear to be conserved throughout the evolution of the Triticeae. The region is more prone to chromosome breakage by gametocidal gene action than gene-poor regions, and evidence for genomic instability was implied by loss of gene collinearity for six loci among the homeologous regions. These data suggest that a unique level of chromatin organization exists within gene-rich recombination hot spots. The many agronomically important genes in this region should be accessible by positional cloning.
In maize (Zea mays L., 2n = 2x = 20), map-based cloning and genome organization studies are often complicated because of the complexity of the genome. Maize chromosome addition lines of hexaploid cultivated oat (Avena sativa L., 2n = 6x = 42), where maize chromosomes can be individually manipulated, represent unique materials for maize genome analysis. Maize chromosome addition lines are particularly suitable for the dissection of a single maize chromosome using radiation because cultivated oat is an allohexaploid in which multiple copies of the oat basic genome provide buffering to chromosomal aberrations and other mutations. Irradiation (gamma rays at 30, 40, and 50 krad) of a monosomic maize chromosome 9 addition line produced maize chromosome 9 radiation hybrids (M9RHs)-oat lines possessing different fragments of maize chromosome 9 including intergenomic translocations and modified maize addition chromosomes with internal and terminal deletions. M9RHs with 1 to 10 radiation-induced breaks per chromosome were identified. We estimated that a panel of 100 informative M9RHs (with an average of 3 breaks per chromosome) would allow mapping at the 0. 5- to 1.0-Mb level of resolution. Because mapping with maize chromosome addition lines and radiation hybrid derivatives involves assays for the presence or absence of a given marker, monomorphic markers can be quickly and efficiently mapped to a chromosome region. Radiation hybrid derivatives also represent sources of region-specific DNA for cloning of genes or DNA markers.
We have constructed a physical map of the human genome by using a panel of 90 whole-genome radiation hybrids (the TNG panel)
in conjunction with 40,322 sequence-tagged sites (STSs) derived from random genomic sequences as well as expressed sequences.
Of 36,678 STSs on the TNG radiation hybrid map, only 3604 (9.8%) were absent from the unassembled draft sequence of the human
genome. Of 20,030 STSs ordered on the TNG map as well as the assembled human genome draft sequence and the Celera assembled
human genome sequence, 36% of the STSs had a discrepant order between the working draft sequence and the Celera sequence.
The TNG map order was identical to one of the two sequence orders in 60% of these discrepant cases.
Univalent chromosomes at meiotic metaphase I have a tendency to misdivide at the centromeres. Fusion of the misdivision products may produce Robertsonian translocations. The fine structure of the centromeres in Robertsonian wheat-rye translocation chromosomes was analyzed by fluorescence in situ hybridization (FISH) using two centromere-specific DNA clones: pRCS1, derived from rice, and pAWRC1, derived from rye. Clone pRCS1 hybridizes to the centromeres of all grasses including wheat and rye, whereas clone pAWRC1 is rye specific and hybridizes only to the centromeres of rye. Four of the six wheat-rye translocations derived from a single centric misdivision event (1st generation translocations) had hybrid centromeres, with approximately half of the centromere derived from rye and half from wheat. In the two other 1st generation translocations, the entire centromere was derived from rye. Among eight reconstructed wheat and rye chromosomes that originated from two consecutive centric misdivision-fusion events (2nd generation translocations), T1BS.1BL (derived from T1BS.1RL and T1RS.1BL) and one of three T2BS.2BL (derived from T2RS.2BL and T2BS.2RL) had hybrid centromeres. T1RS.1RL (derived from T1BS.1RL and T1RS.1BL), two of three T2BS.2BL, and all three T2RS.2RL (derived from T2RS.2BL and T2BS.2RL) had rye centromeres. All three 3rd generation translocations had hybrid centromeres with approximately half of the centromere derived from rye. There were no indications that the composite structure of the centromere in these chromosomes affected their behavior in mitosis or meiosis. These observations support the notion of a compound structure of the centromere in higher organisms, and indicate that during the centric breakage-fusion event, centromere breakage may occur in different positions along the segment of the chromosome that interacts with the spindle fibers. Normal behavior of the 1st, 2nd, and 3rd generation centric translocations in mitosis and meiosis indicates that, at least in wheat and rye, centromeres are not chromosome specific.
A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human-mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.
Radiation hybrid (RH) mapping is based on radiation-induced chromosome breakage and analysis of chromosome segment retention or loss using molecular markers. In durum wheat (Triticum turgidum L., AABB), an alloplasmic durum line [(lo) durum] has been identified with chromosome 1D of T. aestivum L. (AABBDD) carrying the species cytoplasm-specific (scsae) gene. The chromosome 1D of this line segregates as a whole without recombination, precluding the use of conventional genome mapping. A radiation hybrid mapping population was developed from a hemizygous (lo) scsae--line using 35 krad gamma rays. The analysis of 87 individuals of this population with 39 molecular markers mapped on chromosome 1D revealed 88 radiation-induced breaks in this chromosome. This number of chromosome 1D breaks is eight times higher than the number of previously identified breaks and should result in a 10-fold increase in mapping resolution compared to what was previously possible. The analysis of molecular marker retention in our radiation hybrid mapping panel allowed the localization of scsae and 8 linked markers on the long arm of chromosome 1D. This constitutes the first report of using RH mapping to localize a gene in wheat and illustrates that this approach is feasible in a species with a large complex genome.
Physical mapping methods that do not rely on meiotic recombination are necessary for complex polyploid genomes such as wheat (Triticum aestivum L.). This need is due to the uneven distribution of recombination and significant variation in genetic to physical distance ratios. One method that has proven valuable in a number of nonplant and plant systems is radiation hybrid (RH) mapping. This work presents, for the first time, a high-resolution radiation hybrid map of wheat chromosome 1D (D genome) in a tetraploid durum wheat (T. turgidum L., AB genomes) background. An RH panel of 87 lines was used to map 378 molecular markers, which detected 2312 chromosome breaks. The total map distance ranged from approximately 3,341 cR(35,000) for five major linkage groups to 11,773 cR(35,000) for a comprehensive map. The mapping resolution was estimated to be approximately 199 kb/break and provided the starting point for BAC contig alignment. To date, this is the highest resolution that has been obtained by plant RH mapping and serves as a first step for the development of RH resources in wheat.