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.     

Development of 240 novel EST-SSRs in Eucalyptus L’Hérit

Simple sequence repeat (SSR) markers derived from expressed sequence tag (EST) resources provide great potential for comparative mapping, direct gene tagging of quantitative trait loci and functional diversity studies. Here we report on the development of 240 novel EST-SSRs for the important tree genus Eucalyptus L’Hérit. Of the 240 EST-SSR loci, 218 (90.8 %) were polymorphic among 12 individuals of E. grandis Hill ex Maiden, with the number of alleles per locus (N _a), observed heterozygosity (H _o), expected heterozygosity (H _e) and polymorphic information content (PIC) averaging at 5.0, 0.403, 0.598 and 0.529, respectively. High rates of cross-species/subgenus amplification were observed. The EST-SSRs developed herein would be a valuable addition of functional markers for genetics and breeding applications in a wide range of eucalypt species. The primer sequences for the 240 EST-SSRs have been deposited in the Probe database of GenBank (IDs Pr016588534–773).     

Genic Microsatellite Markers in Brassica rapa: Development, Characterization, Mapping, and Their Utility in Other Cultivated and Wild Brassica Relatives

Genic microsatellite markers, also known as functional markers, are preferred over anonymous markers as they reveal the variation in transcribed genes among individuals. In this study, we developed a total of 707 expressed sequence tag-derived simple sequence repeat markers (EST-SSRs) and used for development of a high-density integrated map using four individual mapping populations of B. rapa. This map contains a total of 1426 markers, consisting of 306 EST-SSRs, 153 intron polymorphic markers, 395 bacterial artificial chromosome-derived SSRs (BAC-SSRs), and 572 public SSRs and other markers covering a total distance of 1245.9 cM of the B. rapa genome. Analysis of allelic diversity in 24 B. rapa germplasm using 234 mapped EST-SSR markers showed amplification of 2 alleles by majority of EST-SSRs, although amplification of alleles ranging from 2 to 8 was found. Transferability analysis of 167 EST-SSRs in 35 species belonging to cultivated and wild brassica relatives showed 42.51% (Sysimprium leteum) to 100% (B. carinata, B. juncea, and B. napus) amplification. Our newly developed EST-SSRs and high-density linkage map based on highly transferable genic markers would facilitate the molecular mapping of quantitative trait loci and the positional cloning of specific genes, in addition to marker-assisted selection and comparative genomic studies of B. rapa with other related species.     

草鱼EST-SSR标记及5个不同地域群体的遗传结构分析

以草鱼(Ctenopharyngodon idella)脑、肌肉、肝等组织构建cDNA文库,经测序获得unigenes序列45 318个,从中筛选微卫星序列共5 556个,据此设计EST-SSR引物118对,其中19对引物能够扩增出带型清晰且多态性较高的谱带。用这19个EST-SSR标记研究3个长江水系群体(石首、监利和长沙)和2个珠江水系群体(清远和肇庆)草鱼的遗传结构。结果表明,5个群体的平均多态信息含量(PIC)为0.415 4～0.460 4,显示草鱼群体的遗传多样性偏低;平均观测杂合度(Ho)为0.415 8～0.501 3,平均期望杂合度为(He)0.450 6～0.502 8,其中,长沙群体平均期望杂合度最高,为0.502 8,监利群体的平均观察杂合度和平均期望杂合度均最低,分别为0.415 8和0.450 6,即长沙群体的遗传多样性最高,监利群体的遗传多样性最低;对数据进行F-检验,结果表明,群体间的遗传分化程度低。Hardy-Weinberg平衡的卡方检验结果表明,5个群体均一定程度上偏离了平衡;聚类分析显示长沙群体、石首群体与监利群体聚成一支;肇庆群体与清远群体聚成一支,这与草鱼群体的流域分布一致。     

Pros and cons of using genomic SSRs and EST-SSRs for resolving phylogeny of the genus Gossypium

The genus Gossypium is comprised of 50 diverse cotton species representing eight different genomes (A through G and K), however, phylogenetic relationship using various DNA marker types such as RAPD and SSRs was determined on limited number of cotton species. In this report, we have demonstrated the application of genomic SSRs (gSSRs) and EST-SSRs, and after combining both the data sets, for resolving the phylogenies of 36 cotton species including seven races. Out of the 100 primer pairs surveyed (50 for gSSRs and 50 for EST-SSRs), 75 produced scorable amplification products in all species. Out of these, 73 were found to be polymorphic and amplified 135 alleles ranging from 1 to 5 alleles per SSR marker (average 2.87 alleles per marker). The gSSRs amplified higher number of alleles (72) compared to the EST-SSRs (63). In total 22 highly informative SSRs with PIC values ≥0.5 were identified. Genomic SSRs containing di-while EST-SSRs containing tri-nucleotide repeats exhibited high polymorphism compared to the other nucleotide repeats containing gSSRs/EST-SSRs. Number of tandem repeats and polymorphism were positively correlated. Neither the type of chromosome nor the location of the SSRs showed association with the polymorphism. Gossypium herbaceum var. africanum (Watt) Hutch. ex and Ghose and Gossypium robinsonii F. Muell. were found the most genetically diverse, while among races of Gossypium hirsutum L. “yucatanense” and G. hirsutum “punctatum” were found genetically diverse. Of the three data sets, clustering analysis based on EST-SSRs and combined data sets, revealed parallel results reported in earlier studies. This study further confirmed that Gossypium darwinii Watt has close relationship with Gossypium barbadense L. Moreover, Gossypium raimondii Ulbr. and G. herbaceum/Gossypium arboreum L. are close living relatives of the ancestor allotetraploid species. Our studies suggest that for resolving phylogenetic relationship among the various plant species EST-SSRs could be a better choice. This information can be instrumental in transferring novel alleles or loci from the wild species into the cultivated cotton species which would set a stage for cultivating genetically diverse cultivars—a way to achieve sustainable cotton production in changing climate.     

Molecular Diversity and Population Structure of a Worldwide Collection of Cultivated Tetraploid Alfalfa (Medicago sativa subsp. sativa L.) Germplasm as Revealed by Microsatellite Markers

Information on genetic diversity and population structure of a tetraploid alfalfa collection might be valuable in effective use of the genetic resources. A set of 336 worldwide genotypes of tetraploid alfalfa (Medicago sativa subsp. sativa L.) was genotyped using 85 genome-wide distributed SSR markers to reveal the genetic diversity and population structure in the alfalfa. Genetic diversity analysis identified a total of 1056 alleles across 85 marker loci. The average expected heterozygosity and polymorphism information content values were 0.677 and 0.638, respectively, showing high levels of genetic diversity in the cultivated tetraploid alfalfa germplasm. Comparison of genetic characteristics across chromosomes indicated regions of chromosomes 2 and 3 had the highest genetic diversity. A higher genetic diversity was detected in alfalfa landraces than that of wild materials and cultivars. Two populations were identified by the model-based population structure, principal coordinate and neighbor-joining analyses, corresponding to China and other parts of the world. However, lack of strictly correlation between clustering and geographic origins suggested extensive germplasm exchanges of alfalfa germplasm across diverse geographic regions. The quantitative analysis of the genetic diversity and population structure in this study could be useful for genetic and genomic analysis and utilization of the genetic variation in alfalfa breeding.     

Development of polymorphic EST-derived SSR markers for the shrimp, Fenneropenaeus chinensis

In our study, we evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of Fenneropenaeus chinensis. Characteristics of 11 EST-SSR loci were investigated using 34 F. chinensis individuals. The number of alleles per locus ranged from 4 to 9. The observed heterozygosity ranged from 0.118 to 0.826, while the expected heterozygosity ranged from 0.623 to 0.847. Two loci (T2 and T12) conformed to the Hardy–Weinberg equilibrium. There was no LD observed between all pairs of EST-SSRs loci. The PIC values of 11 microsatellite loci were higher than 0.5. These loci and markers will be useful for population genetics and systemic evolution of F. chinensis.     

Novel and Stress Relevant EST Derived SSR Markers Developed and Validated in Peanut

With the aim to increase the number of functional markers in resource poor crop like cultivated peanut (Arachis hypogaea), large numbers of available expressed sequence tags (ESTs) in the public databases, were employed for the development of novel EST derived simple sequence repeat (SSR) markers. From 16424 unigenes, 2784 (16.95%) SSRs containing unigenes having 3373 SSR motifs were identified. Of these, 2027 (72.81%) sequences were annotated and 4124 gene ontology terms were assigned. Among different SSR motif-classes, tri-nucleotide repeats (33.86%) were the most abundant followed by di-nucleotide repeats (27.51%) while AG/CT (20.7%) and AAG/CTT (13.25%) were the most abundant repeat-motifs. A total of 2456 EST-SSR novel primer pairs were designed, of which 366 unigenes having relevance to various stresses and other functions, were PCR validated using a set of 11 diverse peanut genotypes. Of these, 340 (92.62%) primer pairs yielded clear and scorable PCR products and 39 (10.66%) primer pairs exhibited polymorphisms. Overall, the number of alleles per marker ranged from 1-12 with an average of 3.77 and the PIC ranged from 0.028 to 0.375 with an average of 0.325. The identified EST-SSRs not only enriched the existing molecular markers kitty, but would also facilitate the targeted research in marker-trait association for various stresses, inter-specific studies and genetic diversity analysis in peanut.     

Genetic diversity in European chestnut populations by means of genomic and genic microsatellite markers

Microsatellite or simple sequence repeats (SSRs) are one of the most used markers in population genetic studies. SSR markers developed from expressed sequence tags (EST) have proved useful to examine functional diversity in relation to adaptive variation. The information provided by both genomic and genic microsatellite markers could offer more accurate indication on the distribution of the genetic diversity among and within populations assuming different evolutionary drivers. This is the first study on chestnut (Castanea sativa Mill.) in which the genetic diversity was evaluated by means of genomic (SSRs) and genic (EST-SSRs) microsatellite markers. We genotyped nine natural European chestnut populations distributed throughout representative areas of contrasting climatic conditions in the Mediterranean basin. Genomic SSRs showed significantly higher levels of diversity in terms of number of alleles, effective number of alleles, expected heterozygosity and level of polymorphism. Furthermore, there were significant differences in the level of differentiation among populations. The UPGMA analysis revealed different clustering pattern between populations, being the grouping according to geographic distances in the case of genomic SSRs and two differentiated groups based on the northern–southern distribution of the populations for EST-SSRs. Furthermore, the EST-SSR transferability among related Castanea and Quercus species was stated. Our results confirm that combining genomic SSRs and EST-SSRs is a useful tool to give complementary information to explain the genetic and adaptive diversity in chestnut.     

Evaluation of Genetic Diversity and Population Structure Analysis among Germplasm of Agaricus bisporus by SSR Markers

Agaricus bisporus is a popular edible mushroom that is cultivated worldwide. Due to its secondary homothallic nature, cultivated A. bisporus strains have low genetic diversity, and breeding novel strains is challenging. The aim of this study was to investigate the genetic diversity and population structure of globally collected A. bisporus strains using simple sequence repeat (SSR) markers. Agaricus bisporus strains were divided based on genetic distance-based groups and model-based subpopulations. The major allele frequency (MAF), number of genotypes (NG), number of alleles (NA), observed heterozygosity (HO), expected heterozygosity (HE), and polymorphic information content (PIC) were calculated, and genetic distance, population structure, genetic differentiation, and Hardy–Weinberg equilibrium (HWE) were assessed. Strains were divided into two groups by distance-based analysis and into three subpopulations by model-based analysis. Strains in subpopulations POP A and POP B were included in Group I, and strains in subpopulation POP C were included in Group II. Genetic differentiation between strains was 99%. Marker AB-gSSR-1057 in Group II and subpopulation POP C was confirmed to be in HWE. These results will enhance A. bisporus breeding programs and support the protection of genetic resources.     

High-molecular-weight glutenin subunit variation in Turkish emmer wheat [Triticum turgidum L. ssp. dicoccon (Schrank) Thell.] landraces

The genetic diversity of high-molecular-weight (HMW) glutenin subunits in 18 cultivated emmer wheat landrace populations, originating from Turkey, was investigated using sodium dodecyl sulphate polyacrylamide gel electrophoresis. The mean number of alleles (n _a) and effective alleles (n _ea) were observed as 3.67 and 1.53, respectively. The mean values of expected heterozygosity (gene diversity) (H _e) and average heterozygosity (H _e,av) were calculated as 0.31 and 0.12, respectively. Actual genetic differentiation (D) and gene flow (N _m) between the different populations were observed as 0.24 and 0.16, respectively. Statistical analysis of Pearson’s correlation, multiple regressions and principal component analysis indicated that eco-geographical variables have a significant effect on HMW-glutenin diversity. Considering the dramatic decrease in genetic diversity of modern high-yielding cultivars, the conservation of genetic diversity in these wheat landraces, and in other old cultivars, is important for improving modern monocultures and their ability to resist biotic and abiotic conditions caused by climate changes, thus generating a wide adaption to a variety of environmental conditions. Adoptation measures for germplasm conservation of Turkish emmer wheat landraces and utilisation of their germplasm for improvement of modern wheat varieties were discussed in this study.     

Comparison of the genetic diversity of common wild rice (Oryza rufipogon Griff.) and cultivated rice (O. sativa L.) using RFLP markers

Forty fourth single-copy RFLP markers were used to evaluate the genetic diversity of 122 accessions of common wild rice (CWR, Oryza rufipogon Griff.) and 75 entries of cultivated rice (Oryza sativa L. ) from more than ten Asian countries. A comparison of the parameters showing genetic diversity, including the percentage of polymorphic loci (P), the average number of alleles per locus (A), the number of genotypes (Ng), the average heterozygosity (Ho) and the average genetic multiplicity (Hs) of CWR and indica and japonica subspecies of cultivated rice from different countries and regions, indicated that CWR from China possesses the highest genetic diversity, followed by CWR from South Asia and Southeast Asia. The genetic diversity of CWR from India is the second highest. Although the average gene diversity (Hs)of the South Asian CWR is higher than that of the Southeast Asian CWR, its percentage of polymorphic loci (P), number of alleles (Na) and number of genotypes (Ng) are all smaller. It was also found that the genetic diversity of cultivated rice is obviously lower than that of CWR. At the 44 loci investigated, the number of polymorphic loci of cultivated rice is only 3/4 that of CWR, while the number of alleles, 60%, and the number of genotypes is about 1/2 that of CWR. Of the two subspecies studied, the genetic diversity of indica is higher than that of japonica. The average heterozygosity of the Chinese CWR is the highest among all the entries studied. The average heterozygosity of CWR is about two-times that of cultivated rice. It is suggested that during the course of evolution from wild rice to cultivated rice, many alleles were lost through natural and human selection, leading to the lower heterozygosity and genetic diversity of the cultivated rice. Received: 19 May 1999 / Accepted: 26 April 2000     

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.     

Development of 13 EST-SSRs for Cerasus jamasakura and their transferability for Japanese flowering cherries

Simple sequence repeat (SSR) markers were developed for the flowering cherry Cerasus jamasakura (also known as Prunus jamasakura) using 31,995 expressed sequence tags (ESTs) from the NCBI database. Out of 96 of designed primer pairs, 63 showed clear PCR amplification and 13 of these revealed polymorphism in eight individuals sampled across the species’ range. The number of alleles detected and expected heterozygosity ranged from 1 to 8 and 0.000 to 0.833, respectively, when these 13 loci were examined in 23 individuals from a single population. For all except one of the lcoi, polymorphism was also detected in at least four of six other taxa of flowering cherries examined. The results show that the developed EST-SSRs are highly transferable, and that these markers are likely to be useful in studies of the population genetics of flowering cherries.     

Development of expressed sequence tag derived-simple sequence repeats in the genus Lilium

Although lily is the second largest flower crop in cutting flower commodity, only six simple sequence repeats SSRs have been reported. Thus, we developed expressed sequence tag derived-SSRs (EST-SSRs) for the Lilium genus. Among 2,235 unique ESTs, 754 ESTs contained SSR motifs, among which 165 ESTs were amenable to primer design. Among these 165 EST-SSRs, 131 EST-SSRs showed amplification in at least one Lilium species, and 76 EST-SSRs showed amplification in at least nine species. Of the 76 EST-SSRs, 47 showed amplification in all Lilium species analyzed. Using 10 breeding lines, we selected 21 EST-SSRs that had the highest number of alleles and polymorphism information content. The polymorphism information content values of these selected EST-SSRs ranged from 0.49 to 0.94 with an average of 0.76, which are higher than other plant species. The phylogenetic dendrogram derived from the amplification profiles of the 21 high polymorphic EST-SSRs was congruent with the genetic background of the 84 selected lily accessions and hybrids, which are available in commerce. Thus, the developed EST-SSRs will be very useful in germplasm management, genetic diversity analysis, cultivar finger printing, and molecular breeding in the lily.     

In silico mining of EST-SSRs in <Emphasis Type="Italic">Arachis hypogaea</Emphasis> L. and their utilization for genetic structure and diversity analysis in cultivars/breeding lines in Odisha,India

A total of 26,685 unutilized public domain expressed sequence tags (ESTs) of Arachis hypogaea L. were analyzed to give a total of 4442 EST-SSRs, in which 517 ESTs contained more than one simple sequence repeat (SSR). Of these EST-SSRs, 2542 were mononucleotide repeats (MNRs), 803 were dinucleotide repeats (DNRs), 1043 were trinucleotide repeats (TNRs), 40 were tetranucleotide repeats (TtNRs), six were pentanucleotide repeats (PNRs) and eight were hexanucleotide repeats (HNRs). Out of these 4442 EST-SSRs, only 1160 were found to be successful in non-redundant primer design; 1060 were simple SSRs, while the remaining 100 were compound forms. Among all the motifs, MNRs were abundant, followed by TNRs and DNRs. The AAG/CTT motif was the most abundant (~33 %) TNR, while AG/CT was the most abundant DNR. For redundancy and novelty, a stringent criterion deploying three different strategies was used and a total of 782 novel EST-SSRs were added to the public domain of peanut. These novel EST-SSR markers will be useful for qualitative and quantitative trait mapping, marker-assisted selection and genetic diversity studies in cultivated peanut as well as related Arachis species. A subset of 30 novel EST-SSRs was further randomly selected for validation and genotyping studies with eight well-known cultivars and 32 advanced breeding lines (ADBX lines, ADBY lines and ADBZ lines) from Odisha state, India. The number of polymorphic markers among accessions of A. hypogaea was low; however, a set of informative EST-SSR markers detected considerable levels of genetic variability in peanut cultivars and uncharacterized breeding lines collected from Odisha. The 30 newly developed EST-SSRs from Arachis spp. showed ~97 % amplification in Cicer arientinum and 93 % in pigeon pea. Thus, the EST-SSRs developed in this study will be a very useful asset for genetic analysis, comparative genome mapping, population genetic structure and phylogenetic inferences among wild and allied species of Arachis.     

In silico mining and characterization of simple sequence repeats from gilthead sea bream (Sparus aurata) expressed sequence tags (EST-SSRs); PCR amplification, polymorphism evaluation and multiplexing and cross-species assays

We screened for simple sequence repeats (SSRs) found in ESTs derived from an EST-database development project ('Marine Genomics Europe' Network of Excellence). Different motifs of di-, tri-, tetra-, penta- and hexanucleotide SSRs were evaluated for variation in length and position in the expressed sequences, relative abundance and distribution in gilthead sea bream (Sparus aurata). We found 899 ESTs that harbor 997 SSRs (4.94%). On average, one SSR was found per 2.95 kb of EST sequence and the dinucleotide SSRs are the most abundant accounting for 47.6% of the total number. EST-SSRs were used as template for primer design. 664 primer pairs could be successfully identified and a subset of 206 pairs of primers was synthesized, PCR-tested and visualized on ethidium bromide stained agarose gels. The main objective was to further assess the potential of EST-SSRs as informative markers and investigate their cross-species amplification in sixteen teleost fish species: seven sparid species and nine other species from different families. Approximately 78% of the primer pairs gave PCR products of expected size in gilthead sea bream, and as expected, the rate of successful amplification of sea bream EST-SSRs was higher in sparids, lower in other perciforms and even lower in species of the Clupeiform and Gadiform orders. We finally determined the polymorphism and the heterozygosity of 63 markers in a wild gilthead sea bream population; fifty-eight loci were found to be polymorphic with the expected heterozygosity and the number of alleles ranging from 0.089 to 0.946 and from 2 to 27, respectively. These tools and markers are expected to enhance the available genetic linkage map in gilthead sea bream, to assist comparative mapping and genome analyses for this species and further with other model fish species and finally to help advance genetic analysis for cultivated and wild populations and accelerate breeding programs.