Identification of resistance gene analogs in cotton (Gossypium hirsutum L.)

Sequence analyses of numerous plant disease resistance genes have revealed the presence of conserved motifs common to this class of genes, namely a nucleotide binding site (NBS) and leucine rich repeat region. In this study, thirty-three resistance gene analogs (RGAs) were cloned and sequenced from cotton (Gossypium hirsutum L.) following PCR with degenerate primers designed from the conserved NBS motif of plant resistance (R) genes. Phylogenetic analysis of the predicted amino acid sequences grouped the RGAs into four distinct classes from which several subgroups were delineated based on nucleic acid sequences. Gene database searches with the consensus protein sequences of each of the four classes and respective subgroups of cotton RGAs revealed their conserved NBS domains and homology to RGAs and known resistance genes from a variety of plant genera. Given the complete lack of knowledge regarding molecular organization of R genes in cotton, the cloned RGAs described here may be useful as probes to map, characterize, and manipulate R genes of the cotton genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance of resistance to Verticillium wilt (Verticillium dahliae) in cotton (Gossypium hirsutum L.)

Verticillium wilt, caused by Verticillium dahliae Kleb., is a major constraint to cotton production in almost all countries where cotton is cultivated. Developing new cotton cultivars resistant to Verticillium wilt is the most effective and feasible way to combat the problem. Little is known about the inheritance of resistance to Verticillium wilt of cotton, especially that caused by the defoliating (D) and nondefoliating (ND) pathotypes of the soil‐borne fungus V. dahliae. The objective of this study was to determine the inheritance of resistance in cotton against both pathotypes of V. dahliae. Crosses were made between the susceptible parent ‘Cukurova 1518’ and each of four resistant parents PAUM 401, PAUM 403, PAUM 405 and PAUM 406 to produce F₂ generations in 2002 and F_2:3 families in 2003. Disease responses of parent and progeny populations to the D and ND pathotypes were scored based on a scale of 0‐4 (0, resistant; 4, susceptible). F₂ populations inoculated with the D pathotype showed a 3 : 1 (resistant : susceptible) plant segregation ratio. Tests of F_2:3 families confirmed that resistance was controlled by a single dominant gene. In contrast, analysis of data from F₂‐ and F₂‐derived F₃ families suggested that resistance to the ND pathotype is controlled by dominant alleles at two loci.     

脱落酸对棉花体细胞胚胎发生的影响

【目的】研究脱落酸(Abscisic acid, ABA)对棉花体细胞胚胎发生过程中下胚轴脱分化和再分化的影响,优化体细胞胚胎发生体系和初步解析脱落酸调控棉花体细胞胚胎发生分子机制。【方法】以棉花品种中棉所24(CCRI 24)下胚轴为外植体,设置5个ABA浓度0、0.02、0.04、0.06、0.08μmol·L^-1,分别以A0、A1、A2、A3、A4表示,添加至MSB(MS培养基+B5维生素)培养基诱导愈伤和胚性愈伤,研究ABA对棉花下胚轴初始细胞脱分化、愈伤组织诱导和胚性愈伤组织诱导的影响。【结果】ABA促进下胚轴初始细胞脱分化;显著提高愈伤组织的脱分化率和增殖率;0.02μmol·L^-1ABA显著提高胚性愈伤分化率,0.04~0.08μmol·L^-1ABA显著降低胚性愈伤分化率。ABA处理后胚性愈伤和非胚性愈伤的增殖率均显著提高且质地受到影响。0.02~0.08μmol ABA处理下,LBD和LBD在愈伤起始期上调表达。0.02μmol·L^-1ABA处理下,在愈伤增殖早期和中期BBM、LEC1和AGL15上调表达,愈伤增殖后期FUS3、LEA、ABI3基因上调表达。【结论】脱落酸调控的棉花体细胞胚胎发生与相关标记基因的时空性表达密切相关,这些基因表达水平的增加是ABA调控愈伤和胚性愈伤分化的分子基础。     

陆地棉种质资源抗旱性状的关联分析

干旱是导致全世界棉花严重减产、纤维品质下降的重要因素,因此获得高产、优质、耐旱的棉花新品种一直是棉花的育种目标。本研究选取217份陆地棉栽培种组成的自然群体为研究对象,采用全生育期处理组灌水量为对照组50%的干旱胁迫处理,并在处理后期对217份材料的株高、衣分、单铃重等18个性状进行2年2点的表型鉴定,干旱胁迫后,群体间响应差异明显,多个表型性状在对照和处理间表现显著差异。通过BLUP分析表型数据并计算各性状的抗旱系数;全基因组范围选取的214对多态性SSR分子标记扫描群体,共检测到393个多态性位点,基因多样性系数平均值为0.402,范围为0.072～0.631, PIC值平均为0.329,范围为0.070～0.560;群体结构分析表明,该群体可分为2个亚群。用上述SSR标记分别对18个性状的抗旱系数进行关联分析,共关联到76个极显著位点(P<0.01),表型变异解释率为2.930%～7.218%,其中共有14个标记位点能同时被2种或以上性状检测到。研究结果可为后期棉花杂交育种亲本选择及抗旱分子标记辅助育种提供理论基础及参考依据。     

Lowering seed gossypol content in glanded cotton (Gossypium hirsutum L.) lines

Cottonseed is a rich source of high quality protein, but its value as an animal feed is limited by gossypol, a toxic polyphenolic compound contained in glands located throughout the plant. This compound helps protect the plant from pests. Totally glandless varieties have been developed, but not adopted as these plants are left vulnerable to pests. This study describes a breeding strategy to decrease the levels of gossypol in the seed while maintaining a high enough concentration of toxin in vegetative plant parts to offer protection from pests. Preliminary studies indicated that crosses between varieties with different gland densities and distributions produced a range of glanding patterns. By selecting within the resulting progeny, we have identified F₇ generation progeny that have <0.30% total gossypol in the seed, while still possessing glands at critical locations on the vegetative plant parts. These new lines will be a valuable source of germplasm for developing low seed gossypol varieties. Seed from these varieties would provide a new source of inexpensive protein for animal feeding rations.     

一个陆地棉类烯醇酶基因的克隆及其表达分析

 采用改良的CTAB法提取陆地棉总RNA,运用RT-PCR技术首次克隆了陆地棉类烯醇酶基因的CDS序列,该基因已经在GenBank上登录,登录号为 EU169604,其开放阅读框为1338 bp,编码445个氨基酸残基。对其序列和进化分析表明,陆地棉烯醇酶基因与其它植物中的烯醇酶基因有较高的相似性,该基因在进化过程中是相当保守的。半定量RT-PCR表达分析结果显示该基因在陆地棉的叶、根、茎中均有表达,但在根中的表达量高于叶和茎。     

转外源凝集素基因棉花对棉蚜的抗性鉴定

转外源凝集素基因棉花工程植株在蚜虫发生期稳定地表现出抗蚜性状,转化一代群体抗、感植株分离比例符合3∶1,抗蚜基因是以单一位点整合到棉花染色体组中。经过 4 个世代的抗蚜鉴定、自交纯合和选择,获得遗传组成纯合的转基因抗蚜虫棉花新品系。对3个转外源凝集素基因棉花品系、2个形态抗蚜品种(系)和 6 个常规品种(系)进行抗蚜性鉴定。结果表明,3个转外源凝集素基因品系对棉蚜的抗性均达到高抗水平;川抗 77 在生育期内均中抗棉蚜,川棉109在苗蚜期和恢复期感蚜,而在伏蚜期中抗棉蚜;6 个常规品种(系)皆高感棉蚜。利用三种接蚜处理对不同类型抗蚜品种(系)进行鉴定的结果相同,繁殖行间自然传播蚜虫省略人工接蚜环节,是开展转基因棉花抗蚜性鉴定的一种简便、高效的方法。     

从基因组学到蛋白质组学：棉花分子生物学研究进展

 花基因组计划正在实施,功能基因组学在棉花研究领域扮演着越来越重要的角色。近年来,国内外的研究者在棉花基因组学及蛋白质组学研究领域内取得了比较大的研究进展,本文从棉花遗传图谱、物理图谱的构建,棉花功能基因组、蛋白质组学技术、棉花蛋白质组学研究等方面入手,就棉花分子生物学研究近况进行概述。     

Response to selective pressure in early generation progenies of upland cotton (Gossypium hirsutum L.)

Summary Combining high fiber strength with high yield in upland cotton (Gossipium hirsutum L.) was been difficult. The cross combination, 69–120 × 6M-10, was chosen for this study because of the divergence of parents for fiber strength, seeds per boll and crop maturity. Forty F₂ plants were selected in 1974 solely on the basis of visual yield and 20 plants were randomly chosen to serve as a bulk check. The F₃ progenies, the bulk check, the parents and a commercial check were tested in 1975. From these results, five groups were established, consisting of four (10%) progenies each to represent high seed/boll, low seeds/boll, high fiber strength, low fiber strength and high yield in the 1976 test of F₄ progenies. This experiment was conducted to (a) compare the contributions of yield components to lint yield among groups of early generation progenies (F₃ and F₄) and (b) determine the phenotypic correlations between yield and quality attributes. The yield components, bolls/m², seeds/boll, fibers/seed, mean fiber length and micronaire, were included as well as fiber strength.No significant lint yield differences were found among the five groups. Progenies within groups did, however, differ in lint yield. The low and high seeds boll groups gave similar yield and fiber quality results. The low and high fiber strength groups gave similar yield but longer fiber was obtained with the high strength group.Stepwise regression analyses estimates show that the number of bolls produced per unit area, although the major contributor to lint yield for all groups, contributed only 66.9% of the total variation in the progeny group selected for high yield, with fibers/seed, mean fiber length, micronaire and seeds/boll accounting for 13.2%, 8.1%, 6.0%, and 5.8%, respectively. Correlations between the various yield components and fiber strength were low and mainly insignificant, indicating that minor alterations might be made in later generations without serious consequences.Potential gains from second-stage selective pressure upon yield-per-boll components following initial selection for lint yield are discussed.     

棉花表型性状基因的SSR标记定位

本文以两个陆地棉多标记基因系T582和T586,以及杂交获得的F_1、F_2及F_3代作为试验材料,利用11对SSR引物对F_2群体的120个单株的DNA样品进行多态性分析,并利用F_2和F_3群体对F_2群体对应单株的13个表型性状进行基因型的判定,结果得到了3个与表型性状基因连锁的SSR标记,分别是红茎基因(R_1)与J178连锁、遗传距离为24.9cM,簇生铃基因(CL_1)与J236连锁,遗传距离为46.0cM,茸毛基因(T_1)与J252连锁,遗传距离为28.5cM,其中R_1、CL_1、J178和J236在同一连锁群上。红茎和植株茸毛是具有抗虫性能的形态性状,用SSR标记这些性状将有助于提高育种家的育种效率。     

转兔角蛋白基因改良棉纤维品质研究

通过花粉管通道转基因技术,将E6启动子驱动的兔角蛋白基因导入高产棉花品种苏棉16号。所用转基因表达载体还含有选择标记基因NPTⅡ(卡那霉素抗性基因)及Gus报告基因。对转化体后代的检测结果表明,T1代有2.1%呈现Gus阳性,在Gus阳性株中84.6%具有卡那霉素抗性。用依据E6启动子序列和兔角蛋白基因序列设计的两对引物,对经过上述筛选的植株进行PCR检测,多次重复,最终确定3株结果稳定的转兔角蛋白基因棉株。从品质分析结果看,这3个株系成熟棉纤维的品质部分得到改良,尤其比强度有较大幅度提高,与转基因受体相比平均提高6.3cN·tex 1。     

Genetic analysis of earliness in upland cotton ( Gossypium hirsutum L.). II. Yield and lint percentage

Inheritance and interrelationships of seed cotton and lint yields were evaluated in a diallel analysis involving seven early maturing parents of different origin and a commercial variety. Lint yield showed relatively little additive variance and low heritability, whereas lint percentage showed the opposite. Highest yields were shown by the least determinate and slowest-maturing genotypes; yields generally decreased as determinacy increased and rate of maturity accelerated. Except for date for first open boll, components of earliness showed no associated with yield.     

Cytogenetics of the 'glandless-seed and glanded-plant' trait from Gossypium sturtianum Willis introgressed into upland cotton (Gossypium hirsutum L.)

In order to introgress the ‘glandless-seed and glanded-plant’ trait from Gossypium sturtianum Willis (2n= 2x= 26, C₁ genome) into the cultivated upland cotton Gossypium hirsutum L. (2n= Ax= 52 (AD), genome), two trispecific hybrids have been created using either Gossypium thurberi Torado (2n= 2x= 26, D₁ genome) or Gossypium raimondii Ulbrich (2n= 2x= 26, D₅ genome) as bridge species. The cross of both trispecific hybrids by G. hirsutum produced the first backcross progenies (BCl). Cytogenetic analysis showed that the trispecific hybrids had 52 chromosomes, their chromosome configurations at metaphase I (Ml) being 15.071 + 15.3411 + 0.93III + 0.69IV + 0.26VI in G. thurberi×G. sturtianum×G. hirsutum (TSH) and 14.421 + 17.0311 + 0.82III + 0.15IV + 0.07VI in G. hirsutum × G. raimondii ×. G. sturtianum (HRS), respectively. Among six BCl plants analysed, the only plant expressing the ‘glandless-seed and glanded-plant’ trait had 52 chromosomes and a meiotic configuration of 5.261 + 20.61II + 0.69III + 0.77IV at MI. Pollen fertility was 2.90% in TSH, 8.97% in HRS, and ranged from 0% to 40.28% in the BCl progenies. The introgressed BCl plant is perennial in growth habit. It can be used in breeding programmes aiming at the introgression of the ‘glandless-seed and glanded-plant’ trait into a cultivar of upland cotton.     

Hybridization between diploid (Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton through ovule culture

Summary With in vitro culture of ovules, interspecific hybrids have been obtained in an otherwise incompatible cross between a diploid (Gossypium arboreum) and a tetraploid (G. hirsutum) cultivated cotton. The early abortion of the embryo was prevented by repeated treatment of the flowers, immediately after pollination with a solution of gibberellic acid and naphthalene acetic acid. The ovules excised three days after pollination and cultured in a liquid medium underwent profuse proliferation, whereas on an agar-solidified medium supplemented with casein hydrolysate, indoleacetic acid and kinetin they germinated to form hybrid plants.     

棉花GhFTL1基因的克隆及初步功能分析

通过RT-PCR结合RACE技术,从新疆陆地棉品种新陆早33中克隆到一个FT类似基因,命名为GhFTLl基因(登录号:HM631972).该基因ORF(Open Reading Frame)全长为525 bp,可编码174个氨基酸,与其它植物中克隆的FT同源蛋白高度相似,含有FT蛋白亚家族两个关键的氨基酸残基及保守氨基...     

Hydrogen peroxide reduces heat‐induced yield losses in cotton (Gossypium hirsutum L.) by protecting cellular membrane damage

We investigated the effect of various growth substances such as hydrogen peroxide, salicylic acid (SA), moringa leaf‐extract (MLE) and ascorbic acid (ASA) on leaf physiology and seed cotton yield (SCY) of heat‐stressed cotton. Cotton plants were exposed to elevated temperatures at three reproductive stages, either by staggering planting time in the field or by increasing growth cabinet temperatures (38/24°C and 45/30°C) in glasshouse. Elevated temperature at any reproductive phase significantly damaged cellular membrane and reduced SCY. Plants exposed to 38/24°C and 45/30°C in glasshouse produced 63% and 22% lower SCY, respectively, compared with plants under optimal temperature ((32/20°C). In response to high temperature, cotton plants up‐regulated activities of anti‐oxidative enzymes e.g. peroxidase and ascorbic acid. However, this defensive system could not protect cellular membrane of stressed plants from extreme temperature (38 and 45°C). In contrast, growth substances such as H2O2, ASA and MLE significantly increased anti‐oxidative enzymes activity to an extent, which reduced heat‐induced damage to cellular membrane. No significant effect of any regulator was observed on SCY under optimum temperatures; although H2O2, MLE and ASA significantly increased SCY of heat‐stressed cotton. Hydrogen peroxide increased SCY of April and May thermal regimes crops by 16% (averaged across both sowing dates) under field, while it caused 14% and 20% increase in SCY of plants exposed to sub (38/24°C) and supra optimal (45/30°C) thermal regimes under glasshouse. We concluded that growth regulators, specifically, H2O2 can protect cotton crops from heat‐induced cellular membrane damage by up‐regulating antioxidant defense system.     

光合作用信号途径调控陆地棉矮化的机理研究

【目的】了解光合作用信号途径基因调控陆地棉矮化的机理。【方法】以现蕾期陆地棉矮化突变体LA-1及其近等基因系LH-1茎尖为材料,通过同位素标记相对和绝对定量(Isobaric tags for relative and absolute quantitation, i TRAQ)结合液相质谱串联(LC-MS/MS)的定量蛋白质组学技术及定量反转录-聚合酶链式反应(Quantitative reverse-polymerase chain reaction, qRT-PCR)技术,分析两种材料中蛋白水平及m RNA水平基因表达情况,并通过生理生化方法检测两种材料光合能力差异。【结果】光合作用信号途径差异表达蛋白PsbO、PsaE、PsaH、PetF-1、PetF-2在LA-1中表达量下调,PetC和delta表达量上调。m RNA水平,除delta基因在两种材料中表达量无显著差别外,其它基因与蛋白水平具有同样的表达趋势。现蕾后,LA-1的净光合速率(P_n)、气孔导度(Cond)显著小于LH-1,蒸腾速率(Tr)极显著小于LH-1,而胞间CO2浓度(Ci)无显著差异。与LH-1相比,LA-1的实际光化学效率(Y_Ⅱ)、光系统Ⅱ(PS Ⅱ)的潜在活性(F_v/F_0)显著减少,叶绿素荧光非光化学猝灭(NPQ)显著增大。【结论】陆地棉矮化突变体LA-1的矮化与光合作用信号途径存在相关性,为进一步研究LA-1矮化的分子机理及棉花的矮化育种提供基础。     

QTL analysis for early-maturing traits in cotton using two upland cotton (Gossypium hirsutum L.) crosses

Making use of the markers linked closely to QTL for early-maturing traits for MAS (Marker-assisted selection) is an effective method for the simultaneous improvement of early maturity and other properties in cotton. In this study, two F₂ populations and their F_2:3 families were generated from the two upland cotton (Gossypium hirsutum L.) crosses, Baimian2 × TM-1 and Baimian2 × CIR12. QTL for early-maturing traits were analyzed using F_2:3 families. A total of 54 QTL (31 suggestive and 23 significant) were detected. Fourteen significant QTL had the LOD scores not only > 3 but also exceeding permutation threshold. At least four common QTL, qBP-17 for bud period (BP), qGP-17a/qGP-17b (qGP-17) for growth period (GP), qYPBF-17a/qYPBF-17b (qYPBF-17) for yield percentage before frost (YPBF) and qHFFBN-17 for height of first fruiting branch node (HFFBN), were found in both populations. These common QTL should be reliable and could be used for MAS to facilitate early maturity. The common QTL, qBP-17, had a LOD score not only > 3 but also exceeding permutation threshold, explaining 12.6% of the phenotypic variation. This QTL should be considered preferentially in MAS. Early-maturing traits of cotton are primarily controlled by dominant and over-dominant effects.     

Regulatory Mechanism of the Photosynthetic Pathway in Dwarfed Upland Cotton (Gossypium hirsutum L.)

[Objective] Here, we examined the mechanism of the photosynthetic pathway in dwarfed upland cotton (Gossypium hirsutum L.). [Methods] The upland cotton dwarf line LA-1 and the near-isogenic line LH-1 were used as research materials. We screened for dwarf-related differentially expressed genes at protein and mRNA levels by applying the isobaric tags for relative and absolute quantitation method in combination with LC-MS/MS quantitative proteomic technology and quantitative reverse-polymerase chain reaction. The difference in the photosynthetic abilities between the two materials were detected using physiological and biochemical methods. [Results] An analysis on the differential expression of proteins encoded by photosynthetic system elements revealed that PsbO, PsaE, PsaH, PetF-1 and PetF-2 were down-regulated, while PetC and delta were up-regulated in LA-1. The expression trends of the mRNA levels were the same as at the protein levels, except for those of the delta gene. The net photosynthetic rate, stomatal conductance and transpiration rate in LA-1 were lower than those LH-1, but the intercellular CO₂ concentration was not significantly different after budding. This indicated that the non-stomatal factor led to a decreased net photosynthetic rate in LA-1. Chlorophyll fluorescence detection revealed that the actual photosynthetic efficiency and potential photochemical activity of photosystem II in LA-1 were significantly decreased, while non-photochemical quenching was significantly increased. [Conclusion] The dwarfism of LA-1 is related to the photosynthetic pathway, and the results lay a foundation for exploring key dwarf-related genes and the molecular basis for dwarfism in upland cotton.     

Construction of a genetic linkage map and QTL analysis of fiber-related traits in upland cotton (Gossypium hirsutum L.)

A genetic linkage map with 70 loci (55 SSR, 12 AFLP and 3 morphological loci) was constructed using 117 F₂ plants obtained from a cross between two upland cotton cultivars Yumian 1 and T586, which have relatively high levels of DNA marker polymorphism and differ remarkably in fiber-related traits. The linkage map comprised of 20 linkage groups, covering 525 cM with an average distance of 7.5 cM between two markers, or approximately 11.8% of the recombination length of the cotton genome. The present genetic linkage map was used to identify and map the quantitative trait loci (QTLs) affecting lint percentage and fiber quality traits in 117 F_2:3 family lines. Sixteen QTLs for lint percentage and fiber quality traits were identified in six linkage groups by multiple interval mapping: four QTLs for lint percentage, two QTLs for fiber 2.5% span length, three QTLs for fiber length uniformity, three QTLs for fiber strength, two QTLs for fiber elongation and two QTLs for micronaire reading. The QTL controlling fiber-related traits were mainly additive, and meanwhile including dominant and overdominant. Several QTLs affecting different fiber-related traits were detected within the same chromosome region, suggesting that genes controlling fiber traits may be linked or the result of pleiotropy.