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.

Screening of drought resistance indices and evaluation of drought resistance in cotton(Gossypium hirsutum L.)

Sixteen cotton cultivars widely planted in China were sowed under five different drought concentrations(0,2.5,5,7.5,and 10%)using PEG6000 to screen the indices of drought resistance identification and explore the drought resistance of different cotton cultivars.Eighteen physiological indices including root,stem,and leaf water contents(RWC,SWC,and LWC),net photosynthetic rate(Pn),the maximum photochemical quantum yield(Fv/Fm),the actual photochemical quantum yield(ΦPSII),non-photochemical quenching coefficient(NPQ),leaf water potential(LWP),osmotic potential(Ψs),leaf relative conductivity(REC),leaf proline content(Pro),leaf and root soluble protein contents(LSPC and RSPC),leaf and root malondialdehyde(MDA)contents(LMDA and RMDA),root superoxide dismutase,peroxidase,and catalase activities(RSOD,RPOD,and RCAT)were measured.Results indicated the 18 physiological indices can be converted into five or six independent comprehensive indices by principal component analysis,and nine typical indices(Fv/Fm,SWC,LWP,Pro,LMDA,RSPC,RMDA,RSOD,and RCAT)screened out by a stepwise regression method could be utilized to evaluate the drought resistance.Moreover,the 16 cotton cultivars were divided into four types:drought sensitive,drought weak sensitive,moderate drought resistant,and drought resistant types.The resistance ability of two selected cotton cultivars(drought resistant cultivar,Dexiamian 1;drought sensitive cultivar,Yuzaomian 9110)with contrasting drought sensitivities were further verified by pot experiment.Results showed that the responses of final cotton biomass,yield,and yield composition to drought were significantly different between the two cultivars.In conclusion,drought resistant cultivar Dexiamian 1 and drought sensitive cultivar Yuzaomian 9110 were screened through hydroponics experiment,which can be used as ideal experimental materials to study the mechanism of different cotton cultivars with contrasting drought sensitivities in response to drought stress.

Multi-Location Investigation of Optimum Planting Density and Boll Distribution of High-Yielding Cotton (G.hirsutum L.) in Hubei Province,China

Cotton yield per unit ground area has stagnated for a dozen years in Hubei Province, China, although a series of new high- yielding varieties have been commercialized. A multi-location investigation was carried out in 2008 and 2009 in 13 counties to determine if increased planting population density （PPD） would break the stagnant yield. The results showed that significant differences among the fields existed in theoretical yield, PPD, and bolls per square meter （BPM）. The lowest yield of 1 641.1 kg ha-I was resulted from the lowest PPD of 1.7 plants m-2 and the lowest BPM of 71.8 bolls m-2, while the highest yield of 2 779.7 kg ha-~ was resulted from the highest PPD of 2.5 plants m-2, and the highest BPM of 129.4 bolls m-z. Plant mapping revealed that boll retention rate （BRR） was maintained over 30 or 40% for the first 17-18 fruiting branches （FBs） and decreased dramatically thereafter, rotten boll rate （RBR） decreased, but open boll rate （OBR） rose first and dropped later with rising FB from the bottom to the top. But BRR, RBR, and OBR were all dropped with the fruiting positions （FPs） extending outwards. The optimum range of plant density would be 2-3 plants m-2 and the proper individual plant structure would be 16-19 FBs with 5-7 FPs for cotton production in Hubei Province.

Relationship between plant canopy characteristics and photosynthetic productivity in diverse cultivars of cotton(Gossypium hirsutum L.)

Genotype and plant type affect photosynthetic production by changing the canopy structure in crops.To analyze the mechanism of action of heterosis and plant type on canopy structure in cotton(Gossypium hirsutum L.),we had selected two cotton hybrids(Shiza 2,Xinluzao 43) and two conventional varieties(Xinluzao 13,Xinluzao 33) with different plant types in this experiment.We studied canopy characteristics and their correlation with photosynthesis in populations of different genotypes and plant types during yield formation in Xinjiang,China.Canopy characteristics including leaf area index(LAI),mean foliage tilt angle(MTA),canopy openness(DIFN),and chlorophyll relative content(SPAD).The results showed that LAI and SPAD peak values were higher and their peak values arrived later,and the adjustment capacity of MTA during the flowering and boll-forming stages was stronger in Xinluzao 43,with the normal-leaf,pagoda plant type,than these values in other varieties.DIFN of Xinluzao 43 remained between0.09 and 0.12 during the flowering and boll-forming stages,but was lower than that in the other varieties during the boll-opening stage.Thus,these characteristics of Xinluzao 43 were helpful for optimizing the light environment and maximizing light interception,thereby increasing photosynthetic capability.The photosynthetic rate and photosynthetic area were thus affected by cotton genotype as changes in the adjustment range of MTA,increases in peak values of LAI and SPAD,and extension of the functional stage of leaves.Available photosynthetic area and canopy light environment were affected by cotton plant type as changes in MTA and DIFN.Heterosis expression and plant type development were coordinated during different growth stages,the key to optimizing the canopy structure and further increasing yield.

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.

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

Introduction of Bacillus thuringiensis(Bt)gene does not reduce potassium use efficiency of Bt transgenic cotton(Gossypium hirsutum L.)

Background:Potassium(K)deficiency has become a common field production problem following the widespread adoption of Bacillus thuringiensis(Bt)transgenic cotton(Gossypium hirsutum L.)worldwide.The purpose of this study was to clarify whether the introduction of Bt gene directly reduces the K-use efficiency of cotton to induce K deficiency.Results:The cotton variety,Jihe 321(wild type,WT)and its two Bt(Cry1Ac)-transgenic overexpression lines(OE-29317,OE-29312)were studied in field with low soil-test K+(47.8 mg·kg^(−1)).In the field with low soil-test K+,only OE-29317 had less biomass and K+accumulation than the WT at some growth stages.Both Bt lines produced similar or even greater seed cotton yield than WT in the field.When the Bt gene(~70%)in OE-29317 and OE-29312 plants was silenced by virus-induced gene silencing(VIGS),the VIGS-Bt plants did not produce more biomass than VIGSgreen fluorescent protein(control)plants.Conclusions:The introduction of Bt gene did not necessarily hinder the K use efficiency of the cotton lines under this study.

Introgression of Gossypium klotzschianum Genome into Cultivated Cotton,G.hirsutum

Exotic Gossypium germplasm is a rich source of useful agronomic traits for improvement programs of cultivated cotton.Efficient use of genetic variation available in the wild relatives depends

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

Evaluation of genetic diversity in short duration cotton(Gossypium hirsutum L.)

Background: Cotton(Gossypium hirsutum L.) is an important fiber crop in Bangladesh. Genetic diversity among the genotypes of a germplasm has a great importance for cotton breeding. An experiment was carried out at the experimental field of Cotton Research, Training and Seed Multiplication Farm, Sreepur, Gazipur during the cropping season of 2015-2016 with 100 genotypes to evaluate genetic diversity of cotton genotypes for short duration using field performance.Results: The genotypes under study were grouped into ten clusters through multivariate analysis using GENSTAT-5.Cluster III contained maximum number of genotypes(16) while cluster X contained the least number of genotypes(7).The inter cluster distances were larger than intra cluster distances in all cases suggesting wider genetic diversity among the genotypes of different clusters. The maximum and minimum inter cluster distances were observed between clusters II and V(10.78) and clusters VIII and IX(3.30), respectively. The results indicated diverse and close relationship among the genotypes of those clusters. Earliness index, single boll weight and days to boll opening showed the higher contribution to the genetic divergence among 19 characters.Conclusion: Based on the results of genetic diversity and earliness index, the genotypes from cluster Ⅱ could be used as parent in hybridization program for the development of short duration cotton variety.

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.

陆地棉（G．hirsutumL．）茎尖分生组织培养及其在基因导入上的应用

陆地棉栽培品种的种子无菌发芽１～３天。将萌动种子的顶端分生组织培养在无激素或附加低浓度激素ＭＳ基本培养基上，直接再生完整小植株。从顶端分生组织到再生植株所需的最短培养时间约１０天。顶端分生组织供体种芽的萌芽天数及其所连接的胚轴长度对植株再生频率有明显影响，但与品种的关系较小，不同品种均能获得再生植株。应用基因枪轰击法将ＣＰＴⅠ基因和ＮＰＴⅡ基因的重组质粒导入到棉花茎尖分生组织，培养获得卡那霉素抗性株。

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。

施氮量对不同开花期棉(Gossypium hirsutum L．)铃纤维细度和成熟度形成的影响

为兼顾试验的重复性和生态区域性,选用高品质棉(科棉1号)和常规棉(美棉33B)品种为材料,于2005年分别在江苏南京(118°50'E,32°02'N,长江流域下游棉区)和江苏徐州(117°11'E,34°15'N,黄河流域黄淮棉区)设置施氮量(低氮N 0 kg/hm2;适氮N 240 kg/hm2;高氮N 480 kg/hm2)试验,研究施氮量对不同开花期棉铃纤维细度、成熟度和马克隆值形成的影响。结果表明,(1)施氮量显著影响棉纤维细度、成熟度和马克隆值的形成过程,但三者在不同开花期对氮素水平的响应不同,施氮量与开花期对棉纤维细度、成熟度和马克隆值的形成存在互作效应。8月10日前开花的棉铃,铃期[花后0～50 d(DPA)]日均温在23.3℃以上,纤维细度、马克隆值以N 0 kg/hm2施氮量下最大,棉纤维马克隆值与纤维细度的相关性较大;8月25日开花的棉铃(铃期日均温在20.8～23.3℃之间),纤维成熟度、马克隆值以N 240 kg/hm2施氮量下最大;9月10日开花棉铃(铃期日均温低于20.8℃),纤维细度、成熟度和马克隆值均以N 480 kg/hm2最大,棉纤维马克隆值与纤维成熟度的相关性增强。(2)影响不同开花期间纤维细度、成熟度和马克隆值的主要因素是铃期日均温;最终纤维细度、成熟度和马克隆值在不同施氮量之间的变异与不同开花期(铃期日均温不同)间的变异比较,前者显著小于后者。综上,因开花期不同而形成的铃期日均温是决定纤维细度、成熟度和马克隆值的最重要因素,施氮量可通过对位叶叶氮浓度NA影响棉纤维细度、成熟度和马克隆值的形成过程,增加施氮量可减小上述指标在不同开花期间的差异。

Evolution of Main Characteristics of the Registered Cotton Cultivars in National Cotton Regional Trials in Yangtze River Valley

[Objective] The aim of this study was to characterize the national regis- tered varieties selected from cotton regional trials in Yangtze River Valley （YaRV） in recent years. [Method] Cotton cultivar classification and comprehensive evaluation index were set up based on national cotton registration standard. GGE biplot method was adopted to analyze the correlation of major breeding target characters of 53 national registered cotton varieties in cotton regional trials in YaRV during 1981-2012. According to the shift of check cultivars in cotton regional trials in the past, the cotton regional trial practice since 1981 was divided into five periods. The dynamic of cultivar type＇s proportion and the evaluation index scores was analyzed across the five periods. [Result] There existed intricate interrelationship among cotton breeding target traits, which constrained it necessary to construct indices for com- prehensive evaluation of cotton varieties. The dynamic of cultivar types in the five periods indicated that type II varieties emerged since Simian 3 period and then its proportion decreased gradually; type Ⅲ varieties maintained a certain proportion in each period and kept on the rise overall; type Ⅳvarieties occupied the majority pro- portion of registered cultivars before 1993, but a minor proportion since Simian 3 period. On the other side, the change trend of the evaluation index demonstrated that the varieties registered before 2003 did not pass the qualified line at present. The peak scores appeared in the varieties registered during 2004-2008. The scores of the varieties registered after 2009 were only slightly over the qualified line. [Conclusion] More attention should be paid to the improvement and evaluation of micronaire, so as to guide the simultaneous development of high yielding and fiber quality in cotton breeding and registration procedure in YaRV.

Effect of N fertilization rate on soil alkalihydrolyzable N, subtending leaf N concentration,fiber yield, and quality of cotton

Soil alkali-hydrolyzable nitrogen, which is sensitive to N fertilization rate, is one of the indicators of soil nitrogen supplying capacity. Two field experiments were conducted in Dongtai(120°19″ E, 32°52″ N), Jiangsu, China in 2009 and Dafeng(120°28″ E, 33°12″ N), Jiangsu province, China in 2010. Six nitrogen rates(0, 150, 300, 375, 450, and 600 kg ha^(-1)) were used to study the effect of N fertilization rate on soil alkali-hydrolyzable nitrogen content(SAHNC), subtending leaf nitrogen concentration(SLNC), yield, and fiber quality. In both Dongtai and Dafeng experiment station, the highest yield(1709 kg ha^(-1)), best quality(fiber length 30.6 mm, fiber strength 31.6 c N tex^(-1), micronaire 4.82), and highest N agronomic efficiency(2.03 kg kg^(-1)) were achieved at the nitrogen fertilization rate of 375 kg ha^(-1). The dynamics of SAHNC and SLNC could be simulated with a cubic and an exponential function,respectively. The changes in SAHNC were consistent with the changes in SLNC. Optimal average rate(0.276 mg day^(-1)) and duration(51.8 days) of SAHNC rapid decline were similar to the values obtained at the nitrogen rate of 375 kg ha^(-1)at which cotton showed highest fiber yield, quality, and N agronomic efficiency. Thus, the levels and strategies of nitrogen fertilization can affect SAHNC dynamics. The N fertilization rate that optimizes soil alkali-hydrolyzable nitrogen content would optimize the subtending leaf nitrogen concentration and thereby increase the yield and quality of the cotton fiber.

Effects of Soil Water Content on Cotton Root Growth and Distribution Under Mulched Drip Irrigation

The relation between soil water content and the growth of cotton root was studied for the scheme of field water and cotton yield under mulched drip irrigation. Based on the field experiments, three treatments of soil water content were conducted with 90%, 75%θf, and 60%θf （θfis field water capacity）. Cotton roots and root-shoot ratio were studied with digging method, and the soil moisture was observed with TDR （time domain reflector）, and cotton yield was measured. The results indicated that the growth of cotton root accorded with Logistic growth curve in the three treatments, the cotton root grew quickly and its weight was very high under 75%θf because of the suitable soil water condition, while grew slowly and its weight was lower under 90%θf due to water moisture beyond the suitable condition, and the root weight was in between under 60%θf For the three water treatments, the cotton root weight decreased with soil depth, and decreased more significantly in deeper soil layer with the soil moisture increasing. And the ratio of cotton root weight in 0-30 cm soil layer to the total root weight was the highest under 75%θf. The cotton root system was distributed mainly in the soil of narrow row and wide row mulched with plastic film, and little in the soil outside plastic film. The weight of cotton root was the highest in the soil of narrow row or wide row mulched with plastic film under 75%θf. Root-shoot ratio decreased with the soil moisture increasing. The soil water content affected cotton yields, and cotton yield was the highest under 75%θf. The higher soil moisture level is unfavorable to the growth of cotton root system and yield of cotton under mulched drip irrigation.

Plant Growth,Yield Components,Economic Responses,and Soil Indigenous K Uptake of Two Cotton Genotypes with Different K-Efficiencies

HG103,a high K-efficiency cotton cultivar with high-yield potential,and LG122,a low K-efficiency cotton cultivar with low-yield potential were used to study the genotypic variation on yield,economic responses,and soil indigenous K exploitation for cotton in pot and field conditions in 2006 and 2007.Results indicated that both the cultivars differed significantly in the rate of translocation of dry matter into reproductive organs and the time of running into reproductive stage.Cotton yield and economic parameters of HG103 were much better than LG122.Potassium content in cotton shells reached the highest level at maturity stage for both genotypes.Results also showed that the K content in each plant part of HG103 was lower than that of LG122.The two genotypes showed different efficiencies and abilities on absorbing soil rapidly available K and slowly available K.LG122 showed higher absorption of K rate than HG103 after K fertilization,but on the contrary,HG103 uptake more soil indigenous K than LG122 in no K fertilization treatment.This result indicated that HG103 could tolerant and absorb more soil indigenous K than LG122,under condition of soil K stress.LG122 could grow well and absorb more K nutrient when soil K supply is sufficient.Our results demonstrated that higher K content in plant tissues and higher K uptake by plant may not an exclusive condition for high yield planting with K fertilization.Earlier translocation of more dry matters into reproductive organs is one of the key mechanisms of high K use efficiency in cotton.

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.