采用基于高分辨率熔解曲线技术的SNP标记定位徐州142无絮的短绒控制基因

【目的】定位徐州142无絮(XZ142w)突变体的短绒控制基因n2。【方法】以陆地棉(Gossypium hirsutum L.)徐州142(XZ142)×XZ142w的F2群体为研究对象,利用108个简单重复序列(Simple sequence repeat,SSR)标记对n2进行初步定位,再根据2个亲本材料中有单核苷酸多态性(Single nucleotide polymorphic,SNP)的差异基因设计50对SNP引物,用高分辨率熔解曲线(High resolution melting,HRM)技术从中筛选在亲本间有多态性的SNP引物,并用于后代的基因分型。【结果】利用108个SSR标记将n2初步定位在26号染色体的20.2c M的遗传区间内;用HRM技术筛选到9对亲本间有多态性的SNP引物,成功实现基因分型;并结合以SSR构建的连锁图谱,将n2的遗传区间缩小为19.5 c M,n2与最近的SNP标记Cricaas20158遗传距离为5.5 c M,且遗传图谱上的标记与四倍体陆地棉测序物理图谱基本一致。【结论】HRM技术可用于棉花中的SNP检测和n2基因的定位。     

SSCP Marker Development and Analysis of Candidate Restorer Gene Rf1 for Cotton Cytoplasmic Male Sterility

[Objective] Locating the cotton cytoplasmic male sterility (CMS) restorer gene Rf₁ is important for investigating restorer gene mechanisms and improving restorer lines. In our previous study, a gene cluster, with nine Pentatricopeptide repeat(PPR) genes and nine other genes, was found within the 160-kb Rf₁ target region in Scaffold 333. The objective here was to improve the density of Rf1-linked markers in the target region and determine the expression profiles of candidate genes. [Method] Using the sequences of the 18 genes, we designed 155 single-strand conformation polymorphism (SSCP) primers covering all of the gene sequences to identify the polymorphic SSCP markers between the fertile and sterile pools. Additionally, real-time polymerase chain reaction(PCR) was performed to analyze the expression profiles of eight candidate genes in the four developmental stages of buds of sterile, maintainer and restoring lines, respectively. [Result] In total, 15 polymorphic primers were identified. A genotype analysis of the F₂ population was conducted using the 15 primers and 3 other polymorphic simple sequence repeats (SSR) markers. The markers were distributed in a 4.8 cM range. In addition, owing to the influence of sterile cytoplasm or restorer genes, most of the genes showed different expression patterns in the four developmental stages of the three lines' buds. [Conclusion] SSCP markers tightly linked to Rf₁ were identified and the expression profiles of candidate genes were determined. This study provides a basis for the further fine mapping of restorer genes and for candidate gene screening.     

与亚洲棉光籽突变体DPL972中光籽基因紧密连锁的SSR分子标记的开发与应用

对棉纤维相关性状基因的克隆及遗传分析可为阐明纤维形成机制奠定良好基础.以野生型材料DPL971及其光籽突变体DPL972为亲本构建F2群体,其中光籽与毛籽性状分离比符合3∶1,表明突变体DPL972中的光籽基因为显性单基因(将其命名为GaFzl).结合棉花基因组数据,合成1567对SSR(Simple sequence repeat)引物,覆盖了A组13条染色体,标记多态性分析和群体连锁分析发现在染色体A08上存在15对与光籽基因GaFzl连锁的标记,其中最近的标记为SSR82,遗传距离为6.6 cM.本实验完成了GaFzl基因的染色体初步定位,开发了15个与其连锁的SSR标记,为下一步图位克隆奠定了基础.     

陆地棉光敏雄性不育基因ys-1的遗传分析与初步定位

【目的】精细定位和克隆陆地棉光敏雄性不育基因。【方法】从陆地棉中040029的航天诱变后代中选育出新型光敏雄性不育系中9106,以中9106与11个陆地棉材料配制杂交组合,初步分析了中9106的杂种优势。以中9106为母本与陆地棉乐土603构建遗传分离群体F1、F2,分析不育性状的遗传特征。利用集团分离分析法筛选简单序列重复(Simple sequence repeat,SSR),并在包含186个单株的中9106×乐土603 F2群体中用上述SSR初步定位不育基因。【结果】中9106为母本×陆地棉乐土603的F1单株育性均表现正常,而F2群体中的可育单株数∶不育单株数符合3∶1的分离比,推测中9106的不育性状受1对隐性核基因控制。利用集团分离分析法从16 544对SSR中筛选到18对与该不育基因连锁的标记。利用中9106×乐土603的F2群体和18对SSR标记,将不育基因定位于D12染色体,位于标记NAU3442与CGR6339之间,遗传距离均为0.2c M,将该不育基因命名为ys-1。【结论】本研究有助于ys-1的精细定位及其克隆。     

基于两个陆地棉低世代群体定位纤维品质相关QTL

【目的】定位棉花纤维品质性状相关的数量性状位点(Quantitative trait locus,QTL)。【方法】以陆地棉高强纤维品系中棉所679和纤维品质一般的农垦5号为亲本构建包含200个单株的F2群体及对应的F2:3家系群体,对2个群体的纤维长度、断裂比强度等5个纤维品质性状进行检测。用6 688对简单重复序列(Simple sequence repeat, SSR)引物在双亲间筛选,得到149对多态性引物,以F2为作图群体,使用QTL IciMapping软件进行连锁图谱构建,并对F2及F2:3群体进行QTL定位。【结果】根据F2群体基因型信息构建了1张包含119个标记、28个连锁群、总长为1 173.5 cM(centiMorgan)的遗传连锁图谱。分别在F_2、F2:3群体中检测到9个和11个与纤维品质性状相关的QTLs,这些QTLs分布在11个连锁群上。其中F2群体的qFL-D11-1、q BT-D11-1与F2:3群体的qFL-D11-1、q MIC-D11-1均定位在标记DPL0062与HAU0423之间,推测这些位点可能是控制纤维品质性状的重要QTL。【结论】利用多个群体进行QTL定位有益于发现稳定的QTL位点,控制纤维品质性状的基因可能成簇存在,为挖掘纤维品质性状相关基因及分子标记辅助育种奠定基础。     

Identification of Resistance to Tetranychus turkestani and Analysis of Leaves Surface Wax of Different Cotton Varieties (Lines) in Xinjiang

[Objective] Based on the representative 236 cotton varieties in Xinjiang, the resistance of cotton to mites was identified, and the waxy content and waxy components of leaves were analyzed to lay a foundation for cotton breeding. [Method] In the cotton seedling stage, the waxy content of the leaves of different cotton varieties was determined, and the waxy components of the leaves were analyzed by gas chromatography-mass spectrometry. [Result] Most cotton varieties are sensitive to the damage of cotton mites. There were 2 high resistance varieties, 3 resistant materials, 22 medium resistant materials, 207 sensitive materials and 2 high-sensitivity materials in 236 cotton materials. The waxy content on the surface of cotton leaves was significantly negatively correlated with leaf damage levels. The waxy components of cotton leaves were mainly alkane compounds, containing a small amount of esters, ethers and fatty acids. The content of alkanes and esters in waxy leaves of high-resistance cotton germplasm materials was higher than that of high-sensitivity materials. The compound with the largest difference in content are C₂₄H₅₀, C₃₅H₇₂ and C₁₇H₃₆. [Conclusion] These results lay a foundation for further study on the effects of waxy content on host selection and mite resistance of cotton mites.     

Analysis on Competitive Heterosis of Hybrid F1 and F2 between Transgenic Bt Varieties of Upland Cotton

[Objective] The purpose of this study is to supply basis for selecting strong heterosis hybrid and the feasibility of F₂ utilization, by evaluating yield heterosis and fiber quality traits of hybrid F₁ and F₂ generated by transgenic Bt varieties in upland cotton. [Method] Ten upland cotton varieties (lines) with good comprehensive characteristics were selected to produce 10 crosses, and four of them with better competitive heterosis (CH) in yield were screened out to analyze the lint yield and fiber traits of F₁ and F₂. [Result] The results indicated there was a strong CH in yield of F₁, but the yearly variation was large. Hybrids performed better in the poor production year. The CH in yield decreased obviously in most of the F₂ combinations. Further analysis showed that the decrease of boll weight was the main cause of drop of yield CH in F₂. The fiber quality index in F₂ showed a downward trend compared with F₁, and the coefficient of range and variation(CV) increased. The coefficient of variation of quality traits was much lower than that of yield components, with a trend of F₁ < SCRC 28 (CK) and F₂ > F₁. The CH of lint yield and fiber traits changed between F₁ and F₂ in different combinations. We screened out a combination with higher CH of yield in both F₁ and F₂, and with less decreased yield CH, fine fiber quality and light separation in F₂. [Conclusion] The hybrids generated by transgenic Bt varieties in upland cotton characterized by strong adversity resistance and yield stability. Fine fiber quality and light separation in F₂ came from crosses with the parents of fine fiber quality and small differences. So, it is possible for breedersto select valuable F₂ combinations in production.     

QTL Mapping of Fiber Quality in Low-Generation Upland Cotton Populations and Functional Annotation of Candidate Genes

[Objective] This article aims to provide a theoretical basis for molecular marker-assisted breeding by quantitative trait loci (QTLs) mapping and gene function annotation of fiber quality. [Method] F₂ populations were constructed using the two cotton cultivars (lines), CCRI 49 and 396289, as parents. Based on high-density genetic maps, the F_2:3 families with three environments were included in the F2 population. The QTL mapping of seven fiber quality traits including fineness and maturity, etc., was performed using the Clusters of orthologous groups (COG), Gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) databases to annotate QTLs for gene function. [Result] A total of 157 QTLs related to fiber quality were obtained and distributed on 20 chromosomes. QTLs with more traits on A03, A04, D02, A11 and D07 chromosomes clustered and may be the key chromosomes controlling fiber quality traits. A total of 13 stable QTLs were obtained, of which qFL-A03-1 and qFin-A11-4 were repeated in three environments, and nine other QTLs were repeated in two environments. And 4 763 candidate genes were annotated, with 2 416, 4 188, and 2 512 genes being annotated in COG, GO, and KEGG, respectively. Among them, 429 genes were annotated in stable QTLs. Some of these genes may be closely related to fiber quality. [Conclusion] The high-density genetic map obtained by high-throughput sequencing can help to obtain more QTLs, which is beneficial to the screening of candidate genes related to fiber quality and the improvement of fiber traits, and improves the breeding efficiency.     

利用马克隆值差异显著的BILs鉴定棉纤维品质相关基因

【目的】马克隆值是衡量棉纤维品质的重要指标之一。利用马克隆值存在显著差异的2个陆海回交近交系材料进行高通量基因芯片分析,旨在筛选和鉴定棉纤维发育相关的关键基因。【方法】从SG747(Gossypium hirsutum L.)和Giza75(Gossypium Barbadense L.)高世代回交近交系(Backcross Inbred Lines,BILs)群体中选取NMGA-140(马克隆值6.2)和NMGA-051(马克隆值4.0)为材料,利用Affymetrix公司的棉花寡聚核苷酸基因芯片对其开花后10 DPA(Days after anthesis)的棉纤维进行了表达谱分析。【结果】获得了2655个差异表达基因,包括功能预测基因(15.57%),翻译、核糖体结构与生物合成相关基因(13.54%)和翻译后修饰、蛋白质转换、分子伴侣相关基因(9.31%)等。推测β-tub10正向调控棉纤维发育过程,β-tub1负向调控棉纤维发育过程,Ghi.3578.1.S1_s_at在棉纤维发育早期起到了作用。【结论】为棉花纤维品质的遗传改良提供了丰富的基因资源,为分子标记辅助育种开发更多新的分子标记。     

Optimization of the Fermentation Medium and Analysis of the Compositions of Antifungal Compounds from Bacillus malacitensis Z-5

[Objective] Bacillus malacitensis Z-5 has a significant biocontrol effect on cotton Verticillium wilt mainly through its production of antifungal substances. This study aimed to optimize the culture medium’s composition. Additionally, it aimed to identify the genes that produce the antifungal substances and determine the components of these antifungal substances. [Methods] Single factor tests were used to determine the optimal carbon and nitrogen sources, as well as the inorganic salt level. The most rapid rise method was used to determine the carbon and nitrogen sources and the inorganic salt’s optimal content area. Box–Behnken response surface methodology was applied, and the composition of the medium that produced the optimum level of antifungal substances was determined using antifungal activity as the screening index. The composition of the antifungal substances and the related genes from the Z-5 strain were identified using liquid chromatography-mass spectrometry and polymerase chain reaction, respectively. [Results] The optimal medium for the generation of antifungal substances by the Z-5 strain consisted of 2.68% (mass fraction) corn flour, 1.45% yeast powder, 0.03% K₂SO₄, 0.8% Na₂HPO₄·12H₂O, and 0.2% NaH2PO4·2H₂O at pH 7.2–7.4. The antifungal zone was 1.34 cm in diameter, which was close to the predicted value, confirming that the model was reliable and effective. Antifungal substances were mainly lipopeptide antibiotics C14 surfactin B, C14–C17 iturin A, and C14, C15, and C17 iturin B. polymerase chain reaction revealed that srfAB, ituC, and ituD occur in the Z-5 genome. [Conclusion] This study optimized the culture medium for producing antifungal substances, which are composed of lipopeptide antibiotics, from the B. malacitensis Z-5 strain. The results provide a theoretical reference and material basis for the prevention of cotton Verticillium wilt using these antifungal substances.     

Identification and Analysis of Fiber Quality and Yield Related Traits of Interspecific (Gossypium hirsutum L. × G. barbadense L.) Hybrids

[Objective] The aim of this study is to study the hereditary of heterosis of fiber quality and yield-related traits in the upland-island interspecific hybrids, and breed new interspecific hybrid varieties with high yield and fine fiber quality. [Method] In this study, 12 upland cotton materials and 5 sea-island cotton materials were selected to determine the fiber quality and yield traits of their parents and F₁ in Lin’an, Zhejiang and Sanya, Hainan. [Result] It was found that fiber length and fiber strength of F₁ (Gossypium hirsutum × G. barbadense) generally had significant mid-parent heterosis (MPH), some hybrid combinations showed strong over-parent heterosis (OPH), fiber length had a small coefficient of variation between the two places and could be stably inherited. And in terms of yield, seed cotton weight, lint weight, and lint percentage of some upland-island hybrids had MPH, but they were still significantly lower than those of upland cotton parents. [Conclusion] Two long-staple cotton hybrid combinations T035 and T044 with 5A grade high-quality were obtained, and an excellent material of G. barbadense Ta10-280 was screened. This study provides valuable data for the genetic law of fiber quality heterosis of upland-island hybrid cotton.     

Construction of RNAi Vector of Cotton (Gossypium hirsutum L.) FAD2-1 Gene and Acquisition of New High Oleic Acid Germplasm

[Objective] Here, the aim was to create a new cotton (Gossypium hirsutum L.) germplasm with high oleic acid and low linoleic acid contents without affecting the fiber yield and quality. [Method] The fatty acid desaturation 2-1 (FAD2-1) gene catalyzes the formation of polyunsaturated linoleic acid from monounsaturated fatty acid in cotton. The protein’s properties, structure and functions were analyzed using the information analysis method. A conserved 381-bp fragment of GhFAD2-1 was cloned to construct an RNA interference vector, which was transformed into cotton by Agrobacterium-mediated hypocotyl infection. The fatty acid compositions and contents of T₂―T₄ transgenic plants were determined using gas chromatography-mass spectrometry. The T₄ transgenic lines were used to investigate the copy number, target gene expression, agronomic traits, and fiber quality. [Results] The RNA interference vector of GhFAD2-1 was successfully constructed and transformed into cotton. The target gene’s expression level was significantly lower in transgenic plants than in controls. The transgenic plants had high oleic acid and low linoleic acid contents, which could be stably inherited by the offspring. The oleic acid contents of transgenic cotton seeds increased by 224.1%, and the linoleic acid content decreased by 237.5%, compared with the control seeds. There were no significant differences in the agronomic traits and fiber quality of the transgenic lines compared with the control. [Conclusion] These results verified the function of GhFAD2-1 in cotton and provide a basis for breeding cotton varieties with high oleic acid contents.     

应用病毒诱导基因沉默技术研究棉花GhAKT1和GhKT2基因的功能

【目的】本研究旨在揭示棉花钾离子(K~+)吸收机制。【方法】以国欣棉3号和中棉所41为材料,应用病毒诱导基因沉默技术研究棉花K~+通道基因GhAKT1和K~+转运体基因GhKT2的功能。【结果】在低水平供K~+(0.1 mmol·L~(-1))和充分供K~+(2.5 mmol·L~(-1))条件下,沉默GhAKT1和GhKT2基因使棉花幼苗的K~+积累量分别降低15%左右和25%以上。此外,沉默GhAKT1和GhKT2基因导致棉花幼苗对K~+的最大吸收速率和亲和能力出现不同程度的下降。药理学试验结果表明,在外界K~+浓度低于250μmol·L~(-1)的条件下,高亲和系统对棉花K~+吸收的贡献大于K~+通道;而且GhAKT1蛋白可能是1个对NH4+比较敏感的组分。【结论】棉花K~+通道蛋白GhAKT1和K~+转运体蛋白GhKT2具有吸收K~+的功能,而且表现出双亲和吸收K~+的特性,可作为改善棉花钾营养的候选基因。     

陆地棉光合相关性状的QTL定位分析

光合作用是棉花产量的主要物质来源。本研究以高光效陆地棉冀优861和低光效陆地棉新陆早25号为亲本组配的196个F₂单株为作图群体,利用SSR（Simple Sequence Repeat）标记构建陆陆杂交遗传连锁图谱,共有30个标记位点连锁,包含4个连锁群,全长244.4cM。利用QTL IciMapping 4.1软件的完备区间作图法对冀优861×新陆早25号F_2:3家系的光合相关性状进行QTL作图分析,共定位到光合相关5个性状的10个QTLs,其中1个光合速率QTL和1个胞间CO₂浓度QTL分别定位在D3和D7染色体上。本研究为棉花光合相关性状QTL的精细定位及分离克隆打下基础,为聚合棉花高光效分子标记辅助育种提供理论依据。     

Molecular mapping of a new red mutant gene (Rs) in upland cotton

In recent years, there has been slow progress in improving cotton yield. It is known that the F₁ generation from the cross of the new red mutant and the normal green leaf plant has high photosynthetic efficiency. Therefore, cloning the new red mutant gene and further introducing it into other crops through transgenic techniques is a promising approach for achieving high photosynthetic efficiency through breeding. To map this new mutant gene, tentatively named R _s , the authors constructed an F₂ generation containing 1214 individual plants from mutant EH083 ( Gossypium hirsutum ) and Hai 7124 ( Gossypium barbadense ). Fifty-five pairs of simple sequence repeats and sequence-related amplified polymorphism (SRAP) primers on chromosome 7 were selected to screen the two parents. Finally, the R _s gene was mapped at the 0.3 cM interval flanked by markers NAU3735 and NAU1048.     

光子陆地棉种质资源主要性状的遗传评价

以102份光子陆地棉材料为母本,分别与遗传标准系TM-1杂交,获得102个F₁群体。采用随机区组设计,设置3个重复,对光子陆地棉材料主要性状进行遗传评价。结果表明,调查的11个性状表型差异均较大,材料间产量性状(株高、果枝数、铃数、铃重、衣分和子指)差异大于纤维品质性状(纤维长度、纤维强度、马克隆值、整齐度和伸长率),特别是衣分、铃数等性状差异更明显; 除果枝数、马克隆值、伸长率以外,光子亲本群体其他性状的平均值都小于F₁群体。而亲本群体所有性状的变异系数均大于F₁,不同光子材料的杂种优势有很大差别,中亲优势和超亲优势也有很大的差别,有些种质某些性状的中亲、超亲优势为负值,其后代性状表现劣势; 纤维品质性状的中亲、超亲优势与毛子程度均呈负相关,而产量性状的中亲、超亲优势与毛子程度均呈正相关,说明可利用光子材料杂种优势改良纤维品质,而其后代产量性状的杂种优势利用受到限制; SSR分子标记遗传相似系数与各个性状的中亲、超亲优势的相关都不显著,说明在光子材料的育种中,杂种优势是不能通过亲本之间的遗传背景相似程度来预测的。