Molecular Marker-assisted Selection for Verticillium Wilt Resistance in Upland Cotton (Gossypium hirsutum)

Verticillium wilt is a global important disease ofcotton,which threatens the development ofcotton production seriously.Recent years,because of the change in climate and croppingpattern,Verticillium wilt was broke out incotton production areas in China,which

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

Genetic Diversity and Its Correlation with Heterosis of Upland Cotton (Gossypium hirsutum L. ) Cultivars of Huang-Huai Region in China Evaluated by RAPD

Sixteen cultivars of Upland cotton（Gossypiumhirsutum L.）cultivars in Huang-Huai Cotton-growing Region were detected by RAPD whilethe F1 heterosis of each hybrid involved thesecultivars were evaluated.The genetic similarity（GS）of the 16 cultivars through analysis of 115polymorphic RAPD loci obtained from 70informative primers were 53%～88%.

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

Agrobacterium-meditated Genetic Transformation of an Upland Cotton(Gossypium hirsutum cv Coker 310) Using a Novel Bt Gene Cry2Ac

The development of transgenic cotton varieties resistant to bollworms has been a major success of applying plant genetic engineering technology to agriculture,evidenced by phenomenal increase in

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.

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.

Cytochemical localization of H_2O_2 in pigment glands of cotton(Gossypium hirsutum L.)

Programmed cel death （PCD） plays a critical role in the development of plant pigment glands, while H2O2, which is a kind of reactive oxygen species （ROS） produced by the aerobic metabolism of cels, acts as an important signal in this process. Here, we investigated the temporal and spatial dynamics of accumulated H2O2 in pigment glands ofGossypium hirsutum L. with 3,3-diaminobenzidine （DAB） staining, 2’,7’-dichlorodihydrolfuorescein diacetate （DCFH2）-DA lfuorescent labeling and CeCl3 cytochemical localization techniques. The results showed that thepigment glandsofG. hirsutum could generate H2O2, and the amount and localization of H2O2 variedat different developmental stages. At the early developmental stage, a smal amount of H2O2 accumulated in the vacuole membrane of pigment gland cels. At the intermediate stage, a large number of H2O2 appeared in the vacuole membrane, while cel wals started to accumulate a smal amount of H2O2. When pigment gland cel degraded, H2O2 mainly accumulated on the chloroplast envelope membrane of inner sheath cels. With the degradation of the sheath cels, H2O2was detected in cel wal and the membrane of secretory vesicles which contains the preliminary contents of pigment gland. With the pigment glands completely maturation, H2O2 would disappeared. The accumulation sites of H2O2are consistent with the process of PCD of individual gland cels, which started from the degra-dation of intracelular membrane and ended with the degradation of cel wals. Thus H2O2 probably plays an important role in the development of pigment glands. In addition, the development of pigment glands and the generation of H2O2 are not associated with the light, and no H2O2 was detected in the secretions of pigment glands.

Cloning of Two Genes Related to Plant Defense Response ofSea Island Cotton (Gossypium barbaden.se L. )

Verticillium wilt,caused by V.dahaliae,is aserious fungus disease of cotton in China.Nearly all cultivated upland cotton(Gossypiumhirsutum)varieties are sensitive to it.Somespecies of island cotton(G.barbadense),however,have a natural resistance to thispathogen.To investigate the mechanism of

Effect of Different Hormone Combinations on Somatic Embryogenesis in Cotton Cultivar Xinluzao 33(Gossypium hirsutum L.)

[Objective] This study was conducted to evaluate the efficiency of somatic embryogenesis and plant regeneration for an upland cotton cultivar Xinluzao33 under the induction of different hormone combinations and thus to determine the optimal hormone combination. [Method] Calli of Xinluzao33(Gossypium hirsutum L.) were induced from seedling hypocotyl tissue by a range of DK and BK combinations. Embryogenic calli and embryos were induced on callus-inducing medium(CIM) without any hormones. Callus appearance and quality were compared to determine which medium was the optimal for callus induction. Embryogenesis ratio was calculated to determine which medium was the best for somatic embryogenesis and plant regeneration. [Result] Callus induction rate was 100% in all the 12 hormone combinations.The calli were yellow or kelly, and their texture was loose or soft under low concentrations of DK combinations, green or white, variably compact under high concentrations of DK combinations. The calli induced by BK combinations were kelly or green, covering creamy white substance. The best medium for callus induction was DK6(0.05 mg/L 2, 4-D and 0.10 mg/L KT). Embryogenic calli were successfully induced from all the combinations. The efficiency of embryogenic callus induction,embryogenesis, and plantlet regeneration were significantly different among the 12 combinations. The result showed that the embryogenesis ratio was the highest in BK3 combination(0.50 mg/L IBA and 0.50 mg/L KT), 72.86% of embryogenic calli differentiated into somatic embryos after being cultured on CIM for 80 d, and80.93% of the somatic embryos finally regenerated into plants on SEM(somatic embryo induction medium). [Conclusion] These results indicate that hormone combination BK3(0.50 mg/LIBA and 0.50 mg/L KT) was the best medium for somatic embryogenesis and plant regeneration from Xinluzao33.

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.

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。

Genome-wide identification and expression analysis of Gossypium RING-H2 finger E3 ligase genes revealed their roles in fiber development,and phytohormone and abiotic stress responses

Background： RING H2 finger E3 ligase （RH2FE3） genes encode cysteine rich proteins that mediate E3 ubiquitin ligase activity and degrade target substrates. The roles of these genes in plant responses to phytohormones and abiotic stresses are well documented in various species, but their roles in cotton fiber development are poorly understood. To date, genome wide identification and expression analyses of Gossypium hirsutum RH2FE3 genes have not been reported. Methods： We performed computational identification, structural and phylogenetic analyses, chromosomal distribution analysis and estimated KJKs values of G hirsutum RH2FE3 genes. Orthologous and paralogous gene pairs were identified by all versus all BLASTP searches. We predicted cis regulatory elements and analyzed microarray data sets to generate heatmaps at different development stages. Tissue specific expression in cotton fiber, and hormonal and abiotic stress responses were determined by quantitative real time polymerase chain reaction （qRT PCR） analysis. Results： We investigated 140 G hirsutum, 80 G. orboreum, and evolutionary mechanisms and compared them with orthologs 89 G. roimondii putative RH2FB genes and their in Arobidopsis and rice. A domain based analysis of the G hirsutum RH2FE3 genes predicted conserved signature motifs and gene structures. Chromosomal localization showed the genes were distributed across all G hirsutum chromosomes, and 60 duplication events （4 tandem and 56 segmental duplications） and 98 orthologs were detected, cis elements were detected in the promoter regions of G hirsutum RH2FE3 genes. Microarray data and qRT PCR analyses showed that G hirsutum RH2FE3 genes were strongly correlated with cotton fiber development. Additionally, almost all the （brassinolide, gibberellic acid （GA）, indole 3-acetic acid drought, and salt）. dentified genes were up regulated in response to phytohormones （IAA）, and salicylic acid （SA）） and abiotic stresses （cold, heat, Conclusions： The genome wide identification, comprehensive analysis, and characterization of conserved domains and gene structures, as well as phylogenetic analysis, cis element prediction, and expression profile analysis of G hirsutum RH2FE3 genes and their roles in cotton fiber development and responses to plant hormones and abiotic stresses are reported here for the first time. Our findings will contribute to the genome wide analysis of putative RH2FE3 genes in other species and lay a foundation for future physiological and functional research on G hirsutum RH2FE3 genes.

Current Status of Cotton Genome Resource in India and Initiative on Utilization of Gene Pool through Molecular Technologies

The genus Gossypium is one of the largest havingnearly 50 species of which two are tetraploidcultivars and two are diploid cultivars with 2n =52 and 26 chromosome respectively and the restare wild to semi-wild at both these ploidy levels.The tetraploids(AD)are supposed to containthe Asiatic cotton(A)and American diploid(D)species genomes.

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 1 170 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.

基于加性-显性-上位性(ADAA)模型与主-多基因模型的陆地棉产量与品质性状的遗传分析

本试验为进一步明确陆地棉(Gossypium hirsutum L.)杂交后代产量、纤维品质性状的遗传规律,以9个陆地棉品种为亲本设计不完全双列杂交(NCⅡ)。对2019-2021年的亲本、18个F_(1)和F_(2)组合及组合TH14-22×B7的127个F2单株分别采用加性-显性-上位性(ADAA)模型与主基因-多基因遗传模型,从基因整体与个体水平进行分析。NCⅡ分析结果表明:所有性状均存在基因型与环境的互作,主要通过显性×环境来体现,上位性×环境不可忽视。多数性状的遗传以加性效应为主,整齐度以显性效应为主,伸长率无显著的基因主效应。上半部平均长度与整齐度具有正向平均优势,其他性状无明显的平均优势。分离分析结果表明:各性状普遍受主基因控制。单株铃数、衣分与比强度的最适遗传模型为2MG-EA,单铃质量与上半部平均长度最适遗传模型为2MG-A,整齐度与马克隆值最适遗传模型为2MG-AD,伸长率最适遗传模型为1MG-AD。单铃质量、衣分、上半部平均长度、整齐度与比强度主基因遗传率较高,单株铃数、马克隆值与伸长率的遗传率较低。ADAA模型分析结果与主基因-多基因分析结果的相互补充,可有效了解各性状的遗传特征,为采用适当的育种改良策略提供参考。

陆地棉粉花花蕾与黄花花蕾的比较转录组分析

【目的】挖掘影响棉花花色多态性的代谢物质和候选基因。【方法】以陆地棉黄色花品种中棉所24和粉色花品系中遗红为研究材料,取花瓣变色前后的花蕾进行花色苷含量测定和转录组测序分析。【结果】在开花前,中棉所24和中遗红的花蕾颜色已表现出差异,随着花蕾的发育,花色差异逐渐增大,其中天竺葵素-3-O-葡萄糖苷、矢车菊素-3-O-半乳糖苷、矢车菊素-3-O-葡萄糖苷和矢车菊素-3-O-(6''-O-丙二酰)-葡萄糖苷在中遗红花蕾中特异性积累。对中棉所24和中遗红花蕾中差异表达基因(differentially expressed gene,DEG)进行联合分析,共获得8790个上调表达基因和8521个下调表达基因,它们在液泡质子ATP酶(V型ATP酶)复合体、类黄酮生物合成过程等通路富集。进一步分析发现,花色苷合成相关的基因在2个材料中的表达水平并无明显差异,而调控花色苷合成、转运以及液泡酸碱平衡的基因Gh_A07G083500、bHLH基因Gh_D11G130400、GST基因Gh_A07G079800和V型ATP酶基因Gh_A09G123000、Gh_A08G012100和Gh_D09G115200在中遗红中高水平表达。【结论】本研究构建了粉花花色形成的调控通路,鉴定了与棉花花色相关的候选基因,为棉花育种提供基因资源。