CMD - Cotton Microsatellite Database

Contributors
Xianlong Zhang
Zhongxu Lin
Lili Tu and Yanxin Zhang

National Key Laboratory of Crop Genetic Improvement
Huazhong Agricultural University
Wuhan, Hubei 430070, China
Project Description (HAU001-HAU119)
Expressed sequence tags (ESTs) available in public databases are invaluable resources for identifying simple sequence repeats (SSRs) through data mining. However, the existing cotton EST-SSR markers are mostly derived from G. arboreum and G. hirsutum. The G. barbadense-derived EST-SSRs are rare. A cDNA library was constructed in our lab with 2 – 25 day post anthesis (DPA) developing fibers from G. barbadense cv. Pima 3-79, which gave us an opportunity to explore EST-SSRs from this library. One hundred and nineteen EST-SSRs (named “HAU ×××”, a short prefix for Huazhong Agricultural University) were developed and evaluated from 98 unique ESTs from the cDNA library. Among these SSRs, the most abundant repeat type was trinucleotide (46.22%), and the motifs AT (9.24%) and AAG (11.76%) appeared in high frequency. Seventy-five ( 63.02%) EST-SSRs successfully amplified DNAs of 36 accessions (those consisted of 13 diploids of A genome, 11 diploids of D genome and 12 allotetraploids of AD genome), totally yielding 312 polymorphic fragments with an average of 4.16 fragments per primer pair. The PIC ( polymorphism information content) of the amplified EST-SSRs ranged from 0.17 – 0.95 with an average of 0.53. Based on Jaccard’s genetic similarity coefficient, these 36 accessions can be directly separated into three groups (AA, DD and AADD group). Twenty-one EST-SSR markers that showed polymorphism between the parents Emian 22 and Pima 3-79, successfully amplified DNAs of 141 individuals from the interspecific BC1 mapping population [(Emian 22 × Pima 3-79) × Emian 22]. Twenty-four polymorphic loci were generated and 20 loci were integrated into our backbone interspecific genetic linkage map (unpublished).
Project Description (HAU120-HAU205)
Sea-island cotton (G. barbadense L.) is one of the most important cultivated cotton species in the world. In order to explore the genetic diversity of this species, microsatellite loci were identified from G. barbadense cv. Pima3-79 (the genetic standard line). Microsatellite markers were developed using two different approaches: (i) cloning of ISSR amplified fragments and (ii) amplification using degenerate primers. Two hundred and thirty-nine unique clones were generated from 1447 recombinants, and 214 unique sequences were obtained. Eighty-six primer pairs were developed from 70 sequences that had flanking regions sufficient for primer design. A total of 105 SSRs were identified from the 70 unique sequences, the most abundant repeat types were trinucleotide and dinucleotide, the most frequent motif type was AT and TC. The 86 SSR primer pairs were used to analyze 48 Sea-island cotton accessions and 4 Upland cotton cultivars, 16 primers had no amplification and 43 primers did not detect polymorphism between all the cultivars. Nineteen primers showed polymorphism between the Sea-island cotton. The PIC values ranged from 0.04 to 0.83 with an average of 0.32. Based on Jaccard's genetic similarity coefficient, these primers could distinctly distinguish Sea-island cotton and Upland cotton, and Sea-island cotton accessions were separated into 4 groups. Nine interspecific polymorphic markers were mapped to the cotton genetic map with 4 mapped to A sub-genome and 5 mapped to D sub-genome. These microsatellite markers will be useful for assessing the genetic diversity patterns within Sea-island cotton as well as aiding in construction of genetic linkage maps (unpublished).