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1.  Transferability and polymorphism of barley EST-SSR markers used for phylogenetic analysis in Hordeum chilense 
BMC Plant Biology  2008;8:97.
Hordeum chilense, a native South American diploid wild barley, is a potential source of useful genes for cereal breeding. The use of this wild species to increase genetic variation in cereals will be greatly facilitated by marker-assisted selection. Different economically feasible approaches have been undertaken for this wild species with limited direct agricultural use in a search for suitable and cost-effective markers. The availability of Expressed Sequence Tags (EST) derived microsatellites or simple sequence repeat (SSR) markers, commonly called as EST-SSRs, for barley (Hordeum vulgare) represents a promising source to increase the number of genetic markers available for the H. chilense genome.
All of the 82 barley EST-derived SSR primer pairs tested for transferability to H. chilense amplified products of correct size from this species. Of these 82 barley EST-SSRs, 21 (26%) showed polymorphism among H. chilense lines. Identified polymorphic markers were used to test the transferability and polymorphism in other Poaceae family species with the aim of establishing H. chilense phylogenetic relationships. Triticum aestivum-H. chilense addition lines allowed us to determine the chromosomal localizations of EST-SSR markers and confirm conservation of the linkage group.
From the present study a set of 21 polymorphic EST-SSR markers have been identified to be useful for diversity analysis of H. chilense, related wild barleys like H. murinum, and for wheat marker-assisted introgression breeding. Across-genera transferability of the barley EST-SSR markers has allowed phylogenetic inference within the Triticeae complex.
PMCID: PMC2569940  PMID: 18822176
2.  High-throughput genotyping of wheat-barley amphiploids utilising diversity array technology (DArT) 
BMC Plant Biology  2013;13:87.
Hordeum chilense, a native South American diploid wild barley, is one of the species of the genus Hordeum with a high potential for cereal breeding purposes, given its high crossability with other members of the Triticeae tribe. Hexaploid tritordeum (×Tritordeum Ascherson et Graebner, 2n=6×=42, AABBHchHch) is the fertile amphiploid obtained after chromosome doubling of hybrids between Hordeum chilense and durum wheat. Approaches used in the improvement of this crop have included crosses with hexaploid wheat to promote D/Hch chromosome substitutions. While this approach has been successful as was the case with triticale, it has also complicated the genetic composition of the breeding materials. Until now tritordeum lines were analyzed based on molecular cytogenetic techniques and screening with a small set of DNA markers. However, the recent development of DArT markers in H. chilense offers new possibilities to screen large number of accessions more efficiently.
Here, we have applied DArT markers to genotype composition in forty-six accessions of hexaploid tritordeum originating from different stages of tritordeum breeding program and to H. chilense-wheat chromosome addition lines to allow their physical mapping. Diversity analyses were conducted including dendrogram construction, principal component analysis and structure inference. Euploid and substituted tritordeums were clearly discriminated independently of the method used. However, dendrogram and Structure analyses allowed the clearest discrimination among substituted tritordeums. The physically mapped markers allowed identifying these groups as substituted tritordeums carrying the following disomic substitutions (DS): DS1D (1Hch), DS2D (2Hch), DS5D (5Hch), DS6D (6Hch) and the double substitution DS2D (2Hch), DS5D (5Hch). These results were validated using chromosome specific EST and SSR markers and GISH analysis.
In conclusion, DArT markers have proved to be very useful to detect chromosome substitutions in the tritordeum breeding program and thus they are expected to be equally useful to detect translocations both in the tritordeum breeding program and in the transference of H. chilense genetic material in wheat breeding programs.
PMCID: PMC3679790  PMID: 23725040
Tritordeum; DArT; Chromosome substitution; GISH; EST; SSR markers
3.  Genetic diversity of peanut (Arachis hypogaea L.) and its wild relatives based on the analysis of hypervariable regions of the genome 
BMC Plant Biology  2004;4:11.
The genus Arachis is native to a region that includes Central Brazil and neighboring countries. Little is known about the genetic variability of the Brazilian cultivated peanut (Arachis hypogaea, genome AABB) germplasm collection at the DNA level. The understanding of the genetic diversity of cultivated and wild species of peanut (Arachis spp.) is essential to develop strategies of collection, conservation and use of the germplasm in variety development. The identity of the ancestor progenitor species of cultivated peanut has also been of great interest. Several species have been suggested as putative AA and BB genome donors to allotetraploid A. hypogaea. Microsatellite or SSR (Simple Sequence Repeat) markers are co-dominant, multiallelic, and highly polymorphic genetic markers, appropriate for genetic diversity studies. Microsatellite markers may also, to some extent, support phylogenetic inferences. Here we report the use of a set of microsatellite markers, including newly developed ones, for phylogenetic inferences and the analysis of genetic variation of accessions of A. hypogea and its wild relatives.
A total of 67 new microsatellite markers (mainly TTG motif) were developed for Arachis. Only three of these markers, however, were polymorphic in cultivated peanut. These three new markers plus five other markers characterized previously were evaluated for number of alleles per locus and gene diversity using 60 accessions of A. hypogaea. Genetic relationships among these 60 accessions and a sample of 36 wild accessions representative of section Arachis were estimated using allelic variation observed in a selected set of 12 SSR markers. Results showed that the Brazilian peanut germplasm collection has considerable levels of genetic diversity detected by SSR markers. Similarity groups for A. hypogaea accessions were established, which is a useful criteria for selecting parental plants for crop improvement. Microsatellite marker transferability was up to 76% for species of the section Arachis, but only 45% for species from the other eight Arachis sections tested. A new marker (Ah-041) presented a 100% transferability and could be used to classify the peanut accessions in AA and non-AA genome carriers.
The level of polymorphism observed among accessions of A. hypogaea analyzed with newly developed microsatellite markers was low, corroborating the accumulated data which show that cultivated peanut presents a relatively reduced variation at the DNA level. A selected panel of SSR markers allowed the classification of A. hypogaea accessions into two major groups. The identification of similarity groups will be useful for the selection of parental plants to be used in breeding programs. Marker transferability is relatively high between accessions of section Arachis. The possibility of using microsatellite markers developed for one species in genetic evaluation of other species greatly reduces the cost of the analysis, since the development of microsatellite markers is still expensive and time consuming. The SSR markers developed in this study could be very useful for genetic analysis of wild species of Arachis, including comparative genome mapping, population genetic structure and phylogenetic inferences among species.
PMCID: PMC491793  PMID: 15253775
4.  Gene-based microsatellites for cassava (Manihot esculenta Crantz): prevalence, polymorphisms, and cross-taxa utility 
BMC Plant Biology  2009;9:118.
Cassava (Manihot esculenta Crantz), a starchy root crop grown in tropical and subtropical climates, is the sixth most important crop in the world after wheat, rice, maize, potato and barley. The repertoire of simple sequence repeat (SSR) markers for cassava is limited and warrants a need for a larger number of polymorphic SSRs for germplasm characterization and breeding applications.
A total of 846 putative microsatellites were identified in silico from an 8,577 cassava unigene set with an average density of one SSR every 7 kb. One hundred and ninety-two candidate SSRs were screened for polymorphism among a panel of cassava cultivars from Africa, Latin America and Asia, four wild Manihot species as well as two other important taxa in the Euphorbiaceae, leafy spurge (Euphorbia esula) and castor bean (Ricinus communis). Of 168 markers with clean amplification products, 124 (73.8%) displayed polymorphism based on high resolution agarose gels. Of 85 EST-SSR markers screened, 80 (94.1%) amplified alleles from one or more wild species (M epruinosa, M glaziovii, M brachyandra, M tripartita) whereas 13 (15.3%) amplified alleles from castor bean and 9 (10.6%) amplified alleles from leafy spurge; hence nearly all markers were transferable to wild relatives of M esculenta while only a fraction was transferable to the more distantly related taxa. In a subset of 20 EST-SSRs assessed by fluorescence-based genotyping the number of alleles per locus ranged from 2 to 10 with an average of 4.55 per locus. These markers had a polymorphism information content (PIC) from 0.19 to 0.75 with an average value of 0.55 and showed genetic relationships consistent with existing information on these genotypes.
A set of 124 new, unique polymorphic EST-SSRs was developed and characterized which extends the repertoire of SSR markers for cultivated cassava and its wild relatives. The markers show high PIC values and therefore will be useful for cultivar identification, taxonomic studies, and genetic mapping. The study further shows that mining ESTs is a highly efficient strategy for polymorphism detection within the cultivated cassava gene pool.
PMCID: PMC2758884  PMID: 19747391
5.  Nonrandom distribution and frequencies of genomic and EST-derived microsatellite markers in rice, wheat, and barley 
BMC Genomics  2005;6:23.
Earlier comparative maps between the genomes of rice (Oryza sativa L.), barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) were linkage maps based on cDNA-RFLP markers. The low number of polymorphic RFLP markers has limited the development of dense genetic maps in wheat and the number of available anchor points in comparative maps. Higher density comparative maps using PCR-based anchor markers are necessary to better estimate the conservation of colinearity among cereal genomes. The purposes of this study were to characterize the proportion of transcribed DNA sequences containing simple sequence repeats (SSR or microsatellites) by length and motif for wheat, barley and rice and to determine in-silico rice genome locations for primer sets developed for wheat and barley Expressed Sequence Tags.
The proportions of SSR types (di-, tri-, tetra-, and penta-nucleotide repeats) and motifs varied with the length of the SSRs within and among the three species, with trinucleotide SSRs being the most frequent. Distributions of genomic microsatellites (gSSRs), EST-derived microsatellites (EST-SSRs), and transcribed regions in the contiguous sequence of rice chromosome 1 were highly correlated. More than 13,000 primer pairs were developed for use by the cereal research community as potential markers in wheat, barley and rice.
Trinucleotide SSRs were the most common type in each of the species; however, the relative proportions of SSR types and motifs differed among rice, wheat, and barley. Genomic microsatellites were found to be primarily located in gene-rich regions of the rice genome. Microsatellite markers derived from the use of non-redundant EST-SSRs are an economic and efficient alternative to RFLP for comparative mapping in cereals.
PMCID: PMC550658  PMID: 15720707
6.  Allelic Variation, Alternative Splicing and Expression Analysis of Psy1 Gene in Hordeum chilense Roem. et Schult 
PLoS ONE  2011;6(5):e19885.
The wild barley Hordeum chilense Roem. et Schult. is a valuable source of genes for increasing carotenoid content in wheat. Tritordeums, the amphiploids derived from durum or common wheat and H. chilense, systematically show higher values of yellow pigment colour and carotenoid content than durum wheat. Phytoene synthase 1 gene (Psy1) is considered a key step limiting the carotenoid biosynthesis, and the correlation of Psy1 transcripts accumulation and endosperm carotenoid content has been demonstrated in the main grass species.
Methodology/Principal findings
We analyze the variability of Psy1 alleles in three lines of H. chilense (H1, H7 and H16) representing the three ecotypes described in this species. Moreover, we analyze Psy1 expression in leaves and in two seed developing stages of H1 and H7, showing mRNA accumulation patterns similar to those of wheat. Finally, we identify thirty-six different transcripts forms originated by alternative splicing of the 5′ UTR and/or exons 1 to 5 of Psy1 gene. Transcripts function is tested in a heterologous complementation assay, revealing that from the sixteen different predicted proteins only four types (those of 432, 370, 364 and 271 amino acids), are functional in the bacterial system.
The large number of transcripts originated by alternative splicing of Psy1, and the coexistence of functional and non functional forms, suggest a fine regulation of PSY activity in H. chilense. This work is the first analysis of H. chilense Psy1 gene and the results reported here are the bases for its potential use in carotenoid enhancement in durum wheat.
PMCID: PMC3095628  PMID: 21603624
7.  Development and Characterization of Polymorphic EST-SSR and Genomic SSR Markers for Tibetan Annual Wild Barley 
PLoS ONE  2014;9(4):e94881.
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.
PMCID: PMC3988095  PMID: 24736399
8.  Comparative genomic analysis and expression of the APETALA2-like genes from barley, wheat, and barley-wheat amphiploids 
BMC Plant Biology  2009;9:66.
The APETALA2-like genes form a large multi-gene family of transcription factors which play an important role during the plant life cycle, being key regulators of many developmental processes. Many studies in Arabidopsis have revealed that the APETALA2 (AP2) gene is implicated in the establishment of floral meristem and floral organ identity as well as temporal and spatial regulation of flower homeotic gene expression.
In this work, we have cloned and characterised the AP2-like gene from accessions of Hordeum chilense and Hordeum vulgare, wild and domesticated barley, respectively, and compared with other AP2 homoeologous genes, including the Q gene in wheat. The Hordeum AP2-like genes contain two plant-specific DNA binding motifs called AP2 domains, as does the Q gene of wheat. We confirm that the H. chilense AP2-like gene is located on chromosome 5Hch. Patterns of expression of the AP2-like genes were examined in floral organs and other tissues in barley, wheat and in tritordeum amphiploids (barley × wheat hybrids). In tritordeum amphiploids, the level of transcription of the barley AP2-like gene was lower than in its barley parental and the chromosome substitutions 1D/1Hch and 2D/2Hch were seen to modify AP2 gene expression levels.
The results are of interest in order to understand the role of the AP2-like gene in the spike morphology of barley and wheat, and to understand the regulation of this gene in the amphiploids obtained from barley-wheat crossing. This information may have application in cereal breeding programs to up- or down-regulate the expression of AP2-like genes in order to modify spike characteristics and to obtain free-threshing plants.
PMCID: PMC2700811  PMID: 19480686
9.  Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea) 
BMC Plant Biology  2008;8:55.
Cultivated peanut or groundnut (Arachis hypogaea L.) is the fourth most important oilseed crop in the world, grown mainly in tropical, subtropical and warm temperate climates. Due to its origin through a single and recent polyploidization event, followed by successive selection during breeding efforts, cultivated groundnut has a limited genetic background. In such species, microsatellite or simple sequence repeat (SSR) markers are very informative and useful for breeding applications. The low level of polymorphism in cultivated germplasm, however, warrants a need of larger number of polymorphic microsatellite markers for cultivated groundnut.
A microsatellite-enriched library was constructed from the genotype TMV2. Sequencing of 720 putative SSR-positive clones from a total of 3,072 provided 490 SSRs. 71.2% of these SSRs were perfect type, 13.1% were imperfect and 15.7% were compound. Among these SSRs, the GT/CA repeat motifs were the most common (37.6%) followed by GA/CT repeat motifs (25.9%). The primer pairs could be designed for a total of 170 SSRs and were optimized initially on two genotypes. 104 (61.2%) primer pairs yielded scorable amplicon and 46 (44.2%) primers showed polymorphism among 32 cultivated groundnut genotypes. The polymorphic SSR markers detected 2 to 5 alleles with an average of 2.44 per locus. The polymorphic information content (PIC) value for these markers varied from 0.12 to 0.75 with an average of 0.46. Based on 112 alleles obtained by 46 markers, a phenogram was constructed to understand the relationships among the 32 genotypes. Majority of the genotypes representing subspecies hypogaea were grouped together in one cluster, while the genotypes belonging to subspecies fastigiata were grouped mainly under two clusters.
Newly developed set of 104 markers extends the repertoire of SSR markers for cultivated groundnut. These markers showed a good level of PIC value in cultivated germplasm and therefore would be very useful for germplasm analysis, linkage mapping, diversity studies and phylogenetic relationships in cultivated groundnut as well as related Arachis species.
PMCID: PMC2416452  PMID: 18482440
10.  Hordeum chilense genome, a useful tool to investigate the endosperm yellow pigment content in the Triticeae 
BMC Plant Biology  2012;12:200.
The wild barley Hordeum chilense fulfills some requirements for being a useful tool to investigate the endosperm yellow pigment content (YPC) in the Triticeae including its diploid constitution, the availability of genetic resources (addition and deletion stocks and a high density genetic map) and, especially, its high seed YPC not silenced in tritordeums (amphiploids derived from H. chilense and wheat). Thus, the aim of this work was to test the utility of the H. chilense genome for investigating the YPC in the Triticeae.
Twelve genes related to endosperm carotenoid content and/or YPC in grasses (Dxr, Hdr [synonym ispH], Ggpps1, Psy2, Psy3, Pds, Zds, e-Lcy, b-Lcy, Hyd3, Ccd1 and Ppo1) were identified, and mapped in H. chilense using rice genes to identify orthologs from barley, wheat, sorghum and maize. Macrocolinearity studies revealed that gene positions were in agreement in H. vulgare and H. chilense. Additionally, three main regions associated with YPC were identified in chromosomes 2Hch, 3Hch and 7Hch in H. chilense, the former being the most significant one.
The results obtained are consistent with previous findings in wheat and suggest that Ggpps1, Zds and Hyd3 on chromosome 2Hch may be considered candidate genes in wheat for further studies in YPC improvement. Considering the syntenic location of carotenoid genes in H. chilense, we have concluded that the Hch genome may constitute a valuable tool for YPC studies in the Triticeae.
PMCID: PMC3534404  PMID: 23122232
Yellow pigment content (YPC); Macrocolinearity; Candidate genes; H. chilense; H. vulgare
11.  Microsatellite isolation and marker development in carrot - genomic distribution, linkage mapping, genetic diversity analysis and marker transferability across Apiaceae 
BMC Genomics  2011;12:386.
The Apiaceae family includes several vegetable and spice crop species among which carrot is the most economically important member, with ~21 million tons produced yearly worldwide. Despite its importance, molecular resources in this species are relatively underdeveloped. The availability of informative, polymorphic, and robust PCR-based markers, such as microsatellites (or SSRs), will facilitate genetics and breeding of carrot and other Apiaceae, including integration of linkage maps, tagging of phenotypic traits and assisting positional gene cloning. Thus, with the purpose of isolating carrot microsatellites, two different strategies were used; a hybridization-based library enrichment for SSRs, and bioinformatic mining of SSRs in BAC-end sequence and EST sequence databases. This work reports on the development of 300 carrot SSR markers and their characterization at various levels.
Evaluation of microsatellites isolated from both DNA sources in subsets of 7 carrot F2 mapping populations revealed that SSRs from the hybridization-based method were longer, had more repeat units and were more polymorphic than SSRs isolated by sequence search. Overall, 196 SSRs (65.1%) were polymorphic in at least one mapping population, and the percentage of polymophic SSRs across F2 populations ranged from 17.8 to 24.7. Polymorphic markers in one family were evaluated in the entire F2, allowing the genetic mapping of 55 SSRs (38 codominant) onto the carrot reference map. The SSR loci were distributed throughout all 9 carrot linkage groups (LGs), with 2 to 9 SSRs/LG. In addition, SSR evaluations in carrot-related taxa indicated that a significant fraction of the carrot SSRs transfer successfully across Apiaceae, with heterologous amplification success rate decreasing with the target-species evolutionary distance from carrot. SSR diversity evaluated in a collection of 65 D. carota accessions revealed a high level of polymorphism for these selected loci, with an average of 19 alleles/locus and 0.84 expected heterozygosity.
The addition of 55 SSRs to the carrot map, together with marker characterizations in six other mapping populations, will facilitate future comparative mapping studies and integration of carrot maps. The markers developed herein will be a valuable resource for assisting breeding, genetic, diversity, and genomic studies of carrot and other Apiaceae.
PMCID: PMC3162538  PMID: 21806822
12.  Development and characterization of microsatellite markers for Morus spp. and assessment of their transferability to other closely related species 
BMC Plant Biology  2013;13:194.
Adoption of genomics based breeding has emerged as a promising approach for achieving comprehensive crop improvement. Such an approach is more relevant in the case of perennial species like mulberry. However, unavailability of genomic resources of co-dominant marker systems has been the major constraint for adopting molecular breeding to achieve genetic enhancement of Mulberry. The goal of this study was to develop and characterize a large number of locus specific genic and genomic SSR markers which can be effectively used for molecular characterization of mulberry species/genotypes.
We analyzed a total of 3485 DNA sequences including genomic and expressed sequences (ESTs) of mulberry (Morus alba L.) genome. We identified 358 sequences to develop appropriate microsatellite primer pairs representing 222 genomic and 136 EST regions. Primers amplifying locus specific regions of Dudia white (a genotype of Morus alba L), were identified and 137 genomic and 51 genic SSR markers were standardized. A two pronged strategy was adopted to assess the applicability of these SSR markers using mulberry species and genotypes along with a few closely related species belonging to the family Moraceae viz., Ficus, Fig and Jackfruit. While 100% of these markers amplified specific loci on the mulberry genome, 79% were transferable to other related species indicating the robustness of these markers and the potential they hold in analyzing the molecular and genetic diversity among mulberry germplasm as well as other related species. The inherent ability of these markers in detecting heterozygosity combined with a high average polymorphic information content (PIC) of 0.559 ranging between 0.076 and 0.943 clearly demonstrates their potential as genomic resources in diversity analysis. The dissimilarity coefficient determined based on Neighbor joining method, revealed that the markers were successful in segregating the mulberry species, genotypes and other related species into distinct clusters.
We report a total of 188 genomic and genic SSR markers in Morus alba L. A large proportion of these markers (164) were polymorphic both among mulberry species and genotypes. A substantial number of these markers (149) were also transferable to other related species like Ficus, Fig and Jackfruit. The extent of polymorphism revealed and the ability to detect heterozygosity among the cross pollinated mulberry species and genotypes render these markers an invaluable genomic resource that can be utilized in assessing molecular diversity as well as in QTL mapping and subsequently mulberry crop improvement through MAS.
PMCID: PMC3879070  PMID: 24289047
13.  Development of new genomic microsatellite markers from robusta coffee (Coffea canephora Pierre ex A. Froehner) showing broad cross-species transferability and utility in genetic studies 
BMC Plant Biology  2008;8:51.
Species-specific microsatellite markers are desirable for genetic studies and to harness the potential of MAS-based breeding for genetic improvement. Limited availability of such markers for coffee, one of the most important beverage tree crops, warrants newer efforts to develop additional microsatellite markers that can be effectively deployed in genetic analysis and coffee improvement programs. The present study aimed to develop new coffee-specific SSR markers and validate their utility in analysis of genetic diversity, individualization, linkage mapping, and transferability for use in other related taxa.
A small-insert partial genomic library of Coffea canephora, was probed for various SSR motifs following conventional approach of Southern hybridisation. Characterization of repeat positive clones revealed a very high abundance of DNRs (1/15 Kb) over TNRs (1/406 kb). The relative frequencies of different DNRs were found as AT >> AG > AC, whereas among TNRs, AGC was the most abundant repeat. The SSR positive sequences were used to design 58 primer pairs of which 44 pairs could be validated as single locus markers using a panel of arabica and robusta genotypes. The analysis revealed an average of 3.3 and 3.78 alleles and 0.49 and 0.62 PIC per marker for the tested arabicas and robustas, respectively. It also revealed a high cumulative PI over all the markers using both sib-based (10-6 and 10-12 for arabicas and robustas respectively) and unbiased corrected estimates (10-20 and 10-43 for arabicas and robustas respectively). The markers were tested for Hardy-Weinberg equilibrium, linkage dis-equilibrium, and were successfully used to ascertain generic diversity/affinities in the tested germplasm (cultivated as well as species). Nine markers could be mapped on robusta linkage map. Importantly, the markers showed ~92% transferability across related species/genera of coffee.
The conventional approach of genomic library was successfully employed although with low efficiency to develop a set of 44 new genomic microsatellite markers of coffee. The characterization/validation of new markers demonstrated them to be highly informative, and useful for genetic studies namely, genetic diversity in coffee germplasm, individualization/bar-coding for germplasm protection, linkage mapping, taxonomic studies, and use as conserved orthologous sets across secondary genepool of coffee. Further, the relative frequency and distribution of different SSR motifs in coffee genome indicated coffee genome to be relatively poor in microsatellites compared to other plant species.
PMCID: PMC2396172  PMID: 18447947
14.  Developmenrt of EST-SSR and genomic-SSR markers to assess genetic diversity in Jatropha Curcas L. 
BMC Research Notes  2010;3:42.
Jatropha curcas L. has attracted a great deal of attention worldwide, regarding its potential as a new biodiesel crop. However, the understanding of this crop remains very limited and little genomic research has been done. We used simple sequence repeat (SSR) markers that could be transferred from Manihot esculenta (cassava) to analyze the genetic relationships among 45 accessions of J. curcas from our germplasm collection.
In total, 187 out of 419 expressed sequence tag (EST)-SSR and 54 out of 182 genomic (G)-SSR markers from cassava were polymorphic among the J. curcas accessions. The EST-SSR markers comprised 26.20% dinucleotide repeats, 57.75% trinucleotide repeats, 7.49% tetranucleotide repeats, and 8.56% pentanucleotide repeats, whereas the majority of the G-SSR markers were dinucleotide repeats (62.96%). The 187 EST-SSRs resided in genes that are involved mainly in biological and metabolic processes. Thirty-six EST-SSRs and 20 G-SSRs were chosen to analyze the genetic diversity among 45 J. curcas accessions. A total of 183 polymorphic alleles were detected. On the basis of the distribution of these polymorphic alleles, the 45 accessions were classified into six groups, in which the genotype showed a correlation with geographic origin. The estimated mean genetic diversity index was 0.5572, which suggests that our J. curcas germplasm collection has a high level of genetic diversity. This should facilitate subsequent studies on genetic mapping and molecular breeding.
We identified 241 novel EST-SSR and G-SSR markers in J. curcas, which should be useful for genetic mapping and quantitative trait loci analysis of important agronomic traits. By using these markers, we found that the intergroup gene diversity of J. curcas was greater than the intragroup diversity, and that the domestication of the species probably occurred partly in America and partly in Hainan, China.
PMCID: PMC2844395  PMID: 20181259
15.  Genetic relationships among seven sections of genus Arachis studied by using SSR markers 
BMC Plant Biology  2010;10:15.
The genus Arachis, originated in South America, is divided into nine taxonomical sections comprising of 80 species. Most of the Arachis species are diploids (2n = 2x = 20) and the tetraploid species (2n = 2x = 40) are found in sections Arachis, Extranervosae and Rhizomatosae. Diploid species have great potential to be used as resistance sources for agronomic traits like pests and diseases, drought related traits and different life cycle spans. Understanding of genetic relationships among wild species and between wild and cultivated species will be useful for enhanced utilization of wild species in improving cultivated germplasm. The present study was undertaken to evaluate genetic relationships among species (96 accessions) belonging to seven sections of Arachis by using simple sequence repeat (SSR) markers developed from Arachis hypogaea genomic library and gene sequences from related genera of Arachis.
The average transferability rate of 101 SSR markers tested to section Arachis and six other sections was 81% and 59% respectively. Five markers (IPAHM 164, IPAHM 165, IPAHM 407a, IPAHM 409, and IPAHM 659) showed 100% transferability. Cluster analysis of allelic data from a subset of 32 SSR markers on 85 wild and 11 cultivated accessions grouped accessions according to their genome composition, sections and species to which they belong. A total of 109 species specific alleles were detected in different wild species, Arachis pusilla exhibited largest number of species specific alleles (15). Based on genetic distance analysis, the A-genome accession ICG 8200 (A. duranensis) and the B-genome accession ICG 8206 (A. ipaënsis) were found most closely related to A. hypogaea.
A set of cross species and cross section transferable SSR markers has been identified that will be useful for genetic studies of wild species of Arachis, including comparative genome mapping, germplasm analysis, population genetic structure and phylogenetic inferences among species. The present study provides strong support based on both genomic and genic markers, probably for the first time, on relationships of A. monticola and A. hypogaea as well as on the most probable donor of A and B-genomes of cultivated groundnut.
PMCID: PMC2826335  PMID: 20089171
16.  Exploiting EST databases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L.) 
BMC Plant Biology  2010;10:278.
The castor bean (Ricinus communis L.), a monotypic species in the spurge family (Euphorbiaceae, 2n = 20), is an important non-edible oilseed crop widely cultivated in tropical, sub-tropical and temperate countries for its high economic value. Because of the high level of ricinoleic acid (over 85%) in its seed oil, the castor bean seed derivatives are often used in aviation oil, lubricants, nylon, dyes, inks, soaps, adhesive and biodiesel. Due to lack of efficient molecular markers, little is known about the population genetic diversity and the genetic relationships among castor bean germplasm. Efficient and robust molecular markers are increasingly needed for breeding and improving varieties in castor bean. The advent of modern genomics has produced large amounts of publicly available DNA sequence data. In particular, expressed sequence tags (ESTs) provide valuable resources to develop gene-associated SSR markers.
In total, 18,928 publicly available non-redundant castor bean EST sequences, representing approximately 17.03 Mb, were evaluated and 7732 SSR sites in 5,122 ESTs were identified by data mining. Castor bean exhibited considerably high frequency of EST-SSRs. We developed and characterized 118 polymorphic EST-SSR markers from 379 primer pairs flanking repeats by screening 24 castor bean samples collected from different countries. A total of 350 alleles were identified from 118 polymorphic SSR loci, ranging from 2-6 per locus (A) with an average of 2.97. The EST-SSR markers developed displayed moderate gene diversity (He) with an average of 0.41. Genetic relationships among 24 germplasms were investigated using the genotypes of 350 alleles, showing geographic pattern of genotypes across genetic diversity centers of castor bean.
Castor bean EST sequences exhibited considerably high frequency of SSR sites, and were rich resources for developing EST-SSR markers. These EST-SSR markers would be particularly useful for both genetic mapping and population structure analysis, facilitating breeding and crop improvement of castor bean.
PMCID: PMC3017068  PMID: 21162723
17.  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.
PMCID: PMC3190952  PMID: 21768136
Brassica rapa; expressed sequence-derived SSRs; integrated map; polymorphism information content; transferability
18.  An annotated genetic map of loblolly pine based on microsatellite and cDNA markers 
BMC Genetics  2011;12:17.
Previous loblolly pine (Pinus taeda L.) genetic linkage maps have been based on a variety of DNA polymorphisms, such as AFLPs, RAPDs, RFLPs, and ESTPs, but only a few SSRs (simple sequence repeats), also known as simple tandem repeats or microsatellites, have been mapped in P. taeda. The objective of this study was to integrate a large set of SSR markers from a variety of sources and published cDNA markers into a composite P. taeda genetic map constructed from two reference mapping pedigrees. A dense genetic map that incorporates SSR loci will benefit complete pine genome sequencing, pine population genetics studies, and pine breeding programs. Careful marker annotation using a variety of references further enhances the utility of the integrated SSR map.
The updated P. taeda genetic map, with an estimated genome coverage of 1,515 cM(Kosambi) across 12 linkage groups, incorporated 170 new SSR markers and 290 previously reported SSR, RFLP, and ESTP markers. The average marker interval was 3.1 cM. Of 233 mapped SSR loci, 84 were from cDNA-derived sequences (EST-SSRs) and 149 were from non-transcribed genomic sequences (genomic-SSRs). Of all 311 mapped cDNA-derived markers, 77% were associated with NCBI Pta UniGene clusters, 67% with RefSeq proteins, and 62% with functional Gene Ontology (GO) terms. Duplicate (i.e., redundant accessory) and paralogous markers were tentatively identified by evaluating marker sequences by their UniGene cluster IDs, clone IDs, and relative map positions. The average gene diversity, He, among polymorphic SSR loci, including those that were not mapped, was 0.43 for 94 EST-SSRs and 0.72 for 83 genomic-SSRs. The genetic map can be viewed and queried at
Many polymorphic and genetically mapped SSR markers are now available for use in P. taeda population genetics, studies of adaptive traits, and various germplasm management applications. Annotating mapped genes with UniGene clusters and GO terms allowed assessment of redundant and paralogous EST markers and further improved the quality and utility of the genetic map for P. taeda.
PMCID: PMC3038140  PMID: 21269494
19.  Characterization of the Soybean Genome Using EST-derived Microsatellite Markers 
We generated a high-density genetic linkage map of soybean using expressed sequence tag (EST)-derived microsatellite markers. A total of 6920 primer pairs (10.9%) were designed to amplify simple sequence repeats (SSRs) from 63 676 publicly available non-redundant soybean ESTs. The polymorphism of two parent plants, the Japanese cultivar ‘Misuzudaizu’ and the Chinese line ‘Moshidou Gong 503’, were examined using 10% polyacrylamide gel electrophoresis. Primer pairs showing polymorphism were then used for genotyping 94 recombinant inbred lines (RILs) derived from a cross between the parents. In addition to previously reported markers, 680 EST-derived microsatellite markers were selected and subjected to linkage analysis. As a result, 935 marker loci were mapped successfully onto 20 linkage groups, which totaled 2700.3 cM in length; 693 loci were detected using the 668 EST-derived microsatellite markers developed in this study, the other 242 loci were detected with 105 RFLP markers, 136 genome-derived microsatellite markers, and one phenotypic marker. We examined allelic variation among 23 soybean cultivars/lines and a wild soybean line using 668 mapped EST-derived microsatellite markers (corresponding to 686 marker loci), in order to determine the transferability of the markers among soybean germplasms. A limited degree of macrosynteny was observed at the segmental level between the genomes of soybean and the model legume Lotus japonicus, which suggests that considerable genome shuffling occurred after separation of the species and during establishment of the paleopolyploid soybean genome.
PMCID: PMC2779906  PMID: 18192281
soybean; microsatellite markers; genetic linkage map; polymorphism information content; comparative map
20.  Development of Genic and Genomic SSR Markers of Robusta Coffee (Coffea canephora Pierre Ex A. Froehner) 
PLoS ONE  2014;9(12):e113661.
Coffee breeding and improvement efforts can be greatly facilitated by availability of a large repository of simple sequence repeats (SSRs) based microsatellite markers, which provides efficiency and high-resolution in genetic analyses. This study was aimed to improve SSR availability in coffee by developing new genic−/genomic-SSR markers using in-silico bioinformatics and streptavidin-biotin based enrichment approach, respectively. The expressed sequence tag (EST) based genic microsatellite markers (EST-SSRs) were developed using the publicly available dataset of 13,175 unigene ESTs, which showed a distribution of 1 SSR/3.4 kb of coffee transcriptome. Genomic SSRs, on the other hand, were developed from an SSR-enriched small-insert partial genomic library of robusta coffee. In total, 69 new SSRs (44 EST-SSRs and 25 genomic SSRs) were developed and validated as suitable genetic markers. Diversity analysis of selected coffee genotypes revealed these to be highly informative in terms of allelic diversity and PIC values, and eighteen of these markers (∼27%) could be mapped on a robusta linkage map. Notably, the markers described here also revealed a very high cross-species transferability. In addition to the validated markers, we have also designed primer pairs for 270 putative EST-SSRs, which are expected to provide another ca. 200 useful genetic markers considering the high success rate (88%) of marker conversion of similar pairs tested/validated in this study.
PMCID: PMC4252042  PMID: 25461752
21.  Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.) 
BMC Plant Biology  2009;9:53.
Despite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species.
Considering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (HE) and observed (Ho) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.
UGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.
PMCID: PMC2693106  PMID: 19426565
22.  Genetic Diversity and Population Structure of Miscanthus sinensis Germplasm in China 
PLoS ONE  2013;8(10):e75672.
Miscanthus is a perennial rhizomatous C4 grass native to East Asia. Endowed with great biomass yield, high ligno-cellulose composition, efficient use of radiation, nutrient and water, as well as tolerance to stress, Miscanthus has great potential as an excellent bioenergy crop. Despite of the high potential for biomass production of the allotriploid hybrid M. ×giganteus, derived from M. sacchariflorus and M. sinensis, other options need to be explored to improve the narrow genetic base of M. ×giganteus, and also to exploit other Miscanthus species, including M. sinensis (2n = 2x = 38), as bioenergy crops. In the present study, a large number of 459 M. sinensis accessions, collected from the wide geographical distribution regions in China, were genotyped using 23 SSR markers transferable from Brachypodium distachyon. Genetic diversity and population structure were assessed. High genetic diversity and differentiation of the germplasm were observed, with 115 alleles in total, a polymorphic rate of 0.77, Nei’s genetic diversity index (He) of 0.32 and polymorphism information content (PIC) of 0.26. Clustering of germplasm accessions was primarily in agreement with the natural geographic distribution. AMOVA and genetic distance analyses confirmed the genetic differentiation in the M. sinensis germplasm and it was grouped into five clusters or subpopulations. Significant genetic variation among subpopulations indicated obvious genetic differentiation in the collections, but within-subpopulation variation (83%) was substantially greater than the between-subpopulation variation (17%). Considerable phenotypic variation was observed for multiple traits among 300 M. sinensis accessions. Nine SSR markers were found to be associated with heading date and biomass yield. The diverse Chinese M. sinensis germplasm and newly identified SSR markers were proved to be valuable for breeding Miscanthus varieties with desired bioenergy traits.
PMCID: PMC3792140  PMID: 24116066
23.  New microsatellite markers developed from Urochloa humidicola (Poaceae) and cross amplification in different Urochloa species 
BMC Research Notes  2011;4:523.
Urochloa humidicola is a forage grass that grows in tropical regions and is recognized for its tolerance to seasonal flooding. It is a polyploid and apomictic species with high phenotypic plasticity. As molecular tools are important in facilitating the development of new cultivars and in the classification of related species, the objectives of this study were to develop new polymorphic microsatellite markers from an enriched library constructed from U. humidicola and to evaluate their transferability to other Urochloa species.
Microsatellite sequences were identified from a previously constructed enriched library, and specific primers were designed for 40 loci. Isolated di-nucleotide repeat motifs were the most abundant followed by tetra-nucleotide repeats. Of the tested loci, 38 displayed polymorphism when screened across 34 polyploid Urochloa sp. genotypes, including 20 accessions and six hybrids of U. humidicola and two accessions each from U. brizantha, U. dictyoneura, U. decumbens and U. ruziziensis. The number of bands per Simple Sequence Repeat (SSR) locus ranged from one to 29 with a mean of 11.5 bands per locus. The mean Polymorphism Information Content (PIC) of all loci was 0.7136, and the mean Discrimination Power (DP) was 0.7873. Six loci amplified in all species tested. STRUCTURE analysis revealed six different allelic pools, and the genetic similarity values analyzed using Jaccard's coefficient ranged from 0.000 to 0.913.
This work reports new polymorphic microsatellite markers that will be useful for breeding programs for Urochloa humidicola and other Urochloa species as well as for genetic map development, germplasm characterization, evolutionary and taxonomic studies and marker-assisted trait selection.
PMCID: PMC3251623  PMID: 22142493
24.  Development and characterization of highly polymorphic long TC repeat microsatellite markers for genetic analysis of peanut 
BMC Research Notes  2012;5:86.
Peanut (Arachis hypogaea L.) is a crop of economic and social importance, mainly in tropical areas, and developing countries. Its molecular breeding has been hindered by a shortage of polymorphic genetic markers due to a very narrow genetic base. Microsatellites (SSRs) are markers of choice in peanut because they are co-dominant, highly transferrable between species and easily applicable in the allotetraploid genome. In spite of substantial effort over the last few years by a number of research groups, the number of SSRs that are polymorphic for A. hypogaea is still limiting for routine application, creating the demand for the discovery of more markers polymorphic within cultivated germplasm.
A plasmid genomic library enriched for TC/AG repeats was constructed and 1401 clones sequenced. From the sequences obtained 146 primer pairs flanking mostly TC microsatellites were developed. The average number of repeat motifs amplified was 23. These 146 markers were characterized on 22 genotypes of cultivated peanut. In total 78 of the markers were polymorphic within cultivated germplasm. Most of those 78 markers were highly informative with an average of 5.4 alleles per locus being amplified. Average gene diversity index (GD) was 0.6, and 66 markers showed a GD of more than 0.5. Genetic relationship analysis was performed and corroborated the current taxonomical classification of A. hypogaea subspecies and varieties.
The microsatellite markers described here are a useful resource for genetics and genomics in Arachis. In particular, the 66 markers that are highly polymorphic in cultivated peanut are a significant step towards routine genetic mapping and marker-assisted selection for the crop.
PMCID: PMC3296580  PMID: 22305491
25.  Genetic diversity in Capsicum germplasm based on microsatellite and random amplified microsatellite polymorphism markers 
A sound knowledge of the genetic diversity among germplasm is vital for strategic germplasm collection, maintenance, conservation and utilisation. Genomic simple sequence repeats (SSRs) and random amplified microsatellite polymorphism (RAMPO) markers were used to analyse diversity and relationships among 48 pepper (Capsicum spp.) genotypes originating from nine countries. These genotypes covered 4 species including 13 germplasm accessions, 30 improved lines of 4 domesticated species and 5 landraces derived from natural interspecific crosses. Out of 106 SSR markers, 25 polymorphic SSR markers (24 %) detected a total of 76 alleles (average, 3.04; range, 2–5). The average polymorphic information content (PIC) was 0.69 (range, 0.29–0.92). Seventeen RAMPO markers produced 87 polymorphic fragments with average PIC of 0.63 (range, 0.44–0.81). Dendrograms based on SSRs and RAMPOs generated two clusters. All 38 Capsicum annuum genotypes and an interspecific landrace clustered together, whereas nine non-annuum (three Capsicum frutescens, one Capsicum chinense, one Capsicum baccatum and four interspecific landraces) genotypes clustered separately. Genetic variation within non-annuum genotypes was greater than the C. annuum genotypes. Distinctness of interspecific derivative landraces grown in northeast India was validated; natural crossing between sympatric Capsicum species has been proposed as the mechanism of their origin.
Electronic supplementary material
The online version of this article (doi:10.1007/s12298-013-0185-3) contains supplementary material, which is available to authorized users.
PMCID: PMC3781276  PMID: 24431527
Capsicum; SSR; RAMPO; Interspecific derivatives; Sympatric population

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