| Cotton comprises of four cultivated species. G. herbaceum, G. arboretum, G. hirsutum (upland cotton) and G.barbadense (sea-island cotton). Upland cotton and sea-island cotton are the main cultivated species and provides more than 95% and about 2%of total production in the World. However, as the raw material of textile industry, the fiber quality of upland cotton is poor than that of sea-island cotton. Therefore, how to breed upland cotton varieties with high quality and yield has been an important goal of cotton breeding. Cotton yield and fiber quality traits are quantitative traits, so the traditional method of genetic improvement has been slow and inefficient. The progress of DNA marker technology provides a rapid and effective method for the genetic improvement of quantitative traits. Based on constructing genetic linkage map and mapping quantitative trait loci (QTL), the utilization of molecular markers that are tightly-linked to QTL for marker-assisted selection can greatly improve breeding efficiency. Currently, the genetic linkage map of tetraploid cotton species is relatively saturated, but it cannot be used for the improvement of upland cotton varieties. It need to construct high-density genetic linkage map of upland cotton for the improve requirements of cotton. Because of the low polymorphism of DNA markers among upland cotton varieties, the marker density and genome coverage of the current upland cotton genetic linkage map can not meet the needs of molecular marker-assisted selection. Therefore, it requires to develop large number of new markers, especially the molecular markers which are tightly-linked genes/QTL and to construct high-density genetic linkage map in upland cotton. The present study designed ET-ISJ (Exon-Targeted Intron-Exon Splice junction) mark which amplify exon region according to the conserved sequence of exon splice site in plant structure. The ET-ISJ and SSR markers were used to construct a genetic linkage map of upland cotton with a recombinant inbred population developed from the high quality variety Yumian land multiple dominant marker gene line T586. The main results are as following: 1. ET-ISJ primer design and its characteristicsThe ET-ISJ primers are designed according to the conserved sequence of exon splice sites in the plan.. Both forward primers and reverse primers comprise of the core sequence, fixed base and selecting sequence, and each primers contains 18 bases. Each forward primer can be combined with any reverse primer. The polymorphism of ET-ISJ marker is high, and it has high stability. The cost of development is low, and it is suitable for genetic mapping.2. Polymorphism analysis of primers and genotyping recombinant inbred populationA total of 1280 ET-ISJ primer combinations and 2988 SSR primer pairs were used to screen polymorphism betwen Yumian 1 and T586, and 69 ET-ISJ primer combinations and 180 SSR pair primers with polymorphism were obtained. The polymorphic primer combinations/pairs for ET-ISJ and SSR are 5.4% and 6.0% respectively.The polymorphic primers were used to detect (Yumian 1×T586) F2:7 recombinant inbred population with 270 lines, and 70 ET-ISJ and 181 SSR loci were obtained.3. Segregation analysis of markersChi-square test showed that 23 (32.9%) ET-ISJ loci deviated from 1:1 Mendelian segregation ratio (P≤0.05), and 7 (10.0%) ET-ISJ severely deviated from Mendelian segregation ratios (P≤0.001). The distortion and severe distortion of SSR were 22.7% and 6.1%.4. Construction of genetic map of upland cottonGenetic linkage analysis was conducted on 70 ET-ISJ and 181 SSR loci, together with 795 loci which were obtained before in our laboratory. A genetic linkage map including 63 linkage groups and 1022 loci was constructed. The map covered 4095.5 cM and the average marker interval is 4.0 cM, accounting for 92.1% of tetraploid cotton genome. Sixty-eight 8 ET-ISJ markers distributed on 20 chromosomes, and 181 SSR markers distributed on all 26 chromosomes.
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