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     

Genetic diversity in three groups of barley germplasm assessed by simple sequence repeats.

Genetic diversity can be measured by several criteria, including phenotype, pedigree, allelic diversity at marker loci, and allelic diversity at loci controlling phenotypes of interest. Abundance, high level of polymorphism, and ease of genotyping make simple sequence repeats (SSRs) an excellent molecular marker system for genetics diversity analyses. In this study, we used a set of mapped SSRs to survey three representative groups of barley germplasm: a sample of crop progenitor (Hordeum vulgare subsp. spontaneum) accessions, a group of mapping population parents, and a group of varieties and elite breeding lines. The objectives were to determine (i) how informative SSRs are in these three sets of barley germplasm resources and (ii) the utility of SSRs in classifying barley germplasm. A total of 687 alleles were identified at 42 SSR loci in 147 genotypes. The number of alleles per locus ranged from 4 to 31, with an average of 16.3. Crop progenitors averaged 10.3 alleles per SSR locus, mapping population parents 8.3 alleles per SSR locus, and elite breeding lines 5.8 alleles per SSR locus. There were many exclusive (unique) alleles. The polymorphism information content values for the SSRs ranged from 0.08 to 0.94. The cluster analysis indicates a high level of diversity within the crop progenitors accessions and within the mapping population parents. It also shows a lower level of diversity within the elite breeding germplasm. Our results demonstrate that this set of SSRs was highly informative and was useful in generating a meaningful classification of the germplasm that we sampled. Our long-term goal is to determine the utility of molecular marker diversity as a tool for gene discovery and efficient use of germplasm.     

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.     

Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat

Nearly 900 SSRs (simple sequence repeats) were identified among 15,000 ESTs (expressed sequence tags) belonging to bread wheat ( Triticum aestivum L.). The SSRs were defined by their minimum length, which ranged from 14 to 21 bp. The maximum length ranged from 24 to 87 bp depending upon the length of the repeat unit itself (1–7 bp). The average density of SSRs was one SSR per 9.2 kb of EST sequence screened. The trinucleotide repeats were the most abundant SSRs detected. As a representative sample, 78 primer pairs were designed, which were also used to screen the dbEST entries for Hordeum vulgare and Triticum tauschii (donor of the D-genome of cultivated wheat) using a cut-off E (expectation) value of 0.01. On the basis of in silico analysis, up to 55.12% of the primer pairs exhibited transferability from Triticum to Hordeum, indicating that the sequences flanking the SSRs are not only conserved within a single genus but also between related genera in Poaceae. Primer pairs for the 78 SSRs were synthesized and used successfully for the study of (1) their transferability to 18 related wild species and five cereal species (barley, oat, rye, rice and maize); and (2) polymorphism between the parents of four mapping populations available with us. A subset of 20 EST-SSR primers was also used to assess genetic diversity in a collection of 52 elite exotic wheat genotypes. This was done with a view to compare their utility relative to other molecular markers (gSSRs, AFLPs, and SAMPL) previously used by us for the same purpose with the same set of 52 bread wheat genotypes. Although only a low level of polymorphism was detected, relative to that observed with genomic SSRs, the study suggested that EST-SSRs can be successfully used for a variety of purposes, and may actually prove superior to SSR markers extracted from genomic libraries for diversity estimation and transferability.Communicated by R. Hagemann     

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.     

普通小麦Genomic-SSR和EST-SSR分子标记遗传差异及其与系谱遗传距离的比较研究

采用Genomic-SSR和EST—SSR标记技术,对来自我国北方冬麦区的18份普通小麦品种(系)的遗传多样性进行了探讨,并与系谱遗传距离进行了比较分析。研究发现,平均每个Genomic—SSR检测到的等位基因数为3．34个,明显高于EST-SSR的2．31个。遗传距离(GD)计算结果显示,18个小麦基因型之间的EST—SSR平均遗传距离较小,仅为0．3996,低于Genomic—SSR的GD平均值0．5458。尽管EST-SSR揭示出的多态性明显低于Genomic-SSR,但系谱分析和聚类结果均表明,与Genomic—SSR相比,EST—SSR标记能更准确地反映出不同小麦基因型之间的遗传和亲缘关系。据此可以认为,EST—SSR是评价小麦遗传多样性的一种理想标记形式。研究还证实,一个骨干亲本与由其衍生出来的品种(系)之间的遗传差异一般较小,并对拓宽普通小麦遗传基础的策略和方法进行了讨论。     

Genetic diversity in a collection of old and new bread wheat cultivars from Iran as revealed by simple sequence repeat-based analysis

Genetic diversity in a collection of 70 bread wheat ( Triticum aestivum ) genotypes was studied using 73 microsatellite [simple sequence repeat (SSR)] loci evenly spaced on wheat chromosomes. A total of 592 alleles with an average of 8.53 allele/locus were detected, of which 185 (31.25%) occurred only in a specific group of genotypes. A set of SSR markers consisted of 22 loci with polymorphic information content values of 0.80 or higher were selected for rapid fingerprinting of many genotypes. Average of gene diversity was 0.74 ± 0.017, and significant difference between observed and maximum theoretical values of gene diversity in the analysed SSR loci was obtained using a paired t -test. Genetic distance-based clustering methods including unweighted pair group method with arithmetic average and neighbour joining (NJ) were used for grouping of genotypes. The resulted dendrogram based on NJ and number of differences coefficient hinted of the existence of three groups. This grouping was in agreement with the pedigree information and confirmed by high within-group bootstrap value. A comparatively higher genetic diversity in the studied wheat collection as revealed by presence of high allelic diversity and large number of specific alleles could be utilised in development of new cultivars with desired characteristics.     

Identification,characterization, validation and cross-species amplification of genic-SSRs in Indian Mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>)

Brassica juncea is an economically important oilseed crop worldwide. It has limited genomic resources at present. We generated 47,962,057 expressed sequence reads which were assembled into 45,280 unigenes. A total of 4108 SSR loci (≥10 bp) were identified in these unigenes. Trinucleotide was the most frequent repeat unit (59.91 %) followed by di- (38.66 %), tetra - (0.71 %), hexa - (0.49 %) and pentanucleotide repeats (0.24 %). Primers were designed for 2863 SSR loci among which 460 were selected for primer synthesis. A total of 339 loci amplified successfully of which 134 (39.5 %) exhibited polymorphism among six B. juncea genotypes with PIC values ranging from 0.18 to 0.81. Further, 25 polymorphic SSRs were used for analysis of genetic variability in 25 genotypes of Brassicas and their wild relatives. Two to five alleles with PIC values 0.22–0.66 were detected at these loci. The dendrogram grouped the genotypes according to their known pedigree/systematic position.     

An evaluation of the utility of SSR loci as molecular markers in maize (Zea mays L.): comparisons with data from RFLPS and pedigree

 The utility of 131 simple sequence repeat (SSR) loci to characterize and identify maize inbred lines, validate pedigree, and show associations among inbred lines was evaluated using a set of 58 inbred lines and four hybrids. Thirteen sets of inbred parent-progeny triplet pedigrees together with four hybrids and their parental lines were used to quantify incidences of scoring that departed from expectations based upon simple Mendelian inheritance. Results were compared to those obtained using 80 restriction fragment length polymorphism (RFLP) probes. Over all inbred triplets, 2.2% of SSRs and 3.6% of RFLP loci resulted in profiles that were scored as having segregated in a non-Mendelian fashion. Polymorphic index content (PIC, a measure of discrimination ability) values ranged from 0.06 to 0.91 for SSRs and from 0.10 to 0.84 for RFLPs. Mean values for PIC for SSRs and RFLPs were similar, approximately 0.62. However, PIC values for nine SSRs exceeded the maximum PIC for RFLPs. Di-repeats gave the highest mean PIC scores for SSRs but this class of repeats can result in “stutter” bands that complicate accurate genotyping. Associations among inbreds were similar for SSR and RFLP data, closely approximating expectations from known pedigrees. SSR technology presents the potential advantages of reliability, reproducibility, discrimination, standardization and cost effectiveness over RFLPs. SSR profiles can be readily interpreted in terms of alleles at mapped loci across a broad range of maize germ plasm. Consequently, SSRs represent the optimum approach for the identification and pedigree validation of maize genotypes compared to other currently available methods. Received: 15 January 1997 / Accepted: 28 February 1997     

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.     

Genetic diversity in basmati rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) germplasm as revealed by microsatellite (SSR) markers

Genetic diversity among rice genotypes, including 15 indica basmati advance lines and 5 basmati improved varieties were investigated by 28 SSR markers including one indel marker. The SSRs covered all the 12 chromosomes that distributed across the rice genomes. The mean number of alleles per locus was 3.60, showing average number of polymorphism information content was 0.48. A total of 101 alleles were also identified from the microsatellite marker loci. A number of SSR markers were also identified that could be utilized to differentiate between rice genotypes. Pair wise Nei’s genetic distance between rice genotypes ranged from 0.07 to 0.95. The dendrogram based on cluster analysis by using SSR polymorphism that grouped the 20 genotypes of rice in to five clusters based on their genetic similarity. The result could be useful for the identification and selection of the diverse genotypes for the future cross breeding program and development of new rice varieties.     

Genetic diversity in basmati rice (Oryza sativa L.) germplasm as revealed by microsatellite (SSR) markers

Genetic diversity among rice genotypes, including 15 indica basmati advance lines and 5 basmati improved varieties were investigated by 28 SSR markets including one indel marker. The SSRs covered all the 12 chromosomes that distributed across the rice genomes. The mean number of alleles per locus was 3.60, showing average number of polymorphism information content was 0.48. A total of 101 alleles were also identified from the microsatellite marker loci. A number of SSR markers were also identified that could be utilized to differentiate between rice genotypes. Pair wise Nei,s genetic distance between rice genotypes ranged from 0.07 to 0.95. The dendrogram based on cluster analysis by using SSR polymorphism that grouped the 20 genotypes of rice in to five clusters based on their genetic similarity. The result could be useful for the identification and selection of the diverse genotypes for the future cross breeding program and development of new rice varieties.     

Assessment of genetic diversity and relatedness among Tunisian almond germplasm using SSR markers

Genetic diversity of 50 Tunisian almond (Prunus dulcis Mill.) genotypes and their relationships to European and American cultivars were studied. In total 82 genotypes were analyzed using ten genomic SSRs. A total of 159 alleles were scored and their sizes ranged from 116 to 227 bp. The number of alleles per locus varied from 12 to 23 with an average of 15.9 alleles per locus. Mean expected and observed heterozygosities were 0.86 and 0.68, respectively. The total value for the probability of identity was 4 × 10(-13) . All SSRs were polymorphic and they were able all together to distinguish unambiguously the 82 genotypes. The Dice similarity coefficient was calculated for all pair wise and was used to construct an UPGMA dendrogram. The results demonstrated that the genetic diversity within local almond cultivars was important, with clear geographic divergence between the northern and the southern Tunisian cultivars. The usefulness of SSR markers for almond fingerprinting, detection of synonyms and homonyms and evaluation of the genetic diversity in the Tunisian almond germplasm was also discussed. The results confirm the potential value of genetic diversity preservation for future breeding programs.     

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     

Haplotype diversity of preharvest sprouting QTLs in wheat.

Preharvest sprouting (PHS) is one of the most important factors affecting wheat production worldwide in environments characterized by rainfall and high humidity at harvest. In such environments, the incorporation of seed dormancy of a limited duration is required to minimize losses associated with PHS. A global collection of 28 PHS-resistant and -susceptible wheat germplasm was characterized with microsatellite markers flanking the genomic regions associated with PHS-resistance quantitative trait loci (QTLs), particularly on chromosomes 3D and 4A. The genetic diversity analysis revealed 380 alleles at 54 microsatellite loci, with an average of 7.0 alleles per locus, among the 28 wheat genotypes. Gower's genetic similarity values among all possible pairs of genotypes varied from 0.44 to 0.97, indicating that there is considerable diversity in the PHS germplasm evaluated. Cluster and principal coordinates analysis of genetic similarity estimates differentiated the genotypes into groups, according to their source of PHS resistance. Three major SSR haplotypes were observed on chromosome 4AL, designated RL4137-type allele, Aus1408-type allele, and synthetic-hexaploid-type allele. The RL4137-type allele was prevalent in Canadian cultivars, mostly in cluster 6, followed by the Aus1408-type and its derivatives in clusters 4 and 5. The Syn36 and Syn37 alleles on chromosome 4AL were rare. On chromosome 3DL, the SSRs haplotypes derived from Syn36 and Syn37 were also rare, and proved unique to the Aegilops tauschii - derived synthetic hexaploids. They are therefore likely carrying resistance genes different from those previously reported. Based on genetic relationships, PHS resistance might be improved by selecting parental genotypes from different clusters.     

Mosaic microecological differential stress causes adaptive microsatellite divergence in wild barley, Hordeum spontaneum, at Neve Yaar, Israel.

Genetic diversity at 38 microsatellite (short sequence repeats (SSRs)) loci was studied in a sample of 54 plants representing a natural population of wild barley, Hordeum spontaneum, at the Neve Yaar microsite in Israel. Wild barley at the microsite was organized in a mosaic pattern over an area of 3180 m2 in the open Tabor oak forest, which was subdivided into four microniches: (i) sun-rock (11 genotypes), (ii) sun-soil (18 genotypes), (iii) shade-soil (11 genotypes), and (iv) shade-rock (14 genotypes). Fifty-four genotypes were tested for ecological-genetic microniche correlates. Analysis of 36 loci showed that allele distributions at SSR loci were nonrandom but structured by ecological stresses (climatic and edaphic). Sixteen (45.7%) of 35 polymorphic loci varied significantly (p < 0.05) in allele frequencies among the microniches. Significant genetic divergence and diversity were found among the four subpopulations. The soil and shade subpopulations showed higher genetic diversities at SSR loci than the rock and sun subpopulations, and the lowest genetic diversity was observed in the sun-rock subpopulation, in contrast with the previous allozyme and RAPD studies. On average, of 36 loci, 88.75% of the total genetic diversity exists within the four microniches, while 11.25% exists between the microniches. In a permutation test, G(ST) was lower for 4999 out of 5000 randomized data sets (p < 0.001) when compared with real data (0.1125). The highest genetic distance was between shade-soil and sun-rock (D = 0.222). Our results suggest that diversifying natural selection may act upon some regulatory regions, resulting in adaptive SSR divergence. Fixation of some loci (GMS61, GMS1, and EBMAC824) at a specific microniche seems to suggest directional selection. The pattern of other SSR loci suggests the operation of balancing selection. SSRs may be either direct targets of selection or markers of selected haplotypes (selective sweep).     

Differentiation of "Candidatus Liberibacter asiaticus" isolates by variable-number tandem-repeat analysis

Four highly polymorphic simple sequence repeat (SSR) loci were selected and used to differentiate 84 Japanese isolates of "Candidatus Liberibacter asiaticus." The Nei's measure of genetic diversity values for these four SSRs ranged from 0.60 to 0.86. The four SSR loci were also highly polymorphic in four isolates from Taiwan and 12 isolates from Indonesia.     

Genetic diversity of African maize inbred lines revealed by SSR markers

Knowledge of genetic diversity (GD) and relationships among maize inbred lines is indispensable in a breeding program. Our objectives were to (1) investigate the level of genetic diversity among maize inbred lines and (2) assess their genetic structures by applying simple sequence repeat (SSR) markers. Fifty-six highland and mid-altitude maize inbred lines obtained from CIMMYT programs in Ethiopia and Zimbabwe were genotyped using 27 SSR loci. All of the genotypes studied could unequivocally be distinguished with the combination of the SSRs used. In total, 104 SSR alleles were identified, with a mean of 3.85 alleles per locus. The average polymorphism information content (PIC) was 0.58. GD expressed as Euclidean distance, varied from 0.28 to 0.73 with an average of 0.59. Cluster analysis using unweighted pair group method with arithmetic average (UPGMA) suggested five groups among the inbred lines. Most of the inbred lines adapted to the highlands and the mid-altitudes were positioned in different clusters with a few discrepancies. The pattern of groupings of the inbred lines was mostly consistent with available pedigree information. The variability detected using SSR markers could potentially contribute towards effective utilization of the inbred lines for the exploitation of heterosis and formation of genetically diverse source populations in Ethiopian maize improvement programs.     

Development and use of anchored‐SSRs to study DNA polymorphism in bread wheat (Triticum aestivum L.)

In bread wheat, 21 anchored simple sequence repeat (SSR) primer pairs detecting SSR length polymorphism and 42 anchored SSR primers detecting microsatellite‐anchored fragment length polymorphisms (MFLPs) are reported. Eight bread wheat genotypes were used for detecting polymorphism. The number of alleles in SSR analysis ranged from two to six, with a mean of 2.9 alleles per SSR. The number of polymorphic bands in MFLP ranged from two to 40, with a mean of 12.74 polymorphic bands/primer combination, the SSRs with CT/GA motifs giving the highest level of polymorphism (a mean of 18.37 bands). The average value of polymorphic information content (PIC) was 0.473 for SSRs and 0.061 for MFLP.