Distant hybridization and polyploidy is an important way for cotton germplasm innovation, and it may provide theoretical evidence and new materials by distantly hybridizing G. gossypioides with G. hirsutum and then chromosome doubling to use the excellent genes of G.gossypioides and to broaden the genetic diversity of G. hirsutum. In this study, we obtained allohexaploid cotton by synthesizing the female parent G. hirsutum with the male parent G. gossypioides, following with chromosome doubling by colchicine. And then we examined the morphology（leaf phenotype）, cytology（stomatal density, stomatal length, chloroplasts number in guard cell pairs, and pollen grain diameter）, and physiological and biochemical indexes（SOD, POD, CAT, soluble protein and chlorophyll fluorescence parameters of the hexaploid）. Three hexaploid plants were selected by flow cytometric identification. The statistical results of hexaploid, triploid and parental traits showed that there was not significant difference for the leaf shape index between hexaploid and G. hirsutum, indicating that the leaf shape of hexaploid was overall preferring to G. hirsutum, while the triploid distant hybrid was more inclined to paternal cotton G. gossypioides. The leaf area and stomatal length of hexaploid increased, and the number of chloroplasts in the guard cells significantly increased. The flower phenotypic traits of hexaploid were intermediate between two parents, but the pollen grain diameter of hexaploid was greater than the triploid, with the normal pollen grain ratio of 73.25% for hexaploid and of 51.13% for triploid, indicating the fertility recovering of hexaploid. The content of SOD, POD, CAT and soluble protein in hexaploid leaves were significantly greater than the other generations. There were significant differences in the initial fluorescence Fo,leaf variable fluorescence Fv, primary light energy conversion efficiency（Fv/Fm）, PS II potential photochemical efficiency（Fv/Fo）and open PS II capture excitation energy efficiency（Fm/Fo）between hexaploid and other generations. The overall results proved that the new germplasm of hexaploid between G. hirsutum and G. gossypioides were successfully synthesized by hybridization and chromosome doubling in our study, and it was tested that hexaploid has excellent performances superior to triploid especially in stress resistance and photosynthesis.

多倍化是物种形成及植物育种的重要途径.以实验室前期合成的草棉同源多倍体后代S1为材料,对其进行流式细胞术、形态学、细胞学和SRAP分子标记鉴定.流式细胞倍性鉴定表明,以二倍体的荧光峰值作为参照,8株后代中有5株三倍体和3株四倍体.形态学鉴定表明,二倍体表现为株高、茎细、叶薄、叶片较小、叶色翠绿、花朵较小,三倍体和四倍体则表现为植株矮小、茎秆粗壮、叶片增厚和皱缩、叶片较大、叶色浓绿、花朵较大.细胞学鉴定表明,随着倍性的增加,气孔密度呈下降趋势,而气孔长度、叶绿体数和花粉粒直径均呈上升趋势;二、三和四倍体草棉在花粉母细胞减数分裂过程中均出现正常和异常行为,包括单分体(0.17％,2.50％和2.17％)、二分体(0.33％,0.67％和0)、三分体(4.17％,10.00％和2.33％)、正常四分体(94.83％,54.00％和73.67％)、异常四分体(0.17％,0.33％和0.83％)和多分体(0.33％,32.50％和21.00％);异常多分体均不能形成正常花粉粒,所以三者的正常花粉粒所占比例分别为95.33％、58.83％和77.67％.SRAP分子标记鉴定表明,三倍体和四倍体不仅扩增出与二倍体完全一致的条带,还出现了条带的缺失与新的特异性条带,其中三倍体的遗传比例分别为80.81％、6.73％和12.46％,四倍体的遗传比例分别为76.59％、10.37％和13.04％,从分子水平证明了多倍体的真实性.

通过转录组测序筛选草棉同源多倍体与二倍体相比差异表达基因,为同源多倍体的表型变化及其染色体加倍过程中遗传物质的变化提供基因表达水平的依据.本研究以草棉二倍体(CK)和同源多倍体后代为材料,通过流式细胞仪鉴定出同源三倍体(T)和同源四倍体(M)植株,比较同源四倍体与二倍体的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、脯氨酸和可溶性蛋白含量生理指标,并进行转录组测序分析.草棉同源四倍体叶片的SOD、POD、CAT、脯氨酸和可溶性蛋白含量均显著高于二倍体.DESeq差异分析结果表明,CK vs T、T vs M、CK vs M分别有4166(2832个上调,1334个下调)、4037(1766个上调,2271个下调)、4184(2679个上调,1505个下调)个DEGs,有205个DEGs是不同倍性植株两两比较的3组间共同表达的.GO功能分析表明,DEGs在膜的固有成分、膜的组成部分、催化活性等途径差异显著.KEGG富集分析表明,DEGs在类黄酮生物合成、苯丙烷生物合成、光合作用-天线蛋白等通路较为活跃.通过富集分析筛选CK vs T和CK vs M中与倍性相关的DEGs发现,多倍体在应激反应(62.46％和54.84％)、逆境反应(57.42％和53.85％)、细胞(70.16％和66.22％)、代谢(72.73％和66.14％)、生物活性物质和抗性(60.33％和63.37％)、光合(58.06％和63.16％)等方面的差异基因大都是上调表达,这可能是导致多倍体优势的关键因子.Nr注释信息CK vs M中共获得27个与抗性相关的基因(19个上调,8个下调),11个与光合相关的基因(9个上调,2个下调).对不同倍性草棉与光合相关的27个DEGs进行聚类分析发现,18个(66.67％)基因在四倍体中表达丰度最高.对3组间共同表达的205个DEGs进行Nr注释和表达情况分析发现,有30个基因都表现为上调表达,且在四倍体中表达量最高,7个基因都表现为下调表达,且在四倍体中表达量最低,这37个基因可能是受DNA剂量影响或是与植株倍性相关的基因.研究结果进一步表明多倍体植株在抗性、生长势、新陈代谢以及光合速率方面均优于二倍体.