【作者】 周宝良；

【导师】 张天真；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 棉花是重要的经济作物,是纺织工业的重要原料。棉属除陆地棉、海岛棉、亚洲棉、草棉四个栽培种外,还有46个野生种,分属于A、B、c、D、E、F、G、K和AD基因组,是改良栽培棉花的重要基因资源。但由于种间存在生物学隔离,陆地棉与野生二倍体种杂交转育,存在杂交不亲和性、杂种F₁的不育性以及杂种后代不易稳定等障碍,给野生棉的育种利用带来了很大困难。为了进一步拓宽陆地棉的遗传基因库,丰富品质育种的基因资源,本研究利用棉属野生种广泛开展了棉花种间杂交研究,包括种间杂交不可交配性和杂种F₁不育性等种间杂交育种障碍的克服,将野生棉的优良基因广泛转入栽培品种,改良栽培棉花品种的纤维品质,为育种利用野生棉架起桥梁。此外,利用细胞遗传学手段对棉花种间关系进行研究。对种间杂交后代进行分子细胞遗传鉴定、外源DNA/染色体的检测鉴定、渐渗系纤维品质性状QTL的分子标记筛选及杂交后代的辅助选择、纤维品质性状的遗传和偏相关分析,为棉花育种利用提供理论依据。主要研究结果如下:1.只要针对棉花不同的生长环境,采取适宜的杂交手段,都可以克服种间杂交的不可交配性,成功获得种间杂种。如在南京,则必须进行栽培环境的控制（主要是温度）并与激素保铃相结合;在海南三亚冬季,棉花开花时适逢旱季,空气湿度较低,天气较凉爽,有利于棉花的种间杂交工作,无需附加其它辅助条件。共获得了细胞学和形态学证实的种间杂种F₁植株组合60个,涉及B、C、D、E、G、K等8个染色体组中的6个,这些种间杂种的获得为棉花种间杂交育种研究奠定了基础。2.采用以海岛棉7124为桥梁亲本法,与瑟伯氏棉、松散棉、拟似棉杂交,然后与陆地棉直接杂交,成功地获得了[（栽培四倍体海岛棉×野生二倍体棉）F₁×陆地棉]F₁的可育三元杂种;采用直接回交法,成功克服了栽培四倍体陆地棉与野生二倍体棉斯特提棉种间三倍体杂种F₁的不育性。共使4个野生二倍体棉成功进入育种程度,为育种利用提供了非常宝贵的中间材料。桥梁亲本法和直接回交法的优点是无需经过染色体加倍、无需经历六倍体和五倍体阶段,可以缩短育种年限,也为今后野生棉的育种应用提供了技术基础。3.对棉属的22个种间杂种F₁花粉母细胞（PMC）减数分裂中期Ⅰ（MⅠ）的染色体行为进行了细胞遗传学研究,涉及到A、B、C、D、E、G、AD等七个染色体组棉种,从细胞学上证实了所获得的杂种为真杂种,获得了它们的染色体构型。这22个种间杂种F₁的PMCMⅠ的染色体行为研究表明,①Gherbaceum与Ganomalum,Gbarbosanum间的亲缘关系非常近,染色体间的同源性非常高,但由于出现较高频率的四价体,表明其存在染色体的易位等结构变异。②A与E、G染色体组棉种间的亲缘关系较远,染色体间的同源性不高。③异源四倍体棉的A、D染色体亚组与C或G染色体的亲缘关系较远,染色体间的同源程度很低。④异源四倍体棉（拟似棉除外）的D染色体亚组与D染色体组棉种的染色体同源性较好,其中与雷蒙德氏棉的亲缘关系最近,它最接近于四倍体D亚组的供体;但拟似棉的D染色体与AD四倍体的D亚组染色体配对频率明显低于其它D染色体组棉种,与四倍体中的D亚组染色体同源性较低。’4.采用陆地棉（G hirsutum L）标准系TM-1体细胞有丝分裂中期染色体为靶标,以野生种斯特提棉（G sturtianum Willis）为探针,通过提高封阻DNA与探针浓度比例、洗脱液中甲酰胺浓度、洗脱温度、延长洗脱时间等,以增强DNA杂交反应的严谨性,使靶标中刚刚没有杂交信号为止。结果,封阻DNA与探针之比为100:1、洗脱液中甲酰胺浓度为60%、洗脱温度为43℃、洗脱时间13 min时,TM-1的靶标染色体上无杂交信号,而以TM.1与斯特提棉杂种F₁（基因组组成为A_tD_tC₁）体细胞有丝分裂中期DNA为靶标,则可清晰地将斯特提棉的C₁基因组染色体与陆地棉基因组染色体鉴定出来,建立了棉花外源染色体鉴定的基因组原位杂交（GISH）技术体系。并在此基础上,以A₁基因组的阿非利加草棉（G herbaceum Lvar.africanum）和C₁基因组的斯特提棉两个棉种的DNA同时为探针,可以清晰地区分A_t、D_t和C₁基因组染色体,且重复性好,建立了棉花多色GISH技术体系,并成功应用于种间杂种的鉴定。对引自澳大利亚CSIRO的陆地棉与澳洲棉（G australe F.v.M.）（G₂基因组）六倍体杂种（基因组组成为A_tA_tD_tD_tG₂G₂）进行的多色GISH,可清晰地区分A₁、D_t和G₂基因组染色体。为渐渗系中的外源DNA片段及其整合方式提供了技术基础。5.对比克氏棉（G基因组）、澳洲棉（G基因组）、斯特提棉（C基因组）、雷蒙德氏棉（D基因组）、异常棉（B基因组）、司笃克氏棉（E基因组）等6个野生棉渐渗后代的22个种质系利用SSR标记进行基于高密度分子图谱的全基因组扫描,发现中遗红9种质系中存在比克氏棉特异的NAU 2628、NAU 2186、NAU 2627、NAU 2620标记的DNA片段,位于陆地棉D₇染色体上,长度为1.6cM。为野生棉DNA片段在栽培棉背景中的遗传效应研究提供了可能。6.通过对异常棉、辣根棉、雷蒙德氏棉与陆地棉种间杂交后代连续定向的纤维品质选择,培育出了一系列的纤维细、长、强的高品质棉花新种质,成为高品质育种的种质基因库和基因组学研究的重要资源。J415等10个种质系,其纤维长度都达到35mm,纤维比强度均在40cN/tex以上,马克隆值在3.6-4.2之间。在上述三个野生棉渐渗后代中,以异常棉后代的纤维品质最优;其次为辣根棉后代种质系;再次为雷蒙德氏棉后代种质系。纤维品质远优于陆地棉常规品种。7.以高品质的异常棉渐渗系J381和J415为母本,分别与苏棉12杂交,开展亲本P₁、P₂,杂种F1,回交后代B₁、B₂等6个世代纤维品质性状偏相关分析。结果表明,有7个性状在所有世代均表现出一致的极显著相关,即长度、整齐度、比强度、反射率、黄度等两两间呈极显著负相关,与马克隆值、纺纱均匀性指数均为极显著正相关;马克隆值与纺纱均匀性指数间为极显著负相关。8.利用野生种异常棉渐渗后代获得的高品质种质系J381和J415为材料,分别与推广品种苏棉12号杂交,构建了亲本P₁、P₂、F₁、B₁、B₂等六个世代群体,采用盖钧镒等提出的主基因-多基因遗传模型进行纤维品质的遗传分析。结果表明:（1）纤维长度存在1对主效基因,多基因的遗传率较高,F₂世代多基因的遗传率分别为34.34%和32.98%;（2）纤维强度存在2对主效基因,其遗传率较高,F₂的主基因遗传率分别30.42%和38.77%;（3）马克隆值存在1对主效基因,F₂的主基因遗传率仅为0.41%和0.15%。9.利用BSA法,对J415×苏棉12的F₂群体及其F_2:3群体根据纤维长度、强度、马克隆值、整齐度建立了4个极端值库,经单标记分析,获得了与纤维强度QTL密切相关的SSR标记4个,所解释的表型变异为3.0-9.2%,与纤维长度OTL密切相关的SSR标记4个,所解释的表型变异为4.5-18.3%,与纤维长度整齐度QTL密切相关的SSR分子标记6个,所解释的表型变异为5.2-11.6%。10.采用从J415中筛选获得的与强度紧密连锁的分子标记,对（苏棉12号xJ415）F_2:4群体,同时采用NAU1125和NAU1336两个强度QTL标记进行强度选择,结果纤维比强度可提高3.9 cN/tex,获得了05-342、05-343、05-344和05-352等4个高强优异种质系,连续三年平均比强度均在37.2 cN/tex以上,最高的达42.8 cN/tex。更多还原

【Abstract】 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_t和C₁,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 RepeatⅠandⅡ,respectively. The results indicated that fiber strength and length in both repeats and micronaire in RepeatⅡwere 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.更多还原