CN111778354B - Molecular marker closely linked with photosensitive male sterility character of cotton PSM4, molecular identification method and application - Google Patents
Molecular marker closely linked with photosensitive male sterility character of cotton PSM4, molecular identification method and application Download PDFInfo
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Abstract
The invention relates to a molecular marker closely linked with photosensitive male sterility character of cotton PSM4, a molecular identification method and application, belonging to the technical field of cotton breeding. The molecular markers of the present invention include SSR232 and SSR 243. The molecular marker disclosed by the invention can accurately identify the individual plants with the photosensitive male sterility property in the PSM4 and breeding progeny materials thereof according to the molecular marker result, and can be used for but not limited to the following aspects: the method is used for molecular marker-assisted breeding of excellent photosensitive male sterile materials derived from PSM4, purity identification of PSM4 and photosensitive male sterile lines derived therefrom, purity identification of hybrid F1 prepared from photosensitive male sterile lines, intellectual property protection of PSM4 and photosensitive male sterile lines derived therefrom and the like.
Description
Technical Field
The invention relates to the technical field of cotton breeding, in particular to a molecular marker closely linked with photosensitive male sterility character of cotton PSM4, a molecular identification method and application.
Background
The cotton fiber is an important raw material in the textile industry, and the cotton seed contains protein and oil, can be used for feed production and food processing, and is an important economic crop in China. With the continuous development of economy and the improvement of living standard of people, the demand of people on the yield and the quality of cotton is higher and higher. Cotton has obvious heterosis, and the utilization of heterosis is also an important method for improving the yield and quality of cotton.
In order to fully utilize the heterosis of cotton and carry out the large-scale production of hybrid seeds, the hybrid seed production method developed by researchers in sequence mainly comprises the following steps: artificial castration, chemical reagent castration, self-incompatibility and male sterility. The artificial emasculation method is the main method for configuring hybrid seeds and producing seeds of cotton at present, but with the annual rise of labor cost, the cost of artificial emasculation seed production is higher and higher, the seed production efficiency is lower, and the popularization and utilization of hybrid cotton are severely restricted. The male sterility method is to utilize the fact that the anther of the male organ of the plant does not crack or pollen does not shed or lose the ability to produce viable pollen on the day of flowering, resulting in the inability of the plant to self-pollinate. The male sterility method can overcome the difficulty of artificial emasculation and is an economic and effective way to utilize heterosis.
The phenomenon of male sterility is ubiquitous in nature, and the causes of the formation of the phenomenon are various, and sterile mutants can be obtained by methods such as spontaneous mutation, physical mutagenesis, chemical mutagenesis, biotechnology and the like. The cotton sterile line can be divided into a cytoplasmic sterile line and a nuclear sterile line. Cytoplasmic sterility refers to a line whose sterility trait is controlled by both genetic material in the cytoplasm and genetic material in the nucleus, and fertility can be restored by a restorer gene (Rf) carried in the nuclear genome. In the case of using a cytoplasmic male sterile line for the arrangement of hybrid, a maintainer line and a restorer line, i.e., a "three-line method", are also required. At present, cytoplasm of the cytoplasmic sterile line with breeding value mainly originates from Hakinisi cotton, the cytoplasm has adverse effect on offspring, and the problems of narrow recovery source and poor recovery capability exist, high-advantage hybrid combination is difficult to breed, and the application of three-line hybrid cotton in production is limited.
The fertility of the nuclear sterile line is controlled by the genes of cell nucleus, and the nuclear sterile line can be divided into dominant male sterility and recessive male sterility according to the recessive character of the genes. In cotton, 7 genic male sterile lines controlled by single recessive genes, 2 genic male sterile lines controlled by double recessive genes and 8 sterile lines controlled by dominant genic genes are found. When the nuclear recessive sterile line is used for seed production, single plants of field cotton need to be identified one by one, about 50 percent of fertile plants are pulled out, and sterile plants are remained for hybrid seed production, thereby causing waste of labor force and land resources. The photo-thermo-sensitive male sterile line is a special nuclear male sterile line, the fertility of which is controlled by nuclear genes and is regulated and controlled by the environment such as the illumination time length, the temperature and the like. At present, a plurality of photosensitive, temperature-sensitive and photo-temperature-sensitive male sterile lines, namely special cotton S-1, a photo-temperature A line with a yellow bud mark, TMS-2, Xiang QB and Zhong9106 and the like are found in cotton. The photosensitive genic male sterile line can be self-bred under the condition of fertile photoperiod, and can be matched with other fertile varieties to prepare hybrid seeds under the condition of sterile photoperiod. Is one of the most economic methods for utilizing the heterosis of crops at present.
A photosensitive male sterile mutant PSM1 with a temperature-sensitive yellowing character is obtained by a cotton research institute of Chinese agricultural academy of sciences in 2012 through a tissue culture method, and a photosensitive male sterile line PSM4 which has a normal phenotype and does not have a temperature-sensitive yellowing marker is separated from the photosensitive male sterile mutant PSM 1. Through the perennial fertility observation of Anyang and Mian experiment base in Henan, the sterile line PSM4 is a photosensitive male sterile line, the fertility of which is only controlled by the illumination period, the sterile line shows male sterility under the condition that the sunshine duration is more than 12 hours, the sterile line restores fertility under the condition that the sunshine duration is less than 11.5 hours, and the sterile line can be self-bred. The cultivation of PSM4 and the establishment of molecular marker assisted breeding technology will powerfully promote the utilization of cotton heterosis and develop new situation in cotton breeding industry. However, no molecular marker related to PSM4 assisted breeding is reported at present.
Disclosure of Invention
The invention provides a molecular marker closely linked with photosensitive male sterility character of cotton PSM4, a molecular identification method and application. The molecular marker which is closely linked with the photosensitive male sterility character of cotton PSM4 is provided, and the method for assisting in breeding the photosensitive male sterility character material in PSM4 and breeding offspring thereof by utilizing the molecular marker is provided. The molecular marker disclosed by the invention is tightly linked with the photosensitive nuclear sterility character, can assist in improving the photosensitive male sterile line PSM4 of cotton and cultivating photosensitive male sterile materials derived from the photosensitive male sterile line, and can be used for the work of purity identification of PSM4 and the photosensitive male sterile line derived from the photosensitive male sterile line, hybrid F1 generation and the like.
The invention provides a molecular marker closely linked with photosensitive male sterility traits of cotton PSM4, wherein the molecular marker comprises SSR232 and/or SSR 243; the nucleotide sequence of the forward primer of the SSR232 is shown as SEQ ID NO.1, the nucleotide sequence of the reverse primer of the SSR232 is shown as SEQ ID NO.2, the nucleotide sequence of the forward primer of the SSR243 is shown as SEQ ID NO.3, and the nucleotide sequence of the reverse primer of the SSR243 is shown as SEQ ID NO. 4.
The invention also provides a molecular identification method based on the molecular marker in the technical scheme, which comprises the following steps: respectively utilizing the primers of the molecular markers in the technical scheme to perform PCR amplification on a cotton genome to be detected, detecting the genotype, and identifying the cotton genome to be detected as the photosensitive male sterile single plant with the fertility characteristic of PSM4 when the primers of the SSR232 molecular markers are used for amplification, no specific band can be amplified in 400-500 bp and no specific band can be amplified in 500-1200 bp and/or the primers of the SSR243 molecular markers are used for amplification, and no specific band can be amplified in 50-100 bp.
The invention also provides application of the molecular marker in screening and/or breeding of the cotton PSM4 photosensitive male sterile line and breeding offspring thereof, wherein the screening and/or breeding comprises the step of applying the molecular marker in screening and/or breeding of the molecular marker with photosensitive male sterile property materials in the PSM4 photosensitive male sterile line and the breeding offspring thereof.
The invention also provides application of the molecular marker in the technical scheme in identifying the purity of the photosensitive male sterile line of cotton PSM4 and a photosensitive male sterile trait line of a breeding progeny thereof.
The invention also provides application of the molecular marker in detection of purity of hybrid derived from the cotton PSM4 photosensitive male sterile line and breeding offspring thereof.
The invention provides a molecular marker closely linked with photosensitive male sterility character of cotton PSM4, which locates PSM4 photosensitive nuclear sterility gene and realizes breeding application of the molecular marker. The invention provides a molecular marker of a cotton PSM4 photosensitive male sterile line and a breeding progeny thereof with photosensitive male sterile property materials. The molecular marker can be used for cotton molecular marker-assisted breeding, hybrid purity early detection and photosensitive male sterile line parent material purity identification related to cotton photosensitive male sterile line PSM4, and can be particularly used for identifying photosensitive nuclear sterile materials in PSM4 and breeding offspring thereof. Aiming at the problems of limited field identification time, long detection time and low efficiency of the detection method, the molecular marker provided by the invention can accelerate the breeding and improvement process of the cotton photosensitive male sterile line (PSM4 related). Firstly, the invention obtains molecular markers SSR232 and SSR243 of PSM4 photosensitive genic male sterile genes; secondly, the molecular marker of the invention can realize the rapid identification of the photosensitive male sterile individual plant in the filial generation of PSM4 and other cotton materials, and can rapidly breed the excellent photosensitive male sterile line derived from PSM 4; thirdly, the molecular marker of the invention can realize purity identification of PSM4 and the derivative photosensitive male sterile line thereof; fourthly, the purity of the hybrid can be identified by utilizing the molecular marker of the invention and combining the molecular marker characteristics of F1 and F2 generations. The test result shows that the SSR marker (SSR232 and/or SSR243) is adopted to carry out molecular level polymorphism detection on the cotton photosensitive male sterile material, and the identified result band is clear, stable and reliable.
Drawings
FIG. 1 is a diagram showing the results of polymorphism screening of SSR markers provided in example 1 of the present invention in "6 + 6" (5 sterile individuals +1 fertile parent in 1 sterile parent and F2 population and 5 fertile individuals in F2 population); wherein, each number represents a polymorphic marker, and the primer corresponding to the white dotted frame is the polymorphic marker obtained by the first screening;
FIG. 2 is a graph showing the result of identifying single plant fertility in the F2 population for 2 pairs of SSR markers provided in example 2 of the present invention; wherein each number represents a single plant, and the white arrows are marked as sterile single plants for molecular identification;
FIG. 3 is a graph showing fertility identification results of SSR232 and SSR243 provided in example 4 of the present invention in different filial generations; wherein each number represents a single plant, each combination is separated by a blank space, the last two samples of each combination are DNA samples of two parents, and the sterile female parent and the fertile male parent are sequentially and respectively arranged from left to right; white arrows mark the sterile individual plants identified by the molecules.
Detailed Description
The invention provides a molecular marker closely linked with photosensitive male sterility traits of cotton PSM4, wherein the molecular marker comprises SSR232 and/or SSR 243; the nucleotide sequence of the forward primer of the SSR232 is shown in SEQ ID NO. 1: 5'-GGTTCCTAGGTGTACAATTTCCC-3', the nucleotide sequence of the reverse primer of SSR232 is shown in SEQ ID NO. 2: 5'-TGGTGAACCCCGAATACACA-3', the nucleotide sequence of the forward primer of SSR243 is shown in SEQ ID NO. 3: 5'-GGTTGCTAGCTCCCTTTGCT-3', the nucleotide sequence of the reverse primer of SSR243 is shown in SEQ ID NO. 4: 5'-TGCAAGATGAGGTGACAGACA-3' are provided. The sequence information of the primer of the present invention is specifically shown in table 1.
TABLE 1 SSR primer sequence information
The invention also provides a molecular identification method based on the molecular marker in the technical scheme, which comprises the following steps: respectively utilizing the primers of the molecular markers in the technical scheme to perform PCR amplification on a cotton genome to be detected, detecting the genotype, and identifying the cotton genome to be detected as the photosensitive male sterile single plant with the fertility characteristic of PSM4 when the primers of the SSR232 molecular markers are used for amplification, no specific band can be amplified in 400-500 bp and no specific band can be amplified in 500-1200 bp and/or the primers of the SSR243 molecular markers are used for amplification, and no specific band can be amplified in 50-100 bp. The molecular marker detection technology is a common laboratory technology. PCR amplification used in the present inventionThe reaction system is preferably: each 10 mu L of the PCR amplification reaction system contains 0.5 mu L of forward primer and reverse primer of SSR232 or 0.5 mu L of forward primer and reverse primer of SSR243, 10 XPAGE Buffer 3 mu L, 1 mu L of 10mM dNTP, 0.4 mu L of PAGE Taqase, 1 mu L of DNA template and ddH2O3.6 mu L; the PCR amplification reaction condition is preferably pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 45s, and extension at 72 ℃ for 30s for 35 cycles; extending for 5min at 72 ℃; the molecular marker detection technology is preferably polyacrylamide gel electrophoresis. Other methods that can distinguish between the results of the molecular marker amplification may be substituted.
In the present invention, the source of the cotton genome to be tested includes plant tissues or seeds which can be sampled at various stages of the growth and development of cotton.
The invention also provides application of the molecular marker in screening and/or breeding of the cotton PSM4 photosensitive male sterile line and breeding offspring thereof, wherein the screening and/or breeding comprises the step of applying the molecular marker in screening and/or breeding of the molecular marker with photosensitive male sterile property materials in the PSM4 photosensitive male sterile line and the breeding offspring thereof. The photosensitive male sterile individual plant separated from the segregating generation of the detection hybrid contains the molecular marker banding pattern characteristic, and the photosensitive male sterile line parent is PSM4 or the derivative line thereof.
The invention also provides application of the molecular marker in the technical scheme in identifying the purity of the photosensitive male sterile line of cotton PSM4 and a photosensitive male sterile trait line of a breeding progeny thereof.
The invention also provides application of the molecular marker in detection of purity of hybrid derived from the cotton PSM4 photosensitive male sterile line and breeding offspring thereof.
The molecular marker closely linked with the photosensitive male sterility trait of cotton PSM4, the molecular identification method and the application of the present invention are described in further detail with reference to the following embodiments, which include but are not limited to the following embodiments.
Example 1
Acquisition of molecular marker closely linked with cotton PSM4 photosensitive male sterility gene
1. Construction of cotton photosensitive male sterility identification genetic population
Hybridizing the photosensitive male sterile mutant PSM4 as female parent and the Zhongmian institute 24 as male parent, and hybridizing the hybrid population F1Selfing to obtain F2Population, total 538 individuals. With F2The sterile single plant in the population is used as a female parent, and the Zhongmiao institute 24 is used as a male parent to obtain BC through hybridization1F1Population, then pair BC1F1Inbreeding of the population to obtain BC1F2And a total of 2118 individuals were obtained.
2. Extraction of genomic DNA
Taking tender leaf, extracting F by CTAB method2And BC1F2Genomic DNA of all individuals and parents of the population was measured for concentration and mass using a Biospec-nano microspectrophotometer.
3.F2And BC1F2Identification of group fertility
By using the Zhongmiao cotton plant 24 as a control, the anthers of the PSM4 and the Zhongmiao cotton plant 24 are observed on the day of flowering, the anthers are cracked, the individual plant with pollen grains scattered on the anthers is a fertile individual plant, the anthers are full, the surface is smooth, and the individual plant without pollen grains scattered is a photosensitive male sterile individual plant.
4. Determination of photosensitive male sterility gene linkage SSR marker
Taking two parents, BC1F25 photosensitive male sterile individuals and 5 fertile individuals in the population carry out polymorphism screening on the SSR marker in 5200 (figure 1, part of the SSR marker is '6 + 6' (1 photosensitive male sterile parent and F)25 photosensitive male sterile individuals in the population +1 fertile parent and F 25 fertile individuals in the population) is subjected to polymorphism screening; FIG. 1 shows: in each white dotted line frame, except the leftmost marker, 12 lanes are in a group, and from left to right, 1 photosensitive male sterile parent and F are respectively arranged25 photosensitive male sterile individuals, 1 fertile parent and F in the population 25 fertile individuals in the population. 1-9 white dotted line frame corresponding primer for screening obtained polymorphism marker; each number indicates a polymorphic marker, and the primer corresponding to the white dotted frame is the secondThe polymorphic markers obtained by one-time screening) to obtain 200 pairs of polymorphic markers (only part of the polymorphic markers are listed in figure 1), and taking F 245 fertile single plants and 45 sterile single plants in the population form a fertile mixed pool and a sterile mixed pool, and the character linkage analysis is carried out on 200 pairs of polymorphic markers to find that two pairs of markers are linked with the sterile character. Through further genome information comparison, two pairs of primer specific bands are positioned on chromosome A12, SSR markers on chromosome A12 are further screened, and two pairs of SSR markers, namely SSR232 and SSR243, are found in F2No crossover occurred in the population. Expanding the population at BC1F2Further detection of the crossover individual in the population, SSR232 and SSR243 at BC1F2Crossover individuals were also not detected in the population. The markers SSR232 and SSR243 are closely linked with the photosensitive male sterile gene.
The PCR amplification and polyacrylamide gel electrophoresis methods used were as described in 4.1 and 4.2 below:
4.1 PCR amplification
PCR amplification System: 10 μ L of reaction system containing 10 XPAGE Buffer (containing Mg)2+)3 μ L, dNTP (10mM)1 μ L, PAGE Taqase 0.4 μ L, primers 0.5 μ L, DNA template 1 μ L and ddH2O3.6μL。
PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 45s, and extension at 72 ℃ for 30s for 35 cycles; extending for 5min at 72 ℃, and storing at 10 ℃.
4.2. Polyacrylamide gel electrophoresis
Adding 10 mul of PCR product into 2 mul of bromophenol blue loading buffer solution, fully mixing uniformly, and centrifuging for later use;
the sample is loaded by a gun with 2 mu L, and after the sample is loaded, the power supply is switched on for 220V 1.5h, and electrophoresis is carried out.
After electrophoresis is finished, carefully taking out the gel in the electrophoresis plate and placing the gel in a special fixing groove;
480ml of pure water, 54ml of absolute ethyl alcohol and 3.6ml of glacial acetic acid are added into a special fixing tank;
slowly fixing on a shaking table for at least 8 minutes;
pouring out the stationary liquid, washing with pure water once, and performing the next infiltration;
weighing 1.2g of silver nitrate, dissolving in 540ml of pure water to prepare penetrating fluid, and injecting the penetrating fluid into a penetrating tank;
slowly fixing on a shaking table for at least 12 minutes;
pouring out penetrating fluid, washing twice with pure water, and performing next color development;
the reaction process is carried out in a fume hood, 24g of sodium hydroxide is weighed and dissolved in 1600ml of pure water to prepare a color developing solution, and after the solution is injected into a color developing groove and mixed uniformly, 10ml of formaldehyde solution is added;
placing the color development tank on a shaking table in a fume hood, and slowly shaking the shaking table (about 38rpm) to develop color until an SSR strip appears;
washing the film with pure water at least twice after the color development is finished;
placing the washed film on a film lamp for photographing, storing and reading; statistical analysis is carried out on the band type, and finally the SSR232 and the SSR243 are found to be closely linked with the photosensitive male sterile gene.
Example 2
SSR232 and SSR243 markers at F2Method for identifying photosensitive male sterile individual plant in seedling-generation period
F constructed by using SSR molecular markers SSR232 and SSR243 in PSM4 and Zhongmiao 24 as parents2Performing fertility identification on the colony at the seedling stage, identifying 1147 single plants in total, identifying 266 sterile single plants in the field fertility phenotype, wherein 261 sterile single plants are consistent with the molecular identification result, and 5 sterile single plants are possible to cause DNA extraction or experiment operation problems when the molecular identification is carried out, and no strip is generated; identifying 261 sterile single plants by using molecular markers, wherein the fertility of the 261 sterile single plants is consistent with the field identification result; the identification accuracy reaches 100 percent, and the molecular marker detection rate of the sterile line reaches 98 percent.
The method for identifying the cotton fertility in seedling stage by using molecular markers SSR232 and SSR243 comprises the following steps:
extracting F to be detected2DNA of individual plants of the population;
performing PCR amplification by taking the extracted DNA as a template and taking molecular markers SSR232 and SSR243 as primers respectively;
performing polypropylene gel electrophoresis on the PCR amplification product, and analyzing the genotype by silver staining(FIGS. 2, 2 for SSR marker at F2Identifying the fertility of a part of single plants in the population; where each number represents an individual and the white arrows are labeled as molecularly identified light sensitive male sterile individuals). 261 photosensitive male sterile single plants are identified by the two molecular markers, and the identification result is consistent with the field surface identification result, which shows that the molecular marker can be used for screening the photosensitive male sterile single plants. The marker SSR232 respectively amplifies a specific strip between 400-500 bp and 500-1200 bp which take DL50maker of a prokaryote as a standard in a normal fertile single plant, and does not amplify the two strips in a photosensitive male sterile single plant; the marker SSR243 is used for amplifying a specific band between 50-100 bp which takes DL50maker of a prokaryote as a standard in a normal fertile single plant, and the band is not amplified in a photosensitive male sterile single plant.
Example 3
Photosensitive male sterile line seed identification and detection method and application
F produced by hybridization of PSM4 and Zhongmiao 24 by SSR molecular markers SSR232 and SSR2432And (5) identifying the seeds. 538 seeds are identified in total, 126 photosensitive male sterile line seeds are identified by the two molecular markers, the 126 seeds are sown and germinate into 84 viable plants, and the fertility phenotype identification is photosensitive male sterile single plants.
The method for identifying the fertility characteristics of cotton seeds by using the molecular markers SSR232 and SSR243 comprises the following steps:
DNA extraction: 1/3 seeds to be detected are cut off from the big head and placed in a 1.5mL centrifuge tube, 200 mu L of DNA extracting solution (100mM Tris-HCl, 1M KCl, 100mM EDTA) is added, the sample is mashed by a sterilized gun head and then incubated in a thermostat at 95 ℃ for 10min, then the obtained liquid is fully shaken, finally, the mixture is centrifuged at 12000rpm for 5min, and the supernatant is taken as a DNA sample for standby.
Performing PCR amplification by taking the extracted DNA as a template and taking the molecular markers SSR232 and SSR243 as primers;
and performing polypropylene gel electrophoresis on the PCR amplification product, and analyzing the genotype by silver staining. The marker SSR232 respectively amplifies a specific strip between 400-500 bp and 500-1200 bp which take DL50maker of organisms in the department of Ongziaceae as a standard in a normal fertile single plant, and the two strips are not amplified in a photosensitive male sterile single plant; the marker SSR243 is used for amplifying a specific band between 50-100 bp which takes DL50maker of a prokaryote as a standard in a normal fertile single plant, and the band is not amplified in a photosensitive male sterile single plant.
Example 4
Molecular identification of photosensitive male sterile individual plant separated from hybrid progeny of PSM4 and other materials
Molecular identification is carried out on hybrid progenies of 15 materials which take PSM4 as a female parent and take 15 materials such as Zhong 99668, Simian No.3, Xinluzao No. 12, Xinluzao No. 62, Xinluzhongzhong 26, Zhongmian 125, Lu-mian No. 28, Shikang 126, Zhongmian 94A915, Zhongmian 94A213, Zhongmian 425, Zhongmian 50, Zhongmian 41, Zhongmian 45 and Zhongmian 100 as male parents by utilizing SSR molecular markers SSR232 and SSR 243. 15 filial generation groups are planted in a test field of an Antang Zhongmiao, 3 rows are planted in each combination, one row is randomly taken to extract DNA samples of single plants for molecular identification, and fertility identification is carried out in a field in a full-bloom stage. 441 individuals are identified in total, 72 photosensitive male sterile individuals are identified in field phenotype, the identification results of the two molecular markers are 70 photosensitive male sterile individuals, the undetected 2 electrophoresis results are recorded as blank, and no band is amplified during DNA detection, so that the DNA detection method belongs to the problem of DNA quality or experimental errors caused by experimental operation. The detection rate of the photosensitive male sterile single plant is 97.2 percent.
The method for identifying the fertility of the progeny cotton of different hybridization groups by using the molecular markers SSR232 and SSR243 comprises the following steps:
extracting F to be detected2Population DNA;
performing PCR amplification by taking the extracted DNA as a template and taking the molecular markers SSR232 and SSR243 as primers;
subjecting PCR amplification product to polypropylene gel electrophoresis, and silver staining to analyze its genotype (FIG. 3, fertility identification result chart of SSR232 and SSR243 in five filial generations, wherein each number represents a single plant, each combination is separated by blank space, the last two samples of each combination are DNA samples of two parents, from left to right are photosensitive male sterile female parent and fertile female parent respectivelyA male parent; marked by white arrows are photosensitive male sterile individuals subjected to molecular identification, and five combined male parents are Zhong 99668, Simian No.3, Xinluzao 62, Zhongmian 125 and Luyan cotton No. 28 in turn; maternal PSM 4). Identification of F in a total of 15 different hybridization combinations2The population comprises 441 individuals, 70 sterile individuals are identified by two molecular markers, 72 sterile individuals are identified by field phenotypes, the individuals identified as photosensitive male sterility by the molecular markers are matched with field identification results, and the undetected 2 electrophoresis results are recorded as blank, namely, no band is amplified during DNA detection. The molecular marker has stability in different parents, and F of different hybridization combinations can be identified2And (4) a group. The marker SSR232 respectively amplifies a specific strip in a normal fertile single plant between 400-500 bp and between 500-1200 bp which take DL50maker of a prokaryote as a standard, but does not amplify the two specific strips in a sterile single plant, and the two specific amplified strips are thinner than the lower non-specific amplified strips; the marker SSR243 is characterized in that a specific band is amplified in a normal fertile single plant between 50-100 bp which takes DL50maker of a prokaryote as a standard, and the specific band is not amplified in a photosensitive male sterile single plant.
Example 5
Purity identification of photosensitive male sterile line
PSM4 and its derived photosensitive male sterile line can be sampled at seed stage or other growth and development stage, and DNA of single plant or single seed is extracted and subjected to molecular identification method by the method in example 1. The proportion of seeds or single plants which can not be amplified to the total number of samples by using the primer of the invention for amplification is the purity of the photosensitive male sterile line.
Example 6
Purity identification of hybrid seeds
F is to be1The DNA is extracted from the single-seed sample of the generation hybrid or the single-plant sample of the field material, and the F can be identified by the molecular marker detection method in the embodiment 11The percentage of individuals with normal fertile individual banding patterns in the individual identification of generations to the total test individuals was taken as F1Index of substitution purity. If purity is highThe index can not meet the purity requirement of the hybrid, which indicates that the F1The generation hybrid is mixed with a higher photosensitive male sterile individual, thereby deducing F1The generation hybrid seeds may be mixed with seeds of separate generation or with photosensitive sterile materials; if F1In the individual plant of (A), a specific banding pattern of a non-photosensitive male-sterile plant is present, in which F2The banding pattern of the photosensitive male sterile plant was detected in the generation, and F was deduced to be1The generation is the first generation of hybrid seeds cultivated by using the photosensitive male sterile line.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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tgcaagatga ggtgacagac a 21
Claims (4)
1. The molecular identification method based on the molecular marker closely linked with the photosensitive male sterility trait of cotton PSM4 comprises the following steps: respectively utilizing primers of molecular markers closely linked with photosensitive male sterility traits of cotton PSM4 to perform PCR amplification on a cotton genome to be detected, detecting the genotype, and identifying a photosensitive male sterile single plant with the fertility characteristic of PSM4 when specific bands cannot be amplified in 400-500 bp and 500-1200 bp and specific bands cannot be amplified by primers of SSR243 molecular markers and specific bands cannot be amplified in 50-100 bp by primers of SSR232 molecular markers;
the molecular markers closely linked with the photosensitive male sterility trait of cotton PSM4 are SSR232 and SSR 243; the nucleotide sequence of the forward primer of the SSR232 is shown as SEQ ID NO.1, the nucleotide sequence of the reverse primer of the SSR232 is shown as SEQ ID NO.2, the nucleotide sequence of the forward primer of the SSR243 is shown as SEQ ID NO.3, and the nucleotide sequence of the reverse primer of the SSR243 is shown as SEQ ID NO. 4.
2. The application of the molecular identification method of claim 1 in screening and/or breeding of cotton PSM4 photosensitive male sterile line and its breeding progeny, wherein the screening and/or breeding comprises applying the molecular marker in screening and/or breeding of PSM4 photosensitive male sterile line and its breeding progeny with photosensitive male sterile property material.
3. The application of the molecular identification method of claim 1 in identifying the purity of the photosensitive male sterile line of cotton PSM4 and the photosensitive male sterile trait line of its breeding progeny.
4. The use of the molecular characterization method of claim 1 in the detection of purity of hybrids derived from cotton PSM4 light sensitive male sterile line and its breeding progeny.
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| WO2019153627A1 (en) * | 2018-02-09 | 2019-08-15 | 中国农业科学院棉花研究所 | Photoperiod sensitive genetic male sterility mutant of cotton and application thereof |
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| WO2019153627A1 (en) * | 2018-02-09 | 2019-08-15 | 中国农业科学院棉花研究所 | Photoperiod sensitive genetic male sterility mutant of cotton and application thereof |
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