Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat

Genetic variation present in 64 durum wheat accessions was investigated by using three sources of microsatellite (SSR) markers: EST-derived SSRs (EST-SSRs) and two sources of SSRs isolated from total genomic DNA. Out of 245 SSR primer pairs screened, 22 EST-SSRs and 20 genomic-derived SSRs were polymorphic and used for genotyping. The EST-SSR primers produced high quality markers, but had the lowest level of polymorphism (25%) compared to the other two sources of genomic SSR markers (53%). The 42 SSR markers detected 189 polymorphic alleles with an average number of 4.5 alleles per locus. The coefficient of similarity ranged from 0.28 to 0.70 and the estimates of similarity varied when different sources of SSR markers were used to genotype the accessions. This study showed that EST-derived SSR markers developed in bread wheat are polymorphic in durum wheat when assaying loci of the A and B genomes. A minumum of ten EST-SSRs generated a very low probability of identity (0.36×10⁻¹²) indicating that these SSRs have a very high discriminatory power. EST-SSR markers directly sample variation in transcribed regions of the genome, which may enhance their value in marker-assisted selection, comparative genetic analysis and for exploiting wheat genetic resources by providing a more-direct estimate of functional diversity. Received: 19 December 2000 / Accepted: 17 April 2001     

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.     

广西普通油茶种质资源遗传多样性的SSR分析

普通油茶( Camellia oleifera)是我国分布最广、产量最多的山茶属中一个重要油料树种。广西是普通油茶的重要分布区,种质资源十分丰富。为深入了解广西普通油茶种质资源的遗传变异,服务于种质保存和品种选育,该研究首先对已开发的SSR分子标记进行多态性筛选和评价,在此基础上利用多态性较高的引物,对97份广西有代表性的普通油茶种质资源进行遗传多样性分析。结果表明：(1)在已开发的10对油茶SSR分子标记中,7对能稳定扩增且表现为共显性,2对扩增不稳定,另外1对无法扩增出产物。(2)7对共显性SSR标记总共检测到33个等位基因,每对标记检测到等位基因数目的变化范围为3~6个,平均每个位点等位基因数为4.7143个,有效等位基因数目的变化范围为2.0842~4.3148,平均有效等位基因数为2.8288;基因多样性变化范围为0.5202~0.7682,平均每个位点基因多样性为0.6281。(3)参试群体中绝大多数位点未处于Hardy-Weinberg平衡,存在遗传结构;观测杂合度和期望杂合度的变化范围分别为0.4130~0.6701和0.5233~0.7724,其平均值分别为0.5698和0.6316。(4)种质资源间遗传距离变化范围为0.05~0.7917,平均遗传距离为0.3545;UPGMA聚类显示相同来源的种质资源无法聚成一类,在同一聚类分支上混有不同来源的种质资源。这表明已开发的油茶SSR分子标记适用于广西普通油茶,广西普通油茶种质资源拥有较丰富的遗传多样性。该研究结果为广西普通油茶资源的深度开发和高效利用提供了科学依据。     

An integrated DArT-SSR linkage map of durum wheat

Genetic mapping in durum wheat (Triticum durum Desf.) is constrained by its large genome and allopolyploid nature. We developed a Diversity Arrays Technology (DArT) platform for durum wheat to enable efficient and cost-effective mapping and molecular breeding applications. Genomic representations from 56 durum accessions were used to assemble a DArT genotyping microarray. Microsatellite (SSR) and DArT markers were mapped on a durum wheat recombinant inbred population (176 lines). The integrated DArT-SSR map included 554 loci (162 SSRs and 392 DArT markers) and spanned 2022 cM (5 cM/marker on average). The DArT markers from durum wheat were positioned in respect to anchor SSRs and hexaploid wheat DArT markers. DArT markers compared favourably to SSRs to evaluate genetic relationships among the durum panel, with 1315 DArT polymorphisms found across the accessions. Combining DArT and SSR platforms provides an efficient and rapid method of generating linkage maps in durum wheat.     

Characterization, development and exploitation of EST-derived microsatellites in Porphyra haitanensis Chang et Zheng (Bangiales, Rhodophyta)

The frequency, type and distribution of simple sequence repeats (SSRs) in Porphyra haitanensis genomes was investigated using expressed sequence tag (EST) data deposited in public databases. A total of 3,489 non-redundant P. haitanensis ESTs were screened for SSRs using SSRhunter software. From those, 224 SSRs in 210 ESTs were identified; trinucleotides were the most common type of SSR (64.29%), followed by dinucleotides (33.48%). Tetranucleotides, pentanucleotides, and hexanucleotides were not common. Among all identified motif types, CGG/CCG had the highest frequency (33.9%), followed by TC/AG (24.6%). From these EST-SSRs, 37 SSR primer-pairs were designed and tested using common SSR reaction conditions with 15 P. haitanensis DNAs as templates. The results showed that 28 SSR primer-pairs gave good amplification patterns. These were used to conduct SSR analyses of genetic variations of the 15 germplasm strains of P. haitanensis. A total of 224 alleles were detected, with the number of alleles ranging from 4 to 15. The effective number of alleles, expected heterozygosity, and polymorphism information content of the 15 germplasm strains of P. haitanensis were 2.81, 0.64, and 0.57, respectively. All of these parameters indicate that the 15 germplasm strains of P. haitanensis harbor rich genetic variation.     

Analysis of simple sequence repeats (SSRs) in wild barley from the Fertile Crescent: associations with ecology,geography and flowering time

Wild barley, Hordeum spontaneum C. Koch, is the progenitor of cultivated barley, Hordeum vulgare. The centre of diversity is in the Fertile Crescent of the Near East, where wild barley grows in a wide range of conditions (temperature, water availability, day length, etc.). The genetic diversity of 39 wild barley genotypes collected from Israel, Turkey and Iran was studied with 33 SSRs of known map location. Analysis of molecular variance (AMOVA) was performed to partition the genetic variation present within from the variation between the three countries of origin. Using classification tree analysis, two (or three) specific SSRs were identified which could correctly classify most of the wild barley genotypes according to country of origin. Associations of SSR variation with flowering time and adaptation to site-of-origin ecology and geography were investigated by two contrasting statistical approaches, linear regression based on SSR length variation and linear regression based on SSR allele class differences. A number of SSRs were significantly associated with flowering time under four different growing regimes (short days, long days, unvernalised and vernalised). Most of the associations observed could be accounted for by close linkage of the SSR loci to earliness per se genes. No associations were found with photoperiodic and vernalisation response genes known to control flowering in cultivated barley suggesting that different genetic factors may be active in wild barley. Novel genomic regions controlling flowering time in wild barley were detected on chromosomes 1HS, 2HL, 3HS and 4HS. Associations of SSRs with site-of-origin ecological and geographic data were found primarily in genomic regions determining plant development. This study shows that the analyses of SSR variation by allele class and repeat length are complementary, and that some SSRs are not necessarily selectively neutral.     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

Population structure and long-range linkage disequilibrium in a durum wheat elite collection

A collection of 134 durum wheat accessions, mainly including cultivars (cvs.) representative of the major gene pools, was assembled and characterized with 70 SSRs for genetic diversity and level of long-range linkage disequilibrium (LD). Results of both a distance-based and a model-based (Bayesian) cluster analysis evidenced the presence of a structured diversity. In the model-based analysis, six to eight main distinct subpopulations were identified based on the molecular data. Only a relatively small portion (20%) of the molecular variation was accounted for by the geographical origin of the accessions. Major differences were detected between the North American and the Mediterranean cvs., while a considerable overlap characterized the cvs. from CIMMYT-ICARDA and Italy. The North American cvs. showed the highest within group mean genetic similarity (GS_m = 0.68). French cvs. revealed sizeable similarities with both the North American as well as the Italian and CIMMYT-ICARDA pools. Considering the germplasm as a whole, high levels of LD were found both at locus pairs with an intrachromosomal distance <50 cM as well as at those with distances more than 50 cM and independent (86, 52 and 54% of SSR pairs at p < 0.01, respectively). After re-evaluating LD within each of the three main subgroups identified through the analysis of the germplasm structure, the LD level remained high for tightly to moderately linked locus pairs (<20 cM apart), but was greatly reduced in the loosely linked (more than 50 cM apart) and independent locus pairs. The implications of these findings as to the possibility of using association mapping for gene/QTL discovery in durum wheat are discussed.     

A genetic map of 1,000 SSR and DArT markers in a wide barley cross

A high-density genetic map was developed from an F1-derived doubled haploid population generated from a cross between cultivated barley (Hordeum vulgare) and the subspecies H. vulgare ssp. spontaneum. The map comprises 1,000 loci, amplified using 536 SSR (558 loci) and 442 DArT markers. Of the SSRs, 149 markers (153 loci) were derived from barley ESTs, and 7 from wheat ESTs. A high level of polymorphism (∼70%) was observed, which facilitated the mapping of 197 SSRs for which genetic assignments had not been previously reported. Comparison with a published composite map showed a high level of co-linearity and telomeric coverage on all seven chromosomes. This map provides access to previously unmapped SSRs, improved genome coverage due to the integration of DArT and EST-SSRs and overcomes locus order issues of composite maps constructed from the alignment of several genetic maps. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Assessment of genetic diversity and population structure of Chinese wild almond, Amygdalus nana, using EST- and genomic SSRs

Amygdalus nana L., commonly known as wild almond, is an endangered wild relative of cultivated almond, which has great potential in almond crop breeding. In this study, we used microsatellite (SSR) loci derived from both expressed sequence tag (EST) and anonymous genomic sequence to explore the genetic diversity and population structure of A. nana in Xinjiang of China. Seven natural populations were collected across the whole distribution of A. nana in China, including populations from both inside (four populations) and outside (three populations) the established protected areas. A total of 22 and 19 alleles were detected from the seven pairs of EST and genomic SSR loci, respectively. Generally, the genomic SSRs showed lower levels of variation than EST-SSRs, which may partially due to the higher cross-species transferability in EST-SSRs than in genomic SSRs. The population-level genetic diversity (A = 1.84, P = 50.00%, H_o = 0.3491, H_E = 0.2271) was lower than cultivated almond and several wild fruit species with similar breeding system. Most of the genetic variation (82.16%) was partitioned within populations. In particular, the population collected from Tacheng County (outside the protected areas) had the highest levels of genetic diversity and had significantly different genetic constitution from other populations.     

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.     

Microsatellite (SSR) marker assisted assessment of population structure and genetic diversity for morpho-physiological traits in guava (<Emphasis Type="Italic">Psidium guajava</Emphasis> L.)

Twenty-four SSR markers were utilized to evaluate the genetic variation across thirty-six guava varieties including wild species. The SSR markers revealed a polymorphism of 95.7% and a great range of diversity among the experimental guava germplasm. Eighty-one alleles were detected, in diversity analysis, with 2–7 alleles with a mean of 3.682 alleles per loci. The SSR loci showcased an allele frequency of 0.306 (mPgCIR251) to 0.861 (mPgCIR227) at a mean value 0.561. An average polymorphic value of 0.490 across was measured for all the 36 germplasm with the range of 0.234 in mPgCIR227 to 0.706 in mPgCIR03. The genetic diversity for SSRs varied between 0.248 (mPgCIR227) and 0.747 (mPgCIR03) with an average of 0.548. Clustering of germplasm distinctly separated pink and white flesh germplasm into two major groups. First three coordinates contributed towards 32.76% of the variation measured using principle coordinate analysis. Molecular variance (AMOVA) study showed 06 and 94% genetic variation among population and individual, respectively with five sub populations. This study provides valuable information for understanding the genetic variability in guava which can be exploited to develop varieties with better fruit yield and nutritional quality.     

从CIMMYT引进的人工合成六倍体小麦D染色体组微卫星分子标记的遗传差异

采用微卫星（SSR）分子标记技术,选用23个D染色体组特异性引的对来自CIMMYT的26份人工合成六倍体小麦D染色体组的遗传多样性进行了分析。研究发现,26份材料在D染色体组上存在丰富的等位基因变异（92个）,平均每个基因座为4个。遗传距离计算结果也显示,26份材料D染色体组之间具有较大的遗传差异,平均遗传距离高达0．4955。因此,人工合成六倍体小麦D染色体组中存在丰富的遗传多样性,可以作为拓宽普通小麦遗传基础的新的遗传变异来源。研究还发现,由同一个粗山羊草基因型与不同硬粒小麦杂交合成的人工合成六倍体小麦（如合成种17和18）在所用检测的23个基因座中有3个存在差异,说明小麦在多倍化后,供体基因组在重复序列区域会发生遗传分化。     

An estimation of the minimum number of SSR loci needed to reveal genetic relationships in wheat varieties: Information from 96 random accessions with maximized genetic diversity

Genetic relationships among common wheat varieties from the 10 wheat growing regions of China were assessed using SSR markers. The wheat varieties included 33 modern varieties and 63 landraces selected from the national gene bank collection of China. One hundred and four pairs of selected primers detected a total of 802 alleles, of which 234 were specific to A genome, 309 to B genome, and 221 to D genome. The average genetic richness per locus (A _ij /loci) for A, B and D genomes were 6.88, 7.92 and 7.62, respectively. Their average genetic dispersion indices (H _t ) were 0.637, 0.694 and 0.656, respectively. The B genome showed the highest genetic diversity among the three wheat genomes. The landraces had a higher genetic diversity than the modern varieties, and the major difference between the landraces and the modern varieties in China existed in the D genome, followed by B and A genomes. The majority of the accessions (65.6%) had heterogeneity at the 112 loci detected. The highest heterogeneity locus percentages were 9.09 and 12.73 in the modern varieties and the landraces, respectively. SSR data were analyzed with NTSYS-pc software. The genetic similarities between accessions were estimated with the DICE coefficient. The accessions clustered into two groups, the modern varieties and the landraces by the un-weighted pair-group method using arithmetic average (UPGMA). The trend of correlation coefficients between genetic similarity matrices based on different numbers of random alleles and that of 802 alleles showed that 550 alleles were sufficient to construct a robust dendrogram. The separated simulations from six sub-samples revealed that 550 alleles were the minimum number required to confidently determine the genetic relationships. It was shown that the number of alleles (loci) needed do not have a strong association with the number of wheat lines in the sample size. These data suggested that 73 loci with good polymorphism are needed to reflect genetic relationships among accessions with more than 90% certainty. In the dendrogram, most accessions from the same wheat region were clustered together, and those from geographically adjacent regions usually appeared in the same small group. This indicated that genetic diversity of Chinese common wheat has a close association with their geographic distribution and ecological environment.     

EST-SSR DNA polymorphism in durum wheat (Triticum durum L.) collections

SSRs derived from EST were molecular markers belonging to the transcribed region of the genome. Therefore, any polymorphism detected using EST-SSRs might reflect the better relationship among species or varieties. Using wheat EST-SSR markers, 60 durum wheat (Triticum durum L.) accessions from seven countries were investigated. Twenty-five primer pairs could amplify successfully in the 60 durum wheat accessions, of which tri-nucleotide repeats were the dominant type, and revealed 26 loci on all seven wheat homologous chromosome groups. A total of 87 eSSR alleles were detected, and the number of alleles detected by a single pair of primers ranged from 1 to 11, with an average of 3.3 alleles per locus. Higher numbers of alleles and PIC were identified on the B genome than those on the A genome.     

Length polymorphism and homologies of microsatellites in several Cucurbitaceae species

The objectives of this research were to assess (1) the degree of Simple Sequence Repeats (SSR) DNA length polymorphism in melon (Cucumis melo L.) and other species within the Cucurbitaceae family and (2) the possibility of utilizing SSRs flanking primers from single species to other genera or species of Cucurbitaceae. Five melon (CT/GA) _n SSRs were isolated from a genomic library. Two cucumber (Cucumis sativus L.) SSRs were detected through a search of DNA sequence databases, one contained a (CT)₈ repeat, the other a (AT)₁₃ repeat. The seven SSRs were used to test a diverse sample of Cucurbitaceae, including 8 melon, 11 cucumber, 5 squash, 1 pumpkin, and 3 watermelon genotypes. Five of the seven SSRs detected length polymorphism among the 8 melon genotypes. PCR amplification revealed between three and five length variants (alleles) for each SSR locus, with gene diversity values ranging from 0.53 to 0.75. Codominant segregation of the alleles among F₂ progeny was demonstrated for each of the five SSR loci. Four of the seven SSRs detected polymorphism among the 11 cucumber genotypes, with gene diversity values ranging between 0.18 and 0.64. Primers specific to SSRs of C. melo and C. sativus also amplified DNA extracted from genotypes belonging to other genera of the Cucurbitaceae family.     

SSR allelic variation in almond (Prunus dulcis Mill.)

Sixteen SSR markers including eight EST-SSR and eight genomic SSRs were used for genetic diversity analysis of 23 Chinese and 15 international almond cultivars. EST- and genomic SSR markers previously reported in species of Prunus, mainly peach, proved to be useful for almond genetic analysis. DNA sequences of 117 alleles of six of the 16 SSR loci were analysed to reveal sequence variation among the 38 almond accessions. For the four SSR loci with AG/CT repeats, no insertions or deletions were observed in the flanking regions of the 98 alleles sequenced. Allelic size variation of these loci resulted exclusively from differences in the structures of repeat motifs, which involved interruptions or occurrences of new motif repeats in addition to varying number of AG/CT repeats. Some alleles had a high number of uninterrupted repeat motifs, indicating that SSR mutational patterns differ among alleles at a given SSR locus within the almond species. Allelic homoplasy was observed in the SSR loci because of base substitutions, interruptions or compound repeat motifs. Substitutions in the repeat regions were found at two SSR loci, suggesting that point mutations operate on SSRs and hinder the further SSR expansion by introducing repeat interruptions to stabilize SSR loci. Furthermore, it was shown that some potential point mutations in the flanking regions are linked with new SSR repeat motif variation in almond and peach. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Genetic Diversity and Core Collection Evaluations in Common Wheat Germplasm from the Northwestern Spring Wheat Region in China

Fluorescence microsatellite markers were employed to reveal genetic diversity of 340 wheat accessions consisting of 229 landraces and 111 modern varieties from the Northwest Spring Wheat Region in China. The 340 accessions were chosen as candidate core collections for wheat germplasm in this region. A core collection representing the genetic diversity of these accessions was identified based on a cluster dendrogram of 78 SSR loci. A total of 967 alleles were detected with a mean of 13.6 alleles (5–32) per locus. Mean PIC was 0.64, ranged from 0.05 to 0.91. All loci were distributed relatively evenly in the A, B and D wheat genomes. Mean genetic richness of A, B and D genomes for both landraces and modern varieties was B > A > D. However, mean genetic diversity indices of landraces changed to B > D > A. As a whole, genetic diversity of the landraces was considerably higher than that of the modern varieties. The big difference of genetic diversity indices in the three genomes suggested that breeding has exerted greater selection pressure in the D than the A or B genomes in this region. Changes of allelic proportions represented in the proposed core collection at different sampling scales suggested that the sampling percentage of the core collection in the Northwest Spring Wheat Region should be greater than 4% of the base collection to ensure that more than 70% of the variation is represented by the core collection. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.