Selection and Characterization of a Novel Glyphosate Tolerant Upland Cotton(Gossypium hirsutum L.) Mutant(R1098)

Stepwise selection approach was adopted to obtain glyphosate-tolerant upland cotton mutant(R1098) from the embryogenic calli of Coker 312(Gossypium hirsutum L.).The calli were transferred to selection medium and multi-step selection pressure process was carried out until the

An Integrated Genetic,Physical and Transcript Map of Homoeologous Chromosomes 12 and 26 in Upland Cotton(Gossypium hirsutum L.)

While Upland cotton(Gossypium hirsutum L.) represents 95% of the world production,its genetic improvement is hindered by the shortage of effective genomic tools and resources.The

Biochemical Genetic Mechanism and QTLs of Early Maturing without Yield Loss in Short-season Upland Cotton(Gossypium hirsutum L.)

The short season cotton(SSC) was important Upland plant ecotype(Gossypium hirsutum L.).The growth of SSC was very short that is 105 ～ 110 days(after planting). SSC could increase

Genome-wide association study of micronaire using a natural population of representative upland cotton(Gossypium hirsutum L.)

Background: Micronaire is a comprehensive index reflecting the fineness and maturity of cotton fiber.Micronaire is one of the important internal quality indicators of the cotton fiber and is closely related to the value of the cotton fiber.Understanding the genetic basis of micronaire is required for the genetic improvement of the trait.However,the genetic architecture of micronaire at the genomic level is unclear.The present genome-wide association study(GWAS)aimed to identify the genetic mechanism of the micronaire trait in 83 representa:tive upland cotton lines grown in multiple environments.Results GWAS of micronaire used 83 upland cotton accessions assayed by a Cotton 63 K Illumina Infinium single nucleotide polymorphism(SNP)array.A total of 11 quantitative trait loci(QTLs)for micronaire were detected on 10 chromosomes.These 11 QTLs included 27 identified genes with specific expression patterns.A novel QTL,qFM-A12–1,included 12 significant SNPs,and GhFLA9 was identified as a candidate gene based on haplotype block analysis and on strong and direct linkage disequilibrium between the significantly related SNPs and gene.GhFLA9 was expressed at a high level during secondary wall thickening at 20∼25 days post-anthesis.The expression level of GhFLA9 was significantly higher in the low micronaire line(Msco-12)than that in the high micronaire line(Chuangyou-9).Conclusions: This study provides a genetic reference for genetic improvement of cotton fiber micronaire and a foundation for verification of the functions of GhFLA9.

Comparative Transcriptome Analysis of Seed Germination of a Cotton Variety with High Tolerance to Low Temperature

Gossypium hirsutum L.is an important cash crop native to the subtropics and is widely cultivated around the world.Low temperature is an important stress that seriously affects seed germination and emergence during planting.In this study,transcriptomic profiles of low-temperature-and normal-temperature-germinated seeds of Xinluzao 25,a variety with low-temperature tolerance and high germination rates,were analyzed and compared.The following results were obtained.(1)A total of 81.06 Gb of clean data were obtained after transcriptome sequencing and assembly,and 76,931 non-redundant Unigene sequences were obtained after data consolidation and concatenation;of these,69,883 Unigene sequences were annotated.In addition,55,463 Unigene transcript sequences(72.2%)were annotated for Gene Ontology(GO)classification,and 26,629 genes were involved in 50 metabolic pathways identified by Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.(2)Three main pathways related to low-temperature tolerance of seed germination were identified:starch and sucrose metabolism,phenylpropanoid biosynthesis,and cysteine and methionine metabolism.Their main molecular functions involve the regulation of abscisic acid and activities of enzymes such as amylase,peroxidase,and oxidoreductase.During germination at low temperature,more genes were down-regulated than up-regulated genes at the protrusion stage(2 mm),and more genes were up-regulated than down-regulated at the germination stage(30 mm)after protrusion.(3)The enzyme activities at the two stages showed that amylase,peroxidase,catalase,and glutathione reductase had higher activities when the seeds germinated at 15℃.In this study,high expression of amylase,peroxidase,catalase,and glutathione reductase genes may be the main cause of increased tolerance to low temperature.

Characterization of Gh SERK2 and its expression associated with somatic embryogenesis and hormones level in Upland cotton

Somatic embryogenesis （SE） is one of the most important steps during regeneration of cotton, but the molecular mechanism of SE remains unclear. SOMATIC EMBRYOGENSIS RECEPTOR KINASE （SERK） gene is known to function in SE. A homolog GhSERK2 （accession number： JF430801） was cloned from Upland cotton and characterized for its functions in SE. GhSERK2 expressed in different tissues and showed higher expression level in floral organs than vegetative ones with the highest levels in ovule and anther. GhSERK2 expressed during SE with a high level at globular embryos stage. Upon treatment with indole-3-butytic acid （IBA）, the transcription level of GhSERK2 was induced and promoted SE subsequently. A 2-day treatment of 2,4-dichlorophenoxyacetic acid （2,4-D） induced the expression of GhSERK2, but treatments of 2,4-D for longer periods sharply inhibited the GhSERK2 transcription level of embryogenic callus （EC）. The levels of hormones, including 3-indoleacetic acid （IAA）, abscisic acid （ABA）, and brassinosteroid （BR）, were increased in the initial calli induced from the over-expression of GhSERK2 cotton. Our results indicated that GhSERK2 expression was associated with induction of SE and closely related to hormone levels during tissue culture in Upland cotton, and the gene might play an important role in regeneration of cotton.

Comparative Analysis of Salinity-Induced Proteomic Changes in Cotton (Gossypium hirsutum L.)

Salt stress on cotton varieties of distinct salinity tolerance can induce expression of different proteins. Zhong 07, a salt-tolerant variety and Zhong s9612, a salt-sensitive variety, were utilized as experimental materials. The leaves of trefoil seedlings treated with or without 0.4% NaCl for 24 h were harvested for whole-protein extraction. Two-dimensional technology, combined with mass spectroscopy (MS) analysis and protein database searching, was employed to detect differentially expressed proteins and determine their identities and biological functions. Compared with the control, Zhong 07 showed 10 differentially expressed proteins under salt stress, of which 6 were upregulated and 4 were downregulated. Meanwhile, 12 differentially expressed proteins were detected in Zhong s9612 under salt stress, of which 10 were upregulated and 2 were downregulated. In the matrix-assisted laser desorption-ionization/time of flight-time of flight/MS analysis, 14 differentially expressed proteins were successfully identified, including the ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisco) large subunit-binding protein subunit alpha (RuBisco α), luminal binding protein (LBP), heat shock protein 70 (Hsp1, 2, 3), pathogenesis-related protein class 10 (PR-10), quinoneoxidoreductase-like protein (QOR), S-adenosylmethioninesyn-thetase (SAMS), enolase (EN), and RuBisco large subunit-binding protein subunit beta (RuBisco β). Cellular function is ultimately executed by proteins, and cotton varieties with different salt tolerance can be influenced by salt stress to various degrees, which can provide certain theoretical foundation for the identification of salt tolerance of cotton varieties. The findings also provide some proteins, such as the RuBisco large subunit binding proteins α and β subunits, OEE2 protein, HSP70, and S-adenosylmethionine synthetase, which can be used as protein markers of salt-to-lerance before- and post-treatment, making a big difference in salt-tolerance identification in cotton.

Intron-Targeted Intron-Exon Splice Conjunction (IT-ISJ) Marker and Its Application in Construction of Upland Cotton Linkage Map

To develop a new DNA maker, which could be used in genetic diversity analysis and genetic map construction in plants, IT-ISJ （intron targeted intron-exon splice junction） primer combinations, which were designed according to the intronexon splice junction conserved sequences, were used to construct cotton genetic linkage map in the present study. 49 out of 704 IT-ISJ primer combinations showed polymorphism between upland cotton high quality cultivar Yumian 1 and multiple dominant gene line T586, and the polymorphic primer combinations accounted for 7.0% of total primer combinations. 49 IT-ISJ primer combinations were used to genotype 270 F2：7 recombinant inbred lines developed from （Yumian 1 × T586） F2, and 58 IT-ISJ loci were obtained. 58 IT-ISJ, together with 150 SSR and 8 morphological loci, were used to conduct linkage analysis, and a linkage map including 22 linkage groups and 113 loci （49 IT-ISJ, 62 SSR, and 2 morphological loci） was constructed. The linkage map covered 714.5 cM with an average interval of 6.3 cM between two markers, accounting for 16.1% of cotton genome. The present study demonstrated that the polymorphism of IT-ISJ marker is high, and it could be effectively applied in plant genetic map construction.

多环境下陆地棉(Gossypium hirsutum L.)重组自交系铃重与衣分性状的QTL分析

本研究利用以SGK9708为母本,0-153为父本构建的196个陆地棉重组自交系(F6:8)构建了包含186个标记,总长827.84cM,标记间平均距离4.45cM,覆盖棉花基因组18.6%的遗传连锁图谱,并对7个环境下的铃重和衣分性状进行QTL定位和上位性互作分析。利用两种分析软件(WinQTLcart2.5和QTLNetwork2.0)共同定位了多个环境下稳定表达的5个主效QTLs(qBW-1-1,qBW-1-2,qLP-2-1,qLP-2-2和qLP-4-2)。利用QTLNetwork2.0分别检测到4对铃重上位性互作QTLs和7对衣分上位性互作QTLs,以背景位点间的互作和加性效应位点与背景位点间的互作为主。除主效QTL外,上位性效应也是陆地棉铃重和衣分性状的重要遗传基础。本研究定位的5个主效QTLs为选择高铃重、高衣分品种的棉花分子标记辅助育种提供了重要依据。

Development of glyphosate-tolerant transgenic cotton plants harboring the G2-aroA gene

Given that glyphosate weed control is an effective strategy to reduce costs and improve economic outcomes of agricultural production in China,the development of glyphosate-resistant cotton holds great promise.Using an Agrobacterium-mediated transformation method,a new G2-aroA gene that encodes 5-enolpyruvylshikimate-3-phosphate synthase（EPSPS）was transformed into cotton cultivar K312.The transgenic cotton plants were regenerated from a callus tissue culture via kanamycin selection.Ten regenerated cotton plants were obtained and allowed to flower normally to produce fruit.The results from polymerase chain reaction（PCR）and Southern and Western blot analyses indicated that the target gene was integrated into the cotton chromosome and was expressed effectively at the protein level.The glyphosate tolerance analysis showed that the transgenic cotton had a high resistance to glyphosate.Further,even cotton treated with 45.0 mmol L^–1 of glyphosate was able to slowly grow,bloom and seed.The transgenic cotton may be used for cotton breeding research of glyphosate-tolerant cotton.

Cloning and Expression Analysis of Chalcone Synthase and Chalcone Isomerase Encoding Genes in Gossypium hirsutum

In this study,chalcone synthase and chalcone isomerase encoding genes(GhCHS and GhCHI)were cloned from upland cotton(Gossypium hirsutum)cultivar HM-40,and analyzed bioinformatically.Their functions were analyzed through virus induced gene silencing(VIGS).The results showed that GhCHS gene has an open reading frame(ORF)of 1170 bp and encodes a protein of 389 amino acids.Many phosphorylation sites were detected in GhCHS protein,suggesting that it may be involved in kinase phosphorylation.The deduced GhCHS protein was most closely related to Theobroma cacao CHS protein according to phylogenetic analysis.The GhCHI ORF was 630 bp and encoded a protein of 209 amino acids.Many phosphorylation sites were found in GhCHI protein,indicating that it may be related to kinase phosphorylation.The GhCHI protein was most closely related to Hibiscus cannabinus CHI protein according to phylogenetic analysis.Quantitative PCR(q PCR)showed that GhCHS and GhCHI were rapidly activated after inoculation with Verticillium dahliae VD07,and then their expression levels kept increasing over time,indicating that the two genes might play an important role in the defense response against Verticillium wilt.Virus induced gene silencing(VIGS)was used to silence endogenous GhCHS and GhCHI genes in upland cotton plants before VD07 was inoculated to identify disease resistance.The results showed that the disease index of plants untreated with Agrobacterium tumefaciens was 31.2,and that of the plants inoculated with empty vector was 30.0.The disease index of GhCHS-silenced plants was 72.5,and that in GhCHI-silenced plants was 67.5.These results confirmed that GhCHS and GhCHI may play an important role in defense response of upland cotton to Verticillium wilt.

Study on the Stability of World Diversity of Cultured Species <i>G. hirsutum</i>L. to Salination

The paper presents the results of a study of salt tolerance in some different eco-geographical samples of the cotton germplasm collection of the Institute of Genetics and Experimental Biology of the Academy of Sciences of the Republic of Uzbekistan. According to the results obtained, the studied samples were divided into several groups depending on their salt tolerance. Salt tolerant and unstable samples were found in all studied ecological and geographical groups, but differed in the frequency of distribution.

QTL mapping for fiber quality and yieldrelated traits across multiple generations in segregating population of CCRI 70

Background:Cotton is a significant economic crop that plays an indispensable role in many domains.Gossypium hirsutum L.is the most important fiber crop worldwide and contributes to more than 95%of global cotto n production.Identifying stable quantitative trait locus(QTLs)controlling fiber quality and yield related traits are necessary prerequisites for marker-assisted selection(MAS).Results:A genetic linkage map was constructed with 312 simple sequence repeat(SSR)loci and 35 linkage groups using JoinMap 4.0;the map spanned 1 929.9 cM,with an average interval between two markers of 6.19 cM,and covered approximately 43.37%of the cotton genome.A total of 74 QTLs controlling fiber quality and 41 QTLs controlling yield-related traits were identified in 4 segregating generations.These QTLs were distributed across 20 chromosomes and collectively explained 1.01%?27.80%of the observed phenotypic variations.In particular,35 stable QTLs could be identified in multiple generations,25 common QTLs were con sistent with those in previous studies,and 15 QTL clusters were found in 11 chromosome segments.Conclusion:These studies provide a theoretical basis for improving cotton yield and fiber quality for molecular marker-assisted selection.

Construction of a linkage map and QTL mapping for fiber quality traits in upland cotton (Gossypium hirsutum L.)

With the development in spinning technology, the improvement of cotton fiber quality is becoming more and more important. The main objective of this research was to construct a high-density genetic linkage map to facilitate marker assisted selection for fiber quality traits in upland cotton (Gossypium hirsutum L.). A genetic linkage map comprising 421 loci and covering 3814.3 cM, accounting for approximately 73.35% of the cotton genome, was constructed using an F2 population derived from cross GX1135 (P 1 )×GX100-2 (P 2 ). Forty-four of 49 linkage groups were assigned to the 26 chromosomes. Fiber quality traits were investigated in F2 population sampled from individuals, and in F2:3 , and F2:4 generations sampled by lines from two sites and one respectively, and each followed a randomized complete block design with two replications. Thirty-nine quantitative trait loci were detected for five fiber quality traits with data from single environments (separate analysis each): 12 for fiber length, five for fiber uniformity, nine for fiber strength, seven for fiber elongation, and six for fiber micronaire, whereas 15 QTLs were found in combined analysis (data from means of different environments in F2:3 generation). Among these QTLs, qFL-chr5-2 and qFL-chr14-2 for fiber length were detected simultaneously in three generations (four environments) and verified further by combined analysis, and these QTLs should be useful for marker assisted selection to improve fiber quality in upland cotton.

棉花MADS-box蛋白基因(GhMADS-13)的克隆和表达分析

一组具有MADS-box结构域的转录因子在控制花器官的诱导与发育中起着重要作,以中棉所36(CCRI)均一化全长cDNA文库为基础,结合EST测序分析,分离获得一个棉花的MADS-box基因全长cDNA。该基因cDNA全长1079bp,包含一个732bp的完整的开放阅读框,编码234个氨基酸,具有典型的MADS-box结构域和完整的K区,命名为GhMADS-13(GenBank:FJ409870)。与拟南芥、葡萄等植物的AGL6类基因具有高度的同源性。实时定量RT-PCR分析表明,GhMADS-13在CCRI36的花器官发育中早期表达量逐步增加,后期有下降趋势。推测GhMADS-13可能在开花诱导和花器官发育过程中起着重要作用。

棉花SUPERMAN类锌指蛋白基因GZFP的启动子及功能分析

GZFP是来源于陆地棉的一个SUPERMAN类锌指蛋白基因。为了进一步了解该基因功能,本研究通过染色体步移(Genome walking)获得其启动子区域,并构建该片段与GUS连接的重组载体pBI-pGZFP-5::GUS,通过花浸染法转化拟南芥。转基因植株的GUS染色结果表明GUS基因主要在根部以及花器官表达,而叶片中的表达量很低,并且根部的表达量在整个生育期都很强。通过半定量RT-PCR分析不同诱导下GZFP基因的表达变化,发现ABA、干旱、盐胁迫诱导可以提高其表达量。构建GZFP过量表达载体并通过叶盘法转化烟草,烟草转化株的表型无明显变化,但是转化株中NtOPBP1和NtERD10的表达量较未转化株明显增强,而这两个基因都参与了植物的抗逆反应,说明GZFP基因可能与植物抗逆相关。

陆地棉吐絮铃数及吐絮率的QTL定位

采用复合区间作图法，对陆地棉sGK9708X0-153组合的重组近交系（RI）及永久F2群体在曲周（2009年）、安阳（2009，2010年）3个环境条件下的吐絮铃数和吐絮率进行QTL检测，共获得18个QTLs。其中，7个与吐絮铃数相关，加性效应在-0．46～0．33之间，可解释的表型变异为5．86％～11．28％；11个与吐絮铃率相关，加性效应在-3．64％～3．20％之间，可解释的表型变异4．68％-9．84％。这些QTLs主要分布在25号（吐絮铃数／吐絮率：3个／6个，下同）、16号（2个／2个）、18号（1个／2个）染色体和LG49（1个／1个）上。这18个QTLs中，qPOB-16-2在R1群体中的3个环境下被稳定检测到，qCOB．16．1、qPOB-25—3和qPOB-25．4在RI中的两个环境下被稳定检测到。这些QTLs可以应用于吐絮铃数和吐絮率的分子标记辅助选择。

棉花盐胁迫应答基因GhEXO70B1功能分析

【目的】为了提高棉花的耐盐性,使棉花能够适应盐碱地的生长环境,挖掘耐盐基因迫在眉睫。【方法】利用生物信息学分析了Gh EXO70B1基因的功能和结构特征;利用定量反转录-聚合酶链式反应(Quantitative reverse transcription-polymerase chain reaction,qRT-PCR)分析该基因在不同部位的表达量和盐胁迫下根、茎、叶的表达情况;利用病毒诱导基因沉默技术(Virus-induced gene silencing,VIGS)对该基因进行沉默并进行功能验证,并通过台盼蓝染色观察和生化指标测定综合分析了受胁迫的程度及其可能的生理机制。【结果】本研究从陆地棉中分离了1个新的介导自噬的基因Gh EXO70B1,生物信息学分析表明该基因编码产物含有1个保守的EXO70结构域,不具有跨膜结构,为非分泌蛋白,进化中功能高度保守。qRT-PCR表明该基因在各个组织部位中均有表达,其中根和茎中表达量较高,盐胁迫处理诱导其上调表达。利用VIGS成功沉默GhEXO70B1的表达,基因沉默后的棉株相较于对照更加不耐盐,出现叶片发黄、萎蔫和干枯的现象,脯氨酸含量显著降低,丙二醛含量升高,台盼蓝染色叶片着色范围较大,表明Gh EXO70B1基因沉默后应对盐胁迫的能力降低,细胞死亡的数量显著增加。【结论】陆地棉Gh EXO70B1基因与自噬相关,暗示该基因通过介导细胞自噬应对逆境胁迫。

陆地棉苗期低温响应基因GhZAT10的克隆及功能研究

【目的】挖掘棉花耐冷相关基因,为培育棉花耐冷品种奠定基础。【方法】克隆陆地棉基因GhZAT10(Zinc finger of Arabidopsis thaliana 10),通过实时荧光定量聚合酶链反应(Quantitative real-time polymerase chain reaction,qRT-PCR)分析冷处理前后该基因在根、茎、叶的表达;通过生物信息学方法分析Gh ZAT10基因结构及其编码的蛋白质性质及进化关系;通过Gateway技术构建蛋白表达载体35S::GhZAT10-GFP进行亚细胞定位;利用病毒诱导的基因沉默技术研究低温胁迫下GhZAT10在棉花耐冷反应中的功能。【结果】GhZAT10基因开放阅读框长度为813 bp(base pair,碱基对),编码270个氨基酸;GhZAT10在根中表达量高于茎和叶,低温胁迫下在3种组织中均上调表达。GhZAT10蛋白无信号肽,不包含跨膜螺旋,且其活性与其磷酸化调控关系密切。陆地棉GhZAT10与可可、拟南芥、甜橙中的ZAT10蛋白亲缘关系较近;GhZAT10蛋白定位在细胞核;沉默GhZAT10基因使棉花的耐冷能力减弱。【结论】GhZAT10蛋白属于C2H2型锌指蛋白,在陆地棉冷胁迫响应信号途径中具有积极作用。

棉花硫氧还蛋白基因GhWCRKC2-5参与开花调控的功能研究

目的FLOWERING LOCUST(FT)位于开花调控网络的中心,编码成花激素,不仅调控植物开花转变,还参与调控植物多方面的生长发育。硫氧还蛋白(thioredoxin,Trx)在植物的逆境胁迫中发挥着重要的生物学功能。方法我们前期克隆了一个棉花FT同源基因GhFT1,利用酵母双杂技术筛选到一个与GhFT1蛋白互作的硫氧还蛋白GhWCRKC2-5。本研究采用RT-PCR技术从陆地棉中扩增了GhWCRKC2-5基因的开放阅读框cDNA。该基因编码一个203个氨基酸的短肽,含有WCRKC保守氨基酸基序,为非典型的Trx。全基因组分析表明棉花中含有46个非典型的Trxs,系统进化分析显示GhWCRKC2-5与Theobroma cacao的TcWCRKC亲缘关系最近。qRT-PCR分析表明GhWCRKC2-5基因在棉花根和花中表达较高,在茎、叶和顶端分生组织中表达较低;在纤维不同发育时期,GhWCRKC2-5基因表达呈现出降低-升高-降低的趋势,在棉花开花后25d时的纤维表达最高。结果在拟南芥中过量表达GhWCRKC2-5基因,转基因拟南芥明显比野生型早花,并且转基因拟南芥的FT及开花通路中关键基因SUPPESSOR OF OVER EXPRESSION OF CONSTANS1(SOC1)明显上调表达。利用病毒介导的基因沉默技术干扰GhWCRKC2-5基因的表达,GhWCRKC2-5沉默的植株比对照植株开花时间晚。结论GhWCRKC2-5基因在棉花的开花转变中起着重要作用,本研究为深入分析棉花开花调控机理奠定基础。