Construction and application for QTL analysis of a Restriction Site Associated DNA (RAD) linkage map in barley

Crop and Soil Science Department, Oregon State University, Corvallis, Oregon, USA.
BMC Genomics (Impact Factor: 4.04). 01/2011; 12(1):4. DOI: 10.1186/1471-2164-12-4
Source: PubMed

ABSTRACT Linkage maps are an integral resource for dissection of complex genetic traits in plant and animal species. Canonical map construction follows a well-established workflow: an initial discovery phase where genetic markers are mined from a small pool of individuals, followed by genotyping of selected mapping populations using sets of marker panels. A newly developed sequence-based marker technology, Restriction site Associated DNA (RAD), enables synchronous single nucleotide polymorphism (SNP) marker discovery and genotyping using massively parallel sequencing. The objective of this research was to assess the utility of RAD markers for linkage map construction, employing barley as a model system. Using the published high density EST-based SNP map in the Oregon Wolfe Barley (OWB) mapping population as a reference, we created a RAD map using a limited set of prior markers to establish linakge group identity, integrated the RAD and prior data, and used both maps for detection of quantitative trait loci (QTL).
Using the RAD protocol in tandem with the Illumina sequence by synthesis platform, a total of 530 SNP markers were identified from initial scans of the OWB parental inbred lines--the "dominant" and "recessive" marker stocks--and scored in a 93 member doubled haploid (DH) mapping population. RAD sequence data from the structured population was converted into allele genotypes from which a genetic map was constructed. The assembled RAD-only map consists of 445 markers with an average interval length of 5 cM, while an integrated map includes 463 RAD loci and 2383 prior markers. Sequenced RAD markers are distributed across all seven chromosomes, with polymorphic loci emanating from both coding and noncoding regions in the Hordeum genome. Total map lengths are comparable and the order of common markers is identical in both maps. The same large-effect QTL for reproductive fitness traits were detected with both maps and the majority of these QTL were coincident with a dwarfing gene (ZEO) and the VRS1 gene, which determines the two-row and six-row germplasm groups of barley.
We demonstrate how sequenced RAD markers can be leveraged to produce high quality linkage maps for detection of single gene loci and QTLs. By combining SNP discovery and genotyping into parallel sequencing events, RAD markers should be a useful molecular breeding tool for a range of crop species. Expected improvements in cost and throughput of second and third-generation sequencing technologies will enable more powerful applications of the sequenced RAD marker system, including improvements in de novo genome assembly, development of ultra-high density genetic maps and association mapping.

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    • "Genomic DNA was extracted from pectoral fin tissue using DNAeasy blood and tissue kits (Qiagen Inc. CA, USA), digested with the restriction enzyme SbfI, and processed into RAD libraries following the work of Chutimanitsakun et al. (2011). Bowtie (Langmead et al. 2009) was used to align reads to the reference cichlid sequence (Metriaclima zebra v.0), and SAMtools was used for SNP calling. "
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    ABSTRACT: Exemplary systems for adaptive divergence are often characterized by their large degrees of phenotypic variation. This variation represents the outcome of generations of diversifying selection. However, adaptive radiations can also contain a hierarchy of differentiation nested within them where species display only subtle phenotypic differences that still have substantial effects on ecology, function, and ultimately fitness. Sexual -dimorphisms are also common in species displaying adaptive divergence and can be the result of differential selection between sexes that produce ecological differences between sexes. Understanding the genetic basis of subtle variation (between certain species or sexes) is therefore important for understanding the process of adaptive divergence. Using cichlids from the dramatic adaptive radiation of Lake Malawi, we focus on understanding the genetic basis of two aspects of relatively subtle phenotypic variation. This included a morphometric comparison of the patterns of craniofacial divergence between two ecologically similar species in relation to the larger adaptive radiation of Malawi, and male-female morphological divergence between their F2 hybrids. We then genetically map craniofacial traits within the context of sex and locate several regions of the genome that contribute to variation in craniofacial shape that is relevant to sexual dimorphism within species, subtle divergence between closely related species, and possibly to craniofacial divergence in the Malawi radiation as a whole. To enhance our search for candidate genes we take advantage of population genomic data and a genetic map that is anchored to the cichlid genome to determine which genes within our QTL regions are associated with SNPs that are alternatively fixed between species. In all, this study provides a holistic understanding of the genetic underpinnings of adaptive divergence in craniofacial shape. Copyright © 2015 Author et al.
    G3-Genes Genomes Genetics 06/2015; DOI:10.1534/g3.115.018226 · 2.51 Impact Factor
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    • "Once a mutant of interest is identified, MutMap enables rapid isolation of the candidate gene even when the phenotypic changes are only subtle. Additionally, novel NGS­based genotyping methods such as restriction­site associated DNA (RAD) se­ quencing (Yada et al. 2011) and genotyping­by­sequencing (GBS) (Robert et al. 2011) have enabled high­throughput genotyping in crops including those with large genome sizes, for which the application of WGS remains difficult and expensive. For instance, GBS has been recently applied to 5,000 recombinant inbred lines (RILs) for large­scale association mapping in maize (Kump et al. 2011), high­ lighting the need for large­scale phenotyping protocols for accurate association of genotype­phenotype data. "
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    ABSTRACT: More accurate, rapid, and easy phenotyping tools are required to match the recent advances in high-throughput genotyping for accelerating breeding and genetic analysis. The conventional data recording in field notebooks and then inputting data to computers for further analysis is inefficient, time-consuming, laborious, and prone to human error. Here, we report WIPPER (for Wireless Plant Phenotyper), a new phenotyping platform that combines field phenotyping and data recording with the aid of Bluetooth communication, thus saving time and labor not only for field data recoding but also for inputting data to computers. Additionally, it eliminates the risk of human error associated with phenotyping and inputting data. We applied WIPPER to 100 individuals of a rice recombinant inbred line (RIL) for measuring leaf width and relative chlorophyll content (SPAD value), and were able to record an accurate data in a significantly reduced time compared with the conventional method of data collection. We are currently using WIPPER for routine management of rice germplasm including recording and documenting information on phenotypic data, seeds, and DNA for their accelerated utilization in crop breeding.
    Breeding Science 06/2015; 65:285-289. DOI:10.1270/jsbbs.65.285 · 1.34 Impact Factor
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    • "RAD markers together with NGS have provided an efficient method that can simultaneously detect thousands of SNPs and provide genotypic data of several hundred samples with no prior genome sequence information. The effectiveness of RAD markers for development of high density genetic map and QTL analysis [34], and association mapping [35] has been successfully demonstrated in several plant species. "
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    ABSTRACT: Background In the whole genome sequencing, genetic map provides an essential framework for accurate and efficient genome assembly and validation. The main objectives of this study were to develop a high-density genetic map using RAD-Seq (Restriction-site Associated DNA Sequencing) genotyping-by-sequencing (RAD-Seq GBS) and Illumina GoldenGate assays, and to examine the alignment of the current map with the kabuli chickpea genome assembly. Results Genic single nucleotide polymorphisms (SNPs) totaling 51,632 SNPs were identified by 454 transcriptome sequencing of Cicer arietinum and Cicer reticulatum genotypes. Subsequently, an Illumina GoldenGate assay for 1,536 SNPs was developed. A total of 1,519 SNPs were successfully assayed across 92 recombinant inbred lines (RILs), of which 761 SNPs were polymorphic between the two parents. In addition, the next generation sequencing (NGS)-based GBS was applied to the same population generating 29,464 high quality SNPs. These SNPs were clustered into 626 recombination bins based on common segregation patterns. Data from the two approaches were used for the construction of a genetic map using a population derived from an intraspecific cross. The map consisted of 1,336 SNPs including 604 RAD recombination bins and 732 SNPs from Illumina GoldenGate assay. The map covered 653 cM of the chickpea genome with an average distance between adjacent markers of 0.5 cM. To date, this is the most extensive genetic map of chickpea using an intraspecific population. The alignment of the map with the CDC Frontier genome assembly revealed an overall conserved marker order; however, a few local inconsistencies within the Cicer arietinum pseudochromosome 1 (Ca1), Ca5 and Ca8 were detected. The map enabled the alignment of 215 unplaced scaffolds from the CDC Frontier draft genome assembly. The alignment also revealed varying degrees of recombination rates and hotspots across the chickpea genome. Conclusions A high-density genetic map using RAD-Seq GBS and Illumina GoldenGate assay was developed and aligned with the existing kabuli chickpea draft genome sequence. The analysis revealed an overall conserved marker order, although some localized inversions between draft genome assembly and the genetic map were detected. The current analysis provides an insight of the recombination rates and hotspots across the chickpea genome. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-708) contains supplementary material, which is available to authorized users.
    BMC Genomics 08/2014; 15(1):708. DOI:10.1186/1471-2164-15-708 · 4.04 Impact Factor
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