WO2021128695A1 - 一种棉花细胞质雄性不育恢复基因的分子标记及其应用 - Google Patents

一种棉花细胞质雄性不育恢复基因的分子标记及其应用 Download PDF

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WO2021128695A1
WO2021128695A1 PCT/CN2020/088745 CN2020088745W WO2021128695A1 WO 2021128695 A1 WO2021128695 A1 WO 2021128695A1 CN 2020088745 W CN2020088745 W CN 2020088745W WO 2021128695 A1 WO2021128695 A1 WO 2021128695A1
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seq
cotton
rfm3
rfm2
rfm1
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French (fr)
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张锐
李妍妍
梁成真
王远
孟志刚
郭三堆
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中国农业科学院生物技术研究所
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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
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the invention belongs to the field of biotechnology, and specifically relates to a molecular marker of a cotton cytoplasmic male sterility restoration gene; and also relates to the application of the molecular marker.
  • cotton As an important economic crop, cotton (Gossypium spp.) not only provides natural fiber for the textile industry, but is also an important source of edible oil. Therefore, it is particularly important to improve the yield and fiber quality of cotton.
  • the utilization of heterosis plays an important role in increasing cotton yield, quality and resistance.
  • the cotton hybrids "Yinmian No. 2" and “Yinmian No. 8" cultivated by the Institute of Biotechnology of the Chinese Academy of Agricultural Sciences have not only increased cotton production by more than 20%, but also greatly improved fiber quality.
  • the "three lines" in cotton hybrid seed production refer to: cytoplasmic male sterile line, maintainer line and restorer line.
  • Cytoplasmic male sterile lines contain sterile cytoplasmic and nuclear sterility genes, but lack nuclear restorer genes (Wu et al., 2007).
  • the maintainer line has normal fertile cytoplasm, but the nuclear genome is the same as the cytoplasmic male sterile line.
  • the restorer line contains the restorer gene, which is used as the male parent to cross with the cytoplasmic male sterile line to produce hybrids.
  • CMS cytoplasmic male sterility
  • the upland cotton sterile line containing the cytoplasmic male sterile cytoplasm (CMS-D2) and restorer lines containing restorer genes are one of the main three-line supporting systems in the utilization of cotton heterosis.
  • CCS-D2 cytoplasmic male sterile cytoplasm
  • restorer lines containing restorer genes are one of the main three-line supporting systems in the utilization of cotton heterosis.
  • some progress has been made in the breeding of cotton hybrids mainly the three-line cytoplasmic male sterile material of Hackenisi cotton.
  • the cloning and breeding of restoration genes The related mechanism of sexual restoration is still unclear, which makes the parent resources of excellent restorer lines scarce, and the selection process of the three-line hybrid combination with strong advantage is relatively slow. Therefore, in the breeding process of three-line hybrid cotton, the selection and improvement of excellent restorer lines with strong resilience is the key and difficult point in the breeding work for a long time.
  • the restoring gene Rf2 can be restored by the restoring gene Rf1 from the restorer line of Hackenisi cotton, but the restoration gene Rf2 cannot restore the fertility of Hackenisi cotton CMS-D2-2, and genetic analysis shows that, Rf1 and Rf2 are closely linked with a distance of only 0.93cM (Zhang JF, 2001).
  • the molecular markers of restorer genes currently used include: 6 EST-SSR markers (NAU2650, NAU2924, NAU3205, NAU3652, NAU3938, and NAU4040) closely linked to Rf1 are co-segregating, and they are all 0.327cM away from the Rf1 gene.
  • Sex markers the other 5 are co-dominant markers ("Nanjing Agricultural University", Yang Luming, 2009); SSR markers BNL3535 and CM042 linked to Rf1 and Rf respectively (Northwestern Journal of Botany, 2007, 27(10): 1937- 1942); the BNL632 and CIR222 marks located on both sides of Rf, the distance between the two marks is 6.7cM, (Xinjiang Agricultural Sciences 2013, 50(6): 1003-1007), etc.
  • the selection and breeding of restoring lines mainly adopts backcross improvement methods. For the transformed offspring, it is necessary to determine fertility through testcross test and field phenotype observation. Not only the breeding cycle is long, but the field phenotype observation is susceptible to environmental influences, which is time-consuming and labor-intensive. , Low efficiency, and the use of molecular marker-assisted breeding can solve this problem.
  • Using molecular marker-assisted breeding technology can directly identify restorer genes at the molecular level, and can be identified early in the seedling stage, without being affected by the developmental stage and external environment, and without the need for testcross to identify the representative type, which greatly shortens the breeding cycle. Therefore, through early assisted selection through molecular markers closely linked to the restorer genes, the restorer genes can be identified and screened stably, reliably and quickly.
  • InDel Insertion-Deletion, abbreviated as InDel
  • InDel label has the advantages of codominance, site specificity, stable amplification products and easy detection.
  • this technology only needs to be detected by simple agarose gel electrophoresis, and does not need to be cumbersome and contaminated by polyacrylamide gel electrophoresis (PAGE) and silver staining.
  • PAGE polyacrylamide gel electrophoresis
  • the technology is used for detection, and there is no need for restriction endonuclease digestion like the CAPS label, thus saving the cost of detection.
  • the purpose of the present invention is to provide a molecular marker with a closer genetic distance to the main restorer gene.
  • the present invention provides a molecular marker of a restorer gene for cotton cytoplasmic male sterility, wherein the molecular markers are one to three of RFM1, RFM2, or RFM3; wherein:
  • the length of the RFM1 fragment is 184 bp, and its nucleotide sequence is shown in SEQ ID No: 1;
  • the length of the RFM2 fragment is 154 bp, and its nucleotide sequence is shown in SEQ ID No: 2;
  • the length of the RFM3 fragment is 521 bp, and its nucleotide sequence is shown in SEQ ID No: 3.
  • the invention also provides the application of the above-mentioned molecular markers in the identification of cotton cytoplasmic male sterility restorer lines.
  • the present invention also provides a primer pair for amplifying the above molecular marker, wherein the primer pair for amplifying the above molecular marker RFM1 is:
  • RFM1-F 5’-AGGTTGGTTTGCTATGAATAGGT-3’ (SEQ ID No: 4),
  • RFM1-R 5'-CATCACTTGCAATGTGAATCAGT-3' (SEQ ID No: 5);
  • the primer pair used to amplify the aforementioned molecular marker RFM2 is:
  • RFM2-F 5’-CACTTAAGGTTGGTTTGCTATGA-3’ (SEQ ID No: 6),
  • RFM2-R 5'-TGCACAGTGATAAAAGATTGTGG-3' (SEQ ID No: 7);
  • the primer pair used to amplify the aforementioned molecular marker RFM3 is:
  • RFM3-F 5’-ATTAGCCTGAACGCGTGGAA-3’ (SEQ ID No: 8),
  • RFM3-R 5'-GGTTGAGGGACGATTCAGCA-3' (SEQ ID No: 9).
  • the invention also provides the application of the above primer pair in the identification of cotton cytoplasmic male sterility restorer lines.
  • the present invention also provides a method for identifying and screening cotton cytoplasmic male sterility restorer genes using the above-mentioned molecular markers, which includes the following steps: PCR amplification of the total DNA of the cotton material to be tested using specific primers, and then performing PCR amplification on the obtained amplification product Agarose electrophoresis gel detection, and then judge according to the size of the amplified product:
  • the cotton sample to be tested contains the restorer gene; wherein the RFM1-F is defined by SEQ ID No : The nucleotide sequence shown in 4 is composed; the RFM1-R is composed of the nucleotide sequence shown in SEQ ID No: 5;
  • the specific primer pair is RFM2-F and RFM2-R
  • the cotton sample to be tested contains the restorer gene
  • the RFM2-F is defined by SEQ ID No : The nucleotide sequence shown in 6 is composed; the RFM2-R is composed of the nucleotide sequence shown in SEQ ID No: 7;
  • the cotton sample to be tested contains the restorer gene; wherein the RFM3-F is defined by SEQ ID No : The nucleotide sequence shown in 8; The RFM3-R is composed of the nucleotide sequence shown in SEQ ID No: 9;
  • the above (1), (2) or (3) are tested independently. As long as the amplified product contains any of the specific bands described in (1), (2) or (3), the cotton material to be tested contains Restorer gene; if no specific band as described in (1), (2) or (3) above is detected in the amplified product, the cotton material to be tested does not contain restorer gene.
  • the PCR amplification reaction system is: forward primer (10uM) 1ul, reverse primer (10uM) 1ul, 2xTaq PCR StarMix with Loading Dye 10ul, ultrapure water 7ul, template DNA (50ng/ul) 1ul , The total volume of the reaction system is 20 ⁇ l.
  • the PCR amplification reaction program is: 94°C pre-denaturation for 3 minutes; 94°C pre-denaturation for 30s, 55°C annealing for 30s; 72°C extension for 30s, 72°C complete extension for 10 minutes; 35 cycles.
  • the amplified product was electrophoresed on a 2% agarose gel and a voltage of 120V for 30 minutes, and the result was recorded in a gel imaging analysis system.
  • the present invention also provides a breeding method for selecting cotton cytoplasmic male sterile restorer lines by using the above molecular markers, including PCR amplification in hybridization or selfing offspring, using specific primer pairs as primers and using the DNA of the cotton material to be tested as a template. Increase, perform agarose gel electrophoresis on the amplified product, and determine according to the size of the amplified product band, keep the cotton material containing the restorer gene; eliminate the cotton material without the restorer gene; among them:
  • the cotton material to be tested contains a restorer gene;
  • the RFM1-F described in SEQ ID No: 4 It is composed of the nucleotide sequence shown;
  • the RFM1-R is composed of the nucleotide sequence shown in SEQ ID No: 5;
  • the cotton material to be tested contains a restorer gene;
  • the RFM3-F is determined by SEQ ID No: 8
  • the RFM3-R is composed of the nucleotide sequence shown in SEQ ID No: 9;
  • the above-mentioned (1), (2) or (3) are tested independently. As long as the amplified product contains any of the specific bands in (1), (2) or (3), the cotton material to be tested contains Restorer gene; if no specific band as described in (1), (2) or (3) above is detected in the amplified product, the cotton material to be tested does not contain restorer gene.
  • the present invention also provides a detection kit for identifying a restorer gene of cotton cytoplasmic male sterility, and the detection kit contains the above-mentioned primer pair.
  • the present invention has the following beneficial effects: (1)
  • the three molecular markers provided by the present invention are closely linked to cotton restorer genes, and the three molecular markers are used to identify cotton restorer genes with high accuracy and strong reliability. .
  • the molecular marker of the present invention is used to identify restoration genes at the molecular level without being affected by factors such as external environment, and the InDel molecular marker of the present invention has the advantages of good co-dominance, good stability, and good reproducibility.
  • the detection speed is fast.
  • the molecular marker of the present invention can be used for large-scale screening and identification of whether a single cotton plant contains a restorer gene at the seedling stage, without the need for a test cross test and the like for identification, which greatly shortens the breeding cycle. (4) Simple and practical. PCR amplification detection is simple and easy to operate, saving a lot of manpower and material resources.
  • Figure 1 shows the electrophoresis patterns of PCR amplified products using RFM1-F and RFM1-R as primers to identify 23 kinds of cotton materials; among them are negative controls in turn, followed by 1 for HaiR, 2 for Y18, 3 for 0-613-2R, 4 It is especially R, 5 is 13HN6-4-6, 6 is Xiangyuan R, 7 is triple-split cotton, 8 is Raymond cotton, 9 is TM-1, 10 is P30A, 11 is P53A, and 12 is An D09A , 13 is Xiang S26A, 14 is Zhong 12, 15 is Xinhai 18, 16 is wool cotton, 17 is Zhongcao No. 1, 18 is Zhongya No. 1, 19 is Seber’s, 20 is pseudo-like cotton, 21 is ratio Hemp cotton, 22 is Darwin cotton, 23 is yellow-brown cotton.
  • Figure 2 shows the electrophoresis pattern of PCR amplification products using RFM2-F and RFM2-R as primers to identify 23 kinds of cotton materials; among them are negative controls in turn, followed by 1 for HaiR, 2 for Y18, 3 for 0-613-2R, 4 It is especially R, 5 is 13HN6-4-6, 6 is Xiangyuan R, 7 is triple-split cotton, 8 is Raymond cotton, 9 is TM-1, 10 is P30A, 11 is P53A, and 12 is An D09A , 13 is Xiang S26A, 14 is Zhong 12, 15 is Xinhai 18, 16 is wool cotton, 17 is Zhongcao No. 1, 18 is Zhongya No. 1, 19 is Seber’s, 20 is pseudo-like cotton, 21 is ratio Hemp cotton, 22 is Darwin cotton, 23 is yellow-brown cotton.
  • Figure 3 shows the electrophoresis pattern of PCR amplification products using RFM3-F and RFM3-R as primers to identify 23 kinds of cotton materials; among them are negative controls in turn, followed by 1 for HaiR, 2 for Y18, 3 for 0-613-2R, 4 It is especially R, 5 is 13HN6-4-6, 6 is Xiangyuan R, 7 is triple-split cotton, 8 is Raymond cotton, 9 is TM-1, 10 is P30A, 11 is P53A, and 12 is An D09A , 13 is Xiang S26A, 14 is Zhong 12, 15 is Xinhai 18, 16 is wool cotton, 17 is Zhongcao No. 1, 18 is Zhongya No. 1, 19 is Seber’s, 20 is pseudo-like cotton, 21 is ratio Hemp cotton, 22 is Darwin cotton, 23 is yellow-brown cotton.
  • Figure 4 shows the electrophoresis pattern of PCR amplification products of the self-cultivating cotton restorer line identified with RFM1-F and RFM1-R as primers; where 1 is the restorer line HaiR (as a positive control), and 2 is the sterile line P30A (as a negative control) ), 3 is maintainer 272B (as a negative control), 4 is P80R2, 5 is D45-4242, 6 is D35-5, 7 is KY18, 8 is WSJ07-2-4, 9 is D29-1-1, 10 It is D24-3.
  • Figure 5 shows the electrophoresis pattern of PCR amplification products of the self-cultivating cotton restorer line identified with RFM2-F and RFM2-R primers; where 1 is the restorer line HaiR (as a positive control), and 2 is the sterile line P30A (as a negative control) ), 3 is maintainer 272B (as a negative control), 4 is P80R2, 5 is D45-4242, 6 is D35-5, 7 is KY18, 8 is WSJ07-2-4, 9 is D29-1-1, 10 It is D24-3.
  • Figure 6 shows the electrophoresis pattern of PCR amplified products of the self-cultivating cotton restorer line identified with RFM3-F and RFM3-R as primers; where 1 is the restorer line HaiR (as a positive control), and 2 is the sterile line P30A (as a negative control) ), 3 is maintainer 272B (as a negative control), 4 is P80R2, 5 is D45-4242, 6 is D35-5, 7 is KY18, 8 is WSJ07-2-4, 9 is D29-1-1, 10 It is D24-3.
  • the InDel molecular markers were designed using the genome data of the restorer line HaiR in the applicant’s laboratory and the genome data of Upland cotton TM-1, and the polymorphisms of the parents (sea island cotton HaiR and upland cotton P30A) were screened, and then the polymorphisms were used. Molecular markers perform genotype detection on the F 2 population. The primers were synthesized by Shanghai Shenggong.
  • the cotton restorer gene Rf1 was located in the 106kb section of D05 chromosome. After the positioning interval, the applicant's sea island cotton HaiR genome data and upland cotton TM-1 genome data were used to design InDel molecular markers (see Table 1).
  • InDel markers were used to screen the parent sea island cotton HaiR and Gossypium hirsutum P30A for polymorphism, and used to screen restorer lines.
  • three molecular markers, Marker3, Marker4, and Marker12 were screened. Markers Marker3, Marker4 and Marker12 are named RFM1, RFM2 and RFM3, respectively.
  • Polymorphic molecular markers are used to distinguish restoring lines from non-restoring lines.
  • the three molecular markers RFM1, RFM2, and RFM3 amplified primer pairs independently amplify the cotton material to be tested. These three molecular markers belong to a mutual verification relationship and represent three positions respectively. Due to the complex structure of the cotton genome, non-specific bands may appear if they are mixed and amplified.
  • the specific primer sequence of each molecular marker and the length of the amplified target fragment are as follows:
  • the primer pair of molecular marker RFM1 is:
  • RFM1-F 5’-AGGTTGGTTTGCTATGAATAGGT-3’ (SEQ ID No: 4),
  • RFM1-R 5'-CATCACTTGCAATGTGAATCAGT-3' (SEQ ID No: 5).
  • a DNA fragment with a length of 184bp can be amplified, and the sequence of the amplified product is shown in SEQ ID No: 1, see the sequence table.
  • RFM2-F 5’-CACTTAAGGTTGGTTTGCTATGA-3’ (SEQ ID No: 6),
  • RFM2-R 5'-TGCACAGTGATAAAAGATTGTGG-3' (SEQ ID No: 7).
  • a DNA fragment with a length of 154 bp can be amplified, and the sequence of the amplified product is shown in SEQ ID No: 2, see the sequence table.
  • RFM3-F 5’-ATTAGCCTGAACGCGTGGAA-3’ (SEQ ID No: 8),
  • RFM3-R 5'-GGTTGAGGGACGATTCAGCA-3' (SEQ ID No: 9);
  • a DNA fragment with a length of 521bp can be amplified, and the amplified product sequence is shown in SEQ ID No: 3, see the sequence table.
  • Example 2 Specific analysis test of the molecular markers of the present invention for identification of cotton restorer lines
  • DNA extraction remove the shells from seeds of 23 different cotton materials (see Table 2), then extract DNA by CTAB method, measure the DNA concentration and dilute to 50ng/ ⁇ l, and store at -20°C for later use.
  • step (2) Use the DNA extracted in step (1) as a template, and use the specific primer pairs of RFM1, RFM2 or RFM3 as primers to perform PCR amplification, detect the band type, and use the genotypes of the restorer line HaiR and the sterile line P30A as Reference.
  • InDel labeled PCR reaction system is 20ul, including forward primer (10uM) 1ul, reverse primer (10uM) 1ul, 2xTaq PCR StarMix with Loading Dye 10ul, ultrapure water 7ul, template DNA (50ng/ul) 1ul.
  • the PCR reaction program 94°C pre-denaturation 3min; 94°C pre-denaturation 30s, 55°C annealing 30s; 72°C extension 30s, 72°C complete extension 10min, 35 cycles.
  • the amplification reaction was carried out on a BIO-RAD PCR amplification instrument. The amplified products were electrophoresed on a 2% agarose gel and a voltage of 120V for 30 minutes, and the results were recorded in the BIO-RAD gel imaging analysis system.
  • test materials are 6 high-generation homozygous cotton cytoplasmic male sterile restorer lines (see No. 4-9 in Table 3) cultivated by the applicant.
  • the cotton seeds are removed from the outer shell, and DNA is extracted according to the CTAB method; DNA concentration is determined After diluting to 50ng/ ⁇ l, store at -20°C for later use.
  • step (2) Using the DNA extracted in step (1) as a template, PCR amplification was carried out with the primer pairs of molecular markers RFM1, RFM2 or RFM3 as primers, and the band pattern was detected to restore line HaiR, sterile line P30A and maintainer line
  • the genotype of 272B was used as a reference.
  • InDel labeled PCR reaction system is 20ul, including forward primer (10uM) 1ul, reverse primer (10uM) 1ul, 2xTaq PCR StarMix with Loading Dye 10ul, ultrapure water 7ul, template DNA (50ng/ul) 1ul.
  • the PCR reaction program 94°C pre-denaturation 3min; 94°C pre-denaturation 30s, 55°C annealing 30s; 72°C extension 30s, 72°C complete extension 10min.
  • the amplification reaction was carried out in BIO-RAD’s T00 TM Thermal cycler, and the amplified products were electrophoresed on a 2% agarose gel at a voltage of 120V for 30 minutes, and then recorded in the BIO-RAD Gel Doc TM XR + gel imaging analysis system result.
  • the present invention has obtained InDel molecular markers RFM1, RFM2 and RFM3 for identifying restorer genes for cotton cytoplasmic male sterility, which can be used for molecular marker-assisted breeding of cotton restorer lines.
  • This molecular marker can be used in the cotton seedling stage. Identification of restorer genes without backcrossing identification and field phenotype observation will greatly shorten the breeding cycle of restorer lines and improve the breeding efficiency of restorer lines, which has broad application prospects.

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Abstract

本发明公开了一种棉花细胞质雄性不育恢复基因的分子标记及其应用，属于生物技术领域。所述的分子标记为RFM1、RFM2或RFM3；其核苷酸序列分别如SEQ ID No：1-3所示。本发明还公开了用于分子标记的特异性引物。此外，还公开了利用上述分子标记鉴定和筛选棉花恢复基因的方法。

Description

一种棉花细胞质雄性不育恢复基因的分子标记及其应用 技术领域
本发明属于生物技术领域，具体涉及一种棉花细胞质雄性不育恢复基因的分子标记；还涉及该分子标记的应用。
背景技术
棉花(Gossypium spp.)作为重要的经济作物,不仅为纺织工业提供天然纤维，也是重要的食用油来源，因此提高棉花的产量和纤维品质尤为重要。杂种优势的利用在提高棉花产量、改进品质、提高抗性等方面发挥着重要作用。如中国农业科学院生物技术研究所等培育的棉花杂交种“银棉2号”和“银棉8号”等，不仅使棉花产量增产20％以上，还极大改善了纤维品质。
棉花杂交种制种中的“三系”是指：细胞质雄性不育系、保持系和恢复系。细胞质雄性不育系含有不育细胞质和核不育基因，但缺乏核恢复基因(Wu et al.,2007)。保持系有正常可育的细胞质，但是核基因组却和细胞质雄性不育系一样，在生产中，细胞质雄性不育系作为母本，保持系作为父本为细胞质雄性不育系繁殖后代。恢复系含有恢复基因，其作为父本与细胞质雄性不育系杂交生产杂交种。但是由于恢复系存在恢复力不强等缺陷，使得棉花细胞质雄性不育(Cytoplasmic Male Sterility，CMS)的应用受到极大的限制。因此，育种家长期围绕强恢复力的优良恢复系选育和改良开展相关研究。
含哈克尼西棉细胞质雄性不育胞质(CMS-D2)的陆地棉不育系和含有恢复基因的恢复系是棉花杂种优势利用中主要的三系配套系统之一。目前，棉花杂交种(以哈克尼西棉细胞质雄性不育三系材料为主)的育种已经取得了一定进展，但由于棉花中恢复基因来源狭窄及恢复能力限制，且恢复基因的克隆和育性恢复的相关机理尚不清楚，使得优良恢复系亲本资源匮乏，造成强优势三系杂交组合的选育过程较为缓慢。因此，在三系杂交棉选育过程中，强恢复力的优良恢复系的选育和改良是育种工作中长期面临的重点和难点。
有些研究认为哈克尼西棉CMS育性恢复受一个部分显性基因控制，并受到一些基因的修饰作用(Weaver J B,1977；Sheetz and Weaver,1980)。还有研究表明，哈克尼西棉CMS育性恢复至少受三对显性基因Rf调控(Silva et  al.,1981)，还有人认为哈克尼西棉CMS育性恢复仅受一对基因Rf控制(Kohel R J,1984)。因此，目前对哈克尼西棉CMS育性恢复的遗传机理仍未有定论；Zhang等人在研究三裂棉CMS的育性恢复中发现，其雄性不育特性既能被三裂棉恢复系的恢复基因Rf2所恢复，又能被来自哈克尼西棉恢复系的恢复基因Rf1所恢复，但恢复基因Rf2不能恢复哈克尼西棉CMS-D2-2的育性，且遗传分析表明，Rf1与Rf2紧密连锁、相距仅0.93cM(Zhang JF,2001)。目前所用恢复基因的分子标记有：6个与Rf1紧密连锁的EST-SSR标记(NAU2650、NAU2924、NAU3205、NAU3652、NAU3938和NAU4040)表现为共分离,均与Rf1基因相距0.327cM，其中NAU3205为显性标记,其它5个都是共显性标记(《南京农业大学》，杨路明，2009)；分别与Rf1、Rf连锁的SSR标记BNL3535、CM042(西北植物学报，2007,27(10)：1937-1942)；位于Rf两侧的BNL632、CIR222标记，两标记相距6.7cM,(新疆农业科学2013,50(6)：1003-1007)等。
对恢复系的选育主要采用回交改良方法，对转育后代需要通过测交试验和田间表型观察来确定育性，不仅育种周期长，且田间表型观察易受环境影响，耗时费力、效率低，而引用分子标记辅助育种可以解决这一问题。利用分子标记辅助育种技术可以直接在分子水平上鉴定恢复基因，可在苗期进行早期鉴定，不受发育阶段和外界环境的影响，无需通过测交鉴定后代表型，极大地缩短了育种周期。因此，通过与恢复基因紧密连锁的分子标记在早期进行辅助选择，可以对恢复基因进行稳定可靠而又快速的鉴定和筛选。
在恢复系选育中常用的分子标记有InDel、CAPS、RFLP、RAPD、SSR、AFLP、STS等。但InDel(插入缺失：Insertion-Deletion，简写为InDel)分子标记以其操作简单、扩增产物易于检测等优点，近年来在分子标记辅助育种中得到广泛应用。InDel标记具备共显性、位点特异性、扩增产物稳定和易于检测等优点。与已报道的恢复基因相关的SSR和CAPS标记等相比，该技术只需通过简单的琼脂糖凝胶电泳检测，不需要通过聚丙烯酰胺凝胶电泳(PAGE)和银染等繁琐且具有污染的技术进行检测，而且也不需要像CAPS标记进行限制性内切酶酶切，从而节约了检测成本。
前人研究中多是利用非恢复系的棉花基因组为数据参考序列。因棉花基因组庞大且恢复区段序列高度重复、结构复杂，目前已发表的棉花基因组数据中在棉花恢复区段未获得高质量的组装序列，并且存在缺失。因此，到目前为止，已报道的分子标记与主效恢复基因遗传距离仍相对较远。
发明内容
为了克服现有技术的不足，针对哈克尼西棉细胞质雄性不育恢复系选育中存在的问题，本发明目的在于提供一种与主效恢复基因遗传距离更近的分子标记。
本发明提供一种棉花细胞质雄性不育恢复基因的分子标记，所述分子标记为RFM1、RFM2或RFM3之1～3种；其中：
所述RFM1的片段长度为184bp，其核苷酸序列如SEQ ID No：1所示；
所述RFM2的片段长度为154bp，其核苷酸序列如SEQ ID No：2所示；
所述RFM3的片段长度为521bp，其核苷酸序列如SEQ ID No：3所示。
本发明还提供了上述分子标记在棉花细胞质雄性不育恢复系鉴定上的应用。
本发明还提供了用于扩增上述分子标记的引物对，其中所述用于扩增上述分子标记RFM1的引物对为：
RFM1-F:5’-AGGTTGGTTTGCTATGAATAGGT-3’(SEQ ID No：4)，
RFM1-R：5’-CATCACTTGCAATGTGAATCAGT-3’(SEQ ID No：5)；
所述用于扩增上述分子标记RFM2的引物对为：
RFM2-F：5’-CACTTAAGGTTGGTTTGCTATGA-3’(SEQ ID No：6)，
RFM2-R：5’-TGCACAGTGATAAAAGATTGTGG-3’(SEQ ID No：7)；
所述用于扩增上述分子标记RFM3的引物对为：
RFM3-F：5’-ATTAGCCTGAACGCGTGGAA-3’(SEQ ID No：8)，
RFM3-R：5’-GGTTGAGGGACGATTCAGCA-3’(SEQ ID No：9)。
本发明还提供了上述引物对在棉花细胞质雄性不育恢复系鉴定上的应用。
本发明还提供了利用上述分子标记鉴定和筛选棉花细胞质雄性不育恢复基因的方法，包括如下步骤：利用特异性引物对对待测棉花材料的总DNA进行PCR扩增，然后对所得扩增产物进行琼脂糖电泳凝胶检测，再根据所得扩增产物大小进行判断：
(1)当特异性引物对为RFM1-F和RFM1-R时，如果电泳条带出现184bp特异性条带，则该待测棉花样品含有恢复基因；其中所述的RFM1-F由SEQ ID No： 4所示的核苷酸序列组成；所述的RFM1-R由SEQ ID No：5所示的核苷酸序列组成；
(2)当特异性引物对为RFM2-F和RFM2-R时，如果电泳条带出现154bp特异性条带，则该待测棉花样品含有恢复基因；其中所述的RFM2-F由SEQ ID No：6所示的核苷酸序列组成；所述的RFM2-R由SEQ ID No：7所示的核苷酸序列组成；
(3)当特异性引物对为RFM3-F和RFM3-R时，如果电泳条带出现521bp特异性条带，则该待测棉花样品含有恢复基因；其中所述的RFM3-F由SEQ ID No：8所示的核苷酸序列组成；所述的RFM3-R由SEQ ID No：9所示的核苷酸序列组成；
上述(1)、(2)或(3)相互独立检测，只要扩增产物中含有(1)、(2)或(3)中任一所述特异性条带，则该待测棉花材料含有恢复基因；如果扩增产物中没有检测到上述(1)、(2)或(3)中任一所述的特异性条带，则该待测棉花材料不含恢复基因。
进一步地，所述PCR扩增的反应体系为：正向引物(10uM)1ul，反向引物(10uM)1ul，2xTaq PCR StarMix with Loading Dye 10ul，超纯水7ul，模板DNA(50ng/ul)1ul，反应体系总体积为20μl。
进一步，所述PCR扩增的反应程序为：94℃预变性3min；94℃预变性30s，55℃退火30s；72℃延伸30s，72℃彻底延伸10min；35个循环。
进一步，所述扩增产物在2％琼脂糖凝胶、电压120V条件下电泳30min，在凝胶成像分析系统中记录结果。
本发明还提供了利用上述分子标记辅助选择棉花细胞质雄性不育恢复系的育种方法，包括在杂交或自交后代，以特异性引物对为引物、以待测棉花材料的DNA为模板进行PCR扩增，对所得扩增产物进行琼脂糖凝胶电泳，根据扩增产物条带大小进行判定，保留含有恢复基因的棉花材料；淘汰不含恢复基因的棉花材料；其中：
(1)当以RFM1-F和RFM1-R为引物时，如果电泳条带出现184bp特异性条带，则该待测棉花材料含有恢复基因；其中所述的RFM1-F由SEQ ID No：4 所示的核苷酸序列组成；所述的RFM1-R由SEQ ID No：5所示的核苷酸序列组成；
(2)当以RFM2-F和RFM2-R为引物时，如果电泳条带出现154bp特异性条带，则该待测棉花材料含有恢复基因；所述的RFM2-F由SEQ ID No：6所示的核苷酸序列组成；所述的RFM2-R由SEQ ID No：7所示的核苷酸序列组成；
(3)当以RFM3-F和RFM3-R为引物时，如果电泳条带出现521bp特异性条带，则该待测棉花材料含有恢复基因；所述的RFM3-F由SEQ ID No：8所示的核苷酸序列组成；所述的RFM3-R由SEQ ID No：9所示的核苷酸序列组成；
上述(1)、(2)或(3)相互独立检测，只要扩增产物中含有(1)、(2)或(3)中任一所述特异性条带，则该待测棉花材料含有恢复基因；如果扩增产物中没有检测到上述(1)、(2)或(3)中任一所述的特异性条带，则该待测棉花材料不含恢复基因。
本发明还提供了用于鉴定棉花细胞质雄性不育恢复基因的检测试剂盒，所述的检测试剂盒中含有上述引物对。
与现有技术相比，本发明的有益效果在于：(1)本发明所提供的3个分子标记与棉花恢复基因连锁紧密，利用该3个分子标记鉴定棉花恢复基因准确性高、可靠性强。(2)利用本发明分子标记在分子水平上对恢复基因进行鉴定，不受外在环境等因素的影响，并且本发明InDel分子标记具有共显性好、稳定性好、重复性好的优点。(3)检测速度快。利用本发明分子标记可在苗期对棉花单株是否含有恢复基因进行大规模筛选鉴定，不需要进行测交试验等进行鉴定，大大缩短了育种周期。(4)简单实用。PCR扩增检测简单易操作，节约了大量的人力和物力。
附图说明
图1为用RFM1-F和RFM1-R为引物鉴定23种棉花材料的PCR扩增产物电泳图谱；其中依次为阴性对照，之后1为HaiR，2为Y18，3为0-613-2R，4为尤R，5为13HN6-4-6，6为湘远R，7为三裂棉，8为雷蒙德式棉，9为TM-1，10为P30A，11为P53A，12为安D09A，13为湘S26A，14为中12，15为新海18，16为毛棉，17为中草1号，18为中亚1号，19为瑟伯氏，20为拟似棉，21为 比麻棉，22为达尔文棉，23为黄褐棉。
图2为用RFM2-F和RFM2-R为引物鉴定23种棉花材料的PCR扩增产物电泳图谱；其中依次为阴性对照，之后1为HaiR，2为Y18，3为0-613-2R，4为尤R，5为13HN6-4-6，6为湘远R，7为三裂棉，8为雷蒙德式棉，9为TM-1，10为P30A，11为P53A，12为安D09A，13为湘S26A，14为中12，15为新海18，16为毛棉，17为中草1号，18为中亚1号，19为瑟伯氏，20为拟似棉，21为比麻棉，22为达尔文棉，23为黄褐棉。
图3为用RFM3-F和RFM3-R为引物鉴定23种棉花材料的PCR扩增产物电泳图谱；其中依次为阴性对照，之后1为HaiR，2为Y18，3为0-613-2R，4为尤R，5为13HN6-4-6，6为湘远R，7为三裂棉，8为雷蒙德式棉，9为TM-1，10为P30A，11为P53A，12为安D09A，13为湘S26A，14为中12，15为新海18，16为毛棉，17为中草1号，18为中亚1号，19为瑟伯氏，20为拟似棉，21为比麻棉，22为达尔文棉，23为黄褐棉。
图4为用RFM1-F和RFM1-R为引物鉴定自育的棉花恢复系的PCR扩增产物电泳图谱；其中1为恢复系HaiR(作为阳性对照)，2为不育系P30A(作为阴性对照)，3为保持系272B(作为阴性对照)，4为P80R2，5为D45-4242,6为D35-5，7为KY18，8为WSJ07-2-4，9为D29-1-1，10为D24-3。
图5为用RFM2-F和RFM2-R为引物鉴定自育的棉花恢复系的PCR扩增产物电泳图谱；其中1为恢复系HaiR(作为阳性对照)，2为不育系P30A(作为阴性对照)，3为保持系272B(作为阴性对照)，4为P80R2，5为D45-4242,6为D35-5，7为KY18、8为WSJ07-2-4，9为D29-1-1，10为D24-3。
图6为用RFM3-F和RFM3-R为引物鉴定自育的棉花恢复系的PCR扩增产物电泳图谱；其中1为恢复系HaiR(作为阳性对照)，2为不育系P30A(作为阴性对照)，3为保持系272B(作为阴性对照)，4为P80R2，5为D45-4242,6为D35-5，7为KY18，8为WSJ07-2-4，9为D29-1-1，10为D24-3。
具体实施方式
实施例1棉花细胞质雄性不育恢复基因的InDel分子标记的筛选
(1)群体构建及数据的获得
以陆地棉不育系P30A为母本、以海岛棉恢复系HaiR(为申请人自育的棉花 恢复系，曾用名ISR，其恢复基因来源于0-613-2R)为父本杂交，得F 1代；然后自交得F 2代，构建F 2代分离群体。具体如下：
2015年5月在北京平谷种植HaiR和P30A各500株。当年7月，以P30A为母本、以HaiR为父本杂交；10月，收获杂交F 1代种子。
2015年10月在海南崖州种植F 1代800株，开花期做自交，2016年3月收获自交种子，得F 2代。
2016年5月在北京平谷种植F 2分离大群体3.4万株，当年7月，对单株进行田间农艺性状的调查及棉花完全可育单株、完全不育单株的标记，按性状单株取样。
(2)采用CTAB法提取上述(1)中单株DNA
用本申请人实验室的恢复系HaiR的基因组数据及陆地棉TM-1的基因组数据设计InDel分子标记，对双亲本(海岛棉HaiR和陆地棉P30A)进行多态性筛选，再用多态性分子标记对F 2群体进行基因型检测。引物由上海生工合成。
(3)棉花恢复基因QTL定位
根据F 2分离大群体的棉花育性性状的表型数据和基因型数据，将棉花恢复基因Rf1定位在D05染色体106kb的区段内，将此区段称为恢复区段，确定了恢复基因的定位区间以后，用本申请人的海岛棉HaiR的基因组数据及陆地棉TM-1的基因组数据设计InDel分子标记(见表1)。
表1 设计的InDel分子标记
Figure PCTCN2020088745-appb-000001
Figure PCTCN2020088745-appb-000002
用上述InDel标记对亲本海岛棉HaiR和陆地棉P30A进行多态性筛选，用于筛选恢复系，结果筛选到Marker3、Marker4和Marker12(见表1)3个分子标记，将所筛选的3个分子标记Marker3、Marker4和Marker12分别命名为RFM1、RFM2和RFM3。
多态性分子标记用于区分恢复系与非恢复系。
此三个分子标记RFM1、RFM2和RFM3的扩增引物对分别独立对待测棉花材料进行扩增。这三个分子标记属于相互验证的关系，分别代表三个位置。因棉花基因组结构比较复杂，如果混在一起扩增，可能会出现非特异条带。
其中，各分子标记的特异性引物序列和扩增的目的片段长度如下：
(1)分子标记RFM1的引物对为：
RFM1-F：5’-AGGTTGGTTTGCTATGAATAGGT-3’(SEQ ID No：4)，
RFM1-R：5’-CATCACTTGCAATGTGAATCAGT-3’(SEQ ID No：5)。
可扩增长度为184bp的DNA片段,扩增产物序列如SEQ ID No：1所示，见序列表。
(2)分子标记RFM2的引物对为：
RFM2-F：5’-CACTTAAGGTTGGTTTGCTATGA-3’(SEQ ID No：6)，
RFM2-R：5’-TGCACAGTGATAAAAGATTGTGG-3’(SEQ ID No：7)。
可扩增长度为154bp的DNA片段,扩增产物序列如SEQ ID No：2所示，见序列表。
(3)分子标记RFM3的引物对为：
RFM3-F：5’-ATTAGCCTGAACGCGTGGAA-3’(SEQ ID No：8),
RFM3-R：5’-GGTTGAGGGACGATTCAGCA-3’(SEQ ID No：9)；
可扩增长度为521bp的DNA片段,扩增产物序列如SEQ ID No：3所示，见序列表。
实施例2：本发明分子标记对棉花恢复系鉴定的特异性分析试验
(1)DNA提取：将23种不同棉花材料(见表2)的种子除去外壳，然后通过CTAB法提取DNA，测定DNA浓度后稀释到50ng/μl，-20℃保存备用。
(2)以步骤(1)中提取的DNA为模板，分别以RFM1、RFM2或RFM3的特异引物对为引物进行PCR扩增，检测带型，以恢复系HaiR和不育系P30A的基因型作为参照。InDel标记的PCR反应体系为20ul，其中正向引物(10uM)1ul,反向引物(10uM)1ul,2xTaq PCR StarMix with Loading Dye 10ul,超纯水7ul,模板DNA(50ng/ul)1ul.InDel标记的PCR反应程序：94℃预变性3min；94℃预变性30s，55℃退火30s；72℃延伸30s，72℃彻底延伸10min，35个循环。扩增反应在BIO-RAD PCR扩增仪上进行，扩增产物在2％琼脂糖凝胶、电压为120V条件下电泳30min，在BIO-RAD凝胶成像分析系统中记录结果。
(3)结果分子标记RFM1、RFM2、RFM3的电泳图谱分别见图1、图2和图3，对电泳结果进行分析(见表2)。含恢复基因的材料与恢复系HaiR的带型一致，不含恢复基因的材料跟P30A一致，无条带。上述结果说明本发明3个分子标记对棉花恢复系的鉴定的特异性强，鉴定结果准确、可靠。
表2 用本发明3个分子标记对23种棉花材料恢复基因的PCR鉴定结果
Figure PCTCN2020088745-appb-000003
Figure PCTCN2020088745-appb-000004
实施例3：用本发明分子标记对棉花细胞质雄性不育恢复系的鉴定试验
(1)DNA提取：试验材料为本申请人培育的6种高代纯合棉花细胞质雄性不育恢复系(见表3编号4-9)，将棉花种子除去外壳，按照CTAB法提取DNA；测定DNA浓度后稀释到50ng/μl，-20℃保存备用。
(2)以步骤(1)中提取的DNA为模板，分别以分子标记RFM1、RFM2或RFM3的引物对为引物进行PCR扩增，检测带型，以恢复系HaiR、不育系P30A和保持系272B的基因型作为参照。InDel标记的PCR反应体系为20ul，其中正向引物(10uM)1ul,反向引物(10uM)1ul,2xTaq PCR StarMix with Loading Dye 10ul,超纯水7ul,模板DNA(50ng/ul)1ul.Indel标记的PCR反应程序：94℃预变性3min；94℃预变性30s，55℃退火30s；72℃延伸30s，72℃彻底延伸10min。扩增反应在BIO-RAD公司T00 TM Thermal cycler进行，扩增产物在2％琼脂糖凝 胶、电压为120V条件下电泳30min，然后在BIO-RAD Gel Doc TMXR +凝胶成像分析系统中记录结果。
(3)结果分子标记RFM1、RFM2、RFM3的PCR扩增产物电泳图谱分别见图4、图5和图6，对检测结果进行分析(见表3)，结果3种分子标记RFM1、RFM2和RFM3仅特异性地存在于恢复系，不育系和保持系都没有扩增产物，说明用分子标记RFM1、RFM2或RFM3特异性强，对棉花细胞质雄性不育恢复系鉴定结果准确、可靠，且方法简单，适于大规模在棉花育种中用于恢复基因的辅助选择。
表3 用本发明3个分子标记对6个自育恢复系的鉴定结果
编号 品种名称 材料特性 RFM1 RFM2 RFM3
1 HaiR A1D1(恢复系) 有 有 有
2 P30A A1D1(不育系) 无 无 无
3 272B A1D1(保持系) 无 无 无
4 P80R2 A1D1(恢复系) 有 有 有
5 D45-4242 A1D1(恢复系) 有 有 有
6 D35-5 A1D1(恢复系) 有 有 有
7 KY18 A1D1(恢复系) 有 有 有
8 WSJ07-2-4 A1D1(恢复系) 有 有 有
9 D29-1-1 A1D1(恢复系) 有 有 有
综上所述，本发明获得了用于鉴定棉花细胞质雄性不育恢复基因的InDel分子标记RFM1、RFM2和RFM3，可用于棉花恢复系的分子标记辅助育种，使用该分子标记在棉花幼苗期即可进行恢复基因鉴定，无需进行回交鉴定以及田间表型观察，将极大缩短恢复系的育种周期，提高恢复系育种效率，具有广阔的应用前景。
以上实施例仅用于说明本发明的技术方案而非对其限制，尽管上述实施例对本发明进行了详细的说明，所属领域的普通技术人员依然可以对本发明的具体实施方式进行修改或者等同替换，而这些未脱离本发明精神和范围的任何修改或者等同替换，均在本发明的权利要求保护范围之内。

Claims (9)

  1. 一种棉花细胞质雄性不育恢复基因的分子标记，其特征在于，所述分子标记为RFM1、RFM2或RFM3之1～3种；其中：
    所述RFM1的片段长度为184bp，其核苷酸序列如SEQ ID No：1所示；
    所述RFM2的片段长度为154bp，其核苷酸序列如SEQ ID No：2所示；
    所述RFM3的片段长度为521bp，其核苷酸序列如SEQ ID No：3所示。
  2. 权利要求1所述的分子标记在棉花细胞质雄性不育恢复系鉴定上的应用。
  3. 用于扩增权利要求1所述分子标记的引物对，其特征在于，其中所述用于扩增分子标记RFM1的引物对为：
    RFM1-F:5’-AGGTTGGTTTGCTATGAATAGGT-3’(SEQ ID No：4)，
    RFM1-R：5’-CATCACTTGCAATGTGAATCAGT-3’(SEQ ID No：5)；
    所述用于扩增分子标记RFM2的引物对为：
    RFM2-F：5’-CACTTAAGGTTGGTTTGCTATGA-3’(SEQ ID No：6)，
    RFM2-R：5’-TGCACAGTGATAAAAGATTGTGG-3’(SEQ ID No：7)；
    所述用于扩增分子标记RFM3的引物对为：
    RFM3-F：5’-ATTAGCCTGAACGCGTGGAA-3’(SEQ ID No：8)，
    RFM3-R：5’-GGTTGAGGGACGATTCAGCA-3’(SEQ ID No：9)。
  4. 权利要求3所述的引物对在棉花细胞质雄性不育恢复系鉴定上的应用。
  5. 利用权利要求1所述分子标记鉴定和筛选棉花细胞质雄性不育恢复基因的方法，包括如下步骤：利用特异性引物对对待测棉花材料的总DNA进行PCR扩增，然后对所得扩增产物进行琼脂糖电泳凝胶检测，再根据所得扩增产物大小进行判断：
    (1)当特异性引物对为RFM1-F和RFM1-R时，如果电泳条带出现184bp特异性条带，则该待测棉花样品含有恢复基因；其中所述的RFM1-F由SEQ ID No：4所示的核苷酸序列组成；所述的RFM1-R由SEQ ID No：5所示的核苷酸序列组成；
    (2)当特异性引物对为RFM2-F和RFM2-R时，如果电泳条带出现154bp特异性条带，则该待测棉花样品含有恢复基因；其中所述的RFM2-F由SEQ ID No： 6所示的核苷酸序列组成；所述的RFM2-R由SEQ ID No：7所示的核苷酸序列组成；
    (3)当特异性引物对为RFM3-F和RFM3-R时，如果电泳条带出现521bp特异性条带，则该待测棉花样品含有恢复基因；其中所述的RFM3-F由SEQ ID No：8所示的核苷酸序列组成；所述的RFM3-R由SEQ ID No：9所示的核苷酸序列组成；
    上述(1)、(2)或(3)相互独立检测，只要扩增产物中含有(1)、(2)或(3)中任一所述的特异性条带，则该待测棉花材料含有恢复基因；如果扩增产物中没有检测到上述(1)、(2)或(3)中任一所述的特异性条带，则该待测棉花材料不含恢复基因。
  6. 根据权利要求5所述的方法，其特征在于，所述PCR扩增的反应体系为：正向引物(10uM)1ul，反向引物(10uM)1ul，2xTaq PCR StarMix with Loading Dye 10ul，超纯水7ul，模板DNA(50ng/ul)1ul，反应体系总体积为20μl。
  7. 根据权利要求5所述的方法，其特征在于，所述PCR扩增的反应程序为：94℃预变性3min；94℃预变性30s，55℃退火30s；72℃延伸30s，72℃彻底延伸10min；35个循环；所述扩增产物在2％琼脂糖凝胶、电压120V条件下电泳30min，在凝胶成像分析系统中记录结果。
  8. 利用权利要求1所述的分子标记辅助选择棉花细胞质雄性不育恢复系的育种方法，其特征在于，在杂交或自交后代，以特异性引物对为引物、以待测棉花材料的DNA为模板进行PCR扩增，对所得扩增产物进行琼脂糖凝胶电泳，根据扩增产物条带大小进行判定，保留含有恢复基因的棉花材料；淘汰不含恢复基因的棉花材料；其中：
    (1)当以RFM1-F和RFM1-R为引物时，如果电泳条带出现184bp特异性条带，则该待测棉花材料含有恢复基因；所述的RFM1-F由SEQ ID No：4所示的核苷酸序列组成；所述的RFM1-R由SEQ ID No：5所示的核苷酸序列组成；
    (2)当以RFM2-F和RFM2-R为引物时，如果电泳条带出现154bp特异性条带，则该待测棉花材料含有恢复基因；所述的RFM2-F由SEQ ID No：6所示的核苷酸序列组成；所述的RFM2-R由SEQ ID No：7所示的核苷酸序列组成；
    (3)当以RFM3-F和RFM3-R为引物时，如果电泳条带出现521bp特异性条带，则该待测棉花材料含有恢复基因；所述的RFM3-F由SEQ ID No：8所示的核苷酸序列组成；所述的RFM3-R由SEQ ID No：9所示的核苷酸序列组成；
    上述(1)、(2)或(3)相互独立检测，只要扩增产物中含有(1)、(2)或(3)中任一所述特异性条带，则该待测棉花材料含有恢复基因；如果扩增产物中没有检测到上述(1)、(2)或(3)中任一所述的特异性条带，则该待测棉花材料不含恢复基因。
  9. 用于鉴定棉花细胞质雄性不育恢复基因的检测试剂盒，其特征在于，所述的检测试剂盒中含有权利要求2所述的引物对。
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