Allelic diversity of simple sequence repeats among elite inbred lines of cultivated sunflower.

Simple sequence repeat (SSR) markers were developed for cultivated sunflower (Helianthus annuus L.) from the DNA sequences of 970 clones isolated from genomic DNA libraries enriched for (CA)n,, (CT)n, (CAA)n, (CATA)n, or (GATA)n. The clones harbored 632 SSRs, of which 259 were unique. SSR markers were developed for 130 unique SSRs by designing and testing primers for 171 unique SSRs. Of the total, 74 SSR markers were polymorphic when screened for length polymorphisms among 16 elite inbred lines. The mean number of alleles per locus was 3.7 for dinucleotide, 3.6 for trinucleotide, and 9.5 for tetranucleotide repeats and the mean polymorphic information content (PIC) scores were 0.53 for dinucleotide, 0.53 for trinucleotide, and 0.83 for tetranucleotide repeats. Cluster analyses uncovered patterns of genetic diversity concordant with patterns produced by RFLP fingerprinting. SSRs were found to be slightly more polymorphic than RFLPs. Several individual SSRs were significantly more polymorphic than RFLP and other DNA markers in sunflower (20% of the polymorphic SSR markers had PIC scores ranging from 0.70 to 0.93). The newly developed SSRs greatly increase the supply of sequence-based DNA markers for DNA fingerprinting, genetic mapping, and molecular breeding in sunflower; however, several hundred additional SSR markers are needed to routinely construct complete genetic maps and saturate the genome.     

Identifying Novel Polymorphic Microsatellites from Cultivated Flax (Linum usitatissimum L.) Following Data Mining

One of the major concerns in genetic characterization and breeding of cultivated flax is the lack of informative microsatellite markers (SSRs). In this regard, the development of SSRs using molecular methods might be time-consuming, laborious, and expensive. On the other hand, using bioinformatics to mine sequences in public databases enables a cost-effective discovery of SSRs. A total of 3,242 Linum usitatissimum genomic sequences were surveyed for the identification of SSRs. Among them, 118 non-redundant sequences containing repeats were selected for designing primers. The most abundant motifs were tri- (72.4%) and dinudeotide (16.6%), within which AGG/CCT and AG/CT were predominant. Primers were tested for polymorphism in 60 L. usitatissimum cultivars/accessions including 57 linseed and three fiber flax. Eighty-eight pairs gave amplifications within the expected size range while 60 pairs were found to be polymorphic. The mean number of alleles amplified per primer was 3.0 (range, 2–8; 180 total alleles). The mean polymorphism information content (PIC) value was 0.39 (range, 0.06–0.87), and the highest average PIC was observed in dinucleotide SSRs (0.41). The SSR data mining presented here demonstrates the usefulness of in silico development of microsatellites. These novel genomic SSR markers could be used in genetic diversity studies, the development of genetic linkage maps, quantitative trait loci mapping, association mapping, and marker-assisted selection.     

Mining and charactering microsatellites in Cucumis melo expressed sequence tags from sequence database

Simple sequence repeats (SSRs) derived from expressed sequence tags (ESTs) are valuable markers because they represent transcribed regions and often have putative functions. We mined and characterized microsatellites in melon ESTs. Three hundred and eighty‐three SSR loci were identified in 309 of 3188 unigenes assembled by 5747 EST and mRNA sequences in GenBank with occurring frequency of 1/4.7 kb. Twenty‐two polymorphic EST‐SSR markers were developed with the mean allele number of 2.9 per locus and mean expected heterozygosity of 0.442. Amplification products were also detected by 15 pairs of primer in Cucumis sativus. Those informative EST‐SSR markers can be used in melon genetic improvement projects.     

Development,Characterization and Cross Species Amplification of Polymorphic Microsatellite Markers from Expressed Sequence Tags of Turmeric (<Emphasis Type="Italic">Curcuma longa</Emphasis> L.)

Expressed sequence tags (ESTs) from turmeric (Curcuma longa L.) were used for the screening of type and frequency of Class I (hypervariable) simple sequence repeats (SSRs). A total of 231 microsatellite repeats were detected from 12,593 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs accounts to one SSR per 17.96 kb of EST. Mononucleotides were the most abundant class of microsatellite repeat in turmeric ESTs followed by trinucleotides. A robust set of 17 polymorphic EST–SSRs were developed and used for evaluating 20 turmeric accessions. The number of alleles detected ranged from 3 to 8 per loci. The developed markers were also evaluated in 13 related species of C. longa confirming high rate (100%) of cross species transferability. The polymorphic microsatellite markers generated from this study could be used for genetic diversity analysis and resolving the taxonomic confusion prevailing in the genus.     

New microsatellite markers for bananas (Musa spp)

Thirty-four microsatellite markers (SSRs) were identified in EST and BAC clones from Musa acuminata burmannicoides var. Calcutta 4 and validated in 22 Musa genotypes from the Banana Germplasm Bank of Embrapa-CNPMF, which includes wild and improved diploids. The number of alleles per locus ranged from 2 to 14. The markers were considered highly informative based on their polymorphism information content values; more than 50% were above 0.5. These SSRs will be useful for banana breeding programs, for studies of genetic diversity, germplasm characterization and selection, development of saturated genetic linkage maps, and marker assisted selection.     

Characterization of 215 simple sequence repeat markers in creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is a versatile, cross-pollinated, temperate and perennial turfgrass species. It occurs naturally in a wide variety of habitats and is also cultivated on golf courses, bowling greens and tennis courts worldwide. Isozymes and amplified fragment length polymorphisms (AFLPs) have been used to determine genetic diversity, and restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA (RAPDs) were used to construct a genetic linkage map of this species. In the current report, we developed and characterized 215 unique genomic simple sequence repeat (SSR) markers in creeping bentgrass. The SSRs reported here are the first available markers in creeping bentgrass to date. Eight hundred and eighteen alleles were amplified by 215 SSR loci, an average of 3.72 alleles per locus. Fifty-nine per cent of those alleles segregated in a 1:1 Mendelian fashion (P > 0.05). Twenty-two per cent had a distorted segregation ratio (P ≤ 0.05). These SSR markers will be useful for assessing genetic diversity in creeping bentgrass and will be important for the development of genetic linkage maps and identifying quantitative trait loci. These markers could enhance breeding programmes by improving the efficiency of selection techniques.     

Utilization of in silico EST–SSR markers for diversity studies in castor (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.)

Castor (Ricinus communis L.) a chief non-edible oilseed crop has numerous industrial applications. Systematic genetic diversity analysis utilizing DNA based markers has been quick and reliable method that ensures selection of diverse parents for exploitation of higher levels of heterosis in breeding programs. From NCBI database, 63,852 EST sequences of castor were mined. One thousand one hundred and five (1105) EST–SSRs and 1652 repeat motifs sequences were identified from 20,495 non-redundant unigene sequences. Repeat motifs consisted of 29.7 % mono nucleotide repeats, 24.8 % di nucleotide repeats, 27.27 % tri nucleotide repeats and 3.94 % tetra nucleotide repeats. Twenty eight primer pairs were chosen from SSR-containing ESTs to determine genetic diversity among 27 castor accessions. Twelve EST–SSRs showed polymorphism. Number of alleles detected were 2–3 with an average of 2.33 per locus. 150–400 bp was the size of an allele. Dendrogram analysis grouped the 27 accessions into two separate clusters. Genetic similarity coefficient of dendrogram ranged from 0.24 to 0.83. The polymorphic information content value of 0.28–0.49 revealed medium level of diversity in castor. Results of present study indicated that EST–SSRs to be efficient markers for genetic diversity studies. Knowledge on level of diversity existing in castor genotypes would be useful for breeders to plan efficient hybrid breeding programme.     

Distribution and characterization of simple sequence repeats in Gossypium raimondii genome

Simple sequence repeats (SSRs) can be derived from the complete genome sequence. These markers are important for gene mapping as well as marker-assisted selection (MAS). To develop SSRs for cotton gene mapping, we selected the complete genome sequence of Gossypium raimondii, which consisted of 4447 non-redundant scaffolds. Out of 775.2 Mb sequence examined, a total of 136,345 microsatellites were identified with a density of 5.69 kb per SSR in the G. raimondii genome leading to development of 112,177 primer pairs. The distributions of SSRs in the genome were non-random. Among the different motifs ranging from 1 to 6 bp, penta-nucleotide repeats were most abundant (30.5%), followed by tetra-nucleotide repeats (18.2%) and di-nucleotide repeats (16.9%). Among all identified 457 motif types, the most frequently occurring repeat motifs were poly-AT/TA, which accounted for 79.8% of the total di-nt SSRs, followed by AAAT/TTTA with 51.5% of the total tetra-nucleotede. Further, 18,834 microsatellites were detected from the protein-coding genes, and the frequency of gene containing SSRs was 46.0% in 40,976 genes of G. raimondii. These genome-based SSRs developed in the present study will lay the groundwork for developing large numbers of SSR markers for genetic mapping, gene discovery, genetic diversity analysis, and MAS breeding in cotton.     

Microsatellite DNA markers in Populus tremuloides.

Markers for eight new microsatellite DNA or simple sequence repeat (SSR) loci were developed and characterized in trembling aspen (Populus tremuloides) from a partial genomic library. Informativeness of these microsatellite DNA markers was examined by determining polymorphisms in 38 P. tremuloides individuals. Inheritance of selected markers was tested in progenies of controlled crosses. Six characterized SSR loci were of dinucleotide repeats (two perfect and four imperfect), and one each of trinucleotide and tetranucleotide repeats. The monomorphic SSR locus (PTR15) was of a compound imperfect dinucleotide repeat. The primers of one highly polymorphic SSR locus (PTR7) amplified two loci, and alleles could not be assigned to a specific locus. At the other six polymorphic loci, 25 alleles were detected in 38 P. tremuloides individuals; the number of alleles ranged from 2 to 7, with an average of 4.2 alleles per locus, and the observed heterozygosity ranged from 0.05 to 0.61, with an average of 0.36 per locus. The two perfect dinucleotide and one trinucleotide microsatellite DNA loci were the most informative. Microsatellite DNA variants of four SSR loci characterized previously followed a single-locus Mendelian inheritance pattern, whereas those of PTR7 from the present study showed a two-locus Mendelian inheritance pattern in controlled crosses. The microsatellite DNA markers developed and reported here could be used for assisting various genetic, breeding, biotechnology, genome mapping, conservation, and sustainable forest management programs in poplars.     

Simple sequence repeats for the genetic analysis of apple

 The development of highly informative markers, such as simple sequence repeats, for tagging genes controlling agronomic characters is essential for apple breeding. Furthermore the use of these markers is fundamental both for variety identification and for the characterisation and management of genetic resources. We have developed 16 reliable simple sequence repeat (SSR) markers that amplify all alleles from a panel of 19 Malus x domestica (Borkh.) cultivars or breeding selections and from Malus floribunda 821. Those markers show a high level of genetic polymorphism, with on average 8.2 alleles per locus and an average heterozygosity of 0.78. Due to this high level of polymorphism, it was possible using two selected SSRs to distinguish all cultivars except Starking and Red Delicious. Ten of the markers we developed have been mapped on a RAPD linkage map, proving their Mendelian segregation as well as their random distribution in the apple genome. Finally, we discuss the importance of using co-dominant markers in outbreeding species. Received: 8 October 1997 / Accepted: 9 December 1997     

Development and characterization of simple sequence repeat (SSR) markers and their use in determining relationships among Lycopersicon esculentum cultivars

The simple sequence repeat (SSR) or microsatellite marker is currently the preferred molecular marker due to its highly desirable properties. The aim of this study was to develop and characterize more SSR markers because the number of SSR markers currently available in tomato is very limited. Five hundred DNA sequences of tomato were searched for SSRs and analyzed for the design of PCR primers. Of the 158 pairs of SSR primers screened against a set of 19 diverse tomato cultivars, 129 pairs produced the expected DNA fragments in their PCR products, and 65 of them were polymorphic with the polymorphism information content (PIC) ranging from 0.09 to 0.67. Among the polymorphic loci, 2-6 SSR alleles were detected for each locus with an average of 2.7 alleles per locus; 49.2% of these loci had two alleles and 33.8% had three alleles. The vast majority (93.8%) of the microsatellite loci contained di- or tri-nucleotide repeats and only 6.2% had tetra- and penta-nucleotide repeats. It was also found that TA/AT was the most frequent type of repeat, and the polymorphism information content (PIC) was positively correlated with the number of repeats. The set of 19 tomato cultivars were clustered based on the banding patterns generated by the 65 polymorphic SSR loci. Since the markers developed in this study are primarily from expressed sequences, they can be used not only for molecular mapping, cultivar identification and marker-assisted selection, but for identifying gene-trait relations in tomato.     

Developing and characterising Ricinus communis SSR markers by data mining of whole-genome sequences

Ricinus communis is a versatile industrial oil crop that is cultivated worldwide. Genetic improvement and marker-assisted breeding of castor bean have been slowed owing to the lack of abundant and efficient molecular markers. As co-dominant markers, simple sequence repeats (SSRs) are useful for genetic evaluation and molecular breeding. The recently released whole-genome sequence of castor bean provides useful genomic resources for developing markers on a genome-wide scale. In the present study, the distribution and frequency of microsatellites in the castor bean genome were characterised and numerous SSR markers were developed using genomic data mining. In total, 18,647 SSR loci at a density of one SSR per 18.89 Kb in the castor bean genome sequence (representing approximately 352.27 Mb) were identified. Dinucleotide repeats were the most frequently observed microsatellites, although the AAT repeat motif was also prevalent. Using six cultivars as screening samples, 670 polymorphic SSR markers from 1,435 primer pairs (46.7 %) were developed. Trinucleotide motif loci contained a higher proportion of polymorphisms (48.5 %) than dinucleotide motif loci (39.2 %). The polymorphism level in the SSR loci was positively correlated with the increasing number of repeat units in the microsatellites. The phylogenetic relationship among 32 varieties was evaluated using the developed SSR markers. Cultivars developed at the same institute clustered together, suggesting that these cultivars have a narrow genetic background. The large number of SSR markers developed in this study will be useful for genetic mapping and for breeding improved castor-oil plants. These markers will also facilitate genetic and genomic studies of Euphorbiaceae.     

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     

Distribution and localization of microsatellites in the Perigord black truffle genome and identification of new molecular markers

The level of genetic diversity and genetic structure in the Perigord black truffle (Tuber melanosporum Vittad.) has been debated for several years, mainly due to the lack of appropriate genetic markers. Microsatellites or simple sequence repeats (SSRs) are important for the genome organisation, phenotypic diversity and are one of the most popular molecular markers. In this study, we surveyed the T. melanosporum genome (1) to characterise its SSR pattern; (2) to compare it with SSR patterns found in 48 other fungal and three oomycetes genomes and (3) to identify new polymorphic SSR markers for population genetics. The T. melanosporum genome is rich in SSRs with 22,425 SSRs with mono-nucleotides being the most frequent motifs. SSRs were found in all genomic regions although they are more frequent in non-coding regions (introns and intergenic regions). Sixty out of 135 PCR-amplified mono-, di-, tri-, tetra, penta, and hexa-nucleotides were polymorphic (44%) within black truffle populations and 27 were randomly selected and analysed on 139 T. melanosporum isolates from France, Italy and Spain. The number of alleles varied from 2 to 18 and the expected heterozygosity from 0.124 to 0.815. One hundred and thirty-two different multilocus genotypes out of the 139 T. melanosporum isolates were identified and the genotypic diversity was high (0.999). Polymorphic SSRs were found in UTR regulatory regions of fruiting bodies and ectomycorrhiza regulated genes, suggesting that they may play a role in phenotypic variation. In conclusion, SSRs developed in this study were highly polymorphic and our results showed that T. melanosporum is a species with an important genetic diversity, which is in agreement with its recently uncovered heterothallic mating system.     

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.     

Characterization,development and mapping of Unigene-derived microsatellite markers in sorghum [<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench]

Molecular variation within known genes controlling specific functions provide candidate gene-based markers which are tightly linked with the trait of interest. Unigene-derived microsatellite markers, with their unique identity and positions, offer the advantage of unraveling variation in the expressed component of the genome. We characterized ≥12-bp-long microsatellite loci from 13,899 unique sequences of sorghum [Sorghum bicolor (L.) Moench] available in the NCBI unigene database for their abundance and possible use in sorghum breeding. Analysis of 12,464 unigenes (≥200-bp) using MISA software identified 14,082 simple sequence repeats (SSRs) in 7,370 unigenes, from which 1,519 unigene SSR markers were developed. The average frequency of SSR was 1 per1.6 kb and 1.0 per 1.1 unigene; hexamers followed by trimers were found in abundance, of which 33.3% AT-rich and CCG repeats were the most abundant. Of the 302 unigene SSRs tested, 60 (19.8%) were polymorphic between the two parents, M35-1 and B35 of a recombinant inbred line (RIL) mapping population. A mapping population consisting of 500 RILs was developed using the above two parents, and a subset of random 245 RILs was used for genotyping with polymorphic SSRs. We developed a linkage map containing 231 markers, of which 228 (174 genomic and 54 genic) were microsatellites and three were morphological markers. Markers were distributed over 21 linkage groups, and spanned a genetic distance of 1235.5 cM. This map includes 81 new SSRs, of which 35 (21 unigene and 14 genomic) were developed in the present study and 46 from other studies. The order of the SSR markers mapped in the present study was confirmed physically by BLAST search against the whole-genome shotgun sequence of sorghum. Many unigene sequences used for marker development in this study include genes coding for important regulatory proteins and functional proteins that are involved in stress-related metabolism. The unigene SSR markers used together with other SSR markers to construct the sorghum genetic map will have applications in studies on comparative mapping, functional diversity analysis and association mapping, and for quantitative trait loci detection for drought and other agronomically important traits in sorghum.     

Identification and characterization of salt responsive miRNA-SSR markers in rice (Oryza sativa)

Salinity is an important abiotic stress that affects agricultural production and productivity. It is a complex trait that is regulated by different molecular mechanisms. miRNAs are non-coding RNAs which are highly conserved and regulate gene expression. Simple sequence repeats (SSRs) are robust molecular markers for studying genetic diversity. Although several SSR markers are available now, challenge remains to identify the trait-specific SSRs which can be used for marker assisted breeding. In order to understand the genetic diversity of salt responsive-miRNA genes in rice, SSR markers were mined from 130 members of salt-responsive miRNA genes of rice and validated among the contrasting panels of tolerant as well as susceptible rice genotypes, each with 12 genotypes. Although 12 miR-SSRs were found to be polymorphic, only miR172b-SSR was able to differentiate the tolerant and susceptible genotypes in 2 different groups. It had also been found that miRNA genes were more diverse in susceptible genotypes than the tolerant one (as indicated by polymorphic index content) which might interfere to form the stem-loop structure of premature miRNA and their subsequent synthesis in susceptible genotypes. Thus, we concluded that length variations of the repeats in salt responsive miRNA genes may be responsible for a possible sensitivity to salinity adaptation. This is the first report of characterization of trait specific miRNA derived SSRs in plants.     

Thirty-four Musa (Musaceae) expressed sequence tag-derived microsatellite markers transferred to Musella lasiocarpa

We assembled 31,308 publicly available Musa EST sequences into 21,129 unigenes; 4944 of them contained 5416 SSR motifs. In all, 238 unigenes flanking SSRs were randomly selected for primer design and then tested for amplification in Musella lasiocarpa. Seventy-eight primer pairs were found to be transferable to this species, and 49 displayed polymorphism. A set of 34 polymorphic SSR markers was analyzed in 24 individuals from four wild M. lasiocarpa populations. The mean number of alleles per locus was 3.0, ranging from 2 to 7. The observed and expected heterozygosities per marker ranged from 0.087 to 0.875 (mean 0.503) and from 0.294 to 0.788 (mean 0.544), respectively. These markers will be of practical use for genetic diversity and quantitative trait loci analysis of M. lasiocarpa.     

Development of soybean aphid genomic SSR markers using next generation sequencing

Simple sequence repeats (SSRs) or microsatellites are very useful molecular markers, owing to their locus-specific codominant and multiallelic nature, high abundance in the genome, and high rates of transferability across species. The soybean aphid (Aphis glycines Matsumura) has become the most damaging insect pest of soybean (Glycine max (L.) Merr.) in North America, since it was first found in the Midwest of the United States in 2000. Biotypes of the soybean aphid capable of colonizing newly developed aphid-resistant soybean cultivars have been recently discovered. Genetic resources, including molecular markers, to study soybean aphids are severely lacking. Recently developed next generation sequencing platforms offer opportunities for high-throughput and inexpensive genome sequencing and rapid marker development. The objectives of this study were (i) to develop and characterize genomic SSR markers from soybean aphid genomic sequences generated by next generation sequencing technology and (ii) to evaluate the utility of the SSRs for genetic diversity or relationship analyses. In total 128 SSR primer pairs were designed from sequences generated by Illumina GAII from a reduced representation library of A. glycines. Nearly 94% (120) of the primer pairs amplified SSR alleles of expected size and 24 SSR loci were polymorphic among three aphid samples from three populations. The polymorphic SSRs were successfully used to differentiate among 24 soybean aphids from Ohio and South Dakota. Sequencing of PCR products of two SSR markers from four aphid samples revealed that the allelic polymorphism was due to variation in the SSR repeats among the aphids. These markers should be particularly useful for genetic differentiation among aphids collected from soybean fields at different localities and regions. These SSR markers provide the soybean aphid research community with the first set of PCR-based codominant markers developed from the genomic sequences of A. glycines.