四个栽培棉种间的杂种F1细胞遗传学与亲缘关系研究

以棉属四个栽培棉种进行种间杂交,产生(亚洲棉×草棉)和(陆地棉×海岛棉)2个二元杂种F1及其[(亚洲棉×草棉)×(陆地棉×海岛棉)]四元杂种F1,观察和测定4个栽培棉种及其2个二元杂种F1和四元杂种F1的花粉母细胞(PMC)减数分裂的染色体行为及其花粉生活力,以研究4个栽培棉种间的亲缘关系和进化关系。结果表明,二元杂种(亚洲棉×草棉)F1的PMC减数分裂中期Ⅰ出现一个四体环,其余为二价体,染色体构型为2n=26=11Ⅱ 1Ⅳ;花粉生活力的测定表明,(亚洲棉×草棉)F1可育型花粉为50.71%,表现为典型的配子半不育特性,说明两个二倍体棉种间发生一次染色体易位。(陆地棉×海岛棉)F1以26个二价体细胞为主,但有少量的单价体、三价体以及四价体,染色体构型为2n=52=0.78Ⅰ 22.24Ⅱ 0.94Ⅲ 0.98Ⅳ。花粉生活力的测定表明,(陆地棉×海岛棉)F1可育型花粉为54.84%,可见2个四倍体棉种间亲缘关系较近,二者之间仅发生了染色体的易位或倒位。而由4个栽培种合成的四元杂种F1,其减数分裂异常,染色体丢失现象普遍,部分染色体不能联会配对,以单价体的形式存在,并出现三价体、四价体、五价体等多价体,染色体构型为2n=52=5.45Ⅰ 14.41Ⅱ 2.44Ⅲ 1.59Ⅳ 0.63Ⅴ 0.15Ⅵ,其可育花粉为6.87%。研究结果表明了4种栽培棉种之间的亲缘关系相对较近,可以通过遗传重组产生综合有4个栽培棉种性状的新种质。     

基于陆地棉茎尖转录组序列的genic-SSR标记的开发与评价

利用软件MISA和SSR Locator 对陆地棉（Gossypium hirsutum L.）Coker312茎尖转录组测序得到的73515条Unigene序列进行分析,查找到4507个SSR位点,分布于4039条转录本序列中,SSR出现频率为5.5%,非编码区的SSR位点要显著高于编码区（2853/1654）。SSR重复基元中,三核苷酸重复基元占主导地位（51.03%）,且AAG/CTT （20.08%）类型最多;其次是二核苷酸重复基元（28.76%）,主要为AG/CT（55.47%）。利用引物批量设计软件共开发1569对SSR引物,在陆地棉TM-1、海岛棉3-79 (G. barbadense L.)、阿非利加棉(G. herbaceum L.)、雷蒙德氏棉(G. raimondii Ulbrich.)四个棉种中进行初步评价,其中1117对引物能在四个棉种间扩增出稳定的条带,扩增率为71.2%。选择650对能稳定扩增出条带的引物,在涉及5个棉种（增加了亚洲棉（G. arboreum L.））的13份材料中进一步筛选,83对引物能在13份材料中扩增出特异性条带, PIC变幅在0.121～0.648之间,平均值为0.422,其中80对引物能在7份陆地棉材料中扩增出特异性条带,平均PIC值为0.336;挑选5对在本实验室作图亲本鲁棉研22和鲁原343间有明显多态的引物进行连锁分析,其中4对成功连锁到本实验室构建的遗传图谱上。本研究丰富了棉属genic-SSR的数量,为棉属遗传作图、性状关联分析提供了更多引物选择。     

Protein markers for identification of different species and varieties of cotton

Reference electrophoretic spectra that allow compiling electrophoretic formulas of certain cotton species and varieties were obtained on the basis of analysis of the electrophoretic spectrum of water-soluble and barely soluble proteins of seeds of diploid cotton species of genomic group A (Gossypium arboreum var. indicum, G. arboreum ssp. obtusifolum, G. herbaceum ssp. africanum, and G. herbaceum Harga), group C (G. australe, G. bickii, G. nelsone, and G. sturtianum), group D (G. davidsonii, G. harknessii, G. klotzschianum, G. raimondii, G. thurberi, and G. trilobum), and amphidiploid species of group AD (G. mustelinum, G. hirsutum ssp. palmeri, G. tricuspidatum Bagota, G. tricuspidatum Mari Galanta, G. barbadense L., and G. hirsutum L.)     

三种野生和四个栽培棉种的核型研究

作者对棉属 D 组的瑟伯氏棉(Gossypium thurberi)、戴维逊氏棉(C.davidsonii)、雷蒙德氏棉(G.raimondii)、A 组的草棉(G.herbaceum)、中棉(G.arboreum)、AD 组的陆地棉(G.htrsutum)和海岛棉(C.barbadense)等7个种的核型进行了研究。各个种的核型可简式为:瑟伯氏棉2n=2x=26=24m 2Sm(2SAT);戴维逊氏棉20=2x=26=20m 6Sm(4SAT);雷蒙德氏棉2n=2x=26=20m 6Sm(2SAT);草棉2n=2x=26=18m 4Sm 4St(4SAT);中棉2n=2x=26=18m 6Sm(2SAT) 2St(2SAT);陆地棉2n=4x=52=32m 18Sm(4SAT) 2St(2SAT);海岛棉20=4x=52=38m 12Sm(2SAT) 2St(2SAT)。此外,作者对四倍体种的 A 组和 D 组的供体问题进行了讨论。     

Studies of Karyotypes in Three Wild and Four Cultivated Species of Gossypium

Karyotypes in seven species of Gossypium, G. thurberi, G. advidsonii, G. raimondii of D group; G. herbaceum, G. arboreum of A group; G. hirsutum and G. ba- rbadense of AD group were studied in 1983. It can be simplified as follows: G. thurberi 2n = 2x = 26 = 24m + 2Sm (2SAT); G. davidsonii 2n= 2x = 26 20m+6Sm(4SAT); G. raimondii 2n=2x= 26= 20m+6Sm(2SAT); G. herbaceum 2n = 2x = 26 = 18m + 4Sm+4St(4SAT); G. arboreum 2n = 2x = 26 = 18m + 6Sm (2SAT) + 2St(2SAT); G. hirsutum 2n = 4x =52 = 32m + 18Sm(4SAT) +2St (2 SAT); G. barbadense 2n = 4x = 52 = 38m + 12Sm (2SAT) + 2St(2SAT). This paper also deals with the supplier in A group and D group of tetraploids.     

Genome-Wide Analysis of the Sus Gene Family in Cotton

Sucrose synthase (Sus) is a key enzyme in plant sucrose metabolism. In cotton, Sus (EC 2.4.1.13) is the main enzyme that degrades sucrose imported into cotton fibers from the phloem of the seed coat. This study demonstrated that the genomes of Gossypium arboreum L., G. raimondii Ulbr., and G. hirsutum L., contained 8, 8, and 15 Sus genes, respectively. Their structural organizations, phylogenetic relationships, and expression profiles were characterized. Comparisons of genomic and coding sequences identified multiple introns, the number and positions of which were highly conserved between diploid and allotetraploid cotton species. Most of the phylogenetic clades contained sequences from all three species, suggesting that the Sus genes of tetraploid G. hirsutum derived from those of its diploid ancestors. One Sus group (Sus I) underwent expansion during cotton evolution. Expression analyses indicated that most Sus genes were differentially expressed in various tissues and had development-dependent expression profiles in cotton fiber cells. Members of the same orthologous group had very similar expression patterns in all three species. These results provide new insights into the evolution of the cotton Sus gene family, and insight into its members' physiological functions during fiber growth and development.     

Physiological and biochemical changes associated with cotton fibre development. II. Auxin oxidising system

Changes in IAA oxidase, and in cytoplasmic and ionically wall-bound peroxidase activities were studied in the developing fibres of three cotton cultivars ( Gossypium hirsutum L. cv. Gujarat-67, cv. Khandwa-2 and G. herbaceum L. cv. Digvijay), designated as long, medium and short staple cultivars, respectively. In all the three cultivars IAA oxidase activity was low during the fibre elongation phase, while the activity increased significantly during the secondary thickening phase. The increase in IAA oxidase activity in the three cultivars showed close correspondence with their respective total period of elongation. No relationship between cytoplasmic peroxidase activity and fibre development was discernible. The ionically bound wall peroxidase activity, however, recorded low levels during the elongation phase and higher levels during the secondary thickening phase. The role of wall peroxidase in cessation of elongation growth is discussed.     

QTL mapping in A-genome diploid Asiatic cotton and their congruence analysis with AD-genome tetraploid cotton in genus Gossypium

Asiatic cotton(Gossypium arboreum L.) is an Old World cultivated cotton species.The sinense race was planted extensively in China.Due to the advances in spinning technology during the last century,the species was replaced by the New World allotetraploid cotton G.hirsutum L.Gossypium arboreum is still grown in India and Pakistan and also used as an elite in current cotton breeding programs.In addition,G.arboreum serves as a model for genomic research in Gossypium.In the present study,we generated an A-genome...     

Characterization and expression of plasma and tonoplast membrane aquaporins in elongating cotton fibers

Cotton fiber (Gossypium hirsutum L. and G. barbadense L.) is a good model for studies of plant cell elongation and cell wall biogenesis. Aquaporins are ancient membrane channel proteins that facilitate the permeation of water across biological membranes. We studied GhPIP1-2, encoding plasma membrane intrinsic protein, and GhgammaTIP1, encoding tonoplast intrinsic protein, during cotton fiber development. The full-length cDNAs of GhPIP1-2 and GhgammaTIP1 were obtained through 5' RACE. The deduced amino acid sequences of GhPIP1-2 and GhgammaTIP1 share high sequence identity with aquaporins from diverse plant species. Phylogenetic analysis of GhPIP1-2 and GhgammaTIP1 with other plant aquaporins showed that GhPIP1-2 belongs to the PIP1 group of the PIP subfamily and GhgammaTIP1 belongs to the gammaTIP group of the TIP subfamily. GhPIP1-2 and GhgammaTIP1 contain three and two introns, respectively. Genomic Southern blot analysis indicated that GhPIP1-2 and GhgammaTIP1 have several copies and multiple homologous genes in allotetraploid cotton. Northern blot analysis with gene-specific probes and real-time PCR demonstrated that GhPIP1-2 and GhgammaTIP1 are predominantly expressed during cotton fiber elongation, with the highest expression levels at 5 days post-anthesis. Moreover, expression patterns of the two genes in G. hirsutum and G. barbadense are similar, whereas the expression levels in G. barbadense are much lower than that in G. hirsutum. The high and preferential expression of GhPIP1-2 and GhgammaTIP1 during fiber cell elongation suggests that they may play important roles in supporting the rapid influx of water into vacuoles during cotton fiber cell expansion.     

Introgression of cotton leaf curl virus-resistant genes from Asiatic cotton (Gossypium arboreum) into upland cotton (G. hirsutum)

Cotton is under the constant threat of leaf curl virus, which is a major constraint for successful production of cotton in the Pakistan. A total of 3338 cotton genotypes belonging to different research stations were screened, but none were found to be resistant against the Burewala strain of cotton leaf curl virus (CLCuV). We explored the possibility of transferring virus-resistant genes from Gossypium arboreum (2n = 26) into G. hirsutum (2n = 52) through conventional breeding techniques. Hybridization was done manually between an artificial autotetraploid of G. arboreum and an allotetraploid G. hirsutum, under field conditions. Boll shedding was controlled by application of exogenous hormones, 50 mg/L gibberellic acid and 100 mg/L naphthalene acetic acid. Percentage pollen viability in F(1) hybrids was 1.90% in 2(G. arboreum) x G. hirsutum and 2.38% in G. hirsutum x G. arboreum. Cytological studies of young buds taken from the F(1) hybrids confirmed that they all were sterile. Resistance against CLCuV in the F(1) hybrids was assessed through grafting, using the hybrid plant as the scion; the stock was a virus susceptible cotton plant, tested under field and greenhouse conditions. All F(1) cotton hybrids showed resistance against CLCuV, indicating that it is possible to transfer resistant genes from the autotetraploid of the diploid donor specie G. arboreum into allotetraploid G. hirsutum through conventional breeding, and durable resistance against CLCuV can then be deployed in the field.     

Esterase Isozyme analyses of the Species and Their Varieties of the Genus Gossypium

The isozyme make up of esterases of the seeds from fifteen species and twenty-three cultivar of Gossypium was analyzed by isoelectrofocusing. The experimental results are summarized as follows: 1. Differeces were observed in the number of esterase isozyme bands among the species of different genome groups. The cultivated species, G. hirsutum (AD)1 gave rise to 46 isozyme bands, the most among the species of the genus Gossypium. G. barba- dense (AD)2, G. arboreum (A2) and G. herbaceum (A1) gave rise to 42, 40 and 38 bands, respectively. In wild species, G. australe (C3) had 20 esterase bands, the least in all species of Gossypium. The bands given rise from other wild species ranged from 26 to 40. 2. Each species of every genome groups had its marker bands. The results were in agreement with the traditional classification and provided some biochemical evidence for modern classification of Gossypium. 3. It was clear that all cotton species of different genome groups contain 5 main isozyme bands, viz. PI=3.85, 4.61, 5.48, 5.73 and 5.91 in the zymograms. In other words, these zymograms are common characters of Gossypium. 4. The esterase of 23 cultivers in four cultivated species studied showed that no variation in isozyme patterns existed within one species, except the disease-resistant variety Hea-7124 which differs from other 4 cultivars of G. barbadense.     

Transgenic cotton resistant to herbicide bialaphos

Resistance to bialaphos, a non-selective herbicide, was intro duced into cotton through genetic engineering. A gene encoding phosphinothric in acetyltransferase (bar) from Streptomyces hygroscopicus was inserted into elite varieties of cotton through particle bombardment. Based on the marker gene, -glucuronidase (gus) expression, a total of 18 Pima (Gossypium barbadense), 45 DP50 (G. hirsutum L.), 20 Coker 312 (G. hirsutum) and 2 El Dorado (G. hirsutum) transgenic plants were recovered. Integration of the bar gene into cotton genomic DNA was confirmed by Southern blot analysis and gene expression was confirmed by northern blot and enzyme assays. Herbicide (Basta®) tolerance up to 15 000 ppm was demonstrated in greenhouse trials. The newly introduced herbicide tolerance trait is inherited in a Mendelian fashion in the progenies of germline transformants. This study demonstrates the potential for particle bombardment to introduce commerically important genes directly into elite varieties of cotton. This mode of gene transfer can expedite the introduction of transgenic cotton products into world markets     

Recent long-distance transgene flow into wild populations conforms to historical patterns of gene flow in cotton (Gossypium hirsutum) at its centre of origin

Over 95% of the currently cultivated cotton was domesticated from Gossypium hirsutum, which originated and diversified in Mexico. Demographic and genetic studies of this species at its centre of origin and diversification are lacking, although they are critical for cotton conservation and breeding. We investigated the actual and potential distribution of wild cotton populations, as well as the contribution of historical and recent gene flow in shaping cotton genetic diversity and structure. We evaluated historical gene flow using chloroplast microsatellites and recent gene flow through the assessment of transgene presence in wild cotton populations, exploiting the fact that genetically modified cotton has been planted in the North of Mexico since 1996. Assessment of geographic structure through Bayesian spatial analysis, BAPS and Genetic Algorithm for Rule-set Production (GARP), suggests that G. hirsutum seems to conform to a metapopulation scheme, with eight distinct metapopulations. Despite evidence for long-distance gene flow, genetic variation among the metapopulations of G. hirsutum is high (He = 0.894 ± 0.01). We identified 46 different haplotypes, 78% of which are unique to a particular metapopulation, in contrast to a single haplotype detected in cotton cultivars. Recent gene flow was also detected (m = 66/270 = 0.24), with four out of eight metapopulations having transgenes. We discuss the implications of the data presented here with respect to the conservation and future breeding of cotton populations and genetic diversity at its centre of crop origin.     

Molecular characteristics of chalcone synthase gene families from two cotton species using the polymerase chain reaction]

Using partial sequence data from a genomic clone and the fact of evolutionary conservation of chalcone synthase genes, two primers, corresponding to C-terminal peptides GGAACTCCCTTTTCTGGATAGCTCACC and CCTGGTCCGAACCCAAACAGGACGCCCC, were used to amplify, via polymerase chain reaction, genomic sequences from two Gossypium species, a diploid Gossypium herbaceum, and a tetraploid Gossypium hirsutum cv. 108F. Amplified DNA was separated into individual sequences by cloning into an M13 vector. Six different sequences were identified in each species. From each set of six, one sequence was found to be identical to the genomic sequence, which we have isolated from a subgenomic library of 108F DNA in lambda NM1149. Comparison of other sequences has allowed to find another pair of identical sequences, as well as to get an evidence, that the set isolated from the tetraploid cotton contained preferentially members of only one of the two subfamilies, probably due to primer specificity in amplification reaction. Comparison of specific amino acid substitutions in homologous sequences of cotton, peanut and soybean also suggested that all of the sequences isolated from cotton are more likely to code for chalcone synthase, that for a similar enzyme resveratrol synthetase.     

Genetic diversity evaluation of some elite cotton varieties by RAPD analysis

Random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity of elite commercial cotton varieties. Twenty two varieties belonging to Gossypium hirsutum L. and one to G. arboreum L. were analyzed with 50 random decamer primers using the polymerase chain reaction (PCR). Forty nine primers detected polymorphism in all 23 cotton varieties, while one produced monomorphic amplification profiles. A total of 349 bands were amplified, 89.1% of which were polymorphic. Cluster analysis by the unweighted pair group method of arithmetic means (UPGMA) showed that 17 varieties can be placed in two groups with a similarity ranging from 81.51% to 93.41%. G. hirsutum L. varieties S-12, V3 and MNH-93 showed a similarity of 78.12, 74.46 and 69.56% respectively with rest of the varieties. One variety, CIM-1100, showed 57.02% similarity and was quite distinct. The diploid cotton G. arboreum L. var. Ravi was also very distinct from rest of its tetraploid counterparts and showed only 55.7% similarity. The analysis revealed that the intervarietal genetic relationships of several varieties is related to their center of origin. As expected, most of the varieties have a narrow genetic base. The results obtained can be used for the selection of possible parents to generate a mapping population. The results also reveal the genetic relationship of elite commercial cotton varieties with some standard “Coker” varieties and the diploid G. arboreum L. var. Ravi (old world cotton). Received: 12 July 1996 / Accepted: 26 July 1996     

利用SSR标记技术研究棉属A、D染色体组的进化

利用SSR分子标记技术,对棉属A、D染色体二倍体及四倍体代表棉种进行了遗传多样性分析。供试的10个二倍体代表棉种间遗传多态性丰富,分子聚类结果与Fryxell棉属分类结果相同。分子水平上进一步揭示出属于D染色体组的拟似棉与其他D染色体组棉种的相似系数最低,A,D染色体组间相似系数很高,该结果支持拟全民族似棉是D染色体组最原始棉种,棉属不同染色体组是共同起源,单元进化的理论,利用栽培的异源四倍体棉种不太适于研究棉属A、D染色体组的进化。     

A melting pot of old world begomoviruses and their satellites infecting a collection of gossypium species in pakistan

CLCuD in southern Asia is caused by a complex of multiple begomoviruses (whitefly transmitted, single-stranded [ss]DNA viruses) in association with a specific ssDNA satellite; Cotton leaf curl Multan betasatellite (CLCuMuB). A further single ssDNA molecule, for which the collective name alphasatellites has been proposed, is also frequently associated with begomovirus-betasatellite complexes. Multan is in the center of the cotton growing area of Pakistan and has seen some of the worst problems caused by CLCuD. An exhaustive analysis of the diversity of begomoviruses and their satellites occurring in 15 Gossypium species (including G. hirsutum, the mainstay of Pakistan's cotton production) that are maintained in an orchard in the vicinity of Multan has been conducted using φ29 DNA polymerase-mediated rolling-circle amplification, cloning and sequence analysis. The non-cultivated Gossypium species, including non-symptomatic plants, were found to harbor a much greater diversity of begomoviruses and satellites than found in the cultivated G. hirsutum. Furthermore an African cassava mosaic virus (a virus previously only identified in Africa) DNA-A component and a Jatropha curcas mosaic virus (a virus occurring only in southern India) DNA-B component were identified. Consistent with earlier studies of cotton in southern Asia, only a single species of betasatellite, CLCuMuB, was identified. The diversity of alphasatellites was much greater, with many previously unknown species, in the non-cultivated cotton species than in G. hirsutum. Inoculation of newly identified components showed them to be competent for symptomatic infection of Nicotiana benthamiana plants. The significance of the findings with respect to our understanding of the role of host selection in virus diversity in crops and the geographical spread of viruses by human activity are discussed.     

The cotton centromere contains a Ty3-gypsy-like LTR retroelement

The centromere is a repeat-rich structure essential for chromosome segregation; with the long-term aim of understanding centromere structure and function, we set out to identify cotton centromere sequences. To isolate centromere-associated sequences from cotton, (Gossypium hirsutum) we surveyed tandem and dispersed repetitive DNA in the genus. Centromere-associated elements in other plants include tandem repeats and, in some cases, centromere-specific retroelements. Examination of cotton genomic survey sequences for tandem repeats yielded sequences that did not localize to the centromere. However, among the repetitive sequences we also identified a gypsy-like LTR retrotransposon (Centromere Retroelement Gossypium, CRG) that localizes to the centromere region of all chromosomes in domestic upland cotton, Gossypium hirsutum, the major commercially grown cotton. The location of the functional centromere was confirmed by immunostaining with antiserum to the centromere-specific histone CENH3, which co-localizes with CRG hybridization on metaphase mitotic chromosomes. G. hirsutum is an allotetraploid composed of A and D genomes and CRG is also present in the centromere regions of other AD cotton species. Furthermore, FISH and genomic dot blot hybridization revealed that CRG is found in D-genome diploid cotton species, but not in A-genome diploid species, indicating that this retroelement may have invaded the A-genome centromeres during allopolyploid formation and amplified during evolutionary history. CRG is also found in other diploid Gossypium species, including B and E2 genome species, but not in the C, E1, F, and G genome species tested. Isolation of this centromere-specific retrotransposon from Gossypium provides a probe for further understanding of centromere structure, and a tool for future engineering of centromere mini-chromosomes in this important crop species.     

High-density Linkage Map of Cultivated Allotetraploid Cotton Based on SSR, TRAP, SRAP and AFLP Markers

A high-density linkage map was constructed for an F2 population derived from an Interspecific cross of cultivated allotetraploid species between Gossypium hirsutum L. and G. barbadense L. A total of 186 F2 individuals from the Interspecific cross of ＂CRI 36 × Hal 7124＂ were genotyped at I 252 polymorphic loci Including a novel marker system, target region amplification polymorphism （TRAP）. The map consists of 1 097 markers, including 697 simple se- quence repeats （SSRs）, 171 TRAPs, 129 sequence-related amplified polymorphisms, 98 amplified fragment length polymorphisms, and two morphological markers, and spanned 4 536.7 cM with an average genetic distance of 4.1 cM per marker. Using 45 duplicated SSR loci among chromosomes, 11 of the 13 pairs of homologous chromosomes were Identified In tetraploid cotton. This map will provide an essential resource for high resolution mapping of quantitative trait loci and molecular breeding in cotton.