Asymmetric evolution and domestication in allotetraploid cotton (Gossypium hirsutum L.)

Polyploidy plays a major role in genome evolution,which corresponds to environmental changes over millions of years.The mechanisms of genome evolution,particularly during the process of domestication,are of broad interest in the fields of plant science and crop breeding.Upland cotton is derived from the hybridization and polyploidization of its ancient A and D diploid ancestors.As a result,cotton is a model for polyploid genome evolution and crop domestication.To explore the genomic mysteries of allopolyploid cotton,we investigated asymmetric evolution and domestication in the A and D subgenomes.Interestingly,more structural rearrangements have been characterized in the A subgenome than in the D subgenome.Correspondingly,more transposable elements,a greater number of lost and disrupted genes,and faster evolution have been identified in the A subgenome.In contrast,the centromeric retroelement(RT-domain related) sequence of tetraploid cotton derived from the D subgenome progenitor was found to have invaded the A subgenome centromeres after allotetrapolyploid formation.Although there is no genome-wide expression bias between the subgenomes,as with expression-level alterations,gene expression bias of homoeologous gene pairs is widespread and varies from tissue to tissue.Further,there are more positively selected genes for fiber yield and quality in the A subgenome and more for stress tolerance in the D subgenome,indicating asymmetric domestication.This review highlights the asymmetric subgenomic evolution and domestication of allotetraploid cotton,providing valuable genomic resources for cotton research and enhancing our understanding of the basis of many other allopolyploids.

Comparative Genetics of Floral Morphology in Diploid and Allotetraploid Gossypium

The cultivated Gossypium A genome diploid species G.arboreum and G.herbaceum and the allotetraploid species G.hirsutum and G.barbadense share common morphology for various floral traits,

Telomere-to-telomere genome of the allotetraploid legume Sesbania cannabina reveals transposon-driven subgenome divergence and mechanisms of alkaline stress tolerance

Alkaline soils pose an increasing problem for agriculture worldwide,but using stress-tolerant plants as green manure can improve marginal land.Here,we show that the legume Sesbania cannabina is very tolerant to alkaline conditions and,when used as a green manure,substantially improves alkaline soil.To understand genome evolution and the mechanisms of stress tolerance in this allotetraploid legume,we generated the first telomere-to-telomere genome assembly of S.cannabina spanning~2,087 Mb.The assembly included all centromeric regions,which contain centromeric satellite repeats,and complete chromosome ends with telomeric characteristics.Further genome analysis distinguished A and B subgenomes,which diverged approximately 7.9 million years ago.Comparative genomic analysis revealed that the chromosome homoeologs underwent large-scale inversion events(>10 Mb)and a significant,transposon-driven size expansion of the chromosome 5A homoeolog.We further identified four specific alkali-induced phosphate transporter genes in S.cannabina;these may function in alkali tolerance by relieving the deficiency in available phosphorus in alkaline soil.Our work highlights the significance of S.cannabina as a green tool to improve marginal lands and sheds light on subgenome evolution and adaptation to alkaline soils.

棉属人工异源四倍体后代性状鉴定及花器转录组学分析

实验室前期通过远缘杂交及加倍人工合成草棉与雷蒙德氏棉异源四倍体,旨在拓宽棉花遗传背景,为棉花种质创新提供新材料。本试验以人工异源四倍体3个世代为材料,将其与亲本及陆地棉标准系TM-1进行纤维性状、生理生化比较、SSR标记鉴定以及花器转录组学分析。结果表明,人工异源四倍体纤维颜色介于亲本之间,呈浅黄色,纤维长度与TM-1接近。人工异源四倍体的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性以及丙二醛(MDA)含量均显著高于亲本和TM-1,表明人工异源四倍体经过远缘杂交及加倍后,抗逆性得到加强。选取11对SSR引物在材料中共检测到55个多态性位点,每对引物变异位点为2~10个,平均为5个;多态信息含量(PIC)变幅为0.498~0.892,平均为0.742;表明SSR标记在试验材料中表现出丰富的遗传多样性。S_(2)、S_(3)、S_(4)同时扩增出亲本互补条带和特异性条带,表明该异源四倍体在分子水平上出现了染色体片段的重组。S_(2)、S_(3)、S_(4)与TM-1相同的条带分别是37条、19条、20条,其中17条是各世代与TM-1共有,表明人工异源四倍体与TM-1有相同的遗传背景。花器转录组学结果表明,人工异源四倍体与TM-1中有5653个DEGs,其中3062个上调, 2591个下调。GO功能分析表明, DEGs在光合作用、光系统II、RNA指导的DNA聚合酶活性等途径差异显著。KEGG富集分析表明, DEGs在光合作用-天线蛋白、光合作用和异黄酮的生物合成等相关代谢通路较为活跃。通过富集分析筛选人工异源四倍体与TM-1中与育性相关的DEGs发现,人工异源四倍体在花粉发育与识别方面的差异基因大都是下调表达,这可能是导致人工异源四倍体结实率低的关键因子。通过聚类从Nr注释信息获得3个G型凝集素类受体丝氨酸/苏氨酸蛋白激酶(基因Gh_D01G067500、Gh_D05G168100、Gh_A01G062500)。研究结果表明人工异源四倍体综合了父母本的优良性状,在纤维颜色、抗逆、光合方面表现出优势。期望通过和陆地棉杂交恢复其育性,进一步育成生产上有价值的新材料。

Karyotype Stability and Unbiased Fractionation in the Paleo-Allotetraploid Cucurbita Genomes

The Cucurbita genus contains several economically important species in the Cucurbitaceae family. Here, we report high-quality genome sequences of C. maxima and C. moschata and provide evidence supporting an allotetraploidization event in Cucurbita. We are able to partition the genome into two homoeologous subgenomes based on different genetic distances to melon, cucumber, and watermelon in the Benincaseae tribe. We estimate that the two diploid progenitors successively diverged from Benincaseae around 31 and 26 million years ago （Mya）, respectively, and the allotetraploidization happened at some point between 26 Mya and 3 Mya, the estimated date when C. maxima and C. moschata diverged. The subgenomes have largely maintained the chromosome structures of their diploid progenitors. Such long-term karyotype stability after polyploidization has not been commonly observed in plant polyploids. The two subgenomes have retained similar numbers of genes, and neither subgenome is globally dominant in gene expression. Allele-specific expression analysis in the C. maxima ×C. moschata interspecific F1 hybrid and their two parents indicates the predominance of trans-regulatory effects underlying expression divergence of the parents, and detects transgressive gene expression changes in the hybrid correlated with heterosis in important agronomic traits. Our study provides insights into polyploid genome evolution and valuable resources for genetic improvement of cucurbit crops.

Black carp growth hormone gene transgenic allotetraploid hybrids of Carassius auratus red var. (♀)×Cyprinus carpio (♂)

Ecological safety is a major consideration in the commercialization of transgenic fish. Development of sterile transgenic triploid fish through hybridization of transgenic tetraploid fish and transgenic diploid fish is a feasible way to solve this problem. The "all-fish" transgene, pbcAbcGHc, containing the black carp β-actin gene promoter and the open reading frame (ORF) of the black carp growth hormone (GH) gene was constructed and introduced into fertilized eggs of allotetraploid fish through microinjection. Contrast cultivation results showed that the growth rate of 150 day-old P 0 black carp GH gene transgenic allotetraploid fish was much higher than that of controls. Sixty 150 day-old transgenic allotetraploid fish were assayed by PCR for transgene integration and 90% of fish were positive for the transgene. The transgene was detected in 13 of 20 sperm samples from male transgenic allotetraploid fish. RT-PCR detected transcription of the exogenous black carp GH gene in the muscle, liver, kidney and ovaries of the largest transgenic allotetraploid fish. This study has developed P 0 black carp GH gene transgenic allotetraploid fish with a highly increased growth rate, which provides a solid foundation for the establishment of a pure line of transgenic allotetraploid fish and for the large scale production of sterile transgenic triploid fish.

Functional diversifications of Gh ERF1 duplicate genes after the formation of allotetraploid cotton

Whole genome duplication, a prevalent force of evolution in plants, results in massive genome restructuring in different organisms. Roles of the resultant duplicated genes are poorly understood both functionally and evolutionarily. In the present study, differentially expressed ethylene responsive factors(GhERF1 s), anchored on Chr-A07 and Chr-D07 were isolated from a high-yielding cotton hybrid(XZM2)and its parents. The GhERF1 was located in the B3 subgroup of the ethylene responsive factors subfamily involved in conferring tolerance to abiotic stress Nucleotide sequence analysis of 524 diverse accessions together with quantitative real-time polymerase chain reaction analysis, elucidated that de-functionalization of GhERF1-7 A occurred due to one base insertion following formation of the allotetraploid cotton. Our quantitative trait loci and association mapping analyses highlighted a role for GhERF1-7 A in conferring high boll number per plant in modern cotton cultivars. Overexpression of GhERF1-7 A in transgenic Arabidopsis resulted in a substantial increase in the number of siliques and total seed yield. Neo-functionalization of GhERF1-7 A was also observed in modern cultivars rather than in races and/or landraces, further supporting its role in the development of high-yielding cotton cultivars. Both de-and neofunctionalization occurred in one of the duplicate genes,thus providing new genomic insight into the evolution of allotetraploid cotton species.

RAPD-based genetic analysis of offsprings from the sexual cross using allotetraploid citrus somatic hybrid as pollen parent

Thirty-one polymorphic decamer primers were selected to genotype 92 progenies from the cross be- tween Yiben No.4, a monoembryonic diploid F1 hybrid of Citrus reticulata Blanco cv Huanongbendizao tangerine and C. ichangensis Swingle, and [Hamlin sweet orange + Rough lemon], an allotetraploid somatic hybrid of Citrus sinensis Osbeck cv. Hamlin and C. jambhiri Lush cv. Rough Lemon. χ2 (Chi-square) analysis of RAPD markers in the progenies indicated they were randomly transmitted from the four donor parents, without significant difference between the diploids and triploids. However, these progenies were clustered into three major groups using dendrogram constructed by UPGMA, skewed to three parents in certain degrees, 15 (13 triploids and 2 diploids) to Hamlin, 16 (9 and 7) to Yiben No. 4, and 61 (57 and 4) to [Hamlin sweet orange + Rough Lemon] from which genomic contribu- tion was predominant in progenies, respectively.

Synthesis of Allotetraploid Cotton With [AG] Complex Chromosome Set

Cotton is not only an important source of textile fibre and edible oil but also a valuable source of high-quality protein and fodder, because 45％—50％ of the cotton seed powder is protein. However, the cotton seed of cultivated cotton usually contains a special polyphenol compound called gossypol, which is poisonous and hinders the utilization of the seeds. Therefore breeding new cotton species with no

Evolutionary analysis of allotetraploid hybrids of red crucian carp×common carp,based on ISSR,AFLP molecular markers and cloning of cyclins genes

The allotetraploid hybrids of red crucian carp × common carp are the first reported artificially cultured polyploid fish with bisexual fertility and stable inheritance in vertebrate. Using ISSR and AFLP markers and the cyclins genes, the genomes and cyclin gene sequence changes were analyzed between the allotetraploid hybrids and their parents. The results indicated that the allotetraploids inherited many genetic characteristics from their parents and the genetic characteristics were stable after 15 generations. However, the allotetraploids had a closer genetic relationship with their original female parents and represented a bias toward the maternal progenitor. DNA fingerprinting analysis showed that the allotetraploids had undergone sequences deletion from their original parents and that the deleted sequences were mostly from the male parent's genome. Some non-parental bands were found in the allotetraploid hybrids. Sequences analysis of the cyclin A1 and B1 genes showed nonsynonymous substitutions of single nucleotides in codons that were different from their original parents, leading to non-parental amino acid loci. We speculate that the non-additivity in the allotetraploids, compared with their progenitors, could be an adjustment to the genomic shock from heterozygosity and polyploidy, allowing maintenance of genetic stability.

Identification of Quantitative Trait Loci (QTLs) Affecting Yield and Fiber Properties in Chromosome 16 in Cotton Using Substitution Line

Gossypium hirsutum L. and G. barbadense L. are the two cultivated tetraploid species of cotton. The first is characterized by a high yield and wide adaptation, and the second by its super fiber property. Substitution line in which a pair of intact chromosomes of TM_1 ( G. hirsutum ) were replaced by a pair of homozygous chromosomes of 3_79 ( G. barbadense ) is an excellent material for genetic research and molecular tagging. In this study, substitution line 16 (Sub 16) was used to evaluate the performance of the 16th chromosome in G. barbadense in TM_1 background. The genetic analysis using the major gene plus polygene mixed inheritance model in F 2∶3 family revealed that there might exist 2 QTLs respectively for boll size, lint percentage, lint index, fiber length and the first fruit branch node, 1 QTL for fiber elongation and flowering date, and no QTL for seed index, fiber strength and Micronaire in chromosome 16. However, 9 QTLs (LOD (logarithm of odds)≥3.0) controlling 6 quantitative traits were significantly identified in linkage group of chromosome 16 constructed in (TM_1×3_79) F 2by interval mapping. Among them, 1 QTL for boll size, fiber length, flowering date and fiber elongation could explain 15.2%, 19.7%, 12.1%, and 11.7% phenotypic variance respectively, 2 QTLs for lint index could explain 11.6% and 41.9%, and 3 QTLs for lint percentage could explain 8.7%, 9.6% and 29.2% phenotypic variance respectively. One unlinked SSR marker was associated with one QTL respectively for boll size and flowering date and they could explain 1.60% and 4.63% phenotypic variance. The traits associated significantly with chromosome 16 from Sub 16 were boll weight, lint percentage, lint index, fiber length, fiber elongation and flowering days.

三倍体湘云鲫繁殖季节的性腺结构观察

在繁殖季节解剖 ３７尾 １龄和 １６尾 ２龄湘云鲫，用光镜和电镜观察了湘云鲫的性腺结构。在湘云鲫的性腺部位有三种类型的结构：（１）精巢型：精巢小叶内分布有许多精子细胞，精子细胞呈退化现象，没有发现有正常成熟精子。（２）卵巢型：该类型性腺的大部分为体积较小、分化程度较低的呈囊状排列的细胞，在这些小细胞中镶嵌着少量初级卵母细胞和体积较大的退化的卵母细胞。（３）脂肪型：该类型的性腺部位仅有两条脂肪组织，既没有卵巢结构，也没有精巢结构。上述三种性腺结构证明三倍体湘云鲫是不育的。

异源四倍体鲫鲤F_9～F_(11)染色体和性腺观察

采用肾细胞染色体制片技术 ,检测了异源四倍体鲫鲤F9～F1 1 代的染色体数目及组型 ,结果表明 :其染色体数目为 4n =2 0 0 ,核型公式为 44m +6 8sm +44st+44t,证明F9～F1 1 继续保持四倍体性。观察异源四倍体鲫鲤F9～F1 1 成熟性腺 ,在这 3代四倍体鱼中仍然保持正常卵巢和精巢 ,分别形成正常二倍体卵子和二倍体精子。在自然环境下 ,观察了异源四倍体鲫鲤自行产卵受精并产生存活后代过程 ,证明该四倍体鱼群体在自然环境下能够自行繁殖传代。异源四倍体鲫鲤稳定的染色体数目和正常的性腺结构以及自然条件下的生殖传代行为 ,说明该异源四倍体鲫鲤已成为一个染色体数目为 4n =2 0 0、遗传性状稳定的新型四倍体鱼群体 ,具备形成新种所需的关键因素。

异源四倍体鲫鲤的性腺发育研究

采用组织切片技术对异源四倍体鲫鲤的性腺进行了研究。结果表明 :异源四倍体鲫鲤的性腺发育程序和普通鲤鲫鱼相似 ,可以分为 6个时期。雌雄个体都能达到性成熟 ,其中雌性个体性成熟年龄为一周年 ;雄性个体比雌性个体成熟早 ,1 5 0日龄即可达到性成熟。成熟卵巢的成熟系数为 9.2 5 %— 1 5 .6 0 % ,成熟精巢的成熟系数为 1 .6 5 %— 5 .1 9%。成熟期卵径为1 6 70 .4— 1 780 .5 μm之间 ,成熟精子头径为 2 .3— 2 .4μm。繁殖期为每年 3— 5月份。该研究证明异源四倍体鲫鲤的性腺发育是正常的 ,雌雄个体都能达到性成熟。

白鲫(♀)×红鲫(♂)异源四倍体鱼的倍性操作及其生殖力的研究

采用热休克调控技术诱导出103尾第一代白鲫（♀）×红鲫（）异源四倍体鱼，并对其生殖力进行了研究。1或2龄的四倍体雄鱼能产生精子，而雌鱼不能产生正常的卵。将异源四倍体雄鱼与二倍体白鲫雌鱼交配产生倍间三倍体鱼，染色体检查证明是整三倍体（3N＝150），但其受精率很低（11．4—51．3％，平均32．4％）．网箱养殖结果表明，倍间三倍体白鲫的生长速度比白鲫快30％以上，雌雄均不育．并用冷休克处理回收异源四倍体4N（）×白鲫2N（♀）受精卵的第二极体产生了新的四倍体鱼．文中还对第一代异源四倍体鱼的批量生产、生殖能力以及异源三倍体鱼的生产应用进行了讨论．

基因组原位杂交辨别芸薹属异源四倍体AA、BB、CC基因组研究

利用基因组原位杂交 (GISH)技术 ,以标记的白菜型油菜 (AA ,2n =2 0 )的基因组总DNA为探针 ,分别同芥菜型油菜 (AABB ,2n =36 )和甘蓝型油菜 (AACC ,2n =38)的中期染色体和间期核杂交 ,结果芥菜型油菜有 2 0条染色体表现大而明亮的杂交信号 ,其它染色体上信号很弱或无 ,可以区分A、B基因组 ;甘蓝型油菜染色体上表现有38个信号 ,A、C基因组不能区分开来。以黑芥 (BB ,2n =16 )的基因组总DNA为探针与埃塞俄比亚芥 (BBCC ,2n =34)的中期染色体和间期核杂交 ,16条染色体上显示明显的杂交信号 ,其它染色体上信号很弱。说明在长期的进化过程中 ,芸薹属中BB与AA和CC基因组间分化程度较大 ,而AA与CC基因组分化较小。基因组原位杂交表明最强的信号多集中分布于着丝粒区 。

三倍体鲫鱼——异源四倍体鲫鲤 (♂ )×金鱼 (♀ )

利用雄性四倍体鲫鲤与雌性二倍体红色双尾金鱼交配 ,制备了 1种新型三倍体鲫鱼 ,并对Ⅰ龄三倍体鲫鱼染色体数目和组型、性腺指数和性腺结构、外形、生长速度等生物学特征进行了系统研究。结果表明 :这种三倍体鲫鱼染色体数目为 3n =15 0 ,核型公式为 33m +5 1sm +33st+33t;在繁殖季节 ,三倍体鲫鱼卵巢和精巢指数明显低于作为对照的红鲫卵巢和精巢指数 ;组织学切片表明三倍体鲫鱼的卵巢和精巢不能产生成熟的卵子和精子 ,证明它们是不育的。三倍体鲫鱼体色为青灰色 ,单尾 ,具有一些介于其父母本———异源四倍体鲫鲤和红色双尾金鱼之间的外形特征 ,如具有 1对肉眼不易观察到的短须 ,侧线鳞式为 31 6 7,背鳍为 18,体高 体长之比为 0 5 ,比湘云鲫的体高 体长之比 (0 4 1)有明显的增加。经过约 8个月的饲养 ,该三倍体鲫鱼平均重量为 35 0g,最大个体达 5 5 0g。在Ⅰ龄鱼中 ,雌性三倍体鲫鱼的个体生长速度快于雄性个体。与三倍体湘云鲫相比 ,该三倍体鲫鱼保留了生长速度快、不育等优点 ,同时新添了体高 体长比值更高、肉质更甜美的特点 ,为

多倍体鲫鲤

经过十多年的研究 ,在世界上首次研制出雌雄两性能育、遗传性状稳定的异源四倍体鲫鲤群体。两性可育四倍体鱼群体连续繁殖了 11代 (F3- F1 3) ,形成了一个染色体数目为 2 0 0的新鱼种。用四倍体鲫鲤群体为父本与二倍体鱼母本交配大规模生产了具有生长速度快、不育等优点的三倍体鱼。本文介绍四倍体、三倍体鱼的生物学特性、生物学意义和产生的影响。

四倍体鲫鲤、三倍体湘云鲫染色体减数分裂观察(英文)

用精巢细胞直接制片法观察了异源四倍体鲫鲤、三倍体湘云鲫和二倍体红鲫、湘江野鲤精母细胞染色体第一次减数分裂中期配对情况 ;作为对照 ,观察了上述四种鱼肾细胞的有丝分裂中期染色体。在精母细胞第一次减数分裂中 ,异源四倍体鲫鲤同源染色体两两配对 ,形成 10 0个二价体 ,没有观察到单价体、三价体和四价体 ;三倍体湘云鲫精母细胞形成 5 0个二价体和 5 0个单价体 ;红鲫和湘江野鲤精母细胞分别形成 5 0个二价体。肾细胞检测表明异源四倍体的染色体数目为 4n =2 0 0 ;湘云鲫为 3n =15 0 ;红鲫和湘江野鲤分别为 2n =10 0。减数分裂时染色体分布情况与肾细胞染色体检测结果相吻合。具有四套染色体的异源四倍体鲫鲤在减数分裂中只形成 10 0个二价体 ,而不形成 2 5个四价体或其它形式 ,为产生稳定一致的二倍体配子提供了重要的遗传保障 ,也为人工培育的异源四倍体鲫鲤群体能够世世代代自身繁衍下去提供了重要的遗传学证据。三倍体湘云鲫在减数分裂过程中出现二价体、单价体共存 ,同源染色体在配对和分离中出现紊乱 ,导致非整倍体生殖细胞的产生 。

转基因异源四倍体鲫鲤F_1的研究

采用显微注射法将含有鲤鱼 β actin基因启动子的草鱼生长激素基因“全鱼”基因pCAgcGHc转入异源四倍体鲫鲤 ,然后使其自交得到转基因异源四倍体鲫鲤F1,对 15 0日龄F1体重和体长进行检测 ,可明显看见转基因异源四倍体鲫鲤F1的生长优势 ;取F12 0尾 ,提取尾鳍基因组DNA ,采用合适的引物 ,PCR方法检测转基因异源四倍体鲫鲤F1是否含有外源生长激素基因 ,结果 15 0日龄F1阳性率达到 90 % ,且有些雄性个体可以挤出少量精液 ,而普通 15 0日龄异源四倍体鲫鲤无此现象。