Analysis Of The Difference Of Gametes Recombination Rates, Segregation Distortion And Construction Of A High Density Genetic Linkage Map In Interspecific Population Of Cotton

Cotton (Gossypium spp.) is an important cash crop in China and many other countries. It is the second largest source of textile fiber and edible oil throughout the world. The prosperity or decline of cotton yield is very important to the income of farmers and the development of textile industries. The new cultivars of cotton contribute up to 30% to cotton industries. For a long time, researchers have been involved in improving yield traits mainly by employing conventional breeding techniques. Tetraploid cotton has a larger and complicated genome, which is the limiting factor of cotton improvement. Modern molecular biological techniques have brought new ways for cotton improvement. With the combination of marker assisted and conventional breeding, cotton breeding process has been accelerated. It has helped cotton breeders to increase yield and quality by improving the efficiency of selection.This study was planned to reveal the following aspects by SSR technology:(1) Genetic evaluation of EST-SSRs derived from Gossypium herbaceum, (2) The difference between male and female gametes recombination rates by interspecific backcross of cotton (3) Analysis of genetic segregation distortion of SSR molecular markers in cotton interspecific population (4) Construction of a high density genetic linkage map from interspecific backcross population of cotton.1. Genetic evaluation of EST-SSR derived from Gossypium herbaceumEST-SSRs were isolated from 247 EST sequences of G. herbaceum documented in GenBank. Twenty-seven perfect SSRs were identified from twenty-five unique ESTs. These SSRs contained 1-6bp nucleotide motifs with high frequency for 2bp and 3bp nucleotide motifs. In order to clarify the transferability of A, D and AD sub-genomes, SSRs were designed from 25 pairs of EST-SSR primers. Twenty-two of them could amplify 24 cotton accessions and produced 92 polymorphic fragments. The PIC (Polymorphism information content) values ranged from 0.49 to 0.91 with an average of 0.81. Among the 25 pairs EST-SSR primers, six pairs of them revealed polymorphism between Emian22 and 3-79 and yielded seven polymorphic loci (five were co-dominant and two dominant) in the BC1 [(Emian22×3-79)×Emian22] population. Only HAU230b showed distorted segregation in the BC1 population. Six polymorphic loci were integrated into six chromosomes of our interspecific BC1 backbone genetic linkage map among which, four loci were mapped on four chromosomes of A sub-genome (Chr.6,10,11,12), and two loci on two chromosomes of D sub-genome (Chr.19 and 20). The development of EST-SSRs derived from Gossypium herbaceum will contribute to the origin, evolution and the genomic structure of the tetraploid cotton.2. Research on the difference between male and female gametes recombination rates by interspecific backcross of cottonTwo linkage maps covered with 313 markers have been established by populations B and C of BC1 which was formed by male and female gametes recombination, based on the BC1 genetic linkage map created by our laboratory. The lengths of B and C linkage maps were 4532.9 cM and 4464.4 cM respectively and the mean distances of markers in the linkage maps were 14.48 cM and 14.26 cM respectively.By analyzing the influence of male and female gametes recombination rates to the whole chromosome, there were no significant effects to the genetic linkage maps caused by the recombination rates of male and female gametes. There 21 genetic linkage groups in population B were much longer than those in population C, and 9 genetic linkage maps were shorter. The T test revealed that there were 6 linkage maps and 2 linkage maps showed significant differences at 0.05 level and high significant differences at 0.01 levels of significance.Then analyzing the recombination rates of SSRs derived from male and female gametes recombination rates, we found there were 17 markers comprised of 4 male gametes and 13 female gametes showed significant differences at 0.05 levels using 2×2 contingency Chi-square test. Further study showed that the male gametes mainly lead to a longer distance of markers in the linkage map of cotton, i.e. increase the recombination rates. Meanwhile a shorter distance of markers in the linkage map of cotton was caused by the female recombination rates, i.e. decrease the recombination rates. There will be different effects with different recombination rates, so we can chose many kinds of combination modes according to the breeding objectives in crop genetics and breeding, as well as molecular marker-assisted selection.3. Analysis of genetic segregation distortion of SSR markers in the interspecific population of cottonInterspecific cross population between Upland cotton and Sea-island cotton is very common in cotton genetic linkage map. Segregation distortion was ubiquitous among interspecific backcross population. In order to study the reasons for segregation distortion, populations of positive and negative crosses were used. A total of 114 SSR markers showed segregation distortion among 1026 marker in BC1 mapping population [(Emian22×3-79)×Emian22] (Pop A), of which 107 segregation distortion markers was located on chromosome. These 97 SSR markers were validated in population B [Emian22×(Emian22×3-79)] (Pop B) and population C [(Emian22×3-79)×3-79] (Pop C). In Pop A, segregation distortion of 61 markers were caused by cross mode and segregation distortion of 36 markers were caused by competitive competence of male or female gametes. Segregation distortion markers caused by competitive competence of male and female gamete were distributed on chromosome 14. These markers were found to be more frequent on "D" sub-genome than "A" sub-genome. Most of segregation distortion markers were distributed on chromosome 2,16 and 18. Segregation distortion markers caused by competitive competence of female gamete were distributed on chromosomes of "A" sub-genome. Most of segregation distortion markers caused by competitive disadvantage of female gamete were distributed on chromosome 18. Most of segregation distortion markers caused by competitive competence of male gamete were distributed on chromosomes of "D" sub-genome. Clusters of segregation distortion markers caused by competitive disadvantage of gamete were found to locate on chromosome 2 and 7. Segregation distortion markers caused by competitive disadvantage of male gametes were distributed on chromosome 16. The research of segregation distortion will be important for parent selection and the hybrid approach in marker-assisted selection.4. Construction of high density genetic linkage map by interspecific backcross population of cottonWith the improvement of molecular marker techniques and the improvement of cotton DNA extraction, researches on the use of molecular markers of genetic linkage maps of cotton have achieved rapid development. Until now, more than one molecular marker genetic linkage map of cotton have been established including inter-specific and intra-specific populations. Advanced hybrid approaches including F2, BC1, DH, natural and RIL populations, et al., more molecular markers, such as RFLP, RAPD, AFLP, SCAR, SSR, SRAP and SNP. And more genetic mapping software: Mapmaker/exp3.0 and Joinmap3.0.Based on the BC1 population established by Zhang (2008) and 1026 polymorphic loci of SSRs. 12722 pairs of primers including published SSRs and EST-SSRs as well as designed in our laboratory have been used to scan the polymorphisms between two parents. Among of these markers,2187 primers showed polymorphism between parents while 2528 primers were confirmed to be polymorphic in BC1 population. There were 1023 and 1505 polymorphic loci generated by 4419 SSRs and 8303 EST-SSRs respectively and polymorphic percentages were 21.2% and 15.6%.Map analysis and construction were performed in Joinmap3.0 software, with supposed LOD value (>5.0). The maximum distance of markers was calculated to be<40 cM. Overall,2318 marker loci had been anchored onto 26 linkage groups of cotton genome and length of map was 4418.9 cM. There were 13 short linkage groups established by 56 markers, yet we were unable to locate them on the cotton chromosomes. Another 154 markers did not anchor on any linkage groups.There were 135 markers on Chr.19, whose polymorphic loci were the most one in 26 linkage groups of cotton genome. Chromosomes with the least markers were found on Chr.02 and Chr04 with an average of 89 markers per chromosome. There were 1044 and 1274 markers located on A and D sub-genomes, respectively. The maximum distance of markers was 2.78 cM on Chr.02, compared to the minimum of 1.12 cM on Chr.14. The mean distance of markers in the map was 1.91 cM.There were 425 markers (16.8%) showed segregation distortion (χ2=3.84, P<0.05), of which 358 markers (14.2%) located in the linkage groups.323 markers located in cotton genome,35 markers located in 13 short linkage groups, and 67 markers (2.6%) unallocated in any linkage groups.The 2318 markers located in 26 chromosomes were analyzed by correspondening EST sequences marked by 1812 molecular functions, biological process and cellular element. Some EST sequences were noted on multi-function; some were not noted on any functions in cotton EST database for the present; all of these EST sequences will be noted on homologous function with further research on cotton function genome. In level 3,1236 SSR-EST sequences were noted molecular function, nucleic acid binding is the maximum class(13.37%).2110 SSR-EST sequences were noted biological process, cellular metabolic process is the maximum class (16.54%).2273 SSR-EST sequences were noted cellular element, intracellular is the maximum class (20.50%)With the high density SSRs linkage maps mainly established by EST-SSR in the research, it will be helpful with the evolutionary studies of Gossypium, and cotton genome structure and function as well as cotton yield and fiber development-related genes. It also has great significance for molecular assisted selection and molecular designed breeding.