CN110760613B - Molecular marker of cotton cytoplasmic male sterility restoring gene and application thereof - Google Patents
Molecular marker of cotton cytoplasmic male sterility restoring gene and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker of a cotton cytoplasmic male sterility restoring gene and application thereof, belonging to the technical field of biology. The molecular markers are 1-3 of RFM1, RFM2 or RFM 3; the nucleotide sequences are respectively shown as SEQ ID No: 1-3. The invention also discloses a specific primer for molecular marking. In addition, a method for identifying and screening cotton restorer genes by using the molecular markers is also disclosed. The molecular marker is closely linked with the cotton restoring gene, the specificity is strong, the identification result is accurate and reliable, and the identification and screening speed is high; the molecular marker provided by the invention is used for carrying out auxiliary selection on the restoring gene without being influenced by external environment, can be carried out in an early stage, greatly shortens the breeding period, obviously reduces the breeding cost, and is suitable for being applied to large-scale breeding.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a molecular marker of a cotton cytoplasmic male sterility restoring gene; also relates to the application of the molecular marker.
Background
Cotton (A)Gossypium spp.) As an important economic crop, the natural fiber is provided for the textile industry, and the natural fiber is also an important edible oil source, so that the improvement of the yield and the fiber quality of cotton is particularly important. The utilization of heterosis plays an important role in improving cotton yield, improving quality, improving resistance and the like. Such as cotton hybrid seeds 'silver cotton No. 2' and 'silver cotton No. 8' cultivated by the institute of biotechnology of Chinese academy of agricultural sciences and the like, not only increases the cotton yield by more than 20%, but also greatly improves the fiber quality.
The three lines in the production of cotton hybrid seeds refer to: cytoplasmic male sterile line, maintainer line and restorer line. Cytoplasmic male sterile lines contain sterile cytoplasm and a nuclear sterility gene, but lack a nuclear restorer gene (Wu et al, 2007). The maintainer line has normal fertile cytoplasm, but the nuclear genome is the same as the cytoplasmic male sterile line, and during production, the cytoplasmic male sterile line is used as a female parent, and the maintainer line is used as a male parent to reproduce progeny of the cytoplasmic male sterile line. The restorer line contains restorer gene as male parent and is crossed with cytoplasmic male sterile line to produce hybrid. However, the use of Cytoplasmic Male Sterility (CMS) in cotton is greatly limited due to the weak restoring force of the restorer line. Therefore, breeders have long conducted relevant research around the breeding and improvement of excellent restoring lines with strong restoring force.
The sterile line of upland cotton containing cytoplasmic male sterile cytoplasm (CMS-D2) of Harknessi cotton and the restoring line containing restoring gene are one of the main three-line matching systems for heterosis utilization of cotton. At present, the breeding of cotton hybrid (mainly the three-line cytoplasmic male sterile material of cotton Hakini) has been advanced to a certain extent, but because the source of the restoring gene in cotton is narrow and the restoring capability is limited, and the related mechanism of the cloning and fertility restoration of the restoring gene is not clear, the parent resource of the excellent restoring line is deficient, and the breeding process of the strong-superiority three-line hybrid combination is slower. Therefore, in the process of breeding the three-line hybrid cotton, the breeding and the improvement of the excellent restoring line with strong restoring force are important points and difficulties faced in the breeding work for a long time.
Some studies suggest that CMS fertility restoration in Cotton Harknessii is controlled by a partially dominant gene and modified by several genes (Weaver J B,1977; Sheetz and Weaver, 1980). Further studies have shown that CMS fertility restoration in Cotton Harknessii is subject to at least three pairs of dominant genesRfRegulation (Silva et al, 1981) it has also been suggested that CMS fertility restoration in Cotton Harknessi is only conferred by a pair of genesRfControl (Kohel R J, 1984). Therefore, at present, the genetic mechanism of CMS fertility restoration of Hakinisia gossypii is not yet established; zhang et al found in the research of CMS fertility restoration of the triple-split cotton that the male sterility property of Zhang et al can be restored by the restoring gene of the triple-split cotton restorerRf2The restored gene can be recovered by the cotton restorer line of HakinisiaRf1Restored, but restored geneRf2The fertility of CMS-D2-2 of Hakinisia cotton cannot be restored,and the genetic analysis shows that the gene expression vector has the characteristics of,Rf1andRf2closely linked, only 0.93cM apart (Zhang JF, 2001). Molecular markers of the currently used restorer genes are: the 6 EST-SSR markers (NAU2650, NAU2924, NAU3205, NAU3652, NAU3938 and NAU4040) closely linked to Rf1 appeared to be co-segregating and all were found to be co-segregating withRf1The distance between the genes is 0.327cM, wherein NAU3205 is a dominant marker, and the other 5 genes are co-dominant markers (Nanjing agriculture university, Yangming, 2009); SSR markers BNL3535 and CM042 linked with Rf1 and Rf respectively (northwest plant journal, 2007,27 (10): 1937-; BNL632 and CIR222 marks positioned at two sides of Rf, the distance between the two marks is 6.7cM, (Xinjiang agricultural science 2013,50 (6): 1003-.
The breeding of the restorer line mainly adopts a backcross improvement method, the fertility of the bred offspring needs to be determined through a test cross test and field phenotype observation, the breeding period is long, the field phenotype observation is easily influenced by the environment, the time and the labor are consumed, the efficiency is low, and the problem can be solved by introducing the molecular marker to assist the breeding. The molecular marker assisted breeding technology can be used for directly identifying the restoring gene on the molecular level, early identification can be carried out in the seedling stage, the influence of the development stage and the external environment is avoided, the test cross is not needed for identifying the phenotype of the offspring, and the breeding period is greatly shortened. Therefore, the restoring gene can be stably, reliably and quickly identified and screened by auxiliary selection of molecular markers closely linked with the restoring gene at an early stage.
The molecular markers commonly used in the breeding of the restorer line are InDel, CAPS, RFLP, RAPD, SSR, AFLP, STS and the like. However, the InDel (Insertion-Deletion, abbreviated as InDel) molecular marker has been widely used in molecular marker-assisted breeding in recent years due to its advantages of simple operation and easy detection of amplified products. The InDel marker has the advantages of co-dominance, site specificity, stable amplification product, easy detection and the like. Compared with the reported SSR related to the restoring gene, CAPS mark and the like, the technology only needs simple agarose gel electrophoresis detection, does not need complex and polluted technologies such as polyacrylamide gel electrophoresis (PAGE) and silver staining, and does not need restriction enzyme digestion like the CAPS mark, so that the detection cost is saved.
Most of the previous studies used the cotton genome of the non-restorer line as the data reference sequence. Because the cotton genome is large, the sequence of the recovery segment is highly repetitive and the structure is complex, high-quality assembly sequences are not obtained in the cotton recovery segment in the published cotton genome data at present, and the cotton recovery segment has deletion. Thus, to date, molecular markers have been reported that are still relatively distant from the major restorer gene.
Disclosure of Invention
Aiming at overcoming the defects of the prior art and solving the problems in the breeding of the cytoplasmic male sterile restorer line of the cotton Harkinsoni, the invention aims to provide a molecular marker which is closer to the genetic distance of a major restorer gene.
The invention provides a molecular marker of cotton cytoplasmic male sterility restoring genes, which is 1-3 of RFM1, RFM2 or RFM 3; wherein:
the fragment length of the RFM1 is 184bp, and the nucleotide sequence is shown as SEQ ID No: 1 is shown in the specification;
the fragment length of the RFM2 is 154bp, and the nucleotide sequence is shown as SEQ ID No: 2 is shown in the specification;
the fragment length of the RFM3 is 521bp, and the nucleotide sequence is shown as SEQ ID No: 3, respectively.
The invention also provides the application of the molecular marker in the identification of the cotton cytoplasmic male sterility restorer line.
The invention also provides a primer pair for amplifying the molecular marker, wherein the primer pair for amplifying the molecular marker RFM1 is as follows:
RFM1-F: 5’-AGGTTGGTTTGCTATGAATAGGT-3’（SEQ ID No：4），
RFM1-R：5’-CATCACTTGCAATGTGAATCAGT-3’（SEQ ID No：5）；
the primer pair for amplifying the molecular marker RFM2 is as follows:
RFM2-F：5’-CACTTAAGGTTGGTTTGCTATGA-3’（SEQ ID No：6），
RFM2-R：5’-TGCACAGTGATAAAAGATTGTGG-3’（SEQ ID No：7）；
the primer pair for amplifying the molecular marker RFM3 is as follows:
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 primer pair in the identification of the cotton cytoplasmic male sterility restoring line.
The invention also provides a method for identifying and screening the cotton cytoplasmic male sterility restoring gene by using the molecular marker, which comprises the following steps: carrying out PCR amplification on the total DNA of the cotton material to be detected by utilizing a specific primer pair, carrying out agarose electrophoresis gel detection on the obtained amplification product, and judging according to the size of the obtained amplification product:
(1) when the specific primer pair is RFM1-F and RFM1-R, if the electrophoresis band has a 184bp specific band, the cotton sample to be detected contains a restoring gene; wherein said RFM1-F consists of SEQ ID No: 4; the RFM1-R is represented by SEQ ID No: 5;
(2) when the specific primer pair is RFM2-F and RFM2-R, if the electrophoresis band has a specific band of 154bp, the cotton sample to be detected contains a restoring gene; wherein said RFM2-F consists of SEQ ID No: 6; the RFM2-R is represented by SEQ ID No: 7;
(3) when the specific primer pair is RFM3-F and RFM3-R, if an electrophoresis band has a 521bp specific band, the cotton sample to be detected contains a restoring gene; wherein said RFM3-F consists of SEQ ID No: 8; the RFM3-R is represented by SEQ ID No: 9;
detecting the above (1), (2) or (3) independently, wherein the cotton material to be detected contains a restoring gene as long as the amplification product contains any one of the specific bands of (1), (2) or (3); and (3) if the specific strip is not detected in the amplification product, the cotton material to be detected does not contain a restoring gene.
Further, the reaction system of the PCR amplification is as follows: 1ul of forward primer (10 uM), 1ul of reverse primer (10 uM), 10ul of 2xTaq PCR StarMix with Loading Dye, 7ul of ultrapure water, 1ul of template DNA (50 ng/ul), and the total volume of the reaction system is 20 mu l.
Further, the reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 3 min; pre-denaturation at 94 ℃ for 30s, and annealing at 55 ℃ for 30 s; extension is carried out for 30s at 72 ℃, and complete extension is carried out for 10min at 72 ℃; 35 cycles.
Further, the amplification product was electrophoresed in 2% agarose gel at 120V for 30min, and the results were recorded in a gel imaging analysis system.
The invention also provides a breeding method for selecting the cotton cytoplasmic male sterility restoring line by utilizing the molecular marker, which comprises the steps of carrying out PCR amplification on hybrid or selfed progeny by taking a specific primer pair as a primer and taking the DNA of a cotton material to be detected as a template, carrying out agarose gel electrophoresis on the obtained amplification product, judging according to the size of an amplification product strip, and reserving the cotton material containing the restoring gene; eliminating cotton material without restoring gene; wherein:
(1) when RFM1-F and RFM1-R are used as primers, if an electrophoresis band has a 184bp specific band, the cotton material to be detected contains a recovery gene; wherein said RFM1-F consists of SEQ ID No: 4; the RFM1-R is represented by SEQ ID No: 5;
(2) when RFM2-F and RFM2-R are used as primers, if an electrophoresis band has a 154bp specific band, the cotton material to be detected contains a recovery gene; the RFM2-F consists of SEQ ID No: 6; the RFM2-R is represented by SEQ ID No: 7;
(3) when RFM3-F and RFM3-R are used as primers, if an electrophoresis band has a 521bp specific band, the cotton material to be detected contains a restoring gene; the RFM3-F consists of SEQ ID No: 8; the RFM3-R is represented by SEQ ID No: 9;
detecting the above (1), (2) or (3) independently, wherein the cotton material to be detected contains a restoring gene as long as the amplification product contains any one of the specific bands of (1), (2) or (3); and (3) if the specific strip is not detected in the amplification product, the cotton material to be detected does not contain a restoring gene.
The invention also provides a detection kit for identifying the cotton cytoplasmic male sterility restoring gene, wherein the detection kit contains the primer pair.
Compared with the prior art, the invention has the beneficial effects that: (1) the 3 molecular markers provided by the invention are closely linked with the cotton recovery gene, and the identification of the cotton recovery gene by using the 3 molecular markers has high accuracy and strong reliability. (2) The molecular marker is used for identifying the restoring gene on the molecular level, is not influenced by factors such as external environment and the like, and has the advantages of good codominance, good stability and good repeatability. (3) The detection speed is high. The molecular marker can be used for carrying out large-scale screening identification on whether a single cotton plant contains the restoring gene or not in the seedling stage without carrying out identification through a test cross test and the like, so that the breeding period is greatly shortened. (4) Is simple and practical. The PCR amplification detection is simple and easy to operate, and a large amount of manpower and material resources are saved.
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FIG. 1 is an electrophoretogram of PCR amplification products for identifying 23 cotton materials using RFM1-F and RFM1-R as primers; wherein the two are negative controls in sequence, then 1 is HaiR, 2 is Y18, 3 is 0-613-2R, 4 is Eucalyptus R, 5 is 13HN6-4-6, 6 is Xiangyuan R, 7 is sanke cotton, 8 is Ramengde cotton, 9 is TM-1, 10 is P30A, 11 is P53A, 12 is an N D09A, 13 is Xiang S26A, 14 is a middle 12, 15 is a new sea 18, 16 is wool cotton, 17 is a Chinese herbal medicine No. 1, 18 is a Zhongya No. 1, 19 is a Thurber, 20 is a pseudocotton, 21 is a Bima cotton, 22 is a Darwin cotton, and 23 is a yellow brown cotton.
FIG. 2 is an electrophoretogram of PCR amplification products for identifying 23 cotton materials using RFM2-F and RFM2-R as primers; wherein the two are negative controls in sequence, then 1 is HaiR, 2 is Y18, 3 is 0-613-2R, 4 is Eucalyptus R, 5 is 13HN6-4-6, 6 is Xiangyuan R, 7 is sanke cotton, 8 is Ramengde cotton, 9 is TM-1, 10 is P30A, 11 is P53A, 12 is an N D09A, 13 is Xiang S26A, 14 is a middle 12, 15 is a new sea 18, 16 is wool cotton, 17 is a Chinese herbal medicine No. 1, 18 is a Zhongya No. 1, 19 is a Thurber, 20 is a pseudocotton, 21 is a Bima cotton, 22 is a Darwin cotton, and 23 is a yellow brown cotton.
FIG. 3 is an electrophoretogram of PCR amplification products for identifying 23 cotton materials using RFM3-F and RFM3-R as primers; wherein the two are negative controls in sequence, then 1 is HaiR, 2 is Y18, 3 is 0-613-2R, 4 is Eucalyptus R, 5 is 13HN6-4-6, 6 is Xiangyuan R, 7 is sanke cotton, 8 is Ramengde cotton, 9 is TM-1, 10 is P30A, 11 is P53A, 12 is an N D09A, 13 is Xiang S26A, 14 is a middle 12, 15 is a new sea 18, 16 is wool cotton, 17 is a Chinese herbal medicine No. 1, 18 is a Zhongya No. 1, 19 is a Thurber, 20 is a pseudocotton, 21 is a Bima cotton, 22 is a Darwin cotton, and 23 is a yellow brown cotton.
FIG. 4 is an electropherogram of PCR amplification products of a self-bred cotton restorer line identified using RFM1-F and RFM1-R as primers; wherein 1 is restorer line HaiR (as positive control), 2 is sterile line P30A (as negative control), 3 is maintainer line 272B (as 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, and 10 is D24-3.
FIG. 5 is an electropherogram of PCR amplification products of a self-bred cotton restorer line identified using RFM2-F and RFM2-R as primers; wherein 1 is restorer line HaiR (as positive control), 2 is sterile line P30A (as negative control), 3 is maintainer line 272B (as 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, and 10 is D24-3.
FIG. 6 is an electropherogram of PCR amplification products of a self-bred cotton restorer line identified using RFM3-F and RFM3-R as primers; wherein 1 is restorer line HaiR (as positive control), 2 is sterile line P30A (as negative control), 3 is maintainer line 272B (as 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, and 10 is D24-3.
Detailed Description
Example 1 screening of InDel molecular markers for cytoplasmic Male sterility restorer genes of Cotton
(1) Group construction and data acquisition
Hybridizing by using upland cotton sterile line P30A as female parent and sea island cotton restorer line HaiR (the cotton restorer line self-bred by the applicant, named ISR, the restorer gene of which is from 0-613-2R) as male parent to obtain F1Generation; then selfing to obtain F2Generation, construction of F2And (4) separating the populations. The method comprises the following specific steps:
500 plants each of HaiR and P30A were planted in beijing flatgrain 5 months in 2015. In the 7 th month of the year, hybridizing by taking P30A as a female parent and HaiR as a male parent; 10 months later, harvest hybrid F1And (5) seed generation.
Planting F in cliff in Hainan 10 months in 20151Generating 800 plants, selfing in flowering period, and harvesting selfed seeds in 2016 and 3 months to obtain F2And (4) generation.
F planted in Beijing Pinggu at 5 months in 201623.4 ten thousand plants of a large group are separated, and in 7 months in the year, the individual plants are subjected to investigation of field agronomic characters and marking of cotton completely fertile individual plants and completely sterile individual plants, and the individual plants are sampled according to characters.
(2) Extraction of DNA of the individual plant in (1) above by the CTAB method
Designing InDel molecular marker by using the genome data of restorer line HaiR and upland cotton TM-1 in the laboratory of the applicant, carrying out polymorphism screening on double parents (sea island HaiR and upland cotton P30A), and then using the polymorphism molecular marker to carry out F2The population is subjected to genotype detection. The primer is synthesized by Shanghai.
(3) Cotton restorer gene QTL location
According to F2Separating the phenotypic data and the genotypic data of the fertility character of the cotton of a large population, restoring the gene of the cottonRf1After the mapping interval of the restorer gene was determined by mapping to a 106kb segment of D05 chromosome, InDel molecular markers were designed using the genome data of HaiR of Kalanchoe and the genome data of TM-1 of Gossypium hirsutum of the present applicant (see Table 1).
InDel molecular markers designed in Table 1
The InDel Marker is used for carrying out polymorphism screening on parent sea island cotton HaiR and upland cotton P30A for screening a restorer line, 3 molecular markers of Marker3, Marker4 and Marker12 (shown in Table 1) are screened as a result, and the screened 3 molecular markers of Marker3, Marker4 and Marker12 are named as RFM1, RFM2 and RFM3 respectively.
Polymorphic molecular markers are used to distinguish between restorer and non-restorer lines.
The three amplification primer pairs of the molecular markers RFM1, RFM2 and RFM3 are used for independently amplifying the cotton material to be detected respectively. The three molecular markers belong to a mutual verification relationship and respectively represent three positions. Due to the complex structure of cotton genome, non-specific bands may appear if amplified together.
Wherein, the specific primer sequence of each molecular marker and the length of the amplified target fragment are as follows:
(1) the primer pair of the molecular marker RFM1 is:
RFM1-F：5’-AGGTTGGTTTGCTATGAATAGGT-3’（SEQ ID No：4），
RFM1-R：5’-CATCACTTGCAATGTGAATCAGT-3’（SEQ ID No：5）。
can amplify DNA fragment with length of 184bp, and the sequence of the amplification product is shown as SEQ ID No: 1, shown in a sequence table.
(2) The primer pair of the molecular marker RFM2 is:
RFM2-F：5’-CACTTAAGGTTGGTTTGCTATGA-3’（SEQ ID No：6），
RFM2-R：5’-TGCACAGTGATAAAAGATTGTGG-3’（SEQ ID No：7）。
can amplify DNA fragments with the length of 154bp, and the sequence of the amplification product is shown as SEQ ID No: 2, shown in a sequence table.
(3) The primer pair of the molecular marker RFM3 is:
RFM3-F：5’-ATTAGCCTGAACGCGTGGAA-3’（SEQ ID No：8）,
RFM3-R：5’-GGTTGAGGGACGATTCAGCA-3’（SEQ ID No：9）；
can amplify DNA segments with the length of 521bp, and the sequence of the amplification product is shown as SEQ ID No: 3, shown in a sequence table.
Example 2: the molecular marker of the invention is used for the specificity analysis test of the identification of the cotton restorer
(1) DNA extraction: seeds of 23 different cotton materials (see Table 2) were hulled, then DNA was extracted by CTAB method, and the DNA concentration was measured and diluted to 50 ng/. mu.l, and stored at-20 ℃ for further use.
(2) And (2) performing PCR amplification by taking the DNA extracted in the step (1) as a template and the specific primer pair of RFM1, RFM2 or RFM3 as primers respectively, detecting the band type, and taking the genotype of the restorer line HaiR and the sterile line P30A as reference. The InDel-labeled PCR reaction system is 20ul, wherein the forward primer (10 uM) is 1ul, the reverse primer (10 uM) is 1ul, the 2xTaq PCR StarMixwith Loading Dye is 10ul, the ultrapure water is 7ul, and the template DNA (50 ng/ul) is 1ul, the InDel-labeled PCR reaction program: pre-denaturation at 94 ℃ for 3 min; pre-denaturation at 94 ℃ for 30s, and annealing at 55 ℃ for 30 s; extension at 72 ℃ for 30s, complete extension at 72 ℃ for 10min, 35 cycles. The amplification reaction is carried out on a BIO-RAD PCR amplification instrument, the amplification product is electrophoresed for 30min under the conditions of 2% agarose gel and 120V voltage, and the result is recorded in a BIO-RAD gel imaging analysis system.
(3) Results the electropherograms of the molecular markers RFM1, RFM2, RFM3 are shown in fig. 1, fig. 2 and fig. 3, respectively, and the results of the electrophoresis are analyzed (see table 2). The material containing the restorer gene was identical in band pattern to the restorer line HaiR, and the material without the restorer gene was identical in band pattern to P30A. The results show that the identification specificity of the 3 molecular markers on the cotton restorer line is strong, and the identification result is accurate and reliable.
Table 2 PCR identification results of 23 cotton material restorer genes with 3 molecular markers of the present invention
Example 3: identification test of cytoplasmic male sterility restoring line of cotton by using molecular marker
(1) DNA extraction: the test material is 6 high-generation homozygous cotton cytoplasmic male sterility restorer lines (see the numbers 4-9 in the table 3) cultivated by the applicant, the shells of the cotton seeds are removed, and DNA is extracted according to a CTAB method; the DNA concentration was determined and diluted to 50 ng/. mu.l and stored at-20 ℃ until use.
(2) And (2) performing PCR amplification by taking the DNA extracted in the step (1) as a template and taking a primer pair of molecular markers RFM1, RFM2 or RFM3 as primers respectively, detecting the banding pattern, and taking the genotypes of the restorer line HaiR, the sterile line P30A and the maintainer line 272B as references. The InDel-labeled PCR reaction system is 20ul, wherein the forward primer (10 uM) is 1ul, the reverse primer (10 uM) is 1ul, the 2xTaqPCR StarMix with Loading Dye is 10ul, the ultrapure water is 7ul, the template DNA (50 ng/ul) is 1ul, Indel-labeled PCR reaction program: pre-denaturation at 94 ℃ for 3 min; pre-denaturation at 94 ℃ for 30s, and annealing at 55 ℃ for 30 s; extension at 72 ℃ for 30s and complete extension at 72 ℃ for 10 min. Amplification reactions were performed in BIO-RAD T00TMPerforming Thermal cycler, electrophoresing the amplification product in 2% agarose Gel at 120V for 30min, and performing BIO-RAD Gel DocTMXR+The results were recorded in a gel imaging analysis system.
(3) The electrophoresis patterns of PCR amplification products of the molecular markers RFM1, RFM2 and RFM3 are respectively shown in FIG. 4, FIG. 5 and FIG. 6, the detection results are analyzed (see Table 3), and the results of 3 analysis that the molecular markers RFM1, RFM2 and RFM3 only exist in the restorer line specifically, and the sterile line and the maintainer line do not have amplification products show that the molecular markers RFM1, RFM2 or RFM3 have strong specificity, the identification result of the cotton cytoplasmic male sterility restorer line is accurate and reliable, and the method is simple and is suitable for large-scale auxiliary selection of restoring genes in cotton breeding.
TABLE 3 identification of 6 self-bred restorer lines with 3 molecular markers of the invention
In conclusion, the InDel molecular markers RFM1, RFM2 and RFM3 for identifying the cotton cytoplasmic male sterility restoring genes are obtained, and can be used for molecular marker-assisted breeding of cotton restoring lines.
Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can make modifications and equivalents to the specific embodiments of the invention without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> institute of biotechnology of Chinese academy of agricultural sciences
<120> molecular marker of cotton cytoplasmic male sterility restoring gene and application thereof
<141>2019-11-22
<160>9
<170>SIPOSequenceListing 1.0
<210>1
<211>184
<212>DNA
<213>Gossypium spp.
<400>1
aggttggttt gctatgaata ggttcgtgag ggtatgatat gtttcaatat gagtgattga 60
agtttgctca aaatagacac taaaccaata atattgaaaa taggcactaa acctcattct 120
agtttccaca atcttttatc actgtgcaat ggttatgtac aactgattca cattgcaagt 180
gatg 184
<210>2
<211>154
<212>DNA
<213>Gossypium spp.
<400>2
cacttaaggt tggtttgcta tgaataggtt cgtgagggta tgatatgttt caatatgagt 60
gattgaagtt tgctcaaaat agacactaaa ccaataatat tgaaaatagg cactaaacct 120
cattctagtt tccacaatct tttatcactg tgca 154
<210>3
<211>521
<212>DNA
<213>Gossypium spp.
<400>3
attagcctga acgcgtggaa aactggaagg gggcaaatgg gaaataatcc cattcttgca 60
aaatgcagag ttccagtagg gactaaattg aaaaggcaaa aaaatccgag gtaaaattaa 120
aagcaaaaaa aatacttgat tatgaaacca ttaaaaagtg gaagggctaa aggtgcaatt 180
agcccttccg tcgaaaacac gcggatcctt caggtgggtc gggttgaaat ccgggttaag 240
agccaaaacg acgccgtttt gtcactggtg accttggctt aaaacgacgc cgtttagtgt 300
tgctataaag cccaattttt ttttcaaaat ttctcatttt tccttctttt tcaaaaaaaa 360
ctctcaaaaa actctcaaaa accctctcct ctccccaaaa ttcggccaag ggtccaaccg 420
agagccacca caccggccgc cggtcgccgg cgccagcgct gctgtgcacg gcggcgggga 480
gaccaaaagt ttcccttttt ttgctgaatc gtccctcaac c 521
<210>4
<211>23
<212>DNA
<213>Gossypium spp.
<400>4
aggttggttt gctatgaata ggt 23
<210>5
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<212>DNA
<213>Gossypium spp.
<400>5
catcacttgc aatgtgaatc agt 23
<210>6
<211>23
<212>DNA
<213>Gossypium spp.
<400>6
cacttaaggt tggtttgcta tga 23
<210>7
<211>23
<212>DNA
<213>Gossypium spp.
<400>7
tgcacagtga taaaagattg tgg 23
<210>8
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<212>DNA
<213>Gossypium spp.
<400>8
<210>9
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Claims (9)
1. A molecular marker of cotton cytoplasmic male sterility restorer gene, wherein the molecular marker is RFM1, RFM2 or RFM 3; wherein:
the fragment length of the RFM1 is 184bp, and the nucleotide sequence is shown as SEQ ID No: 1 is shown in the specification;
the fragment length of the RFM2 is 154bp, and the nucleotide sequence is shown as SEQ ID No: 2 is shown in the specification;
the fragment length of the RFM3 is 521bp, and the nucleotide sequence is shown as SEQ ID No: 3, respectively.
2. Use of the molecular marker of claim 1 for identification of cytoplasmic male sterility restorer line of cotton.
3. The primer pair for amplifying the molecular marker of claim 1, wherein the primer pair for amplifying the 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 for amplifying the molecular marker RFM2 is as follows:
RFM2-F：5’-CACTTAAGGTTGGTTTGCTATGA-3’（SEQ ID No：6），
RFM2-R：5’-TGCACAGTGATAAAAGATTGTGG-3’（SEQ ID No：7）；
the primer pair for amplifying the molecular marker RFM3 is as follows:
RFM3-F：5’-ATTAGCCTGAACGCGTGGAA-3’（SEQ ID No：8），
RFM3-R：5’-GGTTGAGGGACGATTCAGCA-3’（SEQ ID No：9）。
4. use of the primer pair of claim 3 for identifying a cytoplasmic male sterility restorer line of cotton.
5. The method for identifying and screening the cytoplasmic male sterility restorer gene of cotton using the molecular marker as set forth in claim 1, comprising the steps of: carrying out PCR amplification on the total DNA of the cotton material to be detected by utilizing a specific primer pair, carrying out agarose electrophoresis gel detection on the obtained amplification product, and judging according to the size of the obtained amplification product:
(1) when the specific primer pair is RFM1-F and RFM1-R, if the electrophoresis band has a 184bp specific band, the cotton sample to be detected contains a restoring gene; wherein said RFM1-F consists of SEQ ID No: 4; the RFM1-R is represented by SEQ ID No: 5;
(2) when the specific primer pair is RFM2-F and RFM2-R, if the electrophoresis band has a specific band of 154bp, the cotton sample to be detected contains a restoring gene; wherein said RFM2-F consists of SEQ ID No: 6; the RFM2-R is represented by SEQ ID No: 7;
(3) when the specific primer pair is RFM3-F and RFM3-R, if an electrophoresis band has a 521bp specific band, the cotton sample to be detected contains a restoring gene; wherein said RFM3-F consists of SEQ ID No: 8; the RFM3-R is represented by SEQ ID No: 9;
detecting the above (1), (2) or (3) independently, wherein the cotton material to be detected contains a restoring gene as long as the amplification product contains the specific band of any one of (1), (2) or (3); and (3) if the specific strip is not detected in the amplification product, the cotton material to be detected does not contain a restoring gene.
6. The method of claim 5, wherein the reaction system of the PCR amplification is: 1ul of 10uM forward primer, 1ul of 10uM reverse primer, 10ul of 2xTaq PCR StarMix with Loading Dye, 7ul of ultrapure water, 1ul of 50ng/ul template DNA, and the total volume of the reaction system is 20 mu l.
7. The method of claim 5, wherein the reaction procedure of the PCR amplification is: pre-denaturation at 94 ℃ for 3 min; pre-denaturation at 94 ℃ for 30s, and annealing at 55 ℃ for 30 s; extension is carried out for 30s at 72 ℃, and complete extension is carried out for 10min at 72 ℃; 35 cycles; and (3) carrying out electrophoresis on the amplification product for 30min under the conditions of 2% agarose gel and 120V voltage, and recording the result in a gel imaging analysis system.
8. A breeding method for selecting a cotton cytoplasmic male sterile restoring line with the aid of a molecular marker according to claim 1, characterized in that, in a hybrid or selfed progeny, a specific primer pair is used as a primer, PCR amplification is carried out by using DNA of a cotton material to be detected as a template, agarose gel electrophoresis is carried out on an obtained amplification product, judgment is carried out according to the size of an amplification product band, and the cotton material containing a restoring gene is reserved; eliminating cotton material without restoring gene; wherein:
(1) when RFM1-F and RFM1-R are used as primers, if an electrophoresis band has a 184bp specific band, the cotton material to be detected contains a recovery gene; the RFM1-F consists of SEQ ID No: 4; the RFM1-R is represented by SEQ ID No: 5;
(2) when RFM2-F and RFM2-R are used as primers, if an electrophoresis band has a 154bp specific band, the cotton material to be detected contains a recovery gene; the RFM2-F consists of SEQ ID No: 6; the RFM2-R is represented by SEQ ID No: 7;
(3) when RFM3-F and RFM3-R are used as primers, if an electrophoresis band has a 521bp specific band, the cotton material to be detected contains a restoring gene; the RFM3-F consists of SEQ ID No: 8; the RFM3-R is represented by SEQ ID No: 9;
detecting the above (1), (2) or (3) independently, wherein the cotton material to be detected contains a restoring gene as long as the amplification product contains any one of the specific bands of (1), (2) or (3); and (3) if the specific strip is not detected in the amplification product, the cotton material to be detected does not contain a restoring gene.
9. A detection kit for identifying a cytoplasmic male sterility restorer gene of cotton, wherein the detection kit contains the primer pair of claim 3.
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| CN201911373852.4A CN110760613B (en) | 2019-12-27 | 2019-12-27 | Molecular marker of cotton cytoplasmic male sterility restoring gene and application thereof |
| PCT/CN2020/088745 WO2021128695A1 (en) | 2019-12-27 | 2020-05-06 | Molecular marker of restoring gene for cytoplasmic male sterility in gossypium spp. and use thereof |
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| CN110760613B (en) * | 2019-12-27 | 2020-04-10 | 中国农业科学院生物技术研究所 | Molecular marker of cotton cytoplasmic male sterility restoring gene and application thereof |
| CN111197103A (en) * | 2020-03-06 | 2020-05-26 | 中国农业科学院棉花研究所 | Cotton leaf honey gland related molecular marker InDel-GaNEC1, primer pair and application thereof |
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| CN101985650A (en) * | 2009-07-29 | 2011-03-16 | 中国农业科学院生物技术研究所 | Molecular markers of male sterile cytoplasm and male fertility cytoplasm of cotton and application thereof |
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| US5981833A (en) * | 1994-11-29 | 1999-11-09 | Iowa State University Research Foundation, Inc. | Nuclear restorer genes for hybrid seed production |
| AU2003222566A1 (en) * | 2002-01-10 | 2003-07-24 | Cornell Research Foundation, Inc. | Genes for altering mitochondrial function and for hybrid seed production |
| CN102154413B (en) * | 2009-07-06 | 2014-07-02 | 中国农业科学院生物技术研究所 | Molecular marker technology based method for evaluating upland cotton cytoplasm male sterile restore gene |
| CN102618647B (en) * | 2012-03-31 | 2014-05-14 | 中国农业科学院棉花研究所 | Molecular identification method for cotton restoring line containing sterile cytoplasm of gossypium harknessii |
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| CN109593875B (en) * | 2019-01-11 | 2021-09-28 | 华中农业大学 | Molecular marker of cotton restorer restoring gene and application thereof |
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| CN110184378A (en) * | 2019-06-05 | 2019-08-30 | 中国农业科学院棉花研究所 | Three split the method for identifying molecules of cotton cell matter fertility restorer gene |
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