Molecular Markers For Genetic Studies Research Articles

Molecular markers in genetic studies of aquatic macrophytes: a systematic review

Molecular markers in genetic studies of aquatic macrophytes: a systematic review

Transcriptome Analysis and Novel EST-SSR Marker Development for Pinus tabuliformis Seedlings from Four Provenances

Chinese pine (Pinus tabuliformis) is a conifer species endemic to the temperate, warm temperate, and semi-arid regions of China. It has important ecological and economic values, but lacks suitable molecular markers for genetic studies. In this study, we collected open-pollinated progeny seeds from four provenances: Ningcheng (Nc), Qinyuan (Qy), Weichang (Wc), and Pingquan (Pq). We sequenced the transcriptomes of open-pollinated progeny seedlings from four provenances and annotated 18,244 unigenes. We analyzed the expression of genes involved in the auxin indole-3-acetic acid (IAA), cytokinin (CTK), and gibberellin (GA) signaling pathways among the provenances. Additionally, we detected 2811 expressed sequence tag simple sequence repeat (EST-SSR) loci in 2360 unigenes, with a frequency of 14.83% and an average of one locus per 14,556 base pairs (bp). We developed 10 polymorphic primers from 67 pairs and tested them on 56 samples from the four provenances. These primers exhibited moderate to high polymorphism and distinguished all samples clearly. Our study reveals variation in growth and development among open-pollinated progeny seedlings from different provenances of Chinese pine and provides novel markers for its genetic diversity study and marker-assisted breeding.

Molecular evolutionary relationships of Vietnamese and global pulmonary Paragonimus species in the family Paragonimidae and suborder Xiphidiata (Platyhelminthes: Trematoda)

Paragonimiasis, caused by Paragonimus species belonging to the family Paragonimidae of the suborder Xiphidiata (Platyhelminthes: Trematoda), often occurs in poor, upland, ethnic minorities, in Vietnam and the world. Asian Paragonimus species are distributed from Japan, South Korea, along with North and Southeast China, North-West and Central Vietnam, the Philippines, Thailand, Bangladesh, India, and Sri Lanka. There are various genetic variants, strains, and genotypes forming different complexes and evolutionary lineages. The 18S, 28S rDNA sequences and the intergenic transcribed spacer regions (ITS-1, ITS-2) of nuclear ribosomal transcription units are commonly used as molecular markers in genetic studies and phylogenetic analyses. We obtained a portion of 28S rDNA (domains D1–D3) of Paragonimus spp. including P. heterotremus (from Vietnam), P. ohirai (Japan), P. iloktsuenensis (Japan), and P. westermani (India and Vietnam) and conducted phylogenetic analysis for molecular evolutionary studies. The results showed that the family Paragonimidae formed the biggest cluster in a phylogenetic tree, which comprises of 46 sequences of 11 species belonging to 11 subgroups, among which the P. westermani complex of strains originating from China, Korea, Japan, India, Philippines, Malaysia, and Vietnam is present. P. westermani complex is arranged in a position of "sister" (sister group) with the subgroup P. siamensis. The P. heterotremus and P. ohirai complexes, and the P. miyazakii, P. harinasutai, P. mexicanus, P. kellicotti, and P. macrorchis complexes are clustered in a common population. P. westermani of Vietnam is in close proximity to the East Asian strains, as of which has been previously reported. P. ohirai and P. iloktsuenensis are considered “sibling” species, sharing the same clade. Phylogenetic analysis using the 28S rDNA sequences directly presented species position and their molecular evolutionary relationships in the families Paragonimidae, Troglotrematidae, Nanophyetidae, and Collyriclidae. Evolutionary analysis has also clarified a number of complex delineation problems and made a clear nomenclature for Paragonimus sp. of Vietnam, in particular, which has scientific grounds merited to recognize as that it is really the P. westermani species.

Tritordeum: Creating a New Crop Species-The Successful Use of Plant Genetic Resources.

Hexaploid tritordeum is the amphiploid derived from the cross between the wild barley Hordeum chilense and durum wheat. This paper reviews the main advances and achievements in the last two decades that led to the successful development of tritordeum as a new crop. In particular, we summarize the progress in breeding for agronomic performance, including the potential of tritordeum as a genetic bridge for wheat breeding; the impact of molecular markers in genetic studies and breeding; and the progress in quality and development of innovative food products. The success of tritordeum as a crop shows the importance of the effective utilization of plant genetic resources for the development of new innovative products for agriculture and industry. Considering that wild plant genetic resources have made possible the development of this new crop, the huge potential of more accessible resources, such as landraces conserved in gene banks, goes beyond being sources of resistance to biotic and abiotic stresses. In addition, the positive result of tritordeum also shows the importance of adequate commercialization strategies and demonstrative experiences aimed to integrate the whole food chain, from producers to end-point sellers, in order to develop new products for consumers.

Application of Molecular Markers and their Utility in Genetic Studies of Floricultural Crops: A Review

Understanding the genetics of floricultural germplasm represents a value added component of managing collections. Molecular approaches collectively represent a potential goldmine of important information that can be applied to programmes of genetic improvement. The development and utilization of molecular markers for the exploitation and identification of plant genetic diversity is one of the most key developments in the field of molecular genetics studies. Molecular markers are popular tool, due to their stability, cost-effectiveness and ease of use for a variety of applications in the field of molecular genetics. To know the role of molecular markers in genetic studies of floricultural crops, a number of molecular marker techniques have been reviewed by many researchers in worldwide. However, all these reviews were meant for researchers with advanced knowledge of molecular markers and their utility in limited aspects under floricultural crops. This review presents an overview of different marker technologies and their variants with a comparative account of their characteristic features and applications especially diverse molecular techniques used in assessing plant genetic diversity, inter-relationship between the genotypes, genetic purity, DNA barcode, tagging of important genes, development of linkage maps, marker assisted selection and application of molecular markers in tissue culture of floricultural crops. Altogether, the characteristics and types of markers with their applications and some recent advancement in genetic studies of floricultural crops are discussed.

Research and partnership in studies of sugarcane using molecular markers: a scientometric approach

In this study, we applied scientometric methods and performed a science mapping analysis to investigate and describe the application of molecular markers in genetic studies for sugarcane. We analyzed 344 papers retrieved from Web of Science Thomson Reuters database, considering the time frame from 1974 to 2017, allowing the analysis of the produced scientific knowledge, temporal trends and research partnership in the subject. To our knowledge, a scientometric evaluation of the use of molecular markers for genetic studies in sugarcane has not yet been reported. Our main finding shows that dominant markers are more commonly used than co-dominant markers, and that when co-dominant markers are applied, most of the retrieved information is underexploited, due to lack of knowledge for the use of dosage information. Our results also show that despite the development of high-throughput sequencing technologies, for sugarcane only 13.33% of the studies reported the use of more than 1000 markers. We also found connectivity among researchers and institutions publishing worldwide, but only few authors represented a bridge between different research groups. Thus, the international research partnership is still low. Our findings show dominance in country publication. Six countries (India, Brazil, USA, Australia, China, and France) were responsible for 80% of the published papers and received most citations. We suggest special reinforcement in building international partnership, since collaborations occur mostly within countries. International collaboration can improve research performance and eventually lead to a better exploitation and more development of knowledge for sugarcane.

Molecular Resources from Transcriptomes in the Brassicaceae Family.

The rapidly falling costs and the increasing availability of large DNA sequence data sets facilitate the fast and affordable mining of large molecular markers data sets for comprehensive evolutionary studies. The Brassicaceae (mustards) are an important species-rich family in the plant kingdom with taxa distributed worldwide and a complex evolutionary history. We performed Simple Sequence Repeats (SSRs) mining using de novo assembled transcriptomes from 19 species across the Brassicaceae in order to study SSR evolution and provide comprehensive sets of molecular markers for genetic studies within the family. Moreover, we selected the genus Cochlearia to test the transferability and polymorphism of these markers among species. Additionally, we annotated Cochlearia pyrenaica transcriptome in order to identify the position of each of the mined SSRs. While we introduce a new set of tools that will further enable evolutionary studies across the Brassicaceae, we also discuss some broader aspects of SSR evolution. Overall, we developed 2012 ready-to-use SSR markers with their respective primers in 19 Brassicaceae species and a high quality annotated transcriptome for C. pyrenaica. As indicated by our transferability test with the genus Cochlearia these SSRs are transferable to species within the genus increasing exponentially the number of targeted species. Also, our polymorphism results showed substantial levels of variability for these markers. Finally, despite its complex evolutionary history, SSR evolution across the Brassicaceae family is highly conserved and we found no deviation from patterns reported in other Angiosperms.

Genetic diversity among Hymenaea courbaril L. genotypes naturally occurring in the north of Mato Grosso State, Brazil

Hymenaea courbaril L. is a tropical tree species with economic and medicinal potential largely used in programs for the recovery of degraded areas. The use of molecular markers in genetic studies can help in the definition of forest restoration strategies. This study was conducted to analyze the genetic diversity among H. courbaril (‘jatoba’) genotypes naturally occurring in the north of Mato Grosso State, Brazil, using ISSR markers, aiming to contribute to the development of seed collection and seedling production strategies. Twenty-four H. courbaril individuals were sampled by collecting leaves for DNA extraction by the CTAB method. The individuals were genotyped using 14 ISSR primers, resulting in the amplification of 87 bands in total, 65 of which were polymorphic. The ISSR markers presented average polymorphism information content of 0.44. Dissimilarity values based on the Jaccard coefficient ranged from 0.12 to 0.52. The most divergent individuals were 3 and 22, whereas 14 and 19 were the most genetically similar. The UPGMA and Tocher clustering methods indicated the formation of four distinct groups, revealing genetic variability among the 24 H. courbaril genotypes. Therefore, all the evaluated genotypes can be used for seed collection and seedling production, but the most divergent individuals should be prioritized.

Identification and characterization of REC66, a Ty1-copia-like retrotransposon in the genome of red flower of Mirabilis jalapa L.

Mirabilis jalapa Lis the most commonly grown ornamental species of Mirabilis and is available in a range of brilliant colors. However, genetic research on Mirabilis jalapa Lis limited. Using fluorescent differential display (FDD) screening, we report the identification of a novel Ty1-copia-like retrotransposon in the genome of the red flower of Mirabilis jalapa L, and we named it REC66based on its sequence homology to the GAG protein from Ty1-copiaretrotransposon. Using degenerate primers based on the DNA sequence of REC66, a total of fourteen different variants in reverse transcriptase (RT) sequence were recovered from the genomic DNA. These RT sequences show a high degree of heterogeneity characterized mainly by deletion mutation; they can be divided into three subfamilies, of which the majority encode defective RT. This is the first report of a Ty1-copiaretrotransposon in Mirabilis jalapa L. The finding could be helpful for the development of new molecular markers for genetic studies, particularly on the origin and evolutionary relationships of M. jalapa L, and the study of Ty1-copiaretrotransposons and plant genome evolution in the genus Mirabilisor family Nyctaginaceae.

Development of INDELs markers in oilseed rape (Brassica napus L.) using re-sequencing data

Insertions/deletions (INDELs), a type of abundant length polymorphisms in the plant genomes, combine the characteristics of both simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNP), and thus can be developed as desired molecular markers for genetic studies and crop breeding. There has been no large-scale characterization of INDELs variations in Brassica napus yet. In this study, we identified a total of 538,691 INDELs in size range of 1–10 bp by aligning whole-genome re-sequencing data of 23 B. napus inbred lines (ILs) to the B. napus genome sequence of ‘Darmor-bzh.’ Of these, 104,190 INDELs were uniquely mapped on the pseudochromosomes of the reference genome. A set of 595 unique INDELs of 2–5 bp in length was selected for experimental validation in the 23 ILs. Of these INDELs, 530 (89.01 %) produced a single PCR product and were single locus. A total of 523 (87.9 %) INDELs were found polymorphic among the 23 ILs. A genetic linkage map containing 108 single-locus INDELs and 89 anchor SSR markers was constructed using 188 recombinant ILs. The majority of INDELs markers on the linkage map showed consistency with the pseudochromosomes of the B. napus cultivar ‘Darmor-bzh.’ The INDELs variations and markers reported here will be valuable resources in future for genetic studies and molecular breeding in oilseed rape.

Genome-Wide Analysis of Simple Sequence Repeats and Efficient Development of Polymorphic SSR Markers Based on Whole Genome Re-Sequencing of Multiple Isolates of the Wheat Stripe Rust Fungus.

The biotrophic parasitic fungus Puccinia striiformis f. sp. tritici (Pst) causes stripe rust, a devastating disease of wheat, endangering global food security. Because the Pst population is highly dynamic, it is difficult to develop wheat cultivars with durable and highly effective resistance. Simple sequence repeats (SSRs) are widely used as molecular markers in genetic studies to determine population structure in many organisms. However, only a small number of SSR markers have been developed for Pst. In this study, a total of 4,792 SSR loci were identified using the whole genome sequences of six isolates from different regions of the world, with a marker density of one SSR per 22.95 kb. The majority of the SSRs were di- and tri-nucleotide repeats. A database containing 1,113 SSR markers were established. Through in silico comparison, the previously reported SSR markers were found mainly in exons, whereas the SSR markers in the database were mostly in intergenic regions. Furthermore, 105 polymorphic SSR markers were confirmed in silico by their identical positions and nucleotide variations with INDELs identified among the six isolates. When 104 in silico polymorphic SSR markers were used to genotype 21 Pst isolates, 84 produced the target bands, and 82 of them were polymorphic and revealed the genetic relationships among the isolates. The results show that whole genome re-sequencing of multiple isolates provides an ideal resource for developing SSR markers, and the newly developed SSR markers are useful for genetic and population studies of the wheat stripe rust fungus.

Preliminary Analysis of Genetic Variability in Montagu's Harrier (Circus pygargus) using Cross-Amplified Microsatellites

Abstract. The aim of our study was to find suitable molecular markers for genetic studies of the population of Montagu's harrier Circus pygargus. We used the crossspecies amplification strategy to test the usefulness of 24 primer pairs, amplifying the microsatellite loci of several other members of Accipitridae. The analysis was performed on 139 Montagu's harriers from breeding populations in Spain and Poland. We found an amplification success of 50%; however, the level of polymorphism in cross-amplified microsatellites was low, especially in terms of heterozygosity. We did not find significant differences in genetic variability, estimated based on microsatellite markers, between breeding populations from Spain and Poland. The level of genetic differentiation between these two populations was low (FST = 0.016), although significant. An analysis of genotypes of nestlings in 10 nests suggested one case of extra-pair paternity.

Molecular markers in genetic studies and breeding

Back in the early decades of genetics, it became clear that genetic markers may be useful in the analysis of complex traits. However, their low occurrence and a number of other disadvantages prevented the wide application of classical genetic markers, and later protein markers, in breeding. The latest generation of genetic markers (molecular or DNA markers) are characterized by frequent occurrence in the genome, and it is based on universal (and hence highly demanded and constantly developing) methods of analysis. This became the key to the rapid development of genetics and breeding areas related to the use of DNA markers. This article provides a brief overview of the main types of molecular markers and the fields of their application.

Genome-wide survey and analysis of microsatellites in the Pacific oyster genome: abundance, distribution, and potential for marker development

Microsatellites are a ubiquitous component of the eukaryote genome and constitute one of the most popular sources of molecular markers for genetic studies. However, no data are currently available regarding microsatellites across the entire genome in oysters, despite their importance to the aquaculture industry. We present the first genome-wide investigation of microsatellites in the Pacific oyster Crassostrea gigas by analysis of the complete genome, resequencing, and expression data. The Pacific oyster genome is rich in microsatellites. A total of 604 653 repeats were identified, in average of one locus per 815 base pairs (bp). A total of 12 836 genes had coding repeats, and 7 332 were expressed normally, including genes with a wide range of molecular functions. Compared with 20 different species of animals, microsatellites in the oyster genome typically exhibited 1) an intermediate overall frequency; 2) relatively uniform contents of (A)n and (C)n repeats and abundant long (C)n repeats (a parts per thousand yen24 bp); 3) large average length of (AG)n repeats; and 4) scarcity of trinucleotide repeats. The microsatellite-flanking regions exhibited a high degree of polymorphism with a heterozygosity rate of around 2.0%, but there was no correlation between heterozygosity and microsatellite abundance. A total of 19 462 polymorphic microsatellites were discovered, and dinucleotide repeats were the most active, with over 26% of loci found to harbor allelic variations. In all, 7 451 loci with high potential for marker development were identified. Better knowledge of the microsatellites in the oyster genome will provide information for the future design of a wide range of molecular markers and contribute to further advancements in the field of oyster genetics, particularly for molecular-based selection and breeding.

Comparative study of different molecular markers for classifying and establishing genetic relationships in Coffea canephora

The genetic variability characterization of the accessions of the germplasm collection, using molecular markers, is being applied as a complementary strategy to the traditional approaches to redefine the plant genetic resources. In this study, we compared the informativeness and efficiency of the molecular markers RAPD, AFLP and SSR in the analysis of 94 accessions of Coffea canephora germplasm held by the breeding program of the Brazilian Agricultural Research Corporation (Embrapa), Rondonia State, Brazil. For this, we considered the marker’s discriminatory power and level of polymorphism detected and also the genetic relationships and clustering (dendrogram) analysis. The RAPD marker yielded low-quality data and problems in the discrimination of some accessions, being less recommended for genetic studies of C. canephora. The SSRs had a higher level of information content and yielded high-quality data, while AFLP was the most efficient marker system because of the simultaneous detection of abundant polymorphism markers per few reactions. Our results indicate that AFLP and SSR, allies to the intrinsic characteristics of each technique, are the most suitable molecular markers for genetic studies of C. canephora. However, the choice of AFLP or SSR in the species characterization should be made in agreement with some characteristics that are discussed in this work.

In Silico chloroplast SSRs mining of Olea species

Filiz E, Koc E. 2012. In Silico chloroplast SSRs mining of Olea species. Biodiversitas 13: 114-117. Simple sequence repeat (SSR) markers are highly informative and have been widely applied as molecular markers in genetic studies. The purpose of present study is to analyze the occurrence and distribution of chloroplast SSRs in genic and intergenic regions from Olea species viz., Olea europaea, Olea europaea subsp. cuspidate, Olea europaea subsp. europaea, Olea europaea subsp. maroccana, Olea woodiana subsp. woodiana by using bioinformatics tools. We identified 1149 chloroplast SSRs (cpSSRs) in all genome and a total of 340 (29.6%) was located in genic regions. It was observed that the most abundant repeat types were found mononucleotide SSR (66.7 %) followed by trinucleotide SSR (28.3 %), dinucleotide (2.7%), tetranucleotide (1.5%) and pentanucleotide (0.8%). cpSSRs located in genic regions were identified only mono- and trinucleotide motifs, the most abundant of which was trinucleotide (16.2%) followed by mononucleotide (14.3%). All types of repeat motif (mono-, di-, tri-, tetra- and pentanucleotide) were detected except hexanucleotide motifs. According to SSRs analysis, the most abundant observed motifs were identified for mono-, di-, tri-, tetra- and pentanucleotide cpSSRs A/T, AT/TA, AAG/CTT, AAAG/AGTTT, and AATCC/ATTGG respectively. This study results provided scientific base for phylogenetics, evolutionary genetics and diversity studies on different Olea species in future.

Exploiting EST databases for the mining and characterization of short sequence repeat (SSR) markers in Catharanthus roseus L.

Periwinkle (Catharanthus roseus L.) (Family: Apocyanaceae) is a ornamental plants with great medicinal properties. Although it is represented by seven species, little work has been carried out on its genetic characterization due to non-availability of reliable molecular markers. Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. With the rapid increase in the deposition of nucleotide sequences in the public databases and advent of bioinformatics tools, it has become a cost effective and fast approach to scan for microsatellite repeats and exploit the possibility of converting it into potential genetic markers. Expressed sequence tags (EST's) from Catharanthus roseus were used for the screening of Class I (hyper variable) simple sequence repeats (SSR's). A total of 502 microsatellite repeats were detected from 21730 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs account to 1 SSR per 10.21 kb of EST. Mononucleotides was the most abundant class of microsatellite motifs. It accounted for 44.02% of the total, followed by the trinucleotide (26.09%) and dinucleotide repeats (14.34%). Among all the repeat motifs, (A/T)n accounted for the highest Proportion (36.25%) followed by (AAG)n. These detected SSRs can be used to design primers that have functional importance and should also facilitate the analysis of genetic diversity, variability, linkage mapping and evolutionary relationships in plants especially medicinal plants.

Mining and characterization of EST derived microsatellites in Curcuma longa L.

Turmeric (Curcuma longa L.) (Family: Zingiberaceae) is a perennial rhizomatous herbaceous plant often used as a spice since time immemorial. Turmeric plants are also widely known for its medicinal applications. Recently EST-derived SSRs (Simple sequence repeats) are a free by-product of the currently expanding EST (Expressed Sequence Tag) databases. SSRs have been widely applied as molecular markers in genetic studies. Development of high throughput method for detection of SSRs has given a new dimension in their use as molecular markers. A software tool SciRoKo was used to mine class I SSR in Curcuma EST database comprising 12953 sequences. A total of 568 non-redundant SSR loci were detected with an average of one SSR per 14.73 Kb of EST. Furthermore, trinucleotide was found to be the most abundant repeat type among 1-6-nucleotide repeat types. It accounted for 41.19% of the total, followed by the mononucleotide (20.07%) and hexanucleotide repeats (15.14%). Among all the repeat motifs, (A/T)n accounted for the highest proportion followed by (AGG)n. These detected SSRs can be greatly used for designing primers that can be used as markers for constructing saturated genetic maps and conducting comparative genomic studies in different Curcuma species.

A fast and cost-effective approach to develop and map EST-SSR markers: oak as a case study

BackgroundExpressed Sequence Tags (ESTs) are a source of simple sequence repeats (SSRs) that can be used to develop molecular markers for genetic studies. The availability of ESTs for Quercus robur and Quercus petraea provided a unique opportunity to develop microsatellite markers to accelerate research aimed at studying adaptation of these long-lived species to their environment. As a first step toward the construction of a SSR-based linkage map of oak for quantitative trait locus (QTL) mapping, we describe the mining and survey of EST-SSRs as well as a fast and cost-effective approach (bin mapping) to assign these markers to an approximate map position. We also compared the level of polymorphism between genomic and EST-derived SSRs and address the transferability of EST-SSRs in Castanea sativa (chestnut).ResultsA catalogue of 103,000 Sanger ESTs was assembled into 28,024 unigenes from which 18.6% presented one or more SSR motifs. More than 42% of these SSRs corresponded to trinucleotides. Primer pairs were designed for 748 putative unigenes. Overall 37.7% (283) were found to amplify a single polymorphic locus in a reference full-sib pedigree of Quercus robur. The usefulness of these loci for establishing a genetic map was assessed using a bin mapping approach. Bin maps were constructed for the male and female parental tree for which framework linkage maps based on AFLP markers were available. The bin set consisting of 14 highly informative offspring selected based on the number and position of crossover sites. The female and male maps comprised 44 and 37 bins, with an average bin length of 16.5 cM and 20.99 cM, respectively. A total of 256 EST-SSRs were assigned to bins and their map position was further validated by linkage mapping. EST-SSRs were found to be less polymorphic than genomic SSRs, but their transferability rate to chestnut, a phylogenetically related species to oak, was higher.ConclusionWe have generated a bin map for oak comprising 256 EST-SSRs. This resource constitutes a first step toward the establishment of a gene-based map for this genus that will facilitate the dissection of QTLs affecting complex traits of ecological importance.

Mining, characterization, and exploitation of EST-derived microsatellites in Gossypium barbadense

Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. However, the number of expressed sequence tags (ESTs) and SSR markers from Gossypium barbadense is fewer than those from other cotton species. In this study, EST-SSR distribution from G. barbadense was characterized and new G. barbadense-derived EST-SSR markers were determined on the basis of the ESTs obtained by randomly sequencing 2 cDNA libraries associated with fiber development in G. barbadense. By mining 9697 non-redundant ESTs, a total of 638 SSR loci derived from 595 ESTs were observed. In G. barbadense, the frequency of ESTs containing SSRs was 6.13%, with an average of 1 SSR in every 10.4 kb of EST sequence. Furthermore, trinucleotide was found to be the most abundant repeat type among 2–6-nucleotide repeat types. It accounted for 26.6% of the total, followed by the hexanucleotide (26.0%) and pentanucleotide repeats (25.9%). Among all the repeat motifs, (AAG)n accounted for the highest proportion. EST-SSR primer pairs were developed using the Primer3 program, and the redundant primers were removed using the virtual PCR approach. As a result, 380 non-redundant EST-SSR primer pairs were developed and used to detect polymorphisms between the mapping parents G. hirsutum ‘TM-1’ and G. barbadense ‘Hai7124’ for constructing linkage groups in cultivated allotetraploid cotton. Out of these, 98 (25.8%) primer pairs detected polymorphisms. Finally, 95 polymorphic loci from 82 primer pairs were integrated into the backbone genetic map; of these, 42 were mapped into the A subgenome and 53 into the D subgenome. The present work provided the foundation for constructing saturated genetic maps and conducting comparative genomic studies on different cotton species.