WO2021017608A1 - 同时鉴定棉花细胞质雄性不育恢复基因Rf1、Rf2的InDel标记 - Google Patents

同时鉴定棉花细胞质雄性不育恢复基因Rf1、Rf2的InDel标记 Download PDF

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WO2021017608A1
WO2021017608A1 PCT/CN2020/093184 CN2020093184W WO2021017608A1 WO 2021017608 A1 WO2021017608 A1 WO 2021017608A1 CN 2020093184 W CN2020093184 W CN 2020093184W WO 2021017608 A1 WO2021017608 A1 WO 2021017608A1
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cotton
cytoplasmic male
male sterility
restorer
indel
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PCT/CN2020/093184
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English (en)
French (fr)
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邢朝柱
冯娟娟
吴建勇
张梦
张学贤
郭立平
戚廷香
唐会妮
王海林
乔秀琴
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中国农业科学院棉花研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/022—Genic fertility modification, e.g. apomixis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/022—Genic fertility modification, e.g. apomixis
    • A01H1/023—Male sterility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/60—Malvaceae, e.g. cotton or hibiscus
    • A01H6/604—Gossypium [cotton]
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the present invention relates to a molecular marker and its application, in particular to an InDel molecular marker linked with two sets of cytoplasmic male sterility three-line material restorer line restorer gene of cotton and its application. Therefore, it can establish a simple and feasible method that can identify two sets at the same time Cotton restores gene type and hybrid purity system.
  • Cotton is an important economic crop in the world and an important fiber energy crop. It has an important position and significance in agricultural production. Breeders have used cotton's heterosis to significantly increase cotton yield, quality, and resistance. Hybrid seed production is an effective way to utilize heterosis, and cotton hybrid varieties increase the yield by about 15% compared with conventional varieties. In production practice, the common methods for cotton hybrid seed production are artificial emasculation and pollination, chemical sterilization and "three-line matching" method.
  • the artificial emasculation and pollination method has become more expensive due to the increasing labor costs year by year, and the purity of hybrids is low, and the seed quality cannot be guaranteed;
  • the chemical emasculation method can reduce the manpower and material input of manual emasculation, but the effect of the emasculation is unstable and easy Cause environmental pollution;
  • the use of cytoplasmic male sterility three-line matching method for seed production has the advantages of simple procedures, low labor input, reduced material consumption, high seed purity, and low cost. It is suitable for large-scale seed production, which will become a commercial hybrid cotton production system.
  • the promotion of three-line hybrid cotton is of great significance for increasing the efficiency of cotton planting, increasing the income of cotton farmers, stabilizing the planting area of hybrid cotton, and developing the cotton processing industry.
  • CCS-D 8 Trifid cotton cytoplasmic male sterility
  • CMS-D 2 Polyfid cotton cytoplasmic male sterility
  • the process of breeding a three-line hybrid combination with a strong advantage is slow. It is difficult to obtain restorer lines with excellent resilience for comprehensive agronomic traits of cotton.
  • the reasons for the lack of germplasm resources of the parents of excellent restorer lines are as follows: Cotton cytoplasmic male sterility restorer genes are narrow and recovered The ability is limited, the mechanism of nucleus-plasma interaction is not clear, and the related physiological mechanism of fertility restoration has not been studied clearly.
  • the restorer genes of cotton come fromhackneysian cotton and it is difficult to locate and clone cotton. Therefore, the long-term goal and important task of three-line hybrid cotton breeding is to breed and improve excellent restorer lines with strong resilience, expand the germplasm resources of three-line hybrid breeding parents, and enrich the parent combination of cotton three-line seed production.
  • Develop the InDel marker which is closely linked and stable inheritance of cotton restorer genes, for the selection of excellent restorer lines and the production of hybrids, to provide markers for molecular-assisted selection breeding to co-segregate with target genes, and to accelerate the accumulation of restorer genes with the help of molecular marker-assisted breeding .
  • the invention provides an InDel molecular marker co-separated with a cotton cytoplasmic male sterility restorer gene and a primer of the InDel molecular marker to solve the problems existing in the selection of restorer lines and improved restorer lines by traditional conventional breeding methods.
  • the invention can distinguish the restorer genes of two sets of cytoplasmic male sterility restorer lines of cotton at the same time by using the same marker, and can identify the hybrids of different three lines and detect the purity of the hybrids.
  • the present invention constructs a system for identifying whether cotton contains restoring genes and distinguishing the types of restoring genes (Rf1/Rf2) to ensure the purity of hybrids.
  • the InDel marker provided by the present invention is not only linked with the cytoplasmic male sterility restoring gene Rf1 of Ralphnesi cotton but also linked to the cytoplasmic male sterility restoring gene Rf2 of trifid cotton.
  • the insertion/deletion length polymorphism of the nucleotide sequence The sex display on the agarose gel electrophoresis chart can identify whether a single cotton plant contains restorer genes and determine the type of restorer genes.
  • the present invention also provides a pair of InDel molecularly labeled primers, characterized in that the upstream primer is: 5'-ACTCAACTGTCTTCCTCTTCTCA-3' (SEQ ID NO. 1); the downstream primer is: 5'-TGGGAGCCTAATATATGTAATGGTG-3' (SEQ ID NO.2).
  • the present invention also provides the application of the above-mentioned InDel molecular marker primers in the identification of the cytoplasmic male sterility restorer gene Rf1 and the trifid cotton cytoplasmic male sterility restorer gene Rf2 of Winney West cotton.
  • the invention also provides the application of the above-mentioned InDel molecular marker primers in the selection of new restorer line materials of cotton with different restorer genes (Rf1/Rf2).
  • the present invention also describes a feasible method for identifying the cytoplasmic male sterility restorer gene Rf1 and the trifid cotton cytoplasmic male sterility restorer gene Rf2 of Ralphenisi cotton by using the above-mentioned InDel molecular marker primer, which is characterized by:
  • the PCR product agarose gel electrophoresis results showed that two bands appeared. If the specific amplified bands are displayed at 376bp and 408bp, it is a material containing the heterozygous site of the Rf2 restorer gene for trifid cotton cytoplasmic male sterility; if it is at 376bp.
  • the specific amplified bands at 562bp and 562bp are the materials containing the heterozygous locus of the cytoplasmic male sterility restorer gene Rf1 of hackney West cotton;
  • the PCR product agarose gel electrophoresis result shows a band. If only a specific amplified band is displayed at 376 bp, it is a material that does not contain restorer gene sites; if only a specific amplified band is displayed at 408 bp, it is a material containing The restorer line material of the Rf2 homozygous locus of the cytoplasmic male sterility restorer gene in trifid cotton; if the specific amplified band is only displayed at 562bp, it is a homozygous locus containing the Rf1 cytoplasmic male sterility restorer gene of
  • the restoration system materials If only a specific amplified band is displayed at 376 bp, it is a material that does not contain restorer gene sites; if only a specific amplified band is displayed at 408 bp, it is a material containing The restorer line material of the Rf2 homozygous locus of the cytoplasmic male sterility restorer gene in trif
  • the identification method provided by the present invention is used for the identification of the restorer genes of the germplasm resources of the two types of cytoplasmic male sterility matching restorer lines of cotton, which can significantly reduce the identification workload of field materials, improve the accuracy, and ensure the restorer line materials And cotton restores the purity of seeds.
  • This method is used for the selection and improvement of restorer lines, can speed up the selection process, reduce breeding costs, and improve identification efficiency.
  • This method is used for three-line hybrid seed production, can ensure the purity of three-line hybrid seeds, and clarify the types and genotypes of restoration genes contained in the materials.
  • This InDel marker can identify two sets of different restorer gene types of cytoplasmic male sterility in cotton, and can also identify whether the restorer gene locus is contained and whether the restorer gene locus is homozygous, so it is better than detecting a set of cytoplasmic male sterility alone.
  • Male sterility has a wider range of applications (especially suitable for the case of two sets of cytoplasmic male sterility matching), and better practicability (there is no need to distinguish which set of cytoplasmic male sterile materials before use, and the identification results can be distinguished Cytoplasmic male sterility restorer line).
  • InDel labeling has the advantages of simplicity, reliability, repeatability, and easy identification. It can be detected only by PCR amplification and agarose gel electrophoresis.
  • the present invention uses a pair of InDel marker primers to distinguish the types of restorer genes (Rf1/Rf2) contained in restorer line materials, three-line hybrids, and materials that do not contain restorer genes at the molecular level, and are independent of plant growth period and environment The influence of factors has improved the accuracy and efficiency of restoration gene identification.
  • InDel marker plays an important role in the selection of new restorer line materials of cotton with different restorer genes (Rf1/Rf2) and the guarantee of the purity of three-line hybrid seeds. Compared with conventional breeding, InDel marker saves a lot of manpower and material resources. In the process of improvement of restorer lines, construction of new restorer lines, and three-line hybrid seed production, the workload of field character investigation and multi-band backcross test can be reduced appropriately.
  • the InDel primers are used for specific amplification. It can be identified by agarose gel electrophoresis. The entire identification and detection process only takes about 3 hours, which is short, efficient and the identification results are more accurate than field character surveys.
  • InDel markers Compared with SSR markers and CAPS markers, InDel markers have the characteristics of large distribution density, higher accuracy, better reproducibility, and easy identification. When PCR amplification is performed with the InDel marker primers of the present invention, there is no Non-specific band amplification.
  • the InDel molecular marker of the present invention has a good prospect in the research of molecular-assisted selection breeding of restorer lines containing different restorer genes in cotton, and the present invention has great practical value in the seed purity identification work of commercial cotton hybrid seed production.
  • Figure 1 shows the sequence alignment results of the three-line cytoplasmic male sterile material of Tri-fission cotton, the three-line cytoplasmic male sterile material of Ralphney West cotton and the reference genome, where TM-1 is the reference genome sequence of upland cotton;
  • A8 , B8, R8, A2, B2, and R2 are the sterile lines, maintainers, and restorer lines that match the cytoplasmic male sterility of trifid cotton, and the sterile lines that match the cytoplasmic male sterility of Hacknesi cotton, Sequences of the corresponding differential fragments on the D05 chromosome in the maintainer and restorer materials;
  • Figure 2 shows the results of PCR amplification and agarose gel electrophoresis for the parents and heterozygotes of the two sets of cytoplasmic male sterility of cotton with InDel labeling.
  • Lanes 1-8 are the cytoplasmic male sterile line, maintainer, restorer line and heterozygote containing the Rf2 restorer gene, and the cytoplasmic male sterile line, maintainer line, Restorer lines and heterozygotes containing the Rf1 restorer gene.
  • Figure 3 shows the identification results of InDel markers on 24 randomly sampled trifid cotton male sterile restorer line backcross generation population materials (BCF1), of which lane 2 is a single plant heterozygous for restorer gene Rf2; lane 1 is no restoration Gene Rf2 single plant.
  • BCF1 trifid cotton male sterile restorer line backcross generation population materials
  • Figure 4 is the identification result of InDel marker on the F2 population material of 24 randomly sampled Ralphenisi cytoplasmic male sterile restorer lines.
  • Lane a is a single plant homozygous for restorer Rf1; lane b is heterozygous for restorer Rf1 Individual plants, lane c is individual plants without restorer gene Rf1.
  • the sequence alignment results of the three-line cytoplasmic male sterile material of Tri-crack cotton, the three-line cytoplasmic male sterile material of Hackenisi cotton and the reference genome are shown in Figure 1. It can be seen from Figure 1: The nucleic acid sequence with indel variation in the cytoplasmic male sterile restorer line (R8), its sequence length is 408 bp; the nucleic acid sequence with indel variation in the Ralphnesi cotton cytoplasmic male sterile restorer line (R2), its sequence length 562bp; the sequence length of the sterile line and the maintainer line are both 376bp.
  • primers were designed upstream and downstream, and they were successfully converted into InDel labeled primers.
  • the primer pairs labeled with the InDel molecule were amplified as shown in Table 1:
  • BC 1 F 1 The restorer and maintainer lines of the backcross generation population (BC 1 F 1 ) constructed by the three-line male sterile cotton (CMS-D8) and the three-line male sterile cotton (CMS-D 2 ) of Farmney West (Sterile line) Extraction of seed DNA of hybrid F 2 population (CTAB method).
  • the PCR program is: pre-denaturation at 94°C for 2min; denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 20s, 35 cycles; then final extension at 72°C for 2min and storage at 4°C.
  • the PCR products were subjected to agarose gel (2.5%) electrophoresis, photographed and stored with a gel imaging system, and band analysis was performed on the electrophoresis results.
  • Trifid cotton cytoplasmic male sterile material In statistics, only the 408bp band appears as a restorer line containing the pure restorer gene Rf2, that is, the trifid cotton cytoplasmic male sterility; if there are both 376bp and 408bp bands, then it contains The materials or hybrids that are heterozygous for the restorer gene Rf2; only the 376bp band appears, that is, the materials (maintainers or sterile lines) that do not contain the restorer gene Rf2.
  • the restoration gene can be identified simply and quickly, the type of restoration gene (Rf2/Rf1), and the genotype of the material can be distinguished. It can ensure the purity of the restorer line in the process of three-line hybrid seed production, can significantly reduce the workload of field material identification, accelerate the work process of conventional hybrid breeding, greatly improve the breeding efficiency and ensure the purity of the three-line hybrid seed, and improve cotton Yield.

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Abstract

本发明公开了一种同时鉴定棉花细胞质雄性不育恢复基因Rf1、Rf2的InDel标记。该InDel标记，不仅与哈克尼西棉细胞质雄性不育恢复基因Rf1连锁而且与三裂棉细胞质雄性不育恢复基因Rf2连锁，该核苷酸序列的插入/缺失长度多态性呈现在琼脂糖凝胶电泳图上可鉴定棉花单株是否含有恢复基因且能判定含有恢复基因的类型，该InDel标记由SEQ ID NO.1-2所示的引物扩增得到。该InDel标记对不同恢复基因(Rf1/Rf2)的棉花新恢复系材料的选育和保证三系杂交种种子纯度方面具有重要作用。本InDel标记可以鉴定棉花的两套细胞质雄性不育的不同的恢复基因类型，其适用范围更广，实用性更好。

Description

同时鉴定棉花细胞质雄性不育恢复基因Rf1、Rf2的InDel标记 技术领域
本发明涉及一种分子标记及其应用，具体涉及一种与棉花的两套细胞质雄性不育三系材料恢复系恢复基因连锁的InDel分子标记及应用，因此可建立简单可行的可同时鉴定两套棉花恢复基因类型和杂交种的纯度体系。
背景技术
棉花是世界上重要的经济作物也是重要的纤维能源作物，在农业生产中具有重要的地位和意义。育种家已经利用棉花的杂种优势显著提高棉花产量、改良品质、提高抗性。杂交制种是利用杂种优势的有效方式，棉花杂交品种比常规品种增产约15％。在生产实践中，棉花杂交制种的常用方法为人工去雄授粉法、化学杀雄法和“三系配套”法。人工去雄授粉法由于人工费用逐年增高成本越来越高，且杂交种纯度低，无法保证种子质量；化学杀雄法，可减少人工去雄的人力物力投入，但杀雄效果不稳定且易造成环境污染；利用细胞质雄性不育三系配套法制种程序简便，人力投入小、减少物力消耗、种子纯度较高、成本较低等优点，适合大面积制种，这将成为杂交棉商业化制种最优途径之一。推广三系杂交棉花，对于增加植棉效益，提高棉农收入，稳定杂交棉种植面积，发展棉花加工业等都具有重要意义。
目前研究较多的两套细胞质雄性不育材料为三裂棉细胞质雄性不育(CMS-D 8)和哈克尼西棉细胞质雄性不育(CMS-D 2)。选育强优势三系杂交组合的过程缓慢，棉花综合农艺性状优良恢复力强的恢复系很难获得，优良恢复系亲本种质资源匮乏的原因如下：棉花细胞质雄性不育恢复基因来源狭窄，恢复能力受限制，质核互作机理还未清晰，育性恢复的相关生理机制还未研究清楚，棉花的恢复基因来源于哈克尼西棉和三裂棉难以定位和克隆。因此，现在三系杂交棉品种选育的长期目标及重要任务是选育和改良强恢复力的优良恢复系和扩充三 系杂交育种亲本的种质资源，丰富棉花三系制种的亲本组合。开发与棉花恢复基因紧密连锁稳定遗传的InDel标记用于优良恢复系的选育和杂交种的生产，为分子辅助选择育种提供与目标基因共分离的标记，借助分子标记辅助育种加速恢复基因的积累。
目前，育种家通常采用常规杂交技术与分子标记辅助育种相结合的方法对优良恢复系进行选育和改良，弥补了传统育种的选育周期长，占地面积大，田间工作量大，效率较低等缺点。而且，在三系杂交制种过程中的关键是保证恢复系的纯度，杂交种是恢复系与不育系杂交获得的F1代，一旦混杂，将会造成杂交种出现不育的后果，降低杂交种纯度，杂交种检验不合格，造成制种农户和制种公司的经济损失。分子标记不受植株发育阶段、生长环境因素以及基因是否表达的影响，具有数量非常丰富，稳定遗传等特点可以从DNA水平反映出核苷酸序列的差异。因此，开发稳定准确易检测的共显性InDel分子标记运用于分子标记辅助选择技术，能够解决传统常规育种过程中田间性状鉴定的耗时、费力、准确性差等缺点，可在种子和植株任何生育时期鉴定单株含有恢复基因的类型和基因型，提高效率，保证恢复系种子的纯度，缩减育种时间，以满足生产实践需求。
发明内容
本发明提供了一种与棉花细胞质雄性不育恢复基因共分离的InDel分子标记与该InDel分子标记的引物，来解决传统常规育种方法选育恢复系和改良恢复系所存在的问题。本发明利用同一标记能够同时区分棉花的两套细胞质雄性不育恢复系的恢复基因，且可以鉴定不同三系配套的杂交种，还可以检测杂交种的纯度。本发明构建了一个鉴定棉花是否含有恢复基因且能区分恢复基因类型(Rf1/Rf2)，保证杂交种纯度的体系。
本发明提供的一种InDel标记，不仅与哈克尼西棉细胞质雄性不育恢复基因Rf1连锁而且与三裂棉细胞质雄性不育恢复基因Rf2连锁，该核苷酸序列的插入/缺失长度多态性呈现在琼脂糖凝胶电泳图上可鉴定棉花单株是否含有恢复基因且能判定含有恢复基因的类型。
本发明还提供了一对InDel分子标记的引物，其特征是，上游引物为：5’-ACTCAACTGTCTTCCTCTTCTCA-3’(SEQ ID NO.1)；下游引物为：5’-TGGGAGCCTAATATATGTAATGGTG-3’(SEQ ID NO.2)。
本发明还提供了上述的InDel分子标记引物在鉴定哈克尼西棉细胞质雄性不育恢复系基因Rf1与三裂棉细胞质雄性不育恢复系基因Rf2中的应用。
本发明还提供了上述的InDel分子标记引物在对不同恢复基因(Rf1/Rf2)的棉花新恢复系材料的选育方面的应用。
本发明还阐述了一种采用上述的InDel分子标记引物鉴定哈克尼西棉细胞质雄性不育恢复系基因Rf1与三裂棉细胞质雄性不育恢复系基因Rf2的可行方法，其特征是：
1)以待测的两套三系棉花材料的基因组DNA为模板，用上述的InDel分子标记引物进行PCR扩增。
2)对上述PCR扩增产物进行琼脂糖凝胶电泳分析；
PCR产物琼脂糖凝胶电泳结果显示出现两条带，如果在376bp和408bp处显示特异扩增条带，则为含三裂棉细胞质雄性不育恢复基因Rf2杂合位点的材料；如果在376bp和562bp处显示特异扩增条带，则为含哈克尼西棉细胞质雄性不育恢复基因Rf1杂合位点和的材料；
PCR产物琼脂糖凝胶电泳结果显示一条带，如果只在376bp处显示特异扩增条带，则为不含恢复基因位点的材料；如果只在408bp处显示特异扩增条带，则为含三裂棉细胞质雄性不育恢复基因Rf2纯合位点的恢复系材料；如果只在562bp处显示特异扩增条带，则为含哈克尼西棉细胞质雄性不育恢复基因Rf1纯合位点的恢复系材料。
本发明提供的鉴定方法，用于棉花的两类细胞质雄性不育相配套的恢复系的种质资源的恢复基因的鉴定，可以显著减少田间材料的鉴定工作量，提高准确性，确保恢复系材料和棉花恢复系种子的纯度。该方法用于恢复系的选育和改良，可以加速选育进程，降低育种成本， 提高鉴定效率。该方法用于三系杂交制种，可以保证三系杂交种种子纯度，且明确材料所包含的恢复基因的类型以及基因型。
与现有技术相比，本发明的有益效果为：
1)多效：本InDel标记可以鉴定棉花的两套细胞质雄性不育的不同的恢复基因类型，还可以鉴定是否含有恢复基因位点以及恢复基因位点是否纯合，因此比单独检测一套细胞质雄性不育，其适用范围更广(尤其适用于两套细胞质雄性不育相配套的杂交的情况)，实用性更好(使用前不用区分是哪一套细胞质雄性不育材料，鉴定结果可区分细胞质雄性不育恢复系)。
2)准确率高：InDel标记具有简单可靠、可重复、易辨别等优点，只需要经PCR扩增、琼脂糖凝胶电泳即可检测。本发明利用一对InDel标记引物，能够从分子水平上区分出恢复系材料所含恢复基因类型(Rf1/Rf2)、三系杂交种以及不含恢复基因的材料，且不受植株生育时期和环境因素的影响，提高了恢复基因鉴定的准确性与高效性。
3)操作简单，鉴定快速：InDel标记对不同恢复基因(Rf1/Rf2)的棉花新恢复系材料的选育和保证三系杂交种种子纯度方面具有重要作用。与常规育种相比，InDel标记节约了大量人力、物力。在恢复系改良、构建新的恢复系以及三系杂交制种过程中，可以适量减少田间性状调查以及多带回交试验的工作量，只需要提取棉花DNA后，用InDel引物特异性扩增后通过琼脂糖凝胶电泳就可鉴定，整个鉴定检测的过程仅需约3小时，短时，高效且鉴定结果比田间性状调查更准确。
4)简单实用：与SSR标记和CAPS标记相比，InDel标记具有分布密度大、准确性较高、重演性较好、易鉴定等特点，用本发明的InDel标记引物进行PCR扩增时，没有非特异性条带扩增。本发明的InDel分子标记应用于棉花含有不同恢复基因的恢复系的分子辅助选择育种研究中有良好前景，本发明用于棉花杂交种商业制种的种子纯度鉴定工作中有很大实用价值。
附图说明
图1为：三裂棉细胞质雄性不育的三系材料、哈克尼西棉细胞质雄性不育的三系材料及参考基因组的序列比对结果，其中TM-1为陆地棉参考基因组序列；A8、B8、R8、A2、B2、和R2分别为三裂棉细胞质雄性不育相配套的不育系、保持系、恢复系，和哈克尼西棉细胞质雄性不育相配套的不育系、保持系和恢复系材料中对应的D05染色体上差异片段的序列；
图2为：InDel标记对棉花的两套细胞质雄性不育三系配套的亲本以及杂合子，PCR扩增，琼脂糖凝胶电泳结果。1-8泳道分别为三裂棉细胞质雄性不育的不育系、保持系、恢复系和含有Rf2恢复基因的杂合子，和哈克尼西棉细胞质雄性不育的不育系、保持系、恢复系和含有Rf1恢复基因的杂合子。
图3为：InDel标记对随机抽样的24份三裂棉雄性不育恢复系回交一代群体材料(BCF1)的鉴定结果，其中2泳道为恢复基因Rf2杂合单株；1泳道为不含恢复基因Rf2单株。
图4为：InDel标记对随机抽样的24份哈克尼西细胞质雄性不育恢复系的F2群体材料的鉴定结果，其中a泳道为恢复基因Rf1纯合单株；b泳道为恢复基因Rf1杂合单株，c泳道为不含恢复基因Rf1单株。
具体实施方式
下面结合具体实施例进一步说明本发明的技术方案。
实施例1：
三裂棉细胞质雄性不育的三系材料、哈克尼西棉细胞质雄性不育的三系材料及参考基因组的序列比对结果如图1所示，从图1可以看出：在三裂棉细胞质雄性不育恢复系(R8)中具有插入缺失变异的核酸序列，其序列长度408bp；在哈克尼西棉细胞质雄性不育恢复系(R2)中具有插入缺失变异的核酸序列，其序列长度562bp；其中不育系和保持系的序列长度均为376bp。
根据该InDel位点、序列和PCR引物设计原则，在其上下游设计引物，成功将其转化为InDel标记引物，扩增该InDel分子标记的引物对如表1：
表1：InDel分子标记引物
Figure PCTCN2020093184-appb-000001
在三裂棉细胞质雄性不育恢复系纯合恢复基因Rf2位点基因组中特异性扩增出发生插入缺失变异的核酸序列为408bp的条带，在Rf2基因杂合材料的基因组中扩增376bp和408bp的条带。在哈克尼西棉细胞质雄性不育恢复系纯合基因Rf1基因组中特异扩增出发生插入缺失序列的562bp的条带，在Rf1杂合材料的基因组中扩增出562bp和376bpbp的条带。不含恢复基因的材料中扩增出没有发生插入缺失序列的376bp的条带。
实施例2
1、三裂棉雄性不育(CMS-D8)三系构建的回交一代群体(BC 1F 1)与哈克尼西棉雄性不育(CMS-D 2)三系的恢复系与保持系(不育系)杂交F 2群体的种子DNA的提取(CTAB法)。
2、利用该InDel分子标记引物对，对提取的DNA进行PCR扩增。PCR反应体系如表2。
表2 20μl PCR反应体系
Figure PCTCN2020093184-appb-000002
Figure PCTCN2020093184-appb-000003
PCR程序为：94℃预变性2min；94℃变性30s，58℃退火30s，72℃延伸20s，35个循环；然后72℃终延伸2min，4℃保存。
对PCR产物进行琼脂糖凝胶(2.5％)电泳，用凝胶成像系统拍照保存，并对电泳结果进行条带分析。
3、检测结果
判断标准：
三裂棉细胞质雄性不育材料：统计中凡是仅出现408bp条带的为含有纯和的恢复基因Rf2即三裂棉细胞质雄性不育的恢复系；若同时出现376bp和408bp条带，则为含有恢复基因Rf2杂合的材料或者杂交种；仅出现376bp条带即为不含有恢复基因Rf2的材料(保持系或者不育系)。
哈克尼西棉雄性不育材料：统计中凡是仅出现562bp条带的为含有纯合的恢复基因Rf1即哈克尼西棉细胞质雄性不育的恢复系；若同时出现376bp和562bp条带，则为含有恢复基因Rf1的杂合材料或者杂交种；仅出现376bp条带即为不含有恢复基因Rf1的材料(保持系或者不育系)。
InDel标记对棉花的两套细胞质雄性不育三系配套的亲本以及杂合子，PCR扩增，琼脂糖凝胶电泳结果如图2所示，图2验证了上述的判断标准。
采用InDel标记对随机抽样的24份三裂棉细胞质雄性不育BCF1材料进行鉴定的结果如图3所示。从图3可以看出：标号为1的泳道为不含恢复基因的单株，在376bp位置出现特异条带；标号为2的泳道为恢复基因位点杂合的单株，同时出现408bp和376bp的目标条带。
采用InDel标记对随机抽样的24份哈克尼西棉细胞质雄性不育恢复系和保持系获得的F2 群体单株进行鉴定的结果如图4所示。从图4可以看出：a泳道为恢复基因Rf1纯合单株；b泳道为恢复基因Rf1杂合单株，c泳道为不含恢复基因Rf1单株。
通过上述步骤即可简单快速的鉴定恢复基因，辨别恢复基因(Rf2/Rf1)类型，区分材料的基因型。能够在三系杂交种制种过程中保证恢复系的纯度，可以显著降低田间材料鉴定的工作量，加快常规杂交育种的工作进程，极大地提高育种效率和保证三系杂交种种子纯度，提高棉花产量。
以上所述，仅为本发明最佳的实施方式，任何熟悉该技术领域的科技人员在本发明披露的技术范围内，可显而易见地获得的技术方案的简易变化或等效替换均属于本发明的保护范围。

Claims (5)

  1. 一种同时鉴定棉花细胞质雄性不育恢复基因Rf1、Rf2的InDel标记，其特征是，该InDel标记，不仅与哈克尼西棉细胞质雄性不育恢复基因Rf1连锁而且与三裂棉细胞质雄性不育恢复基因Rf2连锁，该核苷酸序列的插入/缺失长度多态性呈现在琼脂糖凝胶电泳图上可鉴定棉花单株是否含有恢复基因且能判定含有恢复基因的类型，该InDel标记，上游引物为：5’-ACTCAACTGTCTTCCTCTTCTCA-3’；下游引物为：5’-TGGGAGCCTAATATATGTAATGGTG-3’。
  2. 一种同时鉴定棉花细胞质雄性不育恢复基因Rf1、Rf2的InDel分子标记引物，其特征是，上游引物为：5’-ACTCAACTGTCTTCCTCTTCTCA-3’；下游引物为：5’-TGGGAGCCTAATATATGTAATGGTG-3’。
  3. 权利要求2所述的InDel分子标记引物在鉴定哈克尼西棉细胞质雄性不育恢复系基因Rf1与三裂棉细胞质雄性不育恢复系基因Rf2中的应用。
  4. 权利要求2所述的InDel分子标记引物在对不同恢复基因Rf1/Rf2的棉花新恢复系材料的选育方面的应用。
  5. 一种采用权利要求2所述的InDel分子标记引物鉴定哈克尼西棉细胞质雄性不育恢复系基因Rf1与三裂棉细胞质雄性不育恢复系基因Rf2的方法，其特征是：
    1)以待测的哈克尼西棉细胞质雄性不育三系棉花材料和/或三裂棉细胞质雄性不育三系棉花材料为模板，用InDel分子标记引物进行PCR扩增；
    2)对上述PCR扩增产物进行琼脂糖凝胶电泳分析；
    PCR产物琼脂糖凝胶电泳结果同时出现两条带，如果在376bp和408bp处显示特异扩增条带，则为含三裂棉细胞质雄性不育恢复基因Rf2杂合位点的材料；如果在376bp和562bp处显示特异扩增条带，则为含哈克尼西棉细胞质雄性不育恢复基因Rf1杂合位点的材料；
    PCR产物琼脂糖凝胶电泳结果显示一条带，如果只在376bp处显示特异扩增条带，则为不含恢复基因位点的材料；如果只在408bp处显示特异扩增条带，则为含三裂棉细胞质雄性不育恢复基因Rf2纯合位点的恢复系材料；如果只在562bp处显示特异扩增条带，则为含哈克尼西棉细胞质雄性不育恢复基因Rf1纯合位点的恢复系材料。
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