Genetic Study And Creation Of Superior Fiber Cotton Germplasm Through Introgression From Wild Species

Cotton is a very important cash crop and cotton fiber was being used as raw materials for textile industry. Gossypium contains about 50 diploid and tetraploid species distributed worldwide in both tropical and subtropical areas. The diploid species (2n=2x=26) fall into eight different cytotypes designated A,B, C, D, E, F, G and K.The tetraploid species (2n=4x=52,AADD) contain two distinct subgenomes which are related to the A genome of the Asiatic cultivated diploid species and the D genome of the American wild diploid species, respectively. Four Gossypium species namely G. arboreum,G. barbadense, G. herbaceum and G. hirsutum are cultivated, the upland cotton (G. hirsutum L.) being the most important. The introduction of alien chromosome from the genus Gossypium wild species through interspecific or intergenomic hybridization into upland cotton is a valuable and proven technique for cotton improvement. A number of economically important traits have been transferred into cotton from wild species in the last century. Successful transfers can be greatly assisted by the precise identification of alien chromosome in the recipient progenies. There are biological barriers existed, such as interspecific incompatibility, hybrid sterility and instability in successive generations,hence, hindering wild cotton usage in breeding.To further expand the cotton genetic basis and enrich the gene pool for breeding, widely interspecific hybridizations were carried out to transfer useful gene into cultivated cotton from wild species, construct gene pools with superior fiber properties, and build the bridge for wild species usage in breeding by overcomeing interspecific incompatibility and hybrid F1 sterility. The relationships among species were also studied by cytogenetics. Multi-FISH technique was established to identify exotic chromosome in hybrid. High density SSR-based maps established in our institute were used to identify exotic DNA of wild species and screen molecular markers linked with fiber QTL for MAS in fiber quality improvement. Genetic and partial correlate coefficients analyses were performed on introgression lines for offering scientific significance in cotton breeding.The main results were as follows. 1.Interspecific hybridization incompatibility could be overcome and hybrids could be obtained when proper measure had been taken under different growing conditions. For example, in Nanjing, the extra measures merit to be taken for hybridization. The extra measures were a combination of three factors, including coordination of vegetative and productive growing, controlling of temperature to 25-28℃/20℃for day/night and adding of 50 mg/L of GA₃ to the flowers pollinated. But in winter at Sanya, Hainan, interspecific hybridizations were also successfully carried out under the natural conditions as intraspecific hybridizations because the natural proper environmental conditions existed. In this study, 60 interspecific hybridization combination, involving 6 geneomes of B,C,D,E,G,K in Gossypium, have been produced through confirmation of cytological and morphological observation.2. Using G. barbadense as bridge parent and to be crossed with G. thurberi, G. laxum, G. australe, G. gossypioides, three triploid F₁s produced were directly crossed with G. hirsutum and fertile tri-species hybrids were obtained. Using direct backcrossing method, sterility of one another triploid F₁S of G. hirsutum×G. stUrtianuM had been overcome under the control conditions of temperature and plant hormone addition. At the present, four wild species have come into breeding procedure. These two techniques may be with the advantages of omitting the chromosome doubling of hybrid F₁ and the stage of undergoing hexaploid and pentaploid and additionally breeding progress will be quickened.3. Cytogenetic observations were made on chromosome configuration of pollen mother cells at metaphase I of 22 interspecific F1 hybrids in Gossypium. These hybrids involved A, B, C, D, E, G and AD genome had been confirmed by cytology.The results of chromosome pairing at 11 pairs of bivalents plus 4 univalents in hybrids A₁B₁and A₁B₃ indicated that there are very close relationships and high chromosome homologus between G herbaceum, G anomalum and G barbosahum.High frequent tetravalents in the intergenomic hybrids between A₂ and B showed there were chromosome translocations. Average 6 to 7 pairs of bivalents (range from 4 to 9) in the intergenomic hybrids between A and E/G may be implied that there were distant relationships and low chromosome homologous. Three to five pairs of bivalents plus 28 to 30 univalnts in intergenomic hybrids of AD and C/G indicated that there were far relationshipd between these species. Chromosome paired as 13 bivalents plus 13 univalents in the F₁ hybrids of tetraploid and diploid D genome (except D₆ genome of G gossypioides) means that D subgenome chromosomes and D chromosomes from diploid have very high homologous and these species have very close relations. Based on cytological observations, among D genome species,D₅ of G. raimondii showing the closet relations with tetraploids, it was presumed that G. raimondii is the closest donor species of D subgenome of tetraploids. More univalents in the F₁ hybrids of tetraploid and G. gossypioides indicated that G.gossypioides has a distinctive origin.4.Increased stringency conditions was employed to establish the GISH technique forcotton by optimizing ratios of block DNA to probe, the formamide concentration in wash solution, and wash temperature and duration in wash solution, after DNA hybridization using mitotic metaphase chromosome of somatic cell of upland cotton standard line TM-1 (G. hirsutum L.) as target, genomic DNA from TM-1 and G. sturtianum Willis originated from Australia as blocking DNA and labeled probe, respectively. The results showed that the proper stringency conditions were the combination of four factors, including the ratios of blocking DNA to labeled probe being 100:1, 60% of formamide wash solution, 43℃of wash temperature and duration of 13 min for wash, under which there is just no probe signal on the target chromosome. Under the proper stringency conditions, GISH technique established was tested to discern chromosomes of G. sturtianum (C₁ genome) from intergenomic triploid hybrid F₁ (2n=3X=A_tD_tC₁=39) between upland cotton and G. sturtianum. The results indicated that every donor chromosomes of the two species showed different color and were readily distinguished. So it is proposed that the GISH technique established was feasible for chromosome identification in intergenomic cotton hybrid. Based on the above results, the Multi-color GISH was developed using two species DNA from G. stuitianum and G. herbaceum L. var.africanum (A₁ genome) as labeled probes simultaneously and DNA from G. davidsonii (D_3-d genome) as blocking DNA, to simultaneously discriminate three genomes of the above intergenomic hybrid. The results also indicated that the three genome, A_t,D_tå’ŒC₁,each set of chromosomes were vividly recognized in different color repetitively. The power of the Multi-color GISH has been proven in analysis hexaploid hybrid (2n=6X=AADDGG=78) from (G.hirsutum×G. australe)F₁ chromosome doubling (kindly provided by CSIRO of Australia) and each of three genomes,A_t,D_t,G₂,of chromosomes display one color. It is believed that the powerful Multi-color GISH technique established in the research could be applied extensively in analysis component in polyploidy and precisely identification of alien chromosome in the recipient progenies, which will greatly assist to transfer much more economically important traits from wild species into upland cotton in the future.5.To identify exotic DNA fragment or alien chromosomal segment, genomes of 22 introgression lines from six species, namely, G. bickii and G. australe (G genome),G. sturtianum (C genome),G. raimondii (D genome),G. anomalum (B genome) and G. stocksii (E genome), were globally scanned using SSR markers based on high density backbone map constructed by our institute. The results revealed that exotic DNA fragment from G. bickii have been transferred into D₇ chromosome of G. hirsutum, covering the genetic distance of 1.2 cM.It will facilitate genetic analysis on effect of exotic DNA of wild species in G. hirsutum.6.After direct selection performed on the introgression lines from G. anomalum,G. armourianum and G. raimondii a series of germplasm with finer, longer and stronger fiber had been developed, becoming the gene pool for breeding and genomic study. Their fiber length was longer than 35 mm, strength stronger than 40cN/tex,micronaire reading ranged from 3.6-4.2 in the best area for J415, J414, J405, J400, J416, J402, J371, J372, J406, J401. These fiber qualities will meet the needs of producing yarns with high yarn number. Among introgression lines from three wild species, the best fiber qualities were in lines from G. anomalum, followed by G. armourianum and G. raimondii. They are similar to that of G. barbadense and much better than that of Upland commercial varieties.7.Germplasm lines J381 and J415 with the desired combination of fiber properties were developed through introgression from G. anomalum into G. hirsutum during the past research.In the present study, partial correlation analysis was carried out to elucidate the relations among fiber properties, namely, length, strength, micronaire, length uniformity, elongation, short fiber index (SFI),reflectance degree (Rd),spinning consistency index (SCI),brownness, fiber maturity, and moist-regain at each generation in J381×Sumian 12 and/or J415×Sumian 12. The results showed that there were significant negative relationship between each two of length, strength, length uniformity, Rd and brownness, and between micronaire and SCI, but significant positive between the former five and the latter two in every generation.The average values indicated that J381 and J415 were much better than Sumian 12; F₁ was being close to the average of their parents and backcross generation leaning to their backcrossing parents. Variation was small in parents but big insegregation generations.8.All of fiber properties studied before showed that they were controlled by minor genes. Further researches indicated that these minor genes had different effects on fiber quality and some of them had played a role as major genes. Therefore, in order to elucidate whether major genes exit and how their role is, the mixed major gene plus polygene genetic model established by the group led by Gai (1997) were employed to research on inheritance of three traits of cotton fiber, namely, fiber strength in two repeats was controlled by two major genes plus polygene, fiber length and micronaire in two repeats were controlled by one major gene plus polygene. The heritability values of major gene were estimated as 30.42% and 38.77% in F₂ and 69.47% and 45.63% in B₁ for fiber strength, 0.007% and 0.15% for fiber length, 0.41% and 0.15% for micronaire in F₂,respectively. That of polygene in F₂ as 12.21% and 19.11% for fiber strength, 34.34% and 32.98% for fiber length, and 8.09% and 36.16% for micronaire in Repeatâ… andâ…¡,respectively. The results indicated that fiber strength and length in both repeats and micronaire in Repeatâ…¡were highly inheritable.9.Using bulked segregation analysis (BSA) on F2 and F2:3 populations of J 415×Sumian 12, 4, 4 and 6 SSR markers related to QTLs of fiber strength, length and length uniformity were screened. They explained 3.0-9.2% of phenotypic variation for strength, 4.5-18.3% for length, and 5.2-11.6% for length uniformity.10.Using two SSR markers assisted selection on the population of Sumian 12×J415, fiber strength of the progenies from it had been increased by 3.9 cN/tex, and four lines (05-342,05-343,05-344 and 05-352) reaching 37.2 cN/tex at the minimum and 42.8 cN/tex at the maximum of three years average.