Genetic diversity,population structure and association analysis in linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.)

The present investigation aimed to explore the level of genetic diversity, determine the population structure in a larger set of germplasm of linseed using microsatellite marker and identify linked markers through association mapping. A total of 168 accessions of linseed were evaluated for major agro-economic traits and SSRs markers deployed for diversity assessment. A total of 337 alleles were amplified by 50 SSRs ranging from 2 to 13 with an average of 6.74 ± 2.8 alleles per loci. The neighbor joining based clustering grouped all the accessions into three major clusters that were also confirmed by scatter plot of PCoA. While model based clustering determined four sub-populations (K = 4). Further, analysis of molecular variance analysis considering three population showed that maximum variation (79%) was within the population. We identified one putative SSR marker (Lu_3043) linked with days to 50% flowering through both GLM and MLM analysis of association mapping. The results of this preliminary study revealed genetic diversity, population structure in linseed and linked marker which could be utilized in future breeding program.     

Features of SNP and SSR diversity in a set of ICARDA barley germplasm collection

Detection and utilization of genetic variation available in the germplasm collection for crop improvement have been the prime activities of breeders. Here a set of ICARDA barley germplasm collection comprising of 185 cultivated (Hordeum vulgare L.) and 38 wild (H. spontaneum L.) genotypes originated from 30 countries of four continents was genotyped with 68 single nucleotide polymorphism (SNP) and 45 microsatellite or simple sequence repeat (SSR) markers derived from genes (expressed sequence tags, ESTs). As two SNP markers provided 2 and 3 datapoints, a total of 71 SNPs were surveyed that yielded a total of 143 alleles. The number of SSR alleles per locus ranged from 3 to 22 with an average of 7.9 per marker. Average PIC (polymorphism information content) value for SSR and SNP markers were recorded as 0.63 and 0.38, respectively. Heterogeneity was recorded at both SNP and SSR loci in an average of 5.72 and 12.42% accessions, respectively. Genetic similarity matrices for SSR and SNP allelic data were highly correlated (r = 0.75, P < 0.005) and therefore allelic data for both markers were combined and analyzed for understanding the genetic relationships among the germplasm surveyed. Majority of clusters/subclusters were found to contain genotypes from the same geographic origins. While comparing the genetic diversity, the accessions coming from Middle East Asia and North East Asia showed more diversity as compared to that of other geographic regions. Majority of countries representing Africa, Middle East Asia, North East Asia and Arabian Peninsula included the genotypes that contained rare alleles. As expected, spontaneum accessions, as compared to vulgare accessions, showed a higher number of total alleles, higher number of alleles per locus, higher effective number of alleles and higher allelic richness and a higher number of rare alleles were observed. In summary, the examined ICARDA germplasm set showed ample natural genetic variation that can be harnessed for future breeding of barley as climate change and sustainability have become important throughout all growing areas of the world, drought/heat tolerance being the most important ones.     

Microsatellite analysis reveals a progressive widening of the genetic basis in the elite durum wheat germplasm

It has been argued that the level of genetic diversity in the modern durum wheat (Triticum turgidum L. var. durum) elite germplasm may have declined due to the high selection pressure applied in breeding programs. In this study, 58 accessions covering a wide spectrum of genetic diversity of the cultivated durum wheat gene pool were characterized with 70 microsatellite loci (or simple sequence repeats, SSRs). On average, SSRs detected 5.6 different allelic variants per locus, with a mean diversity index (DI) equal to 0.56, thus revealing a diversity content comparable to those previously observed with SSRs in other small-grain cereal gene pools. The mean genetic similarity value was equal to 0.44. A highly diagnostic SSR set has been identified. A high variation in allele size was detected among SSR loci, suggesting a different suitability of these loci for estimating genetic diversity. The B genome was characterized by an overall polymorphism significantly higher than that of the A genome. Genetic diversity is organised in well-distinct sub-groups identified by the corresponding foundation-genotypes. A large portion (92.7%) of the molecular variation detected within the group of 45 modern cvs was accounted for by SSR alleles tracing back to ten foundation-genotypes; among those, the most recent CIMMYT-derived founders were genetically distant from the old Mediterranean ones. On the other hand, rare alleles were abundant, suggesting that a large number of genetic introgressions contributed to the foundation of the well-diversified germplasm herein considered. The profiles of recently released varieties indicate that the level of genetic diversity present in the modern durum wheat germplasm has actually increased over time.Communicated by F. Salamini     

Development and Characterization of Polymorphic EST-SSR and Genomic SSR Markers for Tibetan Annual Wild Barley

Tibetan annual wild barley is rich in genetic variation. This study was aimed at the exploitation of new SSRs for the genetic diversity and phylogenetic analysis of wild barley by data mining. We developed 49 novel EST-SSRs and confirmed 20 genomic SSRs for 80 Tibetan annual wild barley and 16 cultivated barley accessions. A total of 213 alleles were generated from 69 loci with an average of 3.14 alleles per locus. The trimeric repeats were the most abundant motifs (40.82%) among the EST-SSRs, while the majority of the genomic SSRs were di-nuleotide repeats. The polymorphic information content (PIC) ranged from 0.08 to 0.75 with a mean of 0.46. Besides this, the expected heterozygosity (He) ranged from 0.0854 to 0.7842 with an average of 0.5279. Overall, the polymorphism of genomic SSRs was higher than that of EST-SSRs. Furthermore, the number of alleles and the PIC of wild barley were both higher than that of cultivated barley, being 3.12 vs 2.59 and 0.44 vs 0.37. Indicating more polymorphism existed in the Tibetan wild barley than in cultivated barley. The 96 accessions were divided into eight subpopulations based on 69 SSR markers, and the cultivated genotypes can be clearly separated from wild barleys. A total of 47 SSR-containing EST unigenes showed significant similarities to the known genes. These EST-SSR markers have potential for application in germplasm appraisal, genetic diversity and population structure analysis, facilitating marker-assisted breeding and crop improvement in barley.     

Population structure and genetic diversity in a commercial maize breeding program assessed with SSR and SNP markers

Information about the genetic diversity and population structure in elite breeding material is of fundamental importance for the improvement of crops. The objectives of our study were to (a) examine the population structure and the genetic diversity in elite maize germplasm based on simple sequence repeat (SSR) markers, (b) compare these results with those obtained from single nucleotide polymorphism (SNP) markers, and (c) compare the coancestry coefficient calculated from pedigree records with genetic distance estimates calculated from SSR and SNP markers. Our study was based on 1,537 elite maize inbred lines genotyped with 359 SSR and 8,244 SNP markers. The average number of alleles per locus, of group specific alleles, and the gene diversity (D) were higher for SSRs than for SNPs. Modified Roger’s distance (MRD) estimates and membership probabilities of the STRUCTURE matrices were higher for SSR than for SNP markers but the germplasm organization in four heterotic pools was consistent with STRUCTURE results based on SSRs and SNPs. MRD estimates calculated for the two marker systems were highly correlated (0.87). Our results suggested that the same conclusions regarding the structure and the diversity of heterotic pools could be drawn from both markers types. Furthermore, although our results suggested that the ratio of the number of SSRs and SNPs required to obtain MRD or D estimates with similar precision is not constant across the various precision levels, we propose that between 7 and 11 times more SNPs than SSRs should be used for analyzing population structure and genetic diversity.     

Evaluation of the genetic diversity and population structure of sesame (Sesamum indicum L.) using microsatellite markers

Sixteen polymorphic microsatellite (SSR) markers, developed from an SSR-enriched genomic DNA library of sesame (Sesamum indicum L.), were used to assess genetic diversity, phylogenetic relationships, and population structure among 150 sesame accessions collected from 22 countries. A total of 121 alleles were detected among the sesame accessions. The number of detected alleles varied from 2 to 18, with an average of 7.6 alleles per locus. Polymorphism information content values ranged from 0.03 to 0.79, with an average of 0.42. These values indicated an excess of heterozygous individuals at 16 loci and an excess of homozygous individuals at three loci. Of these, 32 genotype-specific alleles were identified at 11 of 16 polymorphic SSR markers. Cluster analyses were performed by accession and population, revealing a complex accession distribution pattern with mean genetic similarity coefficient of 0.45 by accession and 0.52 by population. The wide variation in genetic similarity among the accessions revealed by SSRs reflected a high level of polymorphism at the DNA level. Model-based structure analysis revealed the presence of three groups that were basically consistent with the clustering results based on genetic distance. These findings may be used to augment the sesame germplasm and to increase the effectiveness of sesame breeding.     

Assessment of genetic variation and differentiation of hop genotypes by microsatellite and AFLP markers.

Microsatellites have many desirable marker properties and have been increasingly used in crop plants in genetic diversity studies. Here we report on the characterisation of microsatellite markers and on their use for the determination of genetic identities and the assessment of genetic variability among accessions from a germplasm collection of hop. Thirty-two polymorphic alleles were found in the 55 diploid genotypes, with an average number of eight alleles (3.4 effective alleles) for four microsatellite loci. Calculated polymorphic information content values classified three loci as informative markers and two loci as suitable for mapping. The average observed heterozygosity was 0.7 and the common probability of identical genotypes was 3.271 x 10(-4). An additional locus, amplified by one primer pair, was confirmed by segregation analysis of two crosses. The locus discovered was heterozygous, with a null allele in the segregating population. The same range of alleles was detected in nine triploid and five tetraploid hop genotypes. Cultivar heterozygosity varied among all 69 accessions, with only one cultivar being homozygous at four loci. Microsatellite allele polymorphisms distinguished 81% of all genotypes; the same allelic profile was found mainly in clonally selected cultivars. Cultivar-specific alleles were found in some genotypes, as well as a specific distribution of alleles in geographically distinct hop germplasms. The genetic relationship among 41 hop accessions was compared on the basis of microsatellite and AFLP polymorphisms. Genetic similarity dendrograms showed low correlation between the two marker systems. The microsatellite dendrogram grouped genetically related accessions reasonably well, while the AFLP dendrogram showed good clustering of closely related accessions and, additionally, separated two geographically distinct hop germplasms. The results of microsatellite and AFLP analysis are discussed from the point of view of the applicability of the two marker systems for different aspects of germplasm evaluation.     

Allelic reduction and genetic shift in the Canadian hard red spring wheat germplasm released from 1845 to 2004

Analysis of genetic diversity changes in existing gene pools of cultivated crops is important for understanding the impact of plant breeding on crop genetic diversity and developing effective indicators for genetic diversity of cultivated plants. The objective of this study was to assess genetic diversity changes in 75 Canadian hard red wheat (Triticum aestivum L.) cultivars released from 1845 to 2004 using 31 simple sequence repeats (SSRs) markers. A total of 267 SSR alleles were detected, and their allelic frequencies ranged from 0.01 to 0.97, with an average of 0.14. Significant allelic reduction was observed at only four SSR loci for the cultivars released from 1970 onwards. However, 51 alleles (about 19%) present in pre-1910 cultivars were undetected in cultivars released after 1990 and were spread over 27 SSR loci. The proportion of SSR variation accounted for by six breeding periods was 12.5%, by four ancestral families, 16.5%, and by eight breeding programs, 8.4%. The average genetic diversity measured by three different band-sharing methods did not change significantly among cultivars released from different breeding periods, breeding programs, and ancestral families. However, genetic shift was obvious in the cultivars released over the six breeding periods, reflecting well the various breeding efforts over years. These results clearly show the allelic reduction and genetic shift in the Canadian hard red spring wheat germplasm released over time. Consequently, more effort needs to be made to broaden the wheat breeding base and conserve wheat germplasm.     

Development and integration of EST–SSR markers into an established linkage map in switchgrass

Switchgrass (Panicum virgatum L.) is a model cellulosic biofuel crop in the United States. Simple sequence repeat (SSR) markers are valuable resources for genetic mapping and molecular breeding. A large number of expressed sequence tags (ESTs) of switchgrass are recently available in our sequencing project. The objectives of this study were to develop new SSR markers from the switchgrass EST sequences and to integrate them into an existing linkage map. More than 750 unique primer pairs (PPs) were designed from 243,600 EST contigs and tested for PCR amplifications, resulting in 538 PPs effectively producing amplicons of expected sizes. Of the effective PPs, 481 amplifying informative bands in NL94 were screened for polymorphisms in a panel consisting of NL94 and its seven first-generation selfed (S1) progeny. This led to the selection of 117 polymorphic EST–SSRs to genotype a mapping population encompassing 139 S1 individuals of NL94. Of 83 markers demonstrating clearly scorable alleles in the mapping population, 79 were integrated into a published linkage map, with three linked to accessory loci and one unlinked. The newly identified EST–SSR loci were distributed in 17 of 18 linkage groups with 27 (32.5 %) exhibiting distorted segregations. The integration of EST–SSRs aided in reducing the average marker interval (cM) to 3.7 from 4.2, and reduced the number of gaps (each >15 cM) to 10 from 23. Developing new EST–SSRs and constructing a higher density linkage map will facilitate quantitative trait locus mapping and provide a firm footing for marker-assisted breeding in switchgrass.     

Identification of unique alleles and assessment of genetic diversity of rabi sorghum accessions using simple sequence repeat markers

Genetic diversity among 42 sorghum accessions representing landraces (19), advanced breeding lines (16), local cultivars (2) and release varieties (5) with 30 simple sequence repeat (SSR) markers revealed 7.6 mean number of alleles per locus showing 93.3% polymorphism and an average polymorphism information content of 0.78 which range from 0.22 (Xtxp12) and 0.91(Xtxp321). The average heterozygosity and effective number of alleles per locus were 0.8 and 6.65 respectively. Cluster analysis based on microsatellite allelic diversity clearly demarcated the accessions into ten clusters. A total of 24 unique alleles were obtained from seven SSR loci in 23 accessions in a size range of 110–380 bp; these unique alleles may serve as diagnostic tools for particular region of the genome of respective genotypes. Selected SSR markers from different linkage groups provided an accurate way of determining genetic diversity at the molecular level.     

Development, characterization and mapping of microsatellite markers in Eucalyptus grandis and E. urophylla

 We report on the development, genetic characterization and linkage mapping of a battery of SSR (simple sequence repeat) loci in Eucalyptus grandis and E. urophylla. This study reveals the abundance of SSRs in Eucalyptus, the very high information content of these markers for mapping and individual identification, and demonstrates the feasibility of constructing a comprehensive microsatellite-based linkage map for Eucalyptus. Primer sequence for a set of 20 highly informative EMBRA (Eucalyptus microsatellites from Brazil) loci are made available together with their map position and estimates of the expected heterozygosity and allele size range in these two species. Using genomic library enrichment and anchored-PCR screening prior to sequencing, the efficiency of SSR marker locus development was 63% from sequencing data to operationally useful SSR loci. Absolute transportability between the two species and very high levels of allelic variability and expected heterozygosity (H) were seen at all SSR loci surveyed. The number of alleles per locus ranged from 9 to 26 with an average of 16.3±4.8. The average H of 15 loci was 0.86±0.04, 0.83±0.08 and 0.89±0.04, respectively, for E. urophylla, E. grandis and the combined two-species estimate. In the mapping analysis 16 out of 20 marker loci segregated in a fully informative configuration, allowing the determination of synteny of six homologous linkage groups between the two species. The availability of transportable, multiallelic, PCR-based co-dominant SSR loci represents a dramatic improvement in our ability to carry out detailed population genetic analysis and to search, understand, and manipulate allelic variation at QTLs (quantitative trait loci) in species of Eucalyptus. Received: 16 March 1998 / Accepted: 22 March 1998     

Detection of favorable alleles for yield and yield components by association mapping in upland cotton

Association mapping based on linkage disequilibrium provides a promising tool for dissecting the genetic basis underlying complex traits. To reveal the genetic variations of yield and yield components traits in upland cotton, 403 upland cotton accessions were collected and analyzed by 560 genome-wide simple sequence repeats (SSRs). A diverse panel consisting of 403 upland cotton accessions was grown in six different environments, and the yield and yield component traits were measured, and 560 SSR markers covering the whole genome were mapped. Association studies were performed to uncover the genotypic and phenotypic variations using a mixed linear model. Favorable alleles and typical accessions for yield traits were identified. A total of 201 markers were polymorphic, revealing 394 alleles. The average gene diversity and polymorphism information content were 0.556 and 0.483, respectively. Based on a population structure analysis, 403 accessions were divided into two subgroups. A mixed linear model analysis of the association mapping detected 43 marker loci according to the best linear unbiased prediction and in at least three of the six environments(??lgP?>?1.30, P?<?0.05). Among the 43 associated markers, five were associated with more than two traits simultaneously and nine were coincident with those identified previously. Based on phenotypic effects, favorable alleles and typical accessions that contained the elite allele loci related to yield traits were identified and are widely used in practical breeding. This study detected favorable quantitative trait loci’s alleles and typical accessions for yield traits, these are excellent genetic resources for future high-yield breeding by marker-assisted selection in upland cotton in China.     

Assessing genetic diversity in <Emphasis Type="Italic">Gossypium arboreum</Emphasis> L. cultivars using genomic and EST-derived microsatellites

The cultivated diploid, Gossypium arboreum L., (A genome) is an invaluable genetic resource for improving modern tetraploid cotton (G. hirsutum L. and G. barbadense L.) cultivars. The objective of this research is to select a set of informative and robust microsatellites for studying genetic relationships among accessions of geographically diverse G. arboreum cultivars. From more than 1,500 previously developed simple sequence repeat (SSR) markers, 115 genomic (BNL) and EST-derived (MUCS and MUSS) markers were used to evaluate the allelic diversity of a core panel of G. arboreum accessions. These SSR data enabled advanced genome analyses. A set of 25 SSRs were selected based both upon their high level of informativeness (PIC ≥ 0.50) and the production of clear PCR bands on agarose gels. Subsequently, 96 accessions representing a wide spectrum of diversity of G. arboreum cultivars were analyzed with these markers. The 25 SSR loci revealed 75 allelic variants (polymorphisms) ranging from 2 to 4 alleles per locus. The Neighborjoining (NJ) method, based on genetic dissimilarities, revealed that cultivars from geographically adjacent countries tend to cluster together. Outcomes of this research should be useful in decreasing redundancy of effort and in constructing a core collection of G. arboreum, important for efficient use of this genetic resource in cotton breeding.     

High SSR diversity but little differentiation between accessions of Nordic timothy (Phleum pratense L.)

A large collection of genebank accessions of the hexaploid outcrossing forage grass species timothy (Phleum pratense L.) was for the first time analysed for SSR diversity on individual, population and regional level. Timothy is the most important forage grass species in the Nordic countries. Eighty-eight timothy accessions from Nordic countries and eight accessions around Europe were analysed with recently developed simple sequence repeat (SSR) markers. Timothy proved to be very polymorphic: the 13 selected SSRs amplified a total of 499 polymorphic alleles, the number of alleles per SSR locus varying from 15 to 74. Taking all SSR alleles together, the observed number in each accession ranged from 95 to 203. Levels of diversity were found to be significantly different between countries, vegetation zones and different cultivar types. However, the differentiation between accessions was low: most of the variation (94%) in the studied timothy material was due to variation within accessions and only 5% was between accessions and 1% between countries. Lack of geographical differentiation may reflect the outcrossing and hexaploid nature of timothy. Our results showed that neutral SSR markers are suitable for demonstrating levels of diversity but not alone adequate to resolve population structure in timothy. Nordic timothy material seems to be diverse enough for breeding purposes and no decline in the level of diversity was observed in varieties compared to wild timothy populations. Challenges in analysing SSR marker data in a hexaploid outcrosser were discussed.     

新疆甜高粱种质资源遗传多样性的SSR分析

选用50对SSR引物对新疆现有72份甜高粱种质资源进行遗传多样性分析。结果表明:有20对引物在72份甜高粱种质资源中表现为多态性。共检测到91个等位基因,每对引物可检测到的等位基因数目为2～5个,平均为3.45个。多态性信息量(PIC)的变动范围为0.2859～0.6652,平均为0.5057。72份甜高粱种质间的遗传相似系数变化范围为0.2001～1.000,平均值为0.5599。UPGMA聚类分析将72份材料划分为A、B两大类群,A群包括69份材料,而A群又被分成从Ⅰ到Ⅺ共11个亚群,B群包括3份材料,农艺性状近似的大多被聚到同一类群。     

Newly developed SSR markers reveal genetic diversity and geographical clustering in spinach (<Emphasis Type="Italic">Spinacia oleracea</Emphasis>)

Spinach is a popular leafy green vegetable due to its nutritional composition. It contains high concentrations of vitamins A, E, C, and K, and folic acid. Development of genetic markers for spinach is important for diversity and breeding studies. In this work, Next Generation Sequencing (NGS) technology was used to develop genomic simple sequence repeat (SSR) markers. After cleaning and contig assembly, the sequence encompassed 2.5% of the 980 Mb spinach genome. The contigs were mined for SSRs. A total of 3852 SSRs were detected. Of these, 100 primer pairs were tested and 85% were found to yield clear, reproducible amplicons. These 85 markers were then applied to 48 spinach accessions from worldwide origins, resulting in 389 alleles with 89% polymorphism. The average gene diversity (GD) value of the markers (based on a GD calculation that ranges from 0 to 0.5) was 0.25. Our results demonstrated that the newly developed SSR markers are suitable for assessing genetic diversity and population structure of spinach germplasm. The markers also revealed clustering of the accessions based on geographical origin with clear separation of Far Eastern accessions which had the overall highest genetic diversity when compared with accessions from Persia, Turkey, Europe, and the USA. Thus, the SSR markers have good potential to provide valuable information for spinach breeding and germplasm management. Also they will be helpful for genome mapping and core collection establishment.     

Comparative analysis of microsatellite DNA polymorphism in landraces and cultivars of rice

Genetic polymorphisms of ten microsatellite DNA loci were examined among 238 accessions of landraces and cultivars that represent a significant portion of the distribution range for both indica and japonica groups of cultivated rice. In all, 93 alleles were identified with these ten markers. The number of alleles varied from a low of 3 or 4 at each of four loci, to an intermediate value of 9–14 at five loci, and to an extra-ordinarily high 25 at one locus. The numbers of alleles per locus are much larger than those detected using other types of markers. The number of alleles detected at a locus is significantly correlated with the number of simple sequence repeats in the targeted microsatellite DNA. Indica rice has about 14% more alleles than japonica rice, and such allele number differences are more pronounced in landraces than in cultivars. The indica-japonica differentiation component accounted for about 10% of the diversity in the total sample, and twice as much differentiation was detected in cultivars as in landraces. About two-thirds as many alleles were observed in cultivars as in landraces; another two-thirds of the alleles in the cultivar group were found in modern elite cultivars or parents of hybrid rice. The majority of the simple sequence repeat (SSR) alleles that were present in high or intermediate frequencies in landraces ultimately survived into modern elite cultivars and hybrids. The greater resolving power and the efficient production of massive amounts of SSR data may be particularly useful for germplasm assessment and evolutionary studies of crop plants.