CN111394509A - Molecular marker linked with pepper reverse temperature-sensitive sterile gene and application thereof - Google Patents
Molecular marker linked with pepper reverse temperature-sensitive sterile gene and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker linked with a pepper reverse temperature-sensitive genic male sterile gene and application thereof. Construction of F from wild-type and mutant Capsicum annuum materials2And (4) a group. Obtaining a chromosome region closely linked with the hot pepper reverse temperature-sensitive sterile gene by using a BSA group positioning method, and developing a molecular marker in a candidate region. Designing 1 KASP molecular marker based on single base mutation, and using the marker pair F2The genotype identification is carried out on 220 single plants in the population, and the coincidence rate reaches 100 percent. The research result is not only beneficial to the identification and the auxiliary breeding of the reverse temperature-sensitive sterility of the hot pepper, but also provides a foundation for the map-based cloning of the reverse temperature-sensitive sterility gene and the analysis of the molecular mechanism of the reverse temperature-sensitive sterility, and has wide popularization value.
Description
Technical Field
The invention belongs to the field of pepper breeding molecular biology, and relates to a molecular marker linked with a pepper (Capsicum annuum L) temperature-sensitive sterile gene and application of the molecular marker in identification and breeding of pepper temperature-sensitive sterility.
Background
The plant male sterility has an important role in heterosis utilization, and is widely applied to the production of commercial hybrid of crops such as rice, wheat, corn, rape, cotton and the like, wherein the temperature-sensitive sterility is one of important types of male sterility, two-line breeding based on the temperature-sensitive male sterility line has obvious advantages, is not restricted by restoration and conservation relation, is matched with one line for two purposes, has simple production process and no potential risk brought by cytoplasmic negative effect and single cytoplasm.
The fertility of the reverse temperature-sensitive male sterile line is opposite to that of the forward temperature-sensitive male sterile line, and the reverse temperature-sensitive male sterile line has the characteristic of high-temperature fertility and low-temperature sterility. The reverse temperature-sensitive male sterile line can be self-propagated under the condition of high temperature, and can be used for seed production in higher latitude or higher altitude areas and low latitude and lower temperature. At present, the rice reverse temperature-sensitive male sterile line mainly comprises Yunnan agriculture S-2, Kunzhi S-1, Heng S-3, IVA, go543S, J207S, DIS and G20S and reverse temperature-sensitive male sterile line FHS obtained by physical mutagenesis. Researches show that the fertility conversion temperature of the reverse temperature-sensitive male sterile line go543S is 28-30.5 ℃, fertility is only controlled by temperature, namely fertility is fertile at the temperature higher than 30.5 ℃ and sterile at the temperature of 28 ℃. The fertility conversion temperature of the reverse temperature-sensitive male sterile line FHS is 25-26 ℃ (Wang Hu, 2005). In wheat, fertility characteristics of the reverse temperature sensitive male sterile lines such as ES series, C49S and C86S series, YM3314, A3017 and 337S are also reported successively. The discovery and the breeding of the reverse temperature-sensitive male sterile line expand a new application space for two-line hybrid breeding and heterosis utilization.
Capsicum (Capsicum annuum L.) Solanaceae, Capsicum annuum, or perennial herb with limited quantity, in recent years, the annual planting area of Capsicum annuum in China is 150-200 ten thousand hm2At present, the sowing area accounts for 8 to 10 percent of the total sowing area of vegetables in China. At present, no report related to reverse temperature-sensitive genic male sterility of pepper exists, so that the pepper temperature-sensitive sterile mutant as an important germplasm resource is favored by breeders in the future. By developing a molecular marker linked with the reverse temperature-sensitive sterile gene of the pepper and adopting the technologies of hybridization, backcross and the like, the temperature-sensitive sterile gene and other excellent character genes are quickly introduced into a plant, thereby providing gene resources for breeding new varieties with high quality. Developing molecular markers linked with the reverse thermo-sensitive sterile gene is beneficial to the breeding of the hot pepper thermo-sensitive male sterility, and lays a foundation for cloning the hot pepper reverse thermo-sensitive sterile gene and researching the molecular mechanism of the reverse thermo-sensitive sterility.
Disclosure of Invention
The invention aims at providing a molecular marker Pe _ K _1200003 (G-to-A at Chr 12: 12,852,132bp of the whole genome of pepper) linked with a pepper reverse temperature-sensitive sterile gene aiming at a male temperature-sensitive sterile phenomenon (fertility is shown at 28 ℃ or above and sterility is shown below 28 ℃) appearing in pepper bud development. Provides a new approach for screening, identifying and assisting in screening the hot pepper temperature-sensitive sterile mutant, breeding hot pepper male temperature-sensitive sterile and the like.
The genotypes corresponding to the molecular markers comprise: g and A G are fertile genotypes with pollen viability, A is sterile genotypes without pollen viability.
The pollen of the invention has pollen activity represented by pollen and sufficient fertility activity (at 28 ℃ and above), and the pollen of the invention has no pollen activity represented by no pollen or very little pollen and no fertility activity (below 28 ℃).
The primers designed aiming at the reverse temperature-sensitive sterile mutation of the hot pepper comprise two forward primers and a reverse primer,
forward primer X: AAAATATCAAACCAATTCCAACTAG, see SEQ ID No. 1;
forward primer Y: AAAATATCAAACCAATTCCAACTAA, see SEQ ID No. 2.
Two forward primers were ligated to different fluorescent linker sequences (FAM or HEX linker sequences from L GC Co.)
Preferably:
FAM: GAAGGTGACCAAGTTCATGCT, see SEQ ID No. 3;
HEX: GAAGGTCGGAGTCAACGGATT, see SEQ ID No. 4.
The primer sequences after connecting different fluorescent linker sequences respectively are as follows:
forward primer Pe _ K _ 1200003X: GAAGGTGACCAAGTTCATGCTAAAATATCAAACCAATTCCAACTAG, see SEQ ID No. 5;
forward primer Pe _ K _ 1200003Y: GAAGGTCGGAGTCAACGGATTAAAATATCAAACCAATTCCAACTAA, see SEQ ID No. 6;
reverse primer Pe _ K _ 1200003C: TCACATGTTAGAAATTGTTTGTCA, see SEQ ID No. 7;
the second purpose of the invention is to provide the application of the molecular marker, which is beneficial to breeding of pepper pollen fertility and lays a foundation for cloning the temperature-sensitive sterility and researching the molecular mechanism of the temperature-sensitive sterility.
The method specifically comprises the following steps: is used for identifying and assisting in screening the fertility of the pepper.
Further, the method is used for identifying, screening or breeding the pepper temperature-sensitive sterility.
When the molecular marker is applied, PCR reaction is adopted for detection.
When the molecular marker is applied, the method specifically comprises the following steps: (1) taking the genome DNA of a sample to be detected as a template, and carrying out amplification by using a molecular-labeled amplification primer to obtain an amplification product; (2) and detecting and analyzing the amplification product.
When the amplified product is subjected to fluorescence detection, if the PCR product of the sample only detects a fluorescence signal corresponding to the primer Pe _ K _1200003X, the detection site is G, namely G genotype, and the pepper is judged to be a single plant with pollen activity phenotype; if only the fluorescent signal corresponding to the primer Pe _ K _1200003Y is detected from the PCR product of the sample, the detection site is A: A genotype, and the individual plant with the pepper pollen sterile phenotype is judged; and if two fluorescent signals are detected simultaneously, determining that the detection site is an A: G genotype, and judging that the pepper is a single plant with pollen activity phenotype.
When the molecular marker is applied, Touchdown PCR is adopted, and the amplification procedure is as follows: 15min at 94 ℃; 20s at 95 ℃; 60s at 65-56 ℃, 10 cycles, and 0.8 ℃ reduction of annealing extension temperature of each cycle; 20s at 94 ℃; 57 ℃ for 60s, 26 cycles.
The invention utilizes a BSA positioning method to position a new SNP variation for controlling the development of pepper pollen, and develops a KASP molecular marker associated with the pepper pollen development gene according to the mutation site of the gene. The molecular marker can be directly used for identifying the fertility and the corresponding genotype of the pepper pollen, and then the molecular marker is used for assisting breeding, so that the problems of long conventional breeding period and easy environmental influence can be effectively solved. Satisfactory plants can be quickly screened by utilizing the molecular marker in the early stage, the planting scale is effectively reduced, and the workload of later-stage identification is reduced. The efficiency and accuracy of selection are improved. Can be used for identifying various hot pepper varieties and has great significance for researching the forming mechanism of reverse temperature-sensitive infertility. Therefore, the invention has important significance in hot pepper male temperature-sensitive sterile breeding practice and temperature-sensitive sterile theory research.
Drawings
FIG. 1 shows F constructed by Pe _ K _1200003 molecular marker in Wild Type (WT) and Mutant (MT) according to the present invention2Partial results of genotyping in the population;
a represents: the PCR product is a fluorescent signal corresponding to the primer Pe _ K _1200003Y and is a sterile homozygous single plant;
b represents: the PCR product is a fluorescent signal corresponding to the primer Pe _ K _1200003X and is a fertile homozygous single plant;
and C represents: the PCR product has two corresponding fluorescent signals of the primer Pe _ K _1200003X, Y and is a fertile heterozygous single strain.
Fig. 2 is a graph of 2 parents located in the BSR population: 6421(a), E6421S (B).
FIG. 3 shows the BSA localization results of 6421 and E6421S constructed populations;
chr01-Chr12 represents chromosome number, and the male temperature-sensitive sterile gene is located on chromosome 12 in the region of 11-20M.
FIG. 4 shows the fine localization of the mutation of the thermo-sensitive sterile gene of the present invention.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples. The materials, reagents, apparatus and methods used in the following examples, which are not specifically illustrated, are all conventional in the art and are commercially available. The pepper germplasm related in the invention is provided by vegetable research institute in Hunan province, and can be sold for at least 20 years.
Example 1 obtaining BSA positioning method of molecular marker BSA molecular marker linked with hot pepper temperature-sensitive sterile gene
1. Construction of the population
Fertile material 6421 '(shown in figure 2A) bred by multi-generation selfing and reverse temperature-sensitive sterile material E6421S' (shown in figure 2B) are used as parents, the fertile parents and the temperature-sensitive sterile parents are hybridized to obtain F1 generation, and F2 population is obtained after F1 generation selfing.
2. Pollen fertility identification
And (4) observing and identifying the existence of pollen when the F2 generation population is in the full-bloom stage, and determining the fertility of the single plant in the population.
3. Primary localization of temperature sensitive sterile gene
F at 6421 × E6421S2Respectively selecting 25 leaves with pollen plants and 25 leaves without pollen plants from a population, equally mixing the leaves of each plant to construct a fertile/sterile DNA mixed pool, extracting total DNA of 4 mixed pools by using a CTAB method, establishing a library for the DNA of 4 mixed pools by using TruSeq DNA L T Sample Prep Kit (Illumina corporation), and performing Illumina HiSeq2000 platformSequencing, performing genome sequencing, extracting polymorphic SNP (single nucleotide polymorphism) from obtained data by utilizing samtools software and an autonomously developed Perl script, calculating the maximum allele type frequency (SNP-index) of a recessive pool and the frequency of the genotype in a dominant pool, calculating the Euclidean distance (ED value) of each SNP locus, filtering out the loci with the sequencing depth of both pools being lower than 10 reads and the SNP-index being greater than 0.7, performing linear regression fitting and mapping after performing power on the ED value, taking 3 times of the sum of median and standard deviation of power-power fitting values of all loci as a threshold line, and taking the section higher than the threshold line as a candidate section, namely positioning the gene for controlling temperature-sensitive sterility in the 11-20M section on the chromosome 12, wherein the section higher than the threshold line is used as a candidate section, namely positioning the gene for controlling the temperature-sensitive sterility in the chromosome 12, namely the chromosome Chr 01-r 12 represents the chromosome number, and the main temperature-sensitive sterility effect QT L is positioned on the chromosome 12.
4. Fine positioning of thermo-sensitive sterile gene
Obtaining a chromosome region for controlling temperature-sensitive sterility through the step 3, analyzing DNA sequence variation in a section in order to further narrow down a candidate region for controlling temperature-sensitive sterility genes, developing InDel and KASP markers, and using the developed InDel and KASP molecular markers to pair F2Individuals of the population are genotyped and crossover individuals are determined. Based on the phenotype survey data of pollen and the determined genotype of the crossover individual plant, the thermo-sensitive sterile gene is positioned in an interval of 11-13M, and KASP marker typing of the pepper is carried out according to SNP in the interval.
Finally obtaining the pepper whole genome Chr12 by sequencing: mutation of G to A at 12,852,132bp, see FIG. 4.
220 individuals of the F2 population constructed by 6421 and E6421S were genotyped with the Pe _ K _1200003 marker. 3 fluorescent signals appeared, with 65 individuals for A, 105 for A, and 50 for G. The phenotype investigation data is combined to find that the genotype is highly consistent with the pollen fertility phenotype, and the coincidence rate reaches 100%. The results fully show that the Pe _ K _1200003 marker has universality and accuracy, and can be applied to prediction, identification and screening of hot pepper temperature-sensitive sterile plants.
When the molecular marker is applied, the method specifically comprises the following steps: (1) taking the genome DNA of a sample to be detected as a template, and carrying out amplification by using a molecular-labeled amplification primer to obtain an amplification product; (2) and detecting and analyzing the amplification product.
Reverse primer Pe _ K _ 1200003C: TCACATGTTAGAAATTGTTTGTCA
Forward primer Pe _ K _ 1200003X: GAAGGTGACCAAGTTCATGCTAAAATATCAAACCAATTCCAACTAG are provided.
Forward primer Pe _ K _ 1200003Y: GAAGGTCGGAGTCAACGGATTAAAATATCAAACCAATTCCAACTAA are provided.
The primer of the invention has specificity through hot pepper whole genome sequence sequencing verification.
When the amplified product is subjected to fluorescence detection, if the PCR product of the sample only detects a fluorescence signal corresponding to the primer Pe _ K _1200003X, the detection site is G, namely G genotype, and the pepper is judged to be a single plant with pollen activity phenotype; if only the fluorescent signal corresponding to the primer Pe _ K _1200003Y is detected from the PCR product of the sample, the detection site is A: A genotype, and the individual plant with the pepper pollen sterile phenotype is judged; and if two fluorescent signals are detected simultaneously, determining that the detection site is an A: G genotype, and judging that the pepper is a single plant with pollen activity phenotype.
When the molecular marker is applied, Touchdown PCR is adopted, and the amplification procedure is as follows: 15min at 94 ℃; 20s at 95 ℃; 60s at 65-56 ℃, 10 cycles, and 0.8 ℃ reduction of annealing extension temperature of each cycle; 20s at 94 ℃; 57 ℃ for 60s, 26 cycles.
The detection sample is a leaf, and the pollen fertility detection is carried out at about 22 ℃.
Table 1 shows the sterility type and genotype of some individuals marked by Pe _ K _1200003 in F2 populations constructed by 6421 and E6421S.
The identification result shows that materials for detecting the fluorescence signal A corresponding to the primer Pe _ K _1200003Y (figure 1) are reserved through molecular marker identification screening in breeding, and then the materials for male temperature-sensitive sterility of the pepper can be bred. The fertile homozygous material can be bred by reserving the material of which the B fluorescent signal corresponding to the primer Pe _ K _1200003X (FIG. 1) is detected. The materials of which the fluorescence signals (including the fluorescence signals corresponding to the first two primers) are detected in the C in the figure 1 are reserved, so that the fertile hybrid materials can be bred, the workload of later screening and identification can be reduced through screening of the early molecular markers, and the breeding process is accelerated.
Sequence listing
<110> vegetable research institute of Hunan province
<120> molecular marker linked with pepper reverse temperature-sensitive sterile gene and application
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aaaatatcaa accaattcca actaa 25
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gaaggtgacc aagttcatgc t 21
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gaaggtcgga gtcaacggat t 21
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gaaggtgacc aagttcatgc taaaatatca aaccaattcc aactag 46
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gaaggtcgga gtcaacggat taaaatatca aaccaattcc aactaa 46
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Claims (10)
1. A molecular marker linked with a pepper reverse temperature-sensitive sterile gene is characterized in that: is pepper Chr 12: mutation of G to A at 12,852,132 bp.
2. The molecular marker of claim 1, wherein the genotype of the molecular marker comprises: g is the genotype with pollen activity, A is the genotype with pollen activity; a is a genotype without pollen viability.
3. The molecular marker according to claim 2, wherein the primers designed for the reverse thermo-sensitive sterile mutation of pepper comprise two forward primers and one reverse primer,
forward primer X: AAAATATCAAACCAATTCCAACTAG, respectively;
forward primer Y: AAAATATCAAACCAATTCCAACTAA are provided.
4. The molecular marker of claim 3, wherein: the two forward primers are respectively connected with different fluorescent joint sequences;
preferably:
FAM：GAAGGTGACCAAGTTCATGCT
HEX：GAAGGTCGGAGTCAACGGATT。
5. use of a molecular marker as claimed in any one of claims 1 to 4 for identifying and aiding in the screening for fertility in capsicum.
6. The use according to claim 5, for identifying, screening or breeding pepper temperature-sensitive sterile mutants.
7. Use according to claim 5, wherein the detection is carried out using a PCR reaction.
8. The use according to claim 7, characterized in that it comprises in particular the following steps:
(1) taking the genome DNA of a sample to be detected as a template, and carrying out PCR amplification by using a molecular-labeled amplification primer to obtain an amplification product;
(2) and detecting and analyzing the amplification product.
9. The use according to claim 8, wherein the amplification product is determined to be a homozygous wild single plant having pollen viability if the amplification product is of G: G genotype when subjected to fluorescence detection; if the A is A, judging that the homozygous mutant single plant does not have pollen activity; and if the genotype is A: G, judging the heterozygous individual with pollen vigor.
10. Use according to claim 8, characterized in that Touchdown PCR is used; the touch hdown PCR amplification procedure is as follows: 15min at 94 ℃; 20s at 95 ℃; 60s at 65-56 ℃, 10 cycles, and 0.8 ℃ reduction of annealing extension temperature of each cycle; 20s at 94 ℃; 57 ℃ for 60s, 26 cycles.
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| CN117385087A (en) * | 2023-11-27 | 2024-01-12 | 北京市农林科学院 | SNP molecular marker Me-5676929 linked with microspore embryogenic gene, application thereof and special primer |
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|---|---|---|---|---|
| CN108754013A (en) * | 2018-06-24 | 2018-11-06 | 华中农业大学 | A kind of and capsicum cytoplasmic male sterility restoring gene close linkage molecular labeling and its preparation method and application |
| CN109207622A (en) * | 2018-10-24 | 2019-01-15 | 湖南省蔬菜研究所 | A kind of molecular labeling and application with the stagnant green gene linkage of capsicum |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108754013A (en) * | 2018-06-24 | 2018-11-06 | 华中农业大学 | A kind of and capsicum cytoplasmic male sterility restoring gene close linkage molecular labeling and its preparation method and application |
| CN109207622A (en) * | 2018-10-24 | 2019-01-15 | 湖南省蔬菜研究所 | A kind of molecular labeling and application with the stagnant green gene linkage of capsicum |
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| CN117385087A (en) * | 2023-11-27 | 2024-01-12 | 北京市农林科学院 | SNP molecular marker Me-5676929 linked with microspore embryogenic gene, application thereof and special primer |
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