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Morgante, M. and Olivieri, A. M.. PCR-amplified microsatellites as markers in plant genetics. Plant J, 3: 175-182
Abstract
In order to assess the feasibility of using microsatellites as markers in plant genetics, a survey of published DNA sequence data for presence, abundance and ubiquity in higher plants of all types of dinucleotide and trinucleotide repeats with a minimum number of 10 and 7 units, respectively, was conducted. This search revealed that such microsatellites are frequent and widely distributed; they were uncovered in 34 species, with a frequency of one every 50 kb. AT repeats were by far the most frequently observed class of dinucleotide microsatellites, whereas AC/TG repeats, which are common in animals, were observed only once. TAT repeats prevailed among trinucleotides. Polymerase chain reaction amplification of (AT)n and (TAT)n microsatellites in soybean (Glycine max (L.) Merr.) revealed that they are highly polymorphic, as a consequence of length variation, somatically stable and inherited in a co-dominant Mendelian manner. The abundance and amount of information derived from such markers, together with the ease by which they can be identified, make them ideal markers for plant genetic linkage and physical mapping, population studies and varietal identification.
... Hence, to avoid misidentification, efficient DNA fingerprinting tools are needed. SSRs (Simple Sequence Repeats) are one of the most powerful marker systems available for such applications because of characteristics such as high levels of polymorphism, codominant inheritance and high discrimination power [16]. Many SSR markers have been developed for different Prunus species [17], including peach and nectarine [18][19][20][21][22][23][24], and apricot [25,26]. ...
... The UPGMA cluster analysis shows the existence of two subclusters in apricot cultivars, with mixed Romanian and North American cultivars (Figures 1 and 2). Subcluster IIb is composed mainly of Romanian validated apricot cultivars (Saturn, Sirena, Comandor and Rares) that were phenotypically selected for fruit quality within the improvement program performed during the period 1983-2006 at the Băneasa Research and Development Station for Fruit Tree Growing [16,57]. Subcluster IIa is composed of Romanian apricot cultivars (Carmela, Viorica) that were selected mainly for resistance to disease by hybridizing with North American varieties obtained from the Rutgers Fruit Research and Extension Center, Rutgers University (CR-2-63-1, CR-2-63-2, CR-24-12-1, CR-24-12-2, NJA13 and NJA20) [37,58]. ...
In Romania, Prunus species have great economic and social importance. With the introduction of new cultivars arises the need to preserve and characterize the local Prunus germplasm. Thus, a set of 24 polymorphic SSRs were selected for the overall characterization, including 10 peach, 11 apricot and 5 nectarine cultivars. The average number of alleles per locus (Na = 1.958), in addition to overall observed (Ho = 0.299) and expected heterozygosity (He = 0.286) were lower or comparable to those reported in similar studies, probably explained by the smaller number of analyzed cultivars restricted to a smaller geographic area. Among 26 genotypes a total of 101 alleles were identified, of which 46 alleles were in peach, 55 in apricot and 40 in nectarine, respectively. Six alleles from six loci (CPPCT-030, Pchgms-003, Pchgms-004, Pchgms-010, UDP97-401, UDP98-405) were common to all taxonomic groups. The most informative loci were BPPCT-025, Pchgms-021 and UDP96-001 in peach; BPPCT-025, BPPCT-001 and UDP96-001 in nectarine; and BPPCT-002, BPPCT-025, Pchgms-004, Pchgms-020 and Pchgms-021 in apricot. Clustering and genetic similarity analysis indicated that the degree of interspecific divergence in peach and nectarine cultivars was less than that in peach and apricot. These results will be useful to prevent confusion between cultivars, to improve breeding strategies and to benefit the management of Prunus cultivars bred in Romania.
... Therefore, when determining the AC or GC, the breeders can predict the range of the remaining parameter. Moreover, within the same AC group, rice varieties with softer GC were preferred (Morgante and Olivieri 1993;Hirano et al. 1998). ...
Amylose content (AC) is one of the key traits related to rice grain quality. It decided the hard or soft grain rice. Consumers generally prefer cooked rice with a soft and sticky texture. AC is a single-gene trait and is controlled by the Waxy gene located on chromosome 6. Genetic studies related to AC in medium-grain rice varieties underpin the identification of genes and linked molecular markers that few previous studies mentioned. The study's purpose is to investigate the genetic characteristics of AC in medium-grain rice on 12 chromosomes. A rice diversity panel of 114 genotypes was applied to evaluate AC and its correlation with the agronomic and quality characteristics. Genome-Wide Association Study (GWAS) was utilized to identify significant SNPs. The results showed that the AC of varieties had a large range from 10.83 to 30.12%, in which, the group of varieties with low AC (less than 20%) accounted for 21.9%. AC was strongly correlated with gel consistency (GC) but not with gelatinization temperature (GT) and agronomic traits. The GWAS analysis of AC identified 9 significant SNPs on chromosomes 1, 2, 4, 8, 9, 11 and 12. The frequency of alleles widely ranged from 0.94 to 56.60%. On chromosome 6, 74 significant SNPs had allele frequencies ranging from 3.8 to 67.0%. In which, two SNPs fd7 (1764762 bp) and fd8 (176707 bp) are located in the Waxy gene region. These findings provided the basic genetic foundation for high-quality medium-grain rice breeding programs.
... PAGE resolution of amplified PCR products PAGE resolution, which separates small DNA fragments, was used (Morgante and Olivieri 1993). After a 30-min pre-run of the polyacrylamide gels, 10 µl of PCR products were loaded into individual wells. ...
Wheat, as a widely cultivated cereal crop, has encountered challenges due to its limited genetic diversity throughout its evolution. To facilitate effective crop improvement and ensure sustainability, a comprehensive understanding of the genetic diversity among wheat cultivars is essential. In this study, 17 bread wheat (Triticum aestivum L.) and 22 durum wheat cultivars (Triticum durum Desf.) were investigated. These cultivars originated from 19 provinces in Iran, encompassing the majority of their natural distribution range. Twenty inter simple sequence repeat (ISSR) molecular markers were utilized to assess the genetic correlations among the different cultivars. The ISSR analyses identified 133 ISSR loci in bread wheat cultivars, out of which 124 loci (93.23%) exhibited polymorphism. In durum wheat cultivars, 104 loci were detected, with 92 loci (88.46%) showing polymorphism. By utilizing the Dice similarity coefficient and the unweighted pair-group method with arithmetic mean (UPGMA) algorithm, a cluster analysis dendrogram was constructed, which successfully divided both bread and durum wheat cultivars into four distinct groups. Furthermore, our results were reinforced through principal component analysis clustering, validating the robustness of the grouping. The dissimilarity matrices derived from the Dice coefficient and the UPGMA analysis provided evidence of distinct genetic diversity between bread and durum wheat. Importantly, this study marks the first attempt to assess origin genetic variation in Iranian indigenous wheat using ISSR markers. This establishes a critical foundation for ex situ conservation and the utilization of genetic resources in wheat breeding endeavors. Overall, this research substantially advances our knowledge of wheat genetic diversity, guiding future crop improvement strategies and sustainable management of genetic resources.
... In oak, polymorphic markers were not evenly distributed among repeat classes, amounted to 58.7%, 44.3% and 36% for di-tri-and hexa-repeats, respectively. These figures confirm the higher level of polymorphism of dinucleotide repeats among plants [49][50][51]. The lower level of polymorphism for tri-and hexa-SSRs is mainly related to their location in translated sequences compared to dimeric SSRs that were preferentially distributed in UTRs. ...
... Therefore, when determining AC or GC, researchers can predict the range of the remaining indicator. Moreover, within the same AC group, the rice varieties with a softer GC are preferred (Morgante & Olivieri, 1993;Hirano et al., 1998;Nguyen Ngoc De, 2008 GT was positively correlated on average with GC, and the correlation coefficient between the two indicators was +0.46. The results showed that many varieties with a high scale of GT (low GT) corresponded with a long GC, however, there were still many exceptions. ...
Rice (Oryza sativa L.) grain attributes, such as grain length and shape, milling quality, and physicochemical quality, are crucial for varietal development and subsequent farm adoption. Thus, it is crucial to comprehend the phenotypic range of these grain attributes and how they relate to one another. Therefore, this study analyzed the main grain quality traits in medium-grain rice, namely the amylose content (AC), gelatinization temperature (GT), gel consistency (GC), ratio of head rice (HR), and percentage of chalkiness (PC). Correlations among the major quality characteristics were then calculated through Pearson correlation matrix analysis. The results showed that AC was highly connected with GC, and PC and HR (GC) showed a strong correlation. The correlation between GT and GC was average. The other quality traits did not correlate significantly. The phenotypes of the grain quality traits provide a basis for improving the quality of medium-grain rice populations.
... Microsatellites, also known as simple sequence repeats (SSR), are tandem repeats two to ten base pairs in length, that are flanked by conserved sequences and occur ubiquitously throughout eukaryotic genomes (Tautz and Renz, 1984). They are highly informative as locus-specific genetic markers due to their high abundance, high reproducibility, co-dominance, and polymorphic nature (Morgante and Olivieri, 1993;Powell et al, 1996). The length of the sequence repeats can be determined through PCR amplification using primers specific to their flanking regions; variation in PCR product length is a function of the number of repeated sequences. ...
Microsatellite primers are a valuable tool to use for both observational and experimental studies in numerous taxa. Here, we develop 18 and 16 microsatellite markers for the widespread duckweeds Lemna minor and Spirodela polyrhiza, respectively. Only four primers were not polymorphic when tested on samples from Europe and Western Pennsylvania, USA.
... Quality of the amplification products and high level of polymorphism was due to the use of preselected primers. The success of ISSR markers is based on the frequency and distribution of microsatellite motifs in the genome (Morgante and Olivieri 1993). Genetic diversity of each cultivar was assessed by calculating Shannons information index (I), polymorphic percentage (P) and Neis genetic diversity (H) ( Table 6). ...
... Microsatellite markers, also known as simple sequence repeats (SSR) or short tandem repeat (STR) data, a segment of DNA consisting of 1-6bp repeat units in tandem, offer several advantages, including high information content, co-dominance, and multiple alleles. Therefore, they are widely used in various genetic studies as an effective molecular marker [20,21]. ...
Suaeda aralocaspica, which is an annual halophyte, grows in saline deserts in Central Asia with potential use in saline soil reclamation and salt tolerance breeding. Studying its genetic diversity is critical for effective conservation and breeding programs. In this study, we aimed to develop a set of polymorphic microsatellite markers to analyze the genetic diversity of S. aralocaspica. We identified 177,805 SSRs from the S. aralocaspica genome, with an average length of 19.49 bp, which were present at a density of 393.37 SSR/Mb. Trinucleotide repeats dominated (75.74%) different types of motifs, and the main motif was CAA/TTG (44.25%). We successfully developed 38 SSR markers that exhibited substantial polymorphism, displaying an average of 6.18 alleles with accompanying average polymorphism information content (PIC) value of 0.516. The markers were used to evaluate the genetic diversity of 52 individuals collected from three populations of S. aralocaspica in Xinjiang, China. The results showed that the genetic diversity was moderate to high, with a mean expected heterozygosity (He) of 0.614, a mean Shannon’s information index (I) of 1.23, and a mean genetic differentiation index (Fst) of 0.263. The SSR markers developed in this study provide a valuable resource for future genetic studies and breeding programs of S. aralocaspica, and even other species in Suaeda.
... SSRs are co-dominant, widely dispersed or confined to certain regions of the chromosomes, highly polymorphic, and easily amplified by PCR. Owing to these special properties, these repeats are useful in linkage mapping, gene tagging, DNA fingerprinting, chromosome identification, molecular karyotyping, and genetic evolution studies [53,54]. ...
... SSRs marker are advantageous to applied plant breeding because they are co-dominant, easily assayed and detected high level of polymorphism [17]. SSRs marker detected genetic variation of flowering time, primers related to candidate genes controlling flowering time generated high polymorphism loci. ...
... The genic SSRs are also important because they present in coding regions. In early 1993, the identification of SSRs was carried out in gene sequences of plant species by Morgante and Olivieri (Morgante & Olivieri, 1993). Microsatellites can be classified depending on repeat units, their size, and their location in the genome. ...
... Moreover, a significant level of polymorphism was found within the group of accessions (54.1%) and among the groups of accession (45.9%) in the species collected from the different geographical locations under study. This was expected since the ISSR markers amplify microsatellite regions that exhibit significant polymorphism [74] and the promising outcome in the present investigation is due to the use of many randomly selected pre-screened highly informative primers. Using SRAP markers, Liang et al. [75] showed that the geographic structure also contributes to genetic differentiation within a species. ...
Cichorium intybus L. (chicory) is an important medicinal plant with immense economic potential and has recently gained rapid momentum in the functional food sector. In the present study, soil chemistry, phytochemical and molecular diversity was assessed for 50 accessions of chicory collected from diverse agro-climatic zones. Mainly 64 common metabolites were identified from the leaves of 7 chicory accessions collected from different altitudes and among them the pre-dominant metabolites include, methyl commate B (6.3-10.14%), gamma sitosterol (2.79-9.3%) and 9, 12, 15-octadecatrienoic-acid (2.55-8.42%). Three terpenoid compounds, viz., betulin, kolavelool and betulinaldehyde which were observed at high altitudes (1790, 1901 and 2172 m) were not observed at low altitudes in which betulin has the highest concentration with an average value of 23.53% followed by kolavelool, 7.37% and betulinaldehyde, 7.21%. For molecular diversity analysis, 12 ISSR primers were selected for PCR amplification and 86 bands were generated with an overall polymorphism percentage of 67.44%. The observed Nei’s genetic diversity (H) and Shannon’s information indices (I) were highest for Pulwama (CIN-PU) group of accessions (H = 0.222 ± 0.018; I = 0.343 ± 0.027) and lowest for the Baramulla (CIN-BM) group of accessions (H = 0.115 ± 0.019; I = 0.173 ± 0.028). The Analysis of Molecular Variance (AMOVA) analysis revealed 56% variation existing within the groups and 44% among the groups of chicory accessions. This study showed that chicory populations vary considerably in terms of their molecular and phytochemical composition as a function of their geographic location. Furthermore, this study demonstrated that chicory phytochemical and molecular diversity was significantly influenced by altitude, soil chemistry, and growing conditions. Using metabolomics and altitudinal variation, cluster analysis showed that geographic origin was correlated with diversity patterns.
... Molecular markers play crucial roles in the in-depth detection of genetic diversity modeling in maize and in determining gene flow from the center of origin to migration pathways [10-11-12-13-14-8]. SSR technique or microsatellite polymorphism, which is one of the PCR-based techniques, can be defined as PCR amplification of tandemly repeated sequences [15][16]. In this co-dominant and multi-allelic technique, the number of repeating units determines the polymorphism for fragment lengths, while heterozygous can be distinguished for different fragments in diploid genomes [17] In the SSR technique, which contains locus-specific side primers that produce a high level of polymorphic banding, genetic variation is quite good due to the differences in the number of consecutively repeated SSR units in a locus. ...
Plant landraces are heterogeneous plant populations that have adapted to local environmental conditions and serve as a source of genetic variations that can be used for the breeding perspectives. Studies regarding genetic diversity assessment are very important as they provide an opportunity for the scientific community to investigate novel genetic variations for the genetic improvement of crops. Maize is one of the most important cereal crops and provides daily calories to millions of people all over the world. This study aimed to investigate the genetic diversity and population structure of 32 local maize genotypes collected from the Black Sea Region of Turkey using SSR markers. Molecular characterization was performed using the 14 most polymorphic primers that yielded a total of 42 bands. An average of 3 alleles per SSR primer was detected, and the number of alleles varied from 1 (phi022) to 6 (umc1571). The unweighted pair-group method with arithmetic means (UPGMA) clustering divided maize accessions into three main populations. According to Nei's genetic distances, DZ-M-145 (Corum) and DZ-M-20 (Trabzon) genotypes were the closest (0.03) genetically related populations, while DZ-M-68 (Artvin) and DZ-M-55 (Rize) were the most genetically distant (0.63) populations. The study identified molecular genetic diversity not mentioned for maize plants from the Black Sea. On this occasion, new opportunities have been created for hybrid maize products.
... Repeat polymorphisms typically result from the addition or deletion of the complete repeat units or motifs triggered by polymerase strand-slippage in DNA replication or recombination errors. Consequently, polymorphisms observed in SSRs for distinct individuals result from differences in the number of repeats of the motifs [206,207]. ...
Sporotrichosis is the most important subcutaneous mycosis that affects humans and animals worldwide. The mycosis is caused after a traumatic inoculation of fungal propagules into the host and may follow an animal or environmental transmission route. The main culprits of sporotrichosis are thermodimorphic Sporothrix species embedded in a clinical clade, including S. brasiliensis, S. schenckii, S. globosa, and S. luriei. Although sporotrichosis occurs worldwide, the etiological agents are not evenly distributed, as exemplified by ongoing outbreaks in Brazil and China, caused by S. brasiliensis and S. globosa, respectively. The gold standard for diagnosing sporotrichosis has been the isolation of the fungus in vitro. However, with the advance in molecular techniques, molecular assays have complemented and gradually replaced the classical mycological tests to quickly and accurately detect and/or differentiate molecular siblings in Sporothrix. Nearly all techniques available for molecular diagnosis of sporotrichosis involve PCR amplification, which is currently moving towards detecting Sporothrix DNA directly from clinical samples in multiplex qPCR assays. From an epidemiological perspective, genotyping is key to tracing back sources of Sporothrix infections, detecting diversity in outbreak areas, and thus uncovering finer-scale epidemiological patterns. Over the past decades, molecular epidemiological studies have provided essential information to policymakers regarding outbreak management. From high-to-low throughput genotyping methods, MLSA, AFLP, SSR, RAPD, PCR-RFLP, and WGS are available to assess the transmission dynamics and sporotrichosis expansion. This review discusses the trends in the molecular diagnosis of sporotrichosis, genotyping techniques applied in molecular epidemiological studies, and perspectives for the near future.
... In addition to all these advantages, the most important disadvantage of the method is that it requires genome information and sequence analysis. (Morgante and Olivieri, 1993). In addition, the development of new SSR markers is not easy and requires high costs. ...
Genetic diversity, refers to the total number of genetic traits in the gene pool of any species and races belonging that species adapted to a particular geographic region. Molecular markers allow us to obtain information about the unique functional genes of the races and the characteristics of these genes. In the detection of genetic diversity are used such as methods polymerase chain reaction (PCR), Amplified Fragment Length Polymorphism (AFLP), Restriction Fragment Length Polymorphism (RFLP), Randomly Amplified Polymorphic DNA (RAPD), Simple Sequence Repeat (SSR), Short Tandem Repeats (STR) and Single Nucleotide Polymorphism (SNP). In poultry such as goose, chicken, quail, duck and turkey; different methods are used depending on the type of animal, its characteristics and the purpose of the study. In the studies conducted to in geese; Microsatellites are more preferred because they require a small amount of DNA, show high polymorphism, are reproducible and provide more information. In this study, it is aimed to compile the researches on the use of molecular markers in other poultry, especially geese.
... Mediterranean publications associated with the second decade, more specifically from 2003 to 2011 (Fig. 2), experienced an increased pace. It could be due to the discovery of new molecular biology technologies worldwide during the first decade of our analysis and implemented into Mediterranean research with a slight delay during the second decade, such as the very popular and efficient microsatellite markers (e.g., simple sequence repeat (SSR) [54]). ...
Purpose of Review
Recognizing that in the context of global change, tree genetic diversity represents a crucial resource for future forest adaptation, we review and highlight the major forest genetics research achievements of the past decades in biodiversity-rich countries of the Mediterranean region. For this, we conducted a bibliometric analysis of the scientific literature spanning the past thirty years (1991–2020). Putting together the representative regionwide expertise of our co-authorship, we propose research perspectives for the next decade.
Recent Findings
Forest genetics research in Mediterranean countries is organized into three different scientific domains of unequal importance. The domain “Population diversity and Differentiation” related to over 62% of all publications of the period, the domain “Environmental conditions, growth and stress response” to almost 23%, and the domain “Phylogeography” to almost 15%. Citation rate was trending the opposite way, indicating a strong and sustained interest in phylogeography and a rising interest for genetics research related to climate change and drought resistance. The share of publications from Asia and Africa to the total within the Mediterranean increased significantly during the 30-year period analyzed, reaching just below 30% during the last decade.
Summary
Describing poorly known species and populations, including marginal populations, using the full potential of genomic methods, testing adaptation in common gardens, and modeling adaptive capacity to build reliable scenarios for forest management remain strategic research priorities. Delineating areas of high and low genetic diversity, for conservation and restoration, respectively, is needed. Joining forces between forest management and forest research, sharing data, experience, and knowledge within and among countries will have to progress significantly, e.g., to assess the potential of Mediterranean genetic resources as assisted migration material worldwide.
Introductory quote
Let us collect with care the facts we can observe, let us consult experience wherever we can, and when this experience is inaccessible to us, let us assemble all the inductions which observation of facts analogous to those which escape us can furnish and let us assert nothing categorically; in this way, we shall be able little by little to discover the causes of a multitude of natural phenomena, and, perhaps, even of phenomena which seem the most incomprehensible... J.B. de Lamarck (Philosophie zoologique, 1809), cited by O. Langlet (1971).
... The genic SSRs are also important because they present in coding regions. In early 1993, the identification of SSRs was carried out in gene sequences of plant species by Morgante and Olivieri (Morgante & Olivieri, 1993). Microsatellites can be classified depending on repeat units, their size, and their location in the genome. ...
... This might be due to the fact that the distribution of these repeats in the Grewia spp genome was beyond the range of amplification by Taq DNA polymerase. However, this is not likely the situation for poly (AT) repeats, as these are reported to be the most abundant motifs in different plant species [49,50]. However, in the present investigation, poly (AT) or poly (TA) repeats gave improper amplifications. ...
This research is conducted to study the genetic diversity of the genus Grewia tenax in Sudan. This plant has a common vernacular Sudanese Arabic name: “Gudeim”. It is mainly cultivated in North Darfur and Western Sudan. Common uses of Grewia species were overviewed in areas of nutrition, folk medicine and famine food. The genetic variation and genetic relationships among Grewia spp populations from different regions were efficiently determined using RAPD and ISSR markers.
The results of genetic analysis were statistically analyzed by STATISTCA and GenALEx 6.5 software. The results of molecular variance revealed that 74% of total genetic diversity was due to within populations variations as opposed to 26% due to variations between populations unweighted pair- group method with arithmetic average (UPGMA) were constructed for and RAPD + ISSR. The UPGMA results showed variability of Grewia genotypes.
It was concluded that both the marker systems RAPD and ISSR combination can be effectively used in determination of genetic relationships among Grewia tenax genotypes.
... Hence, understanding the genetic resistance mechanisms and discovering genes or QTLs are essential for developing MYMV disease resistance in blackgram. SSRs as DNA markers have many advantages over other markers and have been used to assess genetic variation (Morgante and Olivieri 1993). Much work has not been done towards the development of genomic resources in this crop, but by using genomic resources from adzuki bean, common bean and mungbean, genetic maps were constructed (Chaitieng et al. 2006;Gupta et al. 2008;Isemura et al. 2012). ...
Mungbean yellow mosaic virus (MYMV) disease is a significant constraint for blackgram production. The present study
employed a mapping population derived from a cross between susceptible (MDU 1) and resistant (TU 68) genotypes to identify quantitative trait loci (QTL) associated with MYMV disease resistance in addition to bruchine resistance loci identified from the previous study. Phenotyping was carried out in F2 generation under the disease spreader row method at field condition. Disease score observations were carried out 60 days after sowing (DAS). The chi-square goodness of fit test revealed inhibitory gene action with two genes controlling the expression of resistance to MYMV disease. However, QTL analysis revealed one major QTL region, i.e. qMYMVD_60 at LG 10
responsible for MYMV disease score at 60 DAS, accounted for 21 per cent of variation. The identified QTL has the flanking markers as
CEDG180 and CEDG116. Hence, the QTL, qMYMVD_60 may be utilized in the breeding of MYMV disease resistance. Further, the
marker-assisted introgression of both the MYMV and bruchine resistance QTLs can be performed in the near future.
... But for complex quantitative traits, integrated methods such as QTL mapping with classical backcrossing are essential by the use of molecular markers. The molecular marker techniques such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) or microsatellite markers (Morgante and Olivieri 1993), and amplified fragment length polymorphism (AFLP) markers (Vos et al. 1995) have been used for characterization of plant genetic resources. Among them, SSR and AFLP markers are the most widely exploited techniques for the characterization of plant genetic resources. ...
With the increase in human population, over exploitation of bioresources and climate change, there is an ever-growing threat to biological resources and biodiversity. Numerous species of plants and animals are under constant pressure of being extinct due to habitat loss and degradation. Loss of biodiversity would not only be disruptive to the delicate functioning of ecosystems but also incur huge losses economically affecting food security and public health among others. Understanding the inter-dependencies between life forms and their microbiomes is one of the many facets of conservation strategies that can aid in sustainable utilisation of bioresources. This chapter highlights the importance of metagenomics in elucidating roles of microbial (bacterial and fungal) communities whether it be in the gut of an animal, the rhizosphere of a plant or soil in a tropical forest. Due to the unculturability of most microbial species, metagenomics proves to be an effective approach in identifying the microbial wealth as well as their role in the ecological niches. In metagenomics, the total DNA of the collected sample is extracted followed by amplification of certain conserved genes, for example, 16S rDNA in bacteria and ITS regions in fungi. Knowledge of these phylogenetic profiles can be integrated with other existing data to assess host physiology and metabolism, which are known indicators of threat to biodiversity.
... Known simple sequence repeats (SSRs, Microsatellites) (Morgante and Olivieri 1993) are much stronger and enlightening markers for fingerprinting analysis and determination of genetic variety, genetic mapping, population structure and marker assisted selection (MAS) in plants due to their abundance, dominance and high repetitivable and high polymorphism in the genome (Wani et al. 2010). SSRs have the very commonly utilization markers for marker assisted breeding for fruit trees and crop improvement in many plants over for 10 years Singh et al. 2008). ...
... DNA markers have been the choice tool for the genetic characterization of rice genotypes (Karkousis et al., 2003). Among them, simple sequence repeats (SSRs) are preferred due to polymorphic nature, abundance in the eukaryotic genome and ease of generating (Tautz, 1989;Morgante and Olivieri, 1993). In rice, SSRs have been used earlier for the assessment of genetic diversity (Rahman et al., 2012;Choudhury et al., 2013;Singh et al., 2016;Suvi et al., 2019), conservation planning (Sharma et al., 2007), marker-assisted selection (Perez-Sackett et al., 2011;Rani and Adilakshmi, 2011), cultivar identification and hybrid purity analysis, besides gene mapping (Weising et al., 1997;Altaf-Khan et al., 2006;Sarao et al., 2010). ...
This paper reports collection of rice landraces and documentation of traditional knowledge associated with rice cultivation in Sikkim Himalayan region. The methodology employed was-field visits accompanied with survey using purposive-cum-random sampling method, particularly of the elderly farmers. A total of thirty seven farmers from twelve selected villages of the four districts of Sikkim state were interviewed randomly as respondents. As many as seventy four different rice landraces were collected as part of this research. The maximum number of Indigenous Traditional Knowledges ITK was documented under the plant protection against various diseases (10 nos.) and only two ITK was recorded in water management. The rationale on the use of indigenous knowledge, presented here, is derived purely based on the free opinion of the respondents. The results suggest that the indigenous knowledge in rice farming in Sikkim Himalayas is built upon the natural features of the region. The innovation/techniques are based on locally available resources and are rooted in their historical experiences which are passed on through generations. The information on ITK presented here could provide vital clues on the suitable alternatives to expensive modern farming system.
... The microsatellite (SSR) markers are the most suitable for rice because of their reproducibility, multiallelic nature, hyper variability, codominant inheritance, relative abundance and genome-wide cov-erage (Tautz, 1989;Morgante and Olivieri, 1993;Powell et al., 1996). Genetic variability at the molecular level could help identifying and developing unique rice germplasm. ...
... Microsatellites, also called simple sequence repeats, are tandem repeat sequences with a basic motif of 1 to 6 base pairs (Morgante and Olivieri, 1993). ...
Fusarium head blight (FHB) is a destructive disease of wheat
(Triticum aestivum L.) with worldwide economic and health impact
caused by a group of trichothecene producing Fusarium species with
Fusarium graminearum being the most prevalent. Breeding wheat
resistant to FHB is one of the best strategies to minimize crop and
grain quality losses due to the pathogen. However, the number of
reported FHB resistance sources and genes is still limited. In the
present study we initiated a study of molecular and genetic
characterization of FHB-resistant winter wheat Centenaire exhibiting
Type II resistance (resistance to spread). The molecular aspect of this
work included identification and characterization of candidate genes
differentially expressed in the resistant genotype due to F.
graminearum challenge followed by time-course transcript expression
study. Using suppression subtracted hybridization forty three
differentially expressed sequences were identified to be up-regulated
in the resistant germplasm due to the infection in the first 72 hours
post-inoculation. The newly identified putative genes involved in
resistance were grouped in functional groups encompassing various
physiological and defense pathways. Time-course expression study
confirmed the differential transcript accumulation between FHB-
resistant and FHB-sensitive genotypes. Thirty out of forty three gene
candidates were assigned to the corresponding wheat chromosomes
and several were localized in the regions known to harbor quantitative
trait loci for head blight resistance. During the infectious process F.
graminearum produced mycotoxins serving as virulent factors and
5
which are harmful for humans and animals. In the present study, we
identified and characterized five new full-size ABCG transporters
differentially expressed between resistant and susceptible wheat. This
work also emphasis the potential involvement of this gene family in
wheat resistance to FHB. In addition, evidence that ABCB and ABCC
transporters may be also involved in resistance reaction was shown in
the present work. The genetic characterization of Centenaire
demonstrated that eight molecular markers on chromosomes 1B, 2B,
3A, 4B, 5A and 6B were linked to quantitative trait loci for FHB
resistance. The results of this work enlighten the possible molecular
mechanisms involved in resistance to head blight. Quantitative trait
loci for FHB resistance identified in the present study makes
Centenaire a novel possible source for FHB resistance.
... Microsatellites are of two types viz, genomic SSR and expressed-sequence-tag-based/ genic SSR (EST-SSR). Being co-dominant in nature, SSR markers have an edge over other markers, such as, high level of polymorphism, high specificity and repeatability [7]. Hence, SSR markers are widely used to study germplasm characterization, QTL mapping, linkage mapping and marker-assisted selection (MAS) [8]. ...
Ginger ( Zingiber officinale Rosc.) is an economically important and valuable spice crop around the world. It is used as food, spice, condiment, and medicine. A considerable extent of genetic diversity in ginger occurs in the Western Ghats and North-Eastern India. However, genetic diversity studies at the molecular level in ginger is limited due to limited availability of genetic and genomic information. In the present study, for the first time, we have identified and validated expressed sequence tag (EST)-simple sequence repeat (SSR) markers from ginger. We obtained 16,790 EST-SSR loci from 78987 unigenes, and 4597 SSR loci in the predicted 76929 coding sequences from RNA-Seq assembled contigs of ginger through Illumina paired-end sequencing. Gene ontology results indicate that the unigenes with SSR loci participate in various biological processes such as metabolism, growth, and development in ginger. One hundred and twenty-five primer pairs were designed from unigenes and coding sequences. These primers were tested for PCR optimization, characterization, and amplification and identified 12 novel EST-SSR markers. Twelve flanking polymorphic EST-SSR primers were validated using 48 ginger genotypes representing North-Eastern India and different eco-geographical adaptations by PCR amplification and allele sizing through capillary electrophoresis. Twelve EST-SSR primers generated a total of 111 alleles with an average of 9.25 alleles per locus and allele sizes ranging between 115-189bp. This study implies that the SSR markers designed from transcriptome sequences provides ample EST-SSR resources, which are helpful for genetic diversity analysis of Zingiberaceae species and molecular verification of ginger genotypes.
... Microsatellites or SSRs (simple sequence repeats) have been applied to genetic diversity and population structure studies in a number of plants such as cassava (Adjebeng-Danquah et al. 2020), garlic (Kumar et al. 2019, and oil palm (Zhou et al. 2015), as well as animals such as chicken (Özdemir and Cassandro 2018), honey bee (Liu et al. 2016), and cattle (Agung et al. 2019) due to their wide distribution throughout the genome, co-dominant nature, multi-allelic, high polymorphism, reproducibility, and transferability (Morgante and Olivieri 1993;Powell et al. 1996). Generally, the transferability success rate depends on the applicability of the markers between species in the same genus, between genera in the same family, between families in the same order, and between orders in the same class (Barbara et al. 2007). ...
Bauhinia winitii Craib is an endemic species native to Thailand. Genetic diversity was assessed using microsatellite markers developed from a library enriched using (AG)20/(CAG)20 oligonucleotide probes. Eight polymorphic loci produced a total of 28 alleles with a mean number of 3.5 alleles per locus from 33 specimens of B. winitii. The averages of the expected (He) and observed (Ho) heterozygosity were 0.418 and 0.394, respectively. The average value of polymorphic information content (PIC) was 0.364, and two SSR loci were highly informative. Significant linkage disequilibrium (LD) was observed between one pair of loci and a significant deviation from Hardy-Weinberg equilibrium (HWE) (P < 0.05) was detected at three loci. The B. winitii population in Thailand has a moderate level of genetic diversity and a low level of inbreeding. The cross-species transferability success rate among Bauhinia species of the SSRs derived from B. winitii was 75% (B. acuminata), 100% (B. aureifolia), and 100% (B. strychnifolia). These newly developed SSR primers provide a useful genetic tool for analyzing the population structure of B. winitii and the genetic diversity of the related species – B. acuminata, B. aureifolia, and B. strychnifolia. Information from this study can be useful for developing conservation and protection plans as well as management of the genetic resources of B. winitii, an endemic plant in Thailand. © 2021, Department of Science and Technology. All rights reserved.
... This Technique became their name for Microsatellite Markers instead of Simple Sequence Repeat or Short Tandem Repeats [28,29]. Found within eukaryotic genomes [30,31] consisting of repeat units with length from two to six bp, nucleotides depending on basic short motifs, polymorphism occurs between specific loci generating variation in microsatellite length [32,33]. Microsatellite assays required small DNA material by using pair specific primers set 20-30 bases in PCR analysis appear multiple shapes of length between species for specific sites. ...
This article refers to viewing the role of molecular markers during analyzing the genome of plants and their importance in plant biotechnology. In recent years, we observed the role of molecular techniques in programs for improving plant breeding and preserving genetic resources has been observed, and molecular and biochemical indicators which represent basic material through determining the diversity between genotypes for indicators it is never affected by external surrounding conditions as always in the phenotype features. Molecular markers of DNA have been widely applied to answer a range of questions related to taxonomy, molecular evolution, population genetics, and genetic diversity, as well as monitoring trade in plants and food products , in addition to its having a role in studying gene expression , genetic mapping, and studies of species evolution providing fast and accurate results. In this work, the advantages and limitations of the molecular techniques applied in plant sciences such as: RAPD (Random Amplification Polymorphic DNA Marker); ISSR (Inter Simple Sequence Repeat Marker); SSR (Simple Sequence Repeat Marker); AFLP (Amplified Fragment Length Polymorphic Marker); RFLP (Restriction Fragment Length Polymorphism Marker); SNP (Single Nucleotide Polymorphism) and Real Time PCR.
Bermudagrass ( Cynodon spp.) is a drought-resistant warm-season turfgrass adapted to the southern and transitional zones in the United States. Multiple hybrid cultivars have been developed and released for use as turfgrass, and others are still undergoing development. Increasing genetic diversity of commercial cultivars is vital to stress tolerance. A DNA profiling study of 21 experimental selections from the Oklahoma State University turfgrass breeding program and 11 cultivars was conducted using 51 simple sequence repeat primer pairs across the bermudagrass genome. A pairwise genetic relationship analysis of the genotypes using 352 polymorphic bands showed genetic similarity coefficients ranging from 0.59 to 0.89. The average pairwise population differentiation values were 0.012 for the 11 cultivars and 0.169 for the 21 selections. A cluster analysis using the unweighted paired group with the arithmetic average method grouped the entries into six clusters. A correlation analysis identified different levels of pairwise genetic relationships among the entries that largely reflected parental relationship. Directional breeding and selection for cold hardiness or drought resistance created progeny that had distinct genetic diversity in the tested bermudagrasses. It is evident that an increase in genetic diversity of the existing cultivar pool with the release of one or more experimental selections for commercial use will strengthen and improve bermudagrass systems.
Simple sequence repeat (SSR) markers are among more reliable markers widely used in population genetics and traditionally need intensive labor to develop. High-throughput sequencing (HTS) has enabled researches in screening whole DNA in order to develop dozens of reliable SSR markers to pave route for future studies. Annual medics (Medicago spp.) are specifically beneficial in agriculture, genetics, and pastureland management plans. In order to facilitate study of annual medics using SSRs and to test their effectiveness, HTS screening of whole DNA resulted in developing of hundreds of primer pairs published in this paper. Subsequent testing of candidate primers on Iranian M. orbicularis germplasm was narrowed down to six more variable primer pairs that generated 28 polymorphic bands with the allele size of 200–310 bp. Eleven populations were examined using our SSR markers. The average polymorphic information content was very high (> 0.90) for the primer combinations. Genetic diversity parameters were measured for all populations. A high level of polymorphism of 75% within populations was observed. We hypothesized that the self-pollinating reproductive system of M. orbicularis has led to the reduction of gene flow between populations and ultimately the low diversity among populations (24%). Different genotypes observed by structure analysis as well as regression and correlation analyses showed that genetical similarity was congruent with the geographical proximity. Our results suggest that our HTS-generated SSR markers can be used for the further study of other annual medics.
Citation: Alhariri, A.; Behera, T.K.; Jat, G.S.; Devi, M.B.; Boopalakrishnan, G.; Hemeda, N.F.; Teleb, A.A.; Ismail, E.; Elkordy, A. Analysis of Genetic Diversity and Population Structure in Bitter Gourd (Momordica charantia L.) Using Morphological and SSR Markers.
With the advancement of technologies in deciphering new molecular markers, the analysis of diversity between sets of germplasm at the molecular level has become easier. An array of molecular markers is available as a tool to assess the genetic diversity in plants, such as restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphism (AFLP), inter simple sequence repeats (ISSR), simple sequence repeats (SSR), sequence-characterized amplified regions (SCAR), sequence-tagged sites (STS), cleaved amplified polymorphic sequences (CAPSs), expressed sequence tags (ESTs), Sequence Tagged Sites (STSs), Single-Nucleotide Polymorphism (SNP), Diversity Arrays Technology (DArT), Start Codon Targeted (SCoT) and Genotyping-By-Sequencing (GBS). These makers are not affected by environmental factors and/or the developmental stage of the plant and detect the variation that arises from different classes of DNA mutations such as point mutations, insertions, and deletions, or errors in the replication of tandemly repeated DNA. Each marker has its own advantages and limitations. The present book chapter presents a basic description of the most widely used molecular markers utilized for the improvement of crops. We believe that this chapter provides useful information to improve the understanding of molecular markers for graduate students and researchers.KeywordsNGSMolecular markerPolymorphism
In this study, we analyzed the genetic diversity of 102 Algerian date palm (Phoenix dactylifera L.) samples from the oases of Biskra region, the oases of Bechar region, the oases of Adrar region, the oases of Oued Souf region and the oases of Ghardaia region, using SSR (Simple Sequence Repeat) markers. They resulted in 56 alleles with an average of 6.22 alleles per locus. Observed heterozygosity (Ho) ranged from 0.222 (DP 171) to 1.000 (mPdCIR 50, mPdCIR 57, DP 159 and DP 169) with a mean of 0.687. The expected heterozygosity (He) ranged from 0.364 (DP 171) to 0.855 (mPdCIR 50) with a mean of 0.694. From the results obtained in the germplasm of various analyzed Algerian date palm oases, a large genetic diversity was revealed.
L'étude de l'adaptation des espèces cultivées est très importance pour comprendre la diversification des cultures. Un des aspects de cette diversification dans l’histoire évolutive desespèces cultivées est celle liée à la saisonnalité. La présente thèse a utilisé le sorgho (Sorghumbicolor (L.) Moench.) comme modèle pour l’étude de l’adaptation saisonnière. Cette céréalecomprend deux types saisonniers (sorghos pluviaux et sorghos de saison sèche). Ledéveloppement des agrosystèmes de saison sèche en Afrique de l'Ouest et du Centre, très peuétudié, offre un cadre intéressant pour l’étude des bases de l’adaptation saisonnière. L’analysede la divergence morpho-physiologique héréditaire et la différenciation génomique des variétésde sorgho de saison sèche par rapport aux variétés de sorgho pluvial a été proposé dans le cadre de cette thèse. Le matériel végétal est constitué de 153 accessions représentant 4 types écosaisonniers de sorgho définis sur la base de la saisonnalité (deux saisons) croisée avec la zone éco-géographique (Vallée du Fleuve Sénégal et Bassin du Lac Tchad). Dans un premier temps, un plan d’expérimentation a été mis en place pour évaluer en conditions comparables lecomportement des quatre type éco-saisonniers, respectivement en saison pluviale puis en saison sèche. 11 caractères agro-morphologiques et 9 caractères agro-physiologiques ont été mesurés. Dans un second temps, les données de séquençage haut-débit (GBS, genotyping by sequencing) ont permis, sur le base de 120 969 SNPs, de décrire la diversité et la structure génomique du panel étudié. Trois caractères agro-morphologiques (date d’épiaison, hauteur de la plante et diamètre de la tige) et quatre caractères agro-physiologiques (nombre de feuilles à maturité, biomasse sèche aérienne, biomasse sèche racinaire et ratio biomasse racinaire/biomasse aérienne) ont permis de mettre en évidence une différenciation fortement marqué du sorgho de saison sèche du Bassin du Lac Tchad comparé aux trois autres groupes éco-saisonniers. Par contre, aucune différenciation significative des caractères n'a été détectée entre les sorghos pluviaux et les sorghos de saison sèche de la Vallée du Fleuve Sénégal. L’analyse génomique a montré que la diversité des 153 accessions de sorgho est structurée en 3 groupes génétiques. Les accessions de sorgho de saison sèche du Bassin du Lac Tchad forment un groupe bien différencié des accessions de sorgho pluvial. Tous ces résultats expérimentaux fournissent de nouvelles indications pour mieux comprendre les scénarios et les bases de l’adaptation saisonnière du sorgho dans un contexte marqué par la diversification de la culture entre des agrosystèmes de saison pluviale et des agrosystèmes de décrue (saison sèche).
Syzygium cumini L. (ver Jamun; BlackBerry) is a native, evergreen multipurpose tree species of India. Besides being a fruit tree and for agroforestry in different regions, it is medicinally important too. This study aimed to determine genetic diversity using molecular and phytochemical markers in sixteen genotypes of Indian S. cumini from different agro-ecological zones. The present study used a combination of ISSR markers and the HPLC technique to explore these genotypes. The results showed a wide genetic diversity range based on the similarity coefficient values observed in S. cumini sixteen accessions from different sites. Four primary phenolic acids were discovered in all the accessions; caffeic acid (CA) was found in high concentrations. The intraspecific association between molecular and phytochemical characteristics was the primary goal of this investigation. By employing gene-specific markers for the route of secondary metabolites (polyphenols) production, it further investigated the progressive research of diversity analysis of polyphenol content in S. cumini accessions, which may also expand its nutraceutical and pharmaceutical utilization.
The genetic polymorphism of thirty Aegilops ventricosa Tausch. accessions sampled from northern Algeria was evaluated using inter-simple sequence repeat (ISSR) markers (into 5'). Six reproducible primers were used, and 53 out of 62 amplified distinct bands were found with a high score of polymorphism (87.48%). A significant number of specific bands was identified which were useful for genotyping. The majority of ISSR primers showed important resolving power with an average of 10.86, reflecting high informative markers tested in this investigation. SIMQUAL coefficient was used to calculate the genetic similarity among the 30 accessions. This similarity ranged from 0.5806 to 1. Based on Unweighted Pair-Group Method using Arithmetic Averages (UPGMA) method, a dendrogram was established to assess the intraspecific relationships via the genetic distances between the 30 accessions. The ISSR results showed a high variability in the studied collection. These markers could be efficiently applied to estimate the genetic diversity in the Ae. venticosa or others species in the Aegilpos genus.
Cotton (Gossypium) is a fiber-producing oil seed crop with significant economic and scientific importance in natural textile fiber and edible oil industries. Cotton is an ideal crop model system for studying polyploidy, evolution, population genomics, and domestication, due to the presence of several species, wild relatives, and progenitors. Population genomics studies can provide key insights into genetic diversity, population structure, evolution, and domestication as well as genotype-phenotype associations and genomics-assisted breeding of Gossypium species. Population genomics research in cotton is lagging behind when compared to other crops with simple genomes due to its large complex genome size and polyploidy. However, modern genomics technologies such as sequencing, resequencing, comparative genomics, and whole-genome sequencing (WGS) have generated reference genomes of diploid and tetraploid cotton, which have addressed key questions about the role of polyploidy in the evolution of cotton. Population genomics studies and reference genomes have advanced understanding of genomic variation across species, gene expression changes, domestication, and evolutionary history of several species of Gossypium, and genetic variation underlying different traits of cotton such as fiber yield, fiber quality, biotic, and abiotic stress resistance/tolerance. These advances are facilitating selection and breeding of cotton for improved traits through genome-wide association studies (GWAS) and genomic selection. GWAS has discovered candidate genes for different quantitative trait loci (QTLs). Hence, population genomics approach will be a key component in studying genetic history of cotton domestication, improvement of different agronomic traits such as fiber quality, oil yield, disease resistance, and tolerance to extreme environmental conditions. This chapter discusses the recent advances made in cotton population genomics and its future perspectives.KeywordsDiversification DomesticationEvolutionGenomic selectionGenomics
Gossypium
GWASPangenomePolyploidyPopulation genetic variation
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Sinapis alba L. (white mustard) is recognized for carrying host resistance against several biotic stresses including, Alternaria brassicae, which is responsible for blight disease in cultivated Brassica. However, another cultivated Brassica has a dearth for genetic resistance for these stresses due to its narrow genetic base. Therefore, we performed introgression of the genomic regions of S. alba into backcross progenies of B. juncea + S. alba somatic hybrids. These advanced generations with S. alba chromosomal segments are named B. juncea-S. alba introgression lines (ILs). In the present study, we developed the S. alba genome-specific microsatellites from the draft genome to track the S. alba genome introgressions and responsible regions for resistance to A. brassicae. For developing these SSR markers, the unique contigs of S. alba draft genome were identified through BLASTN with B. juncea, B. rapa, B. nigra, and B. oleracea reference genome assemblies, including mitochondrial and chloroplast genomes, and further used for marker development. Out of 403,423 contigs, we have identified 65,343 non-hit contigs of S. alba that yielded a total of 1231 genome-specific microsatellites, out of which 1107 were expected to produce a single allele upon amplification. Out of the total SSRs, 234 primer pairs were randomly picked from whole-genome and validated between B. juncea and S. alba genomes for their specificity. In the validation experiment, these markers gave a single amplicon into S. alba, while they did not amplify in B. juncea genome. Of these, 59 microsatellites were used to track S. alba introgressions in 80 BC2F3 lines. To the best of our knowledge, this is the first time that these two genetic resources are developed in the form of B. juncea-S. alba ILs and S. alba-specific markers. Therefore, both the resources unlock a new avenue of Brassica breeding for biotic and abiotic stresses along with quality traits.
Supplementary information:
The online version contains supplementary material available at 10.1007/s13205-022-03402-0.
Since the developing drought tolerant varieties in arid and semi arid
environmental conditions has been accepted as essential factor for increasing
crop potential, yield improvement and stability, therefore the identification of
effective physiological parameters associated with stable yield under water deficit
conditions is very important. In order to examine the effect of drought on
different physiological characters, yield and yield components two groups of
wheat varieties: susceptible and tolerant were grown under raifed conditions in
two different agro-ecological zones (1st and 2nd settlement zone) in order to expose
these two groups to different rainfall and ultimately water deficit conditions
particularly in the second settlement zone where the annually rainfall is about
199 mm compared with 399 mm in the 1st settlement zone. Chlorophyll content,
membrane stability index, relative water content and chlorophyll fluorescence
were studied at vegetative, anthesis and grain filling stage. Remarkable reduction
in all physiological parameters were recorded in both groups of varieties in the
2nd settlement zone, however tolerant group showed good performance and
maintained better stability compared with susceptible group at all growth stages.
Yield and yield component of both groups were affected negatively by low
rainfall in the 2nd settlement zone. Despite some high yield values maintained by
the susceptible group in the 2nd settlement zone compared to tolerant group, the
susceptible group showed more reduction in all yield data in the 2nd compared to
1st settlement zone. These finding indicate the importance of physiological
parameters for screening and selection tolerant varieties and its great role in the
breeding programs. Looking overall results, it is clear that these parameters
could explain some of the mechanisms which indicate tolerance to drought;
however their relevance in describing tolerant and susceptible group's variability
is significant.
Apple scab caused by the ascomycete Venturia inaequalis is an economically significant disease worldwide. The annual sexual reproduction of V. inaequalis leads to high variation, changes in the population’s genetic structure and adaptations to the changing environment, including overcoming the host’s resistance. The objective of this study is to characterise and assess the genetic diversity of V. inaequalis populations in two main apple-growing regions in Latvia. In total, 143 V. inaequalis isolates were collected from Latvia, six reference strains with known virulence were obtained from other countries, and all strains were genotyped by 12 SSR markers. The SSR markers were highly variable and informative, identifying 158 alleles that ranged from two to 29 per locus. The Bayesian clustering identified three genetic lineages among the Latvian isolates that did not correlate to the geographic origin, host genotype, organ (leaves or fruits) from which the pathogen was isolated, time of collection, and type of isolation (single conidium or ascospore). The possible relatedness to virulence was detected when reference strains with known virulence were included in the analysis. Our findings correspond with previous studies demonstrating that V. inaequalis in Europe has a high genetic diversity within populations, but low diversity among the populations.
An efficient direct in vitro plant regeneration protocol has been established for medicinally important red-colored ginger (Zingiber officinale Rosc.) genotypes, namely, northeast red ginger and exotic red ginger. Shoot, root initiation, and growth response of these two genotypes to five different concentrations of 6-benzylaminopurine (BAP; 1.0, 2.0, 3.0, 4.0, and 5.0 mg L−1) and a control using Murashige and Skoog (MS) medium were studied. The two red ginger genotypes responded differently to five concentrations of hormone. Significantly higher shoot multiplication was observed for northeast red ginger cultured on medium containing 3.0 mg L−1 BAP. In the case of exotic red ginger, maximum shoot multiplication was on medium containing 5.0 mg L−1 BAP and was statistically on par with medium supplemented with 4.0 mg L−1 BAP. Maximum shoot length was measured when cultured on medium containing 3.0 mg L−1 BAP in northeast red ginger and medium with 2.0 mg L−1 BAP in exotic red ginger. In exotic red ginger, the highest number of roots was documented on medium containing 5.0 mg L−1 BAP and medium with 3.0 mg L−1 BAP in northeast red ginger, which was on par with medium containing 2.0 mg L−1. The exotic red ginger had significantly longer root length on medium containing 2.0 mg L−1 BAP. However, root length was on par when northeast red ginger was cultured on medium containing 2.0 to 5.0 mg L−1 BAP. Inter-simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers confirmed the genetic fidelity of the regenerated plantlets. The direct in vitro regeneration protocol may contribute significantly to the development of improved red ginger genotypes using contemporary biotechnological tools.
Brassica species were domesticated as oil producing crops during different periods at many sites throughout the world. Animal fat being pricier, the poor used vegetable oil as a source of their nutrition. Accordingly, world production of vegetable oil has been incremental chiefly due to increased production of soybean, palm and oilseed rape. Rapeseed (Brassica napus L.), also known as Canola or Oilseed rape, has thus become an important source of vegetable oil worldwide, and ranks third after soybean and palm. The world population is expected to cross the 9 billion mark by 2050, and to assure food and nutritional security for our soaring future generations, we need to necessarily double the production of food crops by then. However, various environmental stresses negate the realization of this target. Rapeseed thrives very well in countries of the northern hemisphere of the planet having cool and humid climates, making it a very important oil- and protein-crop, since no other crop can produce such high yields of both oil and protein under these climatic conditions. In the coming decades, it has the potential of achieving the rank numero uno as the cheapest source of nutritious vegetable oil for the impoverished of the world. Nevertheless, it is prone to various abiotic stresses which not only affect normal growth rate of the plant but also decrease crop productivity by alarming proportions. It is, therefore, imperative to develop new stress tolerant varieties having higher productivity and better adaptation to the abiotic stresses abounding because of climate change. This chapter summarizes the various abiotic stresses afflicting rapeseed; the classical, genetic and molecular approaches that have been employed for breeding for abiotic stress tolerance, together with biotechnological and synthetic biology research breakthroughs aimed at creating abiotic stress-resistant climate-resilient varieties. The combination of classical and molecular breeding, being assisted by integrated omics and genome editing breakthroughs, can lead to speed up breeding of the crop and alter the rate of production of rapeseed worldwide, making it feasible to achieve the target of being number one in meeting the demands for vegetable oil of a soaring population.Keywords
Brassica napus
Oilseed rapeRapeseedCanolaAbiotic stressTemperature stressDrought stressSalt stress
Soybean is an agronomically important crop which is rich in seed protein (about 40%) and oil (about 20%), enriches the soil by fixing nitrogen through symbiosis with bacteria. It is widely used as food, feed, and for industrial purpose. In addition to human consumption, soybean is a major protein source in animal feeds. Soybean is also becoming a major crop for biodiesel production. In soybean, abiotic stresses including drought, temperature extremes, floods, salinity, acidity, mineral toxicity and nutrient deficiency have emerged as the major challenges for achieving the increased productivity. Breeding for tolerance to abiotic stresses is cumbersome due to their genetic control by multiple genes and are also very much influenced by the environment. The novel genomic designing approaches have enabled the improvement of soybean at a faster pace than traditional approaches. Genomic-assisted breeding, genomic selection, genome sequencing, marker-assisted selection, genetic engineering approaches, and genomics tools have been used to improve tolerance to biotic and abiotic stresses, yield and seed composition traits. Genomic designing overcomes the limitations of traditional breeding methods and accelerate the development of climate-smart soybean crops. Developing abiotic stress-tolerant soybean varieties have become convenient with the availability of a complete genomic sequence of soybean and functional genomics studies. This chapter discusses the major milestones in soybean genetics, genome mapping and recent developments in comparative and functional genomics and genome editing related to abiotic stresses.KeywordsSoybeanAbiotic stressDrought toleranceGWASFunctional genomics
Castor, Ricinus communis, is one of the top ten oil crops in the world. It has been paid more and more attention because of its high economic value. In the process of growth and development, it is subjected to a variety of abiotic stresses from the environment. In this chapter, the stresses on castor are discussed in consideration of heat tolerances, cold tolerance, drought tolerance, flooding and submergence tolerance, nutrient use efficiency, water use efficiency, salt-alkali stress and metal ion toxicity. It is suggested that more attention should be paid to the physiological adaptation mechanisms of castor to these stresses.
Soybean is an agro-economically leading crop of the world. Soybean is rich in seed protein (about 40%) and oil (about 20%) and enriches the soil by fixing nitrogen through symbiosis with bacteria. It is widely used as food, feed, and for industrial purposes. In soybean, biotic stresses such as insects-pests and diseases have emerged as the major challenge for increasing production. Breeding for tolerance to biotic stresses has made excellent progress however application of novel approaches such as genomic technologies are imperative to meet the challenges. Genomic crop designing and approaches have enabled the rapid improvement of soybean than traditional approaches. Genomic designing overcomes the limitations of traditional breeding methods and accelerates the development of climate-smart soybean crops. Genomic-assisted breeding, genomic selection, genome sequencing, marker-assisted selection, genetic engineering approaches, and genomics tools have been utilized to improve tolerance to biotic stresses, yield and seed composition traits. Developing biotic stress-tolerant soybean varieties have become convenient with the availability of genome sequences of soybean and functional genomics studies. This chapter discusses the major milestones in soybean genetics, genome mapping and recent developments in comparative and functional genomics related to biotic stresses.
Chamaecyparis formosensis is an endemic species of Taiwan, threatened from intensive use and illegal felling. An individual identification system for C. formosensis is required to provide scientific evidence for court use and deter illegal felling. In this study, 36 polymorphic simple sequence repeat markers were developed. By applying up to 28 non-linked of the developed markers, it is calculated that the cumulative random probability of identity (CPI) is as low as 1.652 × 10–12, and the identifiable population size is up to 60 million, which is greater than the known C. formosensis population size in Taiwan. Biogeographical analysis data show that C. formosensis from four geographic areas belong to the same genetic population, which can be further divided into three clusters: SY (Eastern Taiwan), HV and GW (Northwestern Taiwan), and MM (Southwestern Taiwan). The developed system was applied to assess the provenance of samples with 88.44% accuracy rate and therefore can serve as a prescreening tool to reduce the range required for comparison. The system developed in this study is a potential crime-fighting tool against illegal felling.
Availability of genetic diversity is the most important prerequisite of all plant breeding activities. A genetically diverse crop is more resilient to climate change and ensures food and nutritional security for an ever-increasing population. However, the outcome of almost all breeding programs is a population that is less genetically diverse than the parents. Plant genetic resource (PGR) conservation and utilization aim at conserving available germplasm around the world and keeping it readily available for utilization. Genomic strategies like the use of molecular markers, genotyping by sequencing (GBS), association mapping, and next-generation sequencing (NGS) in all the activities of a gene bank have made sharing and utilization of genetic resources easier. There is a scope of utilization of molecular strategies in improving genotype identification and utilization for adaptation to climate change, pre-breeding of valuable PGRs, and fear-free exchange of germplasm across boundaries. However, poor infrastructure, limited funding, lack of sophisticated facilities, and unavailability of staff trained in skills like genomics, computational biology, and population genetics make the vigorous utilization of genomic strategies in most of the gene banks a distant reality.KeywordsGenotyping by sequencing (GBS)Next-generation sequencing (NGS)Plant genetic resources (PGRs)Quantitative trait loci
Saccharina japonica is an economically and ecologically important alga. In recent decades, cytogenetic researches have promoted the large-scale farming of this kelp. However, no reliable cytological landmarks have been developed to distinguish chromosomes and help establish the precise karyotype of S. japonica. In this study, the simple sequence repeat (SSR) (GCT)7 motif with high occupancy level and polymorphism, and a specific SSR (GCT)7 were screened from the genome of S. japonica with all together bioinformatics tools. The existence of (GCT)7 motif and the specific SSR (GCT)7 in the genomic DNA of S. japonica were verified by PCR and southern blot analyses. Fluorescence in situ hybridization (FISH) analysis of the specific SSR (GCT)7 demonstrated that it was stably inherited and co-dominant. With the combined probes of SSR (GCT)7 motif and telomere DNA repeats, dual FISH was conducted for the karyotyping analysis of S. japonica. In agreement with the bioinformatics analysis results, FISH signals of the probe of (GCT)7 motif distributed on all the metaphase chromosomes of the gametophyte of S. japonica with different patterns, and 31 chromosomes were identified. Correspondingly, 31 pairs of homologous chromosomes were distinguished in the sporophyte of S. japonica on the basis of the co-localization of probes of (GCT)7 motif and telomere DNA repeats, as well as the size of metaphase chromosomes. This is the first report of the precise karyotype of S. japonica, which would contribute to the genomic physical mapping and the further genetic breeding of this important brown alga.
Analysis of intra- and inter-population diversity has become important for defining the genetic status and distribution patterns of a species and a powerful tool for conservation programs, as high levels of inbreeding could lead into whole population extinction in few generations. Microsatellites (SSR) are commonly used in population studies but discovering highly variable regions across species’ genomes requires demanding computation and laboratorial optimization. In this work, we combine next generation sequencing (NGS) with automatic computing to develop a genomic-oriented tool for characterizing SSRs at the population level. Herein, we describe a new Python pipeline, named Micro-Primers, designed to identify, and design PCR primers for amplification of SSR loci from a multi-individual microsatellite library. By combining commonly used programs for data cleaning and microsatellite mining, this pipeline easily generates, from a fastq file produced by high-throughput sequencing, standard information about the selected microsatellite loci, including the number of alleles in the population subset, and the melting temperature and respective PCR product of each primer set. Additionally, potential polymorphic loci can be identified based on the allele ranges observed in the population, to easily guide the selection of optimal markers for the species. Experimental results show that Micro-Primers significantly reduces processing time in comparison to manual analysis while keeping the same quality of the results. The elapsed times at each step can be longer depending on the number of sequences to analyze and, if not assisted, the selection of polymorphic loci from multiple individuals can represent a major bottleneck in population studies.
X-linked spinal and bulbar muscular atrophy (Kennedy's disease) is an adult-onset form of motorneuron disease which may be associated with signs of androgen insensitivity. We have now investigated whether the androgen receptor gene on the proximal long arm of the X chromosome is a candidate gene for this disease. In patient samples we found androgen receptor gene mutations with increased size of a polymorphic tandem CAG repeat in the coding region. These amplified repeats were absolutely associated with the disease, being present in 35 unrelated patients and none of 75 controls. They segregated with the disease in 15 families, with no recombination in 61 meioses (the maximum log likelihood ratio (lod score) is 13.2 at a recombination rate of 0). The association is unlikely to be due to linkage disequilibrium, because 11 different disease alleles were observed. We conclude that enlargement of the CAG repeat in the androgen receptor gene is probably the cause of this disorder.
Polymers of random 14 mer oligonucleotides are shown to detect discrete loci in the human genome. Eighteen different synthetic
tandem repeats of random 14 base-pair units (STRs) have been generated and all of them turn out to detect polymorphic loci
on southern blots of human DNA samples, presumably corresponding to a variable number of tandem repeats (VNTR). This finding
suggests that minisatellites are a major component of the human genome and are strongly associated with the generation of
genetic variability. In addition, it should open new strategies to make new polymorphic probes available.
Interspersed DNA elements of the form (dC-dA)n.(dG-dT)n constitute one of the most abundant human repetitive DNA families. We report that specific human (dC-dA)n.(dG-dT)n blocks are polymorphic in length among individuals and therefore represent a vast new pool of potential genetic markers. Comparison of sequences from the literature for (dC-dA)n.(dG-dT)n blocks cloned two or more times revealed length polymorphisms in seven of eight cases. Variations in the lengths of 10 (dC-dA)n.(dG-dT)n blocks were directly demonstrated by amplifying the DNA within and immediately flanking the repeat blocks by using the polymerase chain reaction and then resolving the amplified DNA on polyacrylamide DNA sequencing gels. Use of the polymerase chain reaction to detect DNA polymorphisms offers improved sensitivity and speed compared with standard blotting and hybridization.
A cDNA clone, pTU04, which hybridizes to two different sizes of mRNA on Northern blots was isolated from soybean suspension culture cell poly(A) RNA. Northern analysis reveals that meristematic tissue produces a 1050-nucleotide mRNA while quiescent mature cells produce primarily a 1220-nucleotide mRNA homologous to pTU04. The cDNA and its corresponding genomic clone have been partially characterized. The nucleotide sequence of the gene predicts a proline-rich protein, designated SbPRP1, which contains a signal peptide sequence and 43 repeats of a sequence consisting primarily of Pro-Pro-Val-Tyr-Lys (CCA-CCA-GTT-TAC-AAG). From nuclease S1 and hybrid-select translation analyses, the cDNA clone pTU04 appears to represent the mRNA for the mature tissue 1220-nucleotide RNA observed on Northern blots. Although there is no direct proof that the encoded protein is a cell wall protein, it has the properties similar to previously isolated cell wall proteins: 1) it is very basic with a high content of Pro, Tyr, and Lys; 2) it has similar hydropathic properties; and 3) its repeating unit shares sequence homology with that of more highly characterized cell wall proteins, generally termed extensin (Chen, J., and Varner, J. E. (1985) EMBO J. 4, 2145-2151; Smith, J. J., Muldoon, E. P., Willard, J. J., and Lamport, D. T. A. (1986) Phytochemistry 25, 1021-1030.
Simple repetitive DNA sequences are a widespread and abundant feature of genomic DNA. The following several features characterize such sequences: (1) they typically consist of a variety of repeated motifs of 1-10 bases--but may include much larger repeats as well; (2) larger repeat units often include shorter ones within them; (3) long polypyrimidine and poly-CA tracts are often found; and (4) tandem arrangements of closely related motifs are often found. We propose that slipped-strand mispairing events, in concert with unequal crossing-over, can readily account for all of these features. The frequent occurrence of long tandem repeats of particular motifs (polypyrimidine and poly-CA tracts) appears to result from nonrandom patterns of nucleotide substitution. We argue that the intrahelical process of slipped-strand mispairing is much more likely to be the major factor in the initial expansion of short repeated motifs and that, after initial expansion, simple tandem repeats may be predisposed to further expansion by unequal crossing-over or other interhelical events because of their propensity to mispair. Evidence is presented that single-base repeats (the shortest possible motifs) are represented by longer runs in mammalian introns than would be expected on a random basis, supporting the idea that SSM may be a ubiquitous force in the evolution of the eukaryotic genome. Simple repetitive sequences may therefore represent a natural ground state of DNA unselected for coding functions.
Soybeans, Glycine max, synthesize a family of low-molecular-weight heat shock (HS) proteins in response to HS. The DNA sequences of two genes encoding 17.5- and 17.6-kilodalton HS proteins were determined. Nuclease S1 mapping of the corresponding mRNA indicated multiple start termini at the 5' end and multiple stop termini at the 3' end. These two genes were compared with two other soybean HS genes of similar size. A comparison among the 5' flanking regions encompassing the presumptive HS promoter of the soybean HS-protein genes demonstrated this region to be extremely homologous. Analysis of the DNA sequences in the 5' flanking regions of the soybean genes with the corresponding regions of Drosophila melanogaster HS-protein genes revealed striking similarity between plants and animals in the presumptive promoter structure of thermoinducible genes. Sequences related to the Drosophila HS consensus regulatory element were found 57 to 62 base pairs 5' to the start of transcription in addition to secondary HS consensus elements located further upstream. Comparative analysis of the deduced amino acid sequences of four soybean HS proteins illustrated that these proteins were greater than 90% homologous. Comparison of the amino acid sequence for soybean HS proteins with other organisms showed much lower homology (less than 20%). Hydropathy profiles for Drosophila, Xenopus, Caenorhabditis elegans, and G. max HS proteins showed a similarity of major hydrophilic and hydrophobic regions, which suggests conservation of functional domains for these proteins among widely dispersed organisms.
Using positional cloning strategies, we have identified a CTG triplet repeat that undergoes expansion in myotonic dystrophy patients. This sequence is highly variable in the normal population. PCR analysis of the interval containing this repeat indicates that unaffected individuals have between 5 and 27 copies. Myotonic dystrophy patients who are minimally affected have at least 50 repeats, while more severely affected patients have expansion of the repeat containing segment up to several kilobase pairs. The CTG repeat is transcribed and is located in the 3' untranslated region of an mRNA that is expressed in tissues affected by myotonic dystrophy. This mRNA encodes a polypeptide that is a member of the protein kinase family.
Fifty-eight soybean accessions from the genus Glycine, subgenus Soja, were surveyed with 17 restriction fragment length polymorphism (RFLP) genetic markers to assess the level of molecular diversity and to evaluate the usefulness of previously identified RFLP markers. In general, only low levels of molecular diversity were observed: 2 of the 17 markers exhibited three alleles per locus, whereas all others had only two alleles. Thirty-five percent of the markers had rare alleles present in only 1 or 2 of the 58 accessions. Molecular diversity was least among cultivated soybeans and greatest between accessions of different soybean species such as Glycine max (L.) Merr. and G. soja Sieb. and Zucc. Principal component analysis was useful in reducing the multidimensional genotype data set and identifying genetic relationships.
Restriction Fragment Length Polymorphisms (RFLP) have been identified between widely distant cultivars (Minsoy and Noir 1 ) of soybean Glycine max (L.) Merrill. Using as probes randomly chosen clones of DNA, one in five probes revealed a polymorphism. More than half of these polymorphisms appear to result from rearrangements of the genomic DNA. Twenty seven markers were analyzed for linkage in F2 plants. Eleven of these markers were contained in four linkage groups. Five cultivars were compared in a search for new alleles. When RFLP markers corresponding to low copy DNA were used to analyze three other cultivars — Sooty, Forrest and Mandarin (Ottawa) — few new alleles were found. Using these probes, five different markers could be used to differentiate the five cultivars. Complex probes, which correspond to repeated DNA, revealed different polymorphisms in different cultivars and a single such probe could be used to distinguish the five cultivars from each other.
The genomes of all eukaryotes appear to contain a special class of loci, termed microsatellites, which can serve, if sequenced and taken as the substrate for the polymerase chain reaction, as highly informative, locus-specific markers. By analogy to the "sequence tagged sites" recently proposed by Olsen et al. for standardizing the human physical gene map, these microsatellite markers are termed "sequence tagged microsatellite sites" (STMS). Genetic maps based on STMS will share with the Olsen physical maps the advantage that mapping vocabularies will be standardized to the DNA sequence base and that access to any particular locus will not require shipping or storing cloned probes. The species map will consist simply of a listing of nucleotide sequences. Reference populations for developing STMS maps can be chosen on the basis of biological or economic interest. It will not be necessary to maximize for genetic divergence.
Using positional cloning strategies, we have identified a CTG triplet repeat that undergoes expansion in myotonic dystrophy patients. This sequence is highly variable in the normal population. PCR analysis of the interval containing this repeat indicates that unaffected individuals have been 5 and 27 copies. Myotonic dystrophy patients who are minimally affected have at least 50 repeats, while more severely affected patients have expansion of the repeat containing segment up to several kilobase pairs. The CTG repeat is transcribed and is located in the 3' untranslated region of an mRNA that is expressed in tissues affected by myotonic dystrophy. This mRNA encodes a polypeptide that is a member of the protein kinase family.
Important new polymerase chain reaction-based techniques have been developed to assist in genome analysis. Applications range from genetic and physical mapping of DNA to sequence analysis. The polymerase chain reaction has played a significant role in increasing the feasibility of many aspects of genome analysis and positional cloning.
Fragile X syndrome results from mutations in a (CGG)n repeat found in the coding sequence of the FMR-1 gene. Analysis of length variation in this region in normal individuals shows a range of allele sizes varying from a low of 6 to a high of 54 repeats. Premutations showing no phenotypic effect in fragile X families range in size from 52 to over 200 repeats. All alleles with greater than 52 repeats, including those identified in a normal family, are meiotically unstable with a mutation frequency of one, while 75 meioses of alleles of 46 repeats and below have shown no mutation. Premutation alleles are also mitotically unstable as mosaicism is observed. The risk of expansion during oogenesis to the full mutation associated with mental retardation increases with the number of repeats, and this variation in risk accounts for the Sherman paradox.
Genetic linkage maps order polymorphic loci (markers) along a chromosome and provide a measure of distance that is an inherently statistical measure of the frequency of meiotic recombination and has no simple relationship to DNA length in Mb. Few accurate and dense maps now exist. Maps with markers spaced every 2 cM providing accurate estimates of distance should be available in the next few years, however.
The high degree of polymorphism displayed by DNA microsatellites makes them useful as DNA markers in linkage studies. A search of the DNA sequence databases revealed that the locations of dinucleotide microsatellites are often conserved among mammalian species, enabling the prediction of the presence of DNA microsatellites using comparative genetic data. In closely related species such as cattle and sheep, this conservation was close enough to allow PCR primers designed for use in one species to be used to analyze microsatellite length polymorphism in the other. A total of 48 sets of primer pairs, flanking bovine microsatellites and giving polymorphic PCR products in that species, were tested with template DNA from sheep, horses, and humans. Specific products were obtained in 27 cases (56%) with ovine DNA, 20 of which (42%) showed polymorphisms. With equine DNA, 3 (6.2%) gave specific but monomorphic products, while no specific products were obtained using human DNA. The ability to use heterologous PCR primers, coupled with comparative mapping information will facilitate the use of DNA microsatellites in gene mapping studies in closely related species such as cattle and sheep, rat and mouse, or primates.
Tandemly reiterated sequences represent a rich source of highly polymorphic markers for genetic linkage, mapping, and personal identification. Human trimeric and tetrameric short tandem repeats (STRs) were studied for informativeness, frequency, distribution, and suitability for DNA typing and genetic mapping. The STRs were highly polymorphic and inherited stably. A STR-based multiplex PCR for personal identification is described. It features fluorescent detection of amplified products on sequencing gels, specific allele identification, simultaneous detection of independent loci, and internal size standards. Variation in allele frequencies were explored for four U.S. populations. The three STR loci (chromosomes 4, 11, and X) used in the fluorescent multiplex PCR have a combined average individualization potential of 1/500 individuals. STR loci appear common, being found every 300-500 kb on the X chromosome. The combined frequency of polymorphic trimeric and tetrameric STRs could be as high as 1 locus/20 kb. The markers should be useful for genetic mapping, as they are sequence based, and can be multiplexed with the PCR. A method enabling rapid localization of STRs and determination of their flanking DNA sequences was developed, thus simplifying the identification of polymorphic STR loci. The ease by which STRs may be identified, as well as their genetic and physical mapping utility, give them the properties of useful sequence tagged sites (STSs) for the human genome initiative.
The dinucleotide repetitive sequence, (GT)n, is highly interspersed in eukaryotic genomes and may have functional roles in genetic recombination or the modulation of transcriptional activity. We have examined the distribution and conservation of position of GT repetitive sequences in several mammalian genomes. The distribution of GT repetitive sequences in the human genome was determined by the analysis of over 3700 cosmid clones containing human insert DNA. On average, a GT repetitive sequence occurs every 30 kb in DNA from euchromatic regions. GT repetitive sequences are significantly underrepresented in centric heterochromatin. The density of GT repetitive sequences in the human genome could also be estimated by analyzing GenBank genomic sequences that include introns and flanking sequences. The frequency of GT repetitive sequences found in GenBank human DNA sequences was in close agreement with that obtained by experimental methods. GenBank genomic sequences also revealed that (GT)n repetitive sequences (n greater than 6) occur every 18 and 21 kb, on average, in mouse and rat genomes. Comparative analysis of 31 homologous sequences containing (GT)n repetitive sequences from several mammals representing four orders revealed that the positions of these repeats have been conserved between closely related species, such as humans and other primates. To a lesser extent, positions of GT repetitive sequences have been conserved between species in distantly related groups such as primates and rodents. The distribution and conservation of GT repetitive sequences is discussed with respect to possible functional roles of the repetitive sequence.
Length polymorphisms within simple-sequence loci occur ubiquitously in non-coding eukaryotic DNA and can be highly informative in the analysis of natural populations. Simple-sequence length polymorphisms (SSLP) in the long-finned pilot whale Globicephala melas (Delphinidae) have provided useful information on the mating system as well as on the genetic structure of populations. We have therefore tested whether the polymerase chain reaction primers designed for Globicephala could also be used to uncover variability in other whale species. Homologous loci could indeed be amplified from a diverse range of whales, including all toothed (Odontoceti) and baleen whales (Mysticeti) tested. Cloning and sequencing these loci from 11 different species revealed an unusually high conservation of sequences flanking the simple-sequence stretches, averaging 3.2% difference over 35-40 Myr. This represents the lowest divergence rate for neutral nucleotide positions found for any species group so far and raises the possible need for a re-evaluation of the age of the modern whales. On the other hand, the high conservation of non-coding sequences in whales simplifies the application of SSLP DNA fingerprinting in cetacean species, as primers designed for one species will often uncover variability in other species.
Abundant human interspersed repetitive DNA sequences of the form (dC-dA)n.(dG-dT)n have been shown to exhibit length polymorphisms. Examination of over 100 human (dC-dA)n.(dG-dT)n sequences revealed that the sequences differed from each other both in numbers of repeats and in repeat sequence type. Using a set of precise classification rules, the sequences were divided into three categories: perfect repeat sequences without interruptions in the runs of CA or GT dinucleotides (64% of total), imperfect repeat sequences with one or more interruptions in the run of repeats (25%), and compound repeat sequences with adjacent tandem simple repeats of a different sequence (11%). Informativeness of (dC-dA)n.(dG-dT)n markers in the perfect sequence category was found to increase with increasing average numbers of repeats. PIC values ranged from 0 at about 10 or fewer repeats to above 0.8 for sequences with about 24 or more repeats. (dC-dA)n.(dG-dT)n polymorphisms in the imperfect sequence category showed lower informativeness than expected on the basis of the total numbers of repeats. The longest run of uninterrupted CA or GT repeats was found to be the best predictor of informativeness of (dC-dA)n.(dG-dT)n polymorphisms regardless of the repeat sequence category.
The human genome contains approximately 50,000 copies of an interspersed repeat with the sequence (dT.dG/dA.dC)n, where n = approximately 10-60. We and others have found that several of these repeats have variable lengths in different individuals, with allelic fragments varying in size by multiples of 2 bp. These "microsatellite" variable number of tandem repeats (VNTRs) may be scored by PCR, using unique flanking primers to amplify the repeat-containing regions and resolving the products on DNA sequencing gels. Since few VNTRs have been found on the X chromosome, we screened a flow-sorted X chromosome-specific genomic library for microsatellites. Approximately 25% of the phage clones hybridized to a poly (dT-dG).poly(dA-dC) probe. Of seven X-linked microsatellites present in positive phages, five are polymorphic and three have both eight or more alleles and heterozygosities exceeding 75%. Using PCR to amplify genomic DNAs from hybrid cell panels, we confirmed the X localization of these VNTRs and regionally mapped four of them. The fifth VNTR was regionally mapped by virtue of its tight linkage to DXS87 in Centre du Polymorphisme Humain families. We conclude that whatever factors limit the occurrence of "classical" VNTRs and RFLPs on the X chromosome do not appear to operate in the case of microsatellite VNTRs.
Fifty sequences from the mouse genome database containing simple sequence repeats or microsatellites have been analysed for
size variation using the polymerase chain reaction and gel electrophoresis. 88% of the sequences, most of which contain the
dinucleotide repeat, CA/GT, showed size variations between different inbred strains of mice and the wild mouse, Mus spretus. 62% of sequences had 3 or more alleles. GA/CT and AT/TA -containing sequences were also variable. About half of these size
variants were detectable by agarose gel electrophoresis. This simple approach is extremely useful in linkage and genome mapping
studies and will facilitate construction of high resolution maps of both the mouse and human genomes.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme,
isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed
at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments
up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule
present only once in a sample of 10(5) cells.
The human genome contains approximately 50,000 copies of an interspersed repeat with the sequence (dT-dG)n, where n = approximately 10-60. In humans, (TG)n repeats have been found in several sequenced regions. Since minisatellite regions with larger repeat elements often display extensive length polymorphisms, we suspected that (TG)n repeats ("microsatellites") might also be polymorphic. Using the polymerase chain reaction to amplify a (TG)n microsatellite in the human cardiac actin gene, we detected 12 different allelic fragments in 37 unrelated individuals, 32 of whom were heterozygous. Codominant Mendelian inheritance of fragments was observed in three families with a total of 24 children. Because of the widespread distribution of (TG)n microsatellites, polymorphisms of this type may be generally abundant and present in regions where minisatellites are rare, making such microsatellite loci very useful for linkage studies in humans.
Short simple sequence stretches occur as highly repetitive elements in all eukaryotic genomes and partially also in prokaryotes
and eubacteria (1–6). They are thought to arise by slippage like events working on randomly occuring internally repetitive
sequence stretches (3, 7, 8). This predicts that they should be generally hypervariable in length. I have used the polymerase
chain reaction (PCR) process to show that several randomly chosen simple sequence loci with different nucleotide composition
and from different species show extensive length polymorphisms. These simple sequence length polymorphisms (SSLP) may be usefully
exploited for identity testing, population studies, linkage analysis and genome mapping.
In a report issued in January 1988, the National Research
Council (NRC) Committee on the Mapping and Sequencing
of the Human Genome, on which the present authors
served, recommended a staged mapping and sequencing project
with early emphases on physical mapping of human DNA, mapping and sequencing of the genomes of model organisms, and the development of sequencing technology (1). As the Committee's recommendations on physical mapping are beginning to be implemented on a substantial scale, it is timely to review these recommendations in the light of recent technical advances. In particular, the polymerase chain reaction (PCR) (2), a method that has only come
into widespread use during the past 2 years, seems to us to offer a path toward a physical map that largely circumvents two problems that were prominent in the NRC Committee's discussions. One of these was the difficulty of merging mapping data gathered by diverse methods in different laboratories into a consensus physical map. The second was the logistics and expense of managing the huge collections
of cloned segments on which the mapping data would depend
almost absolutely.
Sequences located several kilobases both 5′ and 3′ of the stably transcribed portion of several genes hybridize to radio-labeled
pure fragments of the alternating sequence poly (dG-dT)(dC-dA) [poly(GT)]. The genes include the ribosornal DNA of mouse,
rat, and human, and also human glucose-6-phosphate dehydrogenase (G6PD) and mouse hypoxanthine-guanine phosphoribosyl transferase
(HPRT). HPRT has additional hybridizing sequences in introns. Fragments that include the hybridizing sequences and up to 300
bp of adjoining DNA show perfect runs of poly(GT) (>30bp) in all but the human 5′ region of rDNA, which shows a somewhat different
alternating purine:pyrimidine sequence, poly(GTAT) (36bp). Within 150 bp of these sequences in various instances are found
a number of other sequences reported to affect DNA conformation in model systems. Most marked is an enhancement of sequences
matching at least 67% to the consensus binding sequence for topoisomerase II. Two to ten-fold less of such sequences were
found in other sequenced portions of the nontranscribed spacer or in the transcribed portion of rDNA. The conservation of
the locations of tracts of alternating purlne:pyrimidine between evolutionarily diverse species is consistent with a possible
functional role for these sequences.
Simple repeated GATA and GACA sequences which were originally isolated from sex-specific snake satellite DNA have been found subsequently in all eukaryotes studied. The organization of these sequences within the mouse genome was investigated here by using synthetic oligonucleotide probes as a novel tool in comparison with conventional hybridization probes. Southern blot hybridization showed sex-specific patterns with both the (GATA)4 and (GACA)4 oligonucleotide probes, as previously described with conventional probes. The quantitative analysis of two mouse DNA phage libraries and of 25 isolated GATA-positive phage clones revealed intensive interspersion of GATA sequences with GACA, and with other repetitive and single-copy sequences. Ubiquitous interspersion and homogeneous genomic distribution of GATA and GACA sequences were confirmed by hybridization in situ of the oligonucleotide probes to metaphase chromosomes. The lengths of the GATA and GACA stretches were found to vary considerably in the individual phage clones. DNA inserts from 20 phages were assigned to autosomes and sex chromosomes and three genomic fragments were found to be confined to the Y chromosome. The organization of GATA and GACA sequences is discussed in the context of their evolutionary potential and possible conservation mechanisms.
Simple sequences are stretches of DNA which consist of only one, or a few tandemly repeated nucleotides, for example poly
(dA).poly (dT) or poly (dG-dT).poly (dC-dA). These two types of simple sequence have been shown to be repetitive and interspersed
in many eukaryotic genomes. Several other types have been found by sequencing eakaryotic DNA. In this report we have undertaken
a systematical survey for simple sequences. We hybridized synthetical simple sequence DNA to genome blots of phylogenetically
different organisms. We found that many, probably even all possible types of simple sequence are repetitive components of
eukaryotic genomes. We propose therefore that they arise by common mechanisms namely slippage replication and unequal crossover
and that they might have no general function with regards to gene expression. This latter inference is supported by the fact
that we have detected simple sequences only in the metabolically inactive micronucleus of the protozoan Stylonychia, but not in the metabolically active macronucleus which is derived from the micronucleus by chromosome diminution.
The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis
and publication of biological sequence data. A group of programs that will interact with each research-article has been developed
for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions
for transfer are described.
Z-DNA-specific antibodies have been used to demonstrate the formation of left-handed Z-DNA in sequences of (dC-dA)32 X (dG-dT)32 in negatively supercoiled plasmids. Z-DNA was found to form at physiological negative superhelical densities (sigma = -0.05) in the absence of high salt concentration or chemical modifications. The binding site of the antibody to the DNA sequences was demonstrated by crosslinking experiments. (dC-dA)n X (dG-dT)n sequences are widespread in eukaryotic DNA, and possible roles for such Z elements in chromatin activation or genome rearrangements are suggested.
Two methods for increasing the length of DNA sequence data that can be read off a polyacrylamide gel are described. We have developed a rapid way to pour a buffer concentration gradient gel that, by altering the vertical band separation on an autoradiograph, allows more sequence to be obtained from a gel. We also show that the use of deoxyadenosine 5'-(alpha-[35S]thio)triphosphate as the label incorporated in dideoxynucleotide sequence reactions increases the sharpness of the bands on an autoradiograph and so increases the resolution achieved.
By Southern blotting and hybridization analysis using 32P-labeled poly(dT-dG) . poly(dC-dA) as a probe, we have found, in eukaryotic genomes, a huge number of stretches of dT-dG alternating sequence, a sequence that has been shown to adopt the Z-DNA conformation under some conditions. This sequence was found in all eukaryotic genomes examined from yeast to human, indicating extraordinary evolutionary conservation. The number of the sequence ranged from about 100 in yeast to tens of thousands in higher eukaryotes. Comparison of nucleotide sequences of dT-dG alternating regions and its flanking regions in several cloned genes showed that the repeated element [the Z(T-G) element]] consists only of dT-dG alternating sequence with variable length. The presence of another purine-pyrimidine alternating sequence was also surveyed in eukaryotic genomes by Southern blot hybridization using 32P-labeled poly(dG-dC) . poly(dG-dC) as the probe. The stretches of dC-dG alternating sequence [the Z(C-G) element] were found to be moderately repetitive in human, mouse, and salmon genomes. However, a few and no copies of the Z(C-G) element were found in yeast and calf genomes, respectively. These results provide evidence for the abundance of potential Z-DNA-forming sequences in nature.
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Restriction fragment length polymorphisms as genetic markers in soybean
- N Apuya
- B Frarier
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Apuya, N., Frarier, B., Keim, P., Roth, E.J. and Lark, K.G. (1988)
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Locations and contexts of sequences that hybridize to poly(dG-dT).(dC-dA) in mammalian ribosomal DNAs and two X-linked genes
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Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox
- Y H Fu
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CGG repeat at the fragile X site results in genetic instability:
resolution of the Sherman paradox. Cell, 67. 1047-1058.