CN112216343A - Screening method of tomato spotted wilt virus resistant hot pepper plants - Google Patents
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Abstract
The invention discloses a screening method of tomato spotted wilt virus resistant hot pepper plants, which comprises the steps of obtaining TSWV resistant phenotype data of a test material; obtaining key QTL sites resisting TSWV and key SNP sites resisting TSWV; cloning and quantitatively expressing the screened candidate genes; on the basis of the correlation analysis and linkage analysis of the pepper TSWV-resistant whole genome, the relation between the polymorphic sites and the TSWV-resistant strength is found, and a molecular marker based on PCR is developed aiming at the significant correlation sites/QTL sites; and (3) breeding a backbone inbred line resisting the tomato spotted wilt virus based on the determined molecular marker, and finally breeding a hybrid variety resisting the tomato spotted wilt virus by using a three-line method. By adopting the screening method, theoretical and material support can be provided for pepper disease-resistant molecule breeding, and a more effective means is provided for new pepper variety breeding, so that a characteristic hybrid combination which is in line with the preparation of high-quality dried peppers is screened out for popularization and application, and the economic benefit and the social benefit are very obvious.
Description
Technical Field
The invention relates to the technical field of crop breeding protection, in particular to a screening method of tomato spotted wilt virus resistant hot pepper plants.
Background
The pepper is a vegetable crop which is popular in daily life of people, the cultivation is general and wide, and in the planting process of the pepper, diseases and insect pests are often easy to occur. Tomato Spotted Wilt Virus (TSWV) is an important plant virus which endangers agricultural production, belongs to a representative species of Tomato spotted wilt virus of bunyaviridae, can infect more than 70 families, more than 1 thousand plants, is a plant virus with the widest known host range at present, is widely distributed in the world, is also the primary virus produced by pepper in North America, Europe and the like, and is reported successively in Brazil, America, Italy, Australia and Korea, so that great loss is brought to pepper production, and in severe cases, the Tomato spotted wilt virus can cause failure. In recent years, tomato spotted wilt virus is found in Yunnan hot pepper in China. Tomato spotted wilt virus is a virus disease specially transmitted by thrips, western flower thrips are found for the first time in China around 2000 years, and then are popular nationwide, and are seriously damaged in open places of Guangdong, Hainan, Guangxi, Yunnan and Guizhou, Shandong, Beijing and other places in the north at present. With the epidemic spread of frankliniella occidentalis, the damage of tomato spotted wilt virus to pepper tends to be aggravated.
Due to the large mobility and the rapid propagation of the toxic insect frankliniella occidentalis, even by improving cultivation measures and planting structures and combining chemical control and biological control, the obvious control effect is difficult to achieve, so that the method is a major problem which troubles the production of hot peppers from discovery to now and is deeply concerned by researchers at home and abroad. From the perspective of 'plant health management', breeding pepper virus-resistant varieties is considered to be the most urgent, most effective and most environment-friendly way, namely, the utilization of the disease resistance of hosts is one of the most effective ways for preventing diseases at present. Screening and evaluating pepper TSWV-resistant germplasm resources, and researching disease-resistant QTL positioning, germplasm innovation and the like based on molecular markers and related mapping populations (F2) are successively carried out at home and abroad. However, due to the strong research dispersity, the disease-resistant germplasm resources are mainly influenced by factors such as foreign research units, different QTL positioning populations, lack of common molecular markers, lack of populations for fine positioning and gene cloning and the like, and the process of breeding new varieties of the tomato spotted wilt virus resistant capsicum is severely restricted and limited.
Disclosure of Invention
The invention aims to solve the technical problem in the background technology and provides a screening method, the method can accurately reflect the disease resistance of the pepper to the tomato spotted wilt virus, and the characteristic hybrid composition which is in line with the preparation of high-quality dry pepper is selected for popularization and application by identifying the anti-infection performance of each variety of pepper seedlings to the tomato spotted wilt virus, in particular to the screening method of the tomato spotted wilt virus resistant hot pepper plants.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a screening method of tomato spotted wilt virus resistant hot pepper plants comprises the following steps:
(1) adopting pepper seedlings of an interspecific BA3 YNXML F2 population and pepper seedlings of an inbred line or a local species to respectively pass single-point and repeated field tests, and carrying out phenotype identification on the pepper seedlings through the field tests to obtain the TSWV-resistant phenotype data of the tested material;
(2) carrying out QTL positioning on pepper TSWV resistance by taking a high-density genetic linkage map constructed by an interspecific BA 3X YNXML F2 population as a basis and combining the TSWV-resistant phenotype data of the test material obtained in the step (1) to obtain key QTL sites of the TSWV resistance; meanwhile, carrying out whole genome association analysis on the TSWV resistance of the pepper by taking SNP genotype data constructed by cross-bred or local species of pepper as the basis and combining the TSWV resistance phenotype data of the test material obtained in the step (1) to obtain key SNP loci of the TSWV resistance;
(3) on the basis of the key QTL sites and key SNP sites obtained in the step (2), determining gene distribution information between sections and in the sections on a genome sequence of the pepper Zunla-1, screening haplotype or allelic variation with important application value by using a comparative genomics and bioinformatics analysis method, screening key QTL and genes determining TSWV resistance, and cloning and quantitatively expressing and analyzing the screened candidate genes;
(4) on the basis of hot pepper TSWV-resistant whole genome association analysis and linkage analysis, selecting 5-8 genome sequences where SNP/QTL (single nucleotide polymorphism/quantitative trait locus) which are significantly associated with TSWV-resistant genes are located, selecting 3-5 candidate genes by combining the selected gene functions and detected TSWV-resistant QTL sites, searching for polymorphic sites of the genes by a PCR (polymerase chain reaction) re-sequencing method, finally detecting the relation between the searched polymorphic sites and the TSWV-resistant strength by the candidate gene association analysis method, and developing PCR-based molecular markers aiming at the significantly associated sites/QTL sites;
(5) and (4) breeding a backbone inbred line resisting the tomato spotted wilt virus based on the molecular marker determined in the step (4), and finally breeding a hybrid variety resisting the tomato spotted wilt virus by using a three-line method.
Further, in the screening method of the tomato spotted wilt virus resistant pepper plants, in the step (4), the relation between the polymorphic site and the TSWV resistance is the disease of the pepper seedlings to be detected, the disease of the disease resistant pepper seedlings to be detected needs to be investigated once every 10 days, the disease incidence A of the pepper seedlings against the tomato spotted wilt virus is calculated, 2-3 times of investigation are needed in total, resistant and sensitive plants are classified, and the highest incidence investigation result is taken as the disease condition of the pepper seedlings of the variety for statistics; the disease rate A = (number of infected plants of the variety/total number of plants of the variety). times.100%.
Furthermore, the screening method of the tomato spotted wilt virus resistant hot pepper plants, disclosed by the invention, is characterized in that the disease investigation adopts a method of serological detection or molecular biological detection; during the serological detection, collecting leaves of a pepper seedling plant to be detected with disease resistance, and performing ELISA detection by using a tomato spotted wilt virus test strip or a tomato spotted wilt virus antibody, wherein if the leaves are positive, the pepper seedling plant is indicated as a disease-sensitive plant, and if the leaves are negative, the pepper seedling plant is indicated as a disease-resistant plant; and during the molecular biological detection, total RNA of plants is extracted, and the tomato spotted wilt virus specific primers are used for RT-PCR detection, wherein if the total RNA is positive, the plant is indicated as a susceptible plant, and if the total RNA is negative, the plant is indicated as a disease-resistant plant.
Furthermore, the screening method of the tomato spotted wilt virus resistant hot pepper plants, disclosed by the invention, comprises the steps of observing symptoms; in the observation of symptoms, the symptoms of the affected plants include: the systemic leaves have necrotic spots, shrinkage, yellowing, short plants and necrosis of the whole plant at the later stage of disease.
Furthermore, the screening method of the tomato spotted wilt virus resistant capsicum plants, provided by the invention, has the advantages that the relation between the polymorphic sites and the TSWV resistant strength is divided into three types according to the disease incidence, wherein the disease incidence A =0 is an immune variety, the disease incidence A is more than 0 and less than or equal to 20% and is a disease-resistant variety, and the disease incidence A is more than 20% and less than or equal to 100% and is a disease-susceptible variety; finally, the disease-resistant variety is used as a molecular marker of PCR.
Compared with the prior art, the screening method of the tomato spotted wilt virus resistant hot pepper plants has the beneficial effects that: the method comprises the steps of obtaining QTL positioning and SNP loci by utilizing phenotypic data, finding the relationship between the polymorphic loci and the strength of TSWV resistance on the basis of TSWV resistance whole genome association analysis and linkage analysis of the hot pepper, developing corresponding molecular markers aiming at the significant association loci/QTL loci, breeding backbone inbred lines of the tomato spotted wilt virus resistance through the determined molecular markers, and finally breeding hybrid varieties of the tomato spotted wilt virus resistance by utilizing a three-line method, so that theory and material support are provided for breeding of hot pepper disease-resistant molecules, more effective means are provided for breeding new hot pepper varieties, and therefore the characteristic hybrid combinations which accord with the preparation of high-quality dry hot peppers are screened out for popularization and application, and the method has very significant economic benefits and social benefits.
Detailed Description
The present invention is further illustrated by the following specific examples, and it should be noted that, for those skilled in the art, variations and modifications can be made without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention.
The reagents used in the examples of the present invention may be commercially available for carrying out the present invention unless otherwise specified, and the technical solutions described in the literature in the art may be applied or may be manipulated according to the product specifications unless otherwise specified.
Example 1
A screening method of tomato spotted wilt virus resistant hot pepper plants comprises the following steps:
(1) the phenotypic identification of the tomato spotted wilt virus resistance of the hot pepper is as follows: 297 parts of interspecific BA 3X YNXML F2 population and 399 parts of pepper inbred line are taken as research objects, and phenotype identification is carried out on the research objects through repeated field tests of single point and 2-year 2-point 4 respectively to obtain the TSWV-resistant phenotype data of the test material; wherein the selfing line is local Zunyi upturned chili.
(2) QTL positioning and whole genome association analysis of tomato spotted wilt virus resistance of pepper: carrying out QTL positioning on the TSWV resistance of the hot pepper by taking a high-density genetic linkage map constructed by an interspecific BA 3X YNXML F2 population as a basis and combining the phenotype data obtained in the step (1) to obtain a key QTL of the TSWV resistance; and (3) carrying out whole genome association analysis on pepper TSWV resistance by taking 399 parts of pepper inbred line or local species SNP genotype data as a basis and combining phenotype data obtained from the research content (1) to obtain key SNP loci of the TSWV resistance.
(3) Identifying and analyzing a key QTL/gene of the tomato spotted wilt virus resistance of pepper: based on the obtained key QTL sites and key SNP sites, the gene distribution information between the sections and in the sections is determined on the genome sequence of the pepper Zunla-1, haplotype or allelic variation with important application value is screened by using comparative genomics and bioinformatics analysis strategies, the key QTL and gene determining TSWV resistance are identified, and candidate genes are cloned and quantitatively expressed and analyzed.
(4) Excavating tomato spotted wilt resistant gene of pepper: on the basis of hot pepper TSWV-resistant whole genome association analysis and linkage analysis, 5 genome sequences where SNP/QTL (single nucleotide polymorphism/quantitative trait loci) which are obviously associated with TSWV are annotated, 3 candidate genes are selected by combining the annotated gene functions and the detected TSWV-resistant QTL, polymorphic sites of the genes are searched by a PCR (polymerase chain reaction) re-sequencing method, the relation between the polymorphic sites and the TSWV resistance is detected by the candidate gene association analysis method, and molecular markers based on PCR are developed aiming at the obvious associated sites/QTL.
(5) The breeding and demonstration display of the new hybrid variety resisting the tomato spotted wilt virus are as follows: based on the anti-molecular marker, a backbone inbred line for resisting the tomato spotted wilt virus is bred, and finally a new hybrid variety (combination) for resisting the tomato spotted wilt virus is bred by a three-line method, so that a new hybrid variety of Zunyi pod pepper for resisting the tomato spotted wilt virus is screened out for demonstration planting.
The relation between the polymorphic site and the anti-TSWV strength is the disease of the pepper seedlings to be detected, the disease of the pepper seedlings to be detected with disease resistance is investigated once every 10 days, the disease incidence A of the pepper seedlings against the tomato spotted wilt virus is calculated, the investigation is carried out for 2 times in total, resistant and sensitive plants are divided, and the disease condition of the pepper seedlings of the variety is counted by taking the highest incidence investigation result as the disease condition of the pepper seedlings of the variety; the disease rate A = (number of infected plants of the variety/total number of plants of the variety). times.100%. The disease incidence is distinguished by the relation between the polymorphic site and the anti-TSWV strength, wherein the disease incidence A =0 is an immune variety, the disease incidence a is more than 0 and less than or equal to 20 percent and is a disease-resistant variety, and the disease incidence a is more than 20 percent and less than or equal to 100 percent and is a disease-susceptible variety; finally, the disease-resistant variety is used as a molecular marker of PCR.
The disease investigation adopts a method of serological detection or molecular biological detection; during the serological detection, collecting leaves of a pepper seedling plant to be detected with disease resistance, and performing ELISA detection by using a tomato spotted wilt virus test strip or a tomato spotted wilt virus antibody, wherein if the leaves are positive, the pepper seedling plant is indicated as a disease-sensitive plant, and if the leaves are negative, the pepper seedling plant is indicated as a disease-resistant plant; and during the molecular biological detection, total RNA of plants is extracted, and the tomato spotted wilt virus specific primers are used for RT-PCR detection, wherein if the total RNA is positive, the plant is indicated as a susceptible plant, and if the total RNA is negative, the plant is indicated as a disease-resistant plant. And in symptom observation, the symptoms of the affected plants comprise: the systemic leaves have necrotic spots, shrinkage, yellowing, short plants and necrosis of the whole plant at the later stage of disease.
Example 2
A screening method of tomato spotted wilt virus resistant hot pepper plants comprises the following steps:
(1) the phenotypic identification of the tomato spotted wilt virus resistance of the hot pepper is as follows: 297 parts of interspecific BA3 YNXML F2 population and 399 parts of local pepper species are taken as research objects, and phenotype identification is carried out on the population through repeated field tests of single point and 2-year 2-point 4 respectively to obtain the TSWV-resistant phenotype data of the tested material; wherein the local species is Zunyi No. 4.
(2) QTL positioning and whole genome association analysis of tomato spotted wilt virus resistance of pepper: carrying out QTL positioning on the TSWV resistance of the hot pepper by taking a high-density genetic linkage map constructed by an interspecific BA 3X YNXML F2 population as a basis and combining the phenotype data obtained in the step (1) to obtain a key QTL of the TSWV resistance; and (3) carrying out whole genome association analysis on pepper TSWV resistance by taking 399 parts of pepper inbred line or local species SNP genotype data as a basis and combining phenotype data obtained from the research content (1) to obtain key SNP loci of the TSWV resistance.
(3) Identifying and analyzing a key QTL/gene of the tomato spotted wilt virus resistance of pepper: based on the obtained key QTL sites and key SNP sites, the gene distribution information between the sections and in the sections is determined on the genome sequence of the pepper Zunla-1, haplotype or allelic variation with important application value is screened by using comparative genomics and bioinformatics analysis strategies, the key QTL and gene determining TSWV resistance are identified, and candidate genes are cloned and quantitatively expressed and analyzed.
(4) Excavating tomato spotted wilt resistant gene of pepper: on the basis of hot pepper TSWV-resistant whole genome association analysis and linkage analysis, 6 genome sequences where SNP/QTL (single nucleotide polymorphism/quantitative trait locus) which are obviously associated with TSWV are annotated, 4 candidate genes are selected by combining the annotated gene functions and the detected TSWV-resistant QTL, polymorphic sites of the genes are searched by a PCR (polymerase chain reaction) re-sequencing method, the relation between the polymorphic sites and the TSWV resistance is detected by the candidate gene association analysis method, and PCR-based molecular markers are developed aiming at the obvious associated sites/QTL.
(5) The breeding and demonstration display of the new hybrid variety resisting the tomato spotted wilt virus are as follows: based on the anti-molecular marker, a backbone inbred line for resisting the tomato spotted wilt virus is bred, and finally a new hybrid variety (combination) for resisting the tomato spotted wilt virus is bred by a three-line method, so that a new hybrid variety of Zunyi pod pepper for resisting the tomato spotted wilt virus is screened out for demonstration planting.
The relation between the polymorphic site and the anti-TSWV strength is the disease of the pepper seedlings to be detected, the disease of the pepper seedlings to be detected with disease resistance is investigated once every 10 days, the disease incidence A of the pepper seedlings against the tomato spotted wilt virus is calculated, the investigation is carried out 3 times in total, resistant and sensitive plants are divided, and the disease condition of the pepper seedlings of the variety is counted by taking the highest incidence investigation result as the disease condition of the pepper seedlings; the disease rate A = (number of infected plants of the variety/total number of plants of the variety). times.100%. The disease incidence is distinguished by the relation between the polymorphic site and the anti-TSWV strength, wherein the disease incidence A =0 is an immune variety, the disease incidence a is more than 0 and less than or equal to 20 percent and is a disease-resistant variety, and the disease incidence a is more than 20 percent and less than or equal to 100 percent and is a disease-susceptible variety; finally, the disease-resistant variety is used as a molecular marker of PCR.
The disease investigation adopts a method of serological detection or molecular biological detection; during the serological detection, collecting leaves of a pepper seedling plant to be detected with disease resistance, and performing ELISA detection by using a tomato spotted wilt virus test strip or a tomato spotted wilt virus antibody, wherein if the leaves are positive, the pepper seedling plant is indicated as a disease-sensitive plant, and if the leaves are negative, the pepper seedling plant is indicated as a disease-resistant plant; and during the molecular biological detection, total RNA of plants is extracted, and the tomato spotted wilt virus specific primers are used for RT-PCR detection, wherein if the total RNA is positive, the plant is indicated as a susceptible plant, and if the total RNA is negative, the plant is indicated as a disease-resistant plant. And in symptom observation, the symptoms of the affected plants comprise: the systemic leaves have necrotic spots, shrinkage, yellowing, short plants and necrosis of the whole plant at the later stage of disease.
Example 3
A screening method of tomato spotted wilt virus resistant hot pepper plants comprises the following steps:
(1) the phenotypic identification of the tomato spotted wilt virus resistance of the hot pepper is as follows: 297 parts of interspecific BA3 YNXML F2 population and 399 parts of local pepper species are taken as research objects, and phenotype identification is carried out on the population through repeated field tests of single point and 2-year 2-point 4 respectively to obtain the TSWV-resistant phenotype data of the tested material; wherein the local species is Zunyi 5.
(2) QTL positioning and whole genome association analysis of tomato spotted wilt virus resistance of pepper: carrying out QTL positioning on the TSWV resistance of the hot pepper by taking a high-density genetic linkage map constructed by an interspecific BA 3X YNXML F2 population as a basis and combining the phenotype data obtained in the step (1) to obtain a key QTL of the TSWV resistance; and (3) carrying out whole genome association analysis on pepper TSWV resistance by taking 399 parts of pepper inbred line or local species SNP genotype data as a basis and combining phenotype data obtained from the research content (1) to obtain key SNP loci of the TSWV resistance.
(3) Identifying and analyzing a key QTL/gene of the tomato spotted wilt virus resistance of pepper: based on the obtained key QTL sites and key SNP sites, the gene distribution information between the sections and in the sections is determined on the genome sequence of the pepper Zunla-1, haplotype or allelic variation with important application value is screened by using comparative genomics and bioinformatics analysis strategies, the key QTL and gene determining TSWV resistance are identified, and candidate genes are cloned and quantitatively expressed and analyzed.
(4) Excavating tomato spotted wilt resistant gene of pepper: on the basis of hot pepper TSWV-resistant whole genome association analysis and linkage analysis, 8 genome sequences where SNP/QTL (single nucleotide polymorphism/quantitative trait loci) are significantly associated with TSWV are annotated, 5 candidate genes are selected by combining the annotated gene functions and the detected TSWV-resistant QTL, polymorphic sites of the genes are searched by a PCR (polymerase chain reaction) re-sequencing method, the relationship between the polymorphic sites and the TSWV resistance is detected by the candidate gene association analysis method, and PCR-based molecular markers are developed aiming at the significantly associated sites/QTL.
(5) The breeding and demonstration display of the new hybrid variety resisting the tomato spotted wilt virus are as follows: based on the anti-molecular marker, a backbone inbred line for resisting the tomato spotted wilt virus is bred, and finally a new hybrid variety (combination) for resisting the tomato spotted wilt virus is bred by a three-line method, so that a new hybrid variety of Zunyi pod pepper for resisting the tomato spotted wilt virus is screened out for demonstration planting.
The relation between the polymorphic site and the anti-TSWV strength is the disease of the pepper seedlings to be detected, the disease of the pepper seedlings to be detected with disease resistance is investigated once every 10 days, the disease incidence A of the pepper seedlings against the tomato spotted wilt virus is calculated, the investigation is carried out 3 times in total, resistant and sensitive plants are divided, and the disease condition of the pepper seedlings of the variety is counted by taking the highest incidence investigation result as the disease condition of the pepper seedlings; the disease rate A = (number of infected plants of the variety/total number of plants of the variety). times.100%. The disease incidence is distinguished by the relation between the polymorphic site and the anti-TSWV strength, wherein the disease incidence A =0 is an immune variety, the disease incidence a is more than 0 and less than or equal to 20 percent and is a disease-resistant variety, and the disease incidence a is more than 20 percent and less than or equal to 100 percent and is a disease-susceptible variety; finally, the disease-resistant variety is used as a molecular marker of PCR.
The disease investigation adopts a method of serological detection or molecular biological detection; during the serological detection, collecting leaves of a pepper seedling plant to be detected with disease resistance, and performing ELISA detection by using a tomato spotted wilt virus test strip or a tomato spotted wilt virus antibody, wherein if the leaves are positive, the pepper seedling plant is indicated as a disease-sensitive plant, and if the leaves are negative, the pepper seedling plant is indicated as a disease-resistant plant; and during the molecular biological detection, total RNA of plants is extracted, and the tomato spotted wilt virus specific primers are used for RT-PCR detection, wherein if the total RNA is positive, the plant is indicated as a susceptible plant, and if the total RNA is negative, the plant is indicated as a disease-resistant plant. And in symptom observation, the symptoms of the affected plants comprise: the systemic leaves have necrotic spots, shrinkage, yellowing, short plants and necrosis of the whole plant at the later stage of disease.
By adopting the screening method, the QTL/gene of the pepper for resisting the tomato spotted wilt virus is determined by constructing a phenotype database, and the influence of candidate genes on the pepper for resisting the tomato spotted wilt virus is utilized, so that a functional PCR marker which can be directly applied to molecular breeding research is developed; determining haplotypes (regulatory elements and genes) or allelic variations within a segment of a chromosome of interest that have significant utility; finally, a new hybrid variety (combination) for resisting the tomato spotted wilt virus is bred by a three-line method, so that a new hybrid variety for resisting the tomato spotted wilt virus of the Gymnodid pepper is screened out for demonstration planting. Therefore, the characteristic hybrid combination which accords with the locally planted high-quality dried pepper can be selected for popularization and application, and more remarkable economic and social benefits can be generated.
Claims (5)
1. A screening method of tomato spotted wilt virus resistant hot pepper plants is characterized by comprising the following steps:
(1) adopting pepper seedlings of an interspecific BA3 YNXML F2 population and pepper seedlings of an inbred line or a local species to respectively pass single-point and repeated field tests, and carrying out phenotype identification on the pepper seedlings through the field tests to obtain the TSWV-resistant phenotype data of the tested material;
(2) carrying out QTL positioning on pepper TSWV resistance by taking a high-density genetic linkage map constructed by an interspecific BA 3X YNXML F2 population as a basis and combining the TSWV-resistant phenotype data of the test material obtained in the step (1) to obtain key QTL sites of the TSWV resistance; meanwhile, carrying out whole genome association analysis on the TSWV resistance of the pepper by taking SNP genotype data constructed by cross-bred or local species of pepper as the basis and combining the TSWV resistance phenotype data of the test material obtained in the step (1) to obtain key SNP loci of the TSWV resistance;
(3) on the basis of the key QTL sites and key SNP sites obtained in the step (2), determining gene distribution information between sections and in the sections on a genome sequence of the pepper Zunla-1, screening haplotype or allelic variation with important application value by using a comparative genomics and bioinformatics analysis method, screening key QTL and genes determining TSWV resistance, and cloning and quantitatively expressing and analyzing the screened candidate genes;
(4) on the basis of hot pepper TSWV-resistant whole genome association analysis and linkage analysis, selecting 5-8 genome sequences where SNP/QTL (single nucleotide polymorphism/quantitative trait locus) which are significantly associated with TSWV-resistant genes are located, selecting 3-5 candidate genes by combining the selected gene functions and detected TSWV-resistant QTL sites, searching for polymorphic sites of the genes by a PCR (polymerase chain reaction) re-sequencing method, finally detecting the relation between the searched polymorphic sites and the TSWV-resistant strength by the candidate gene association analysis method, and developing PCR-based molecular markers aiming at the significantly associated sites/QTL sites;
(5) and (4) breeding a backbone inbred line resisting the tomato spotted wilt virus based on the molecular marker determined in the step (4), and finally breeding a hybrid variety resisting the tomato spotted wilt virus by using a three-line method.
2. The method for screening tomato spotted wilt virus resistant hot pepper plants as claimed in claim 1, wherein the method comprises the following steps: in the step (4), the relation between the polymorphic site and the anti-TSWV strength is the disease of the pepper seedlings to be detected, the disease of the pepper seedlings to be detected is investigated once every 10 days, the disease incidence A of the pepper seedlings against the tomato spotted wilt virus is calculated, the investigation is carried out for 2-3 times in total, resistant and sensitive plants are divided, and the highest incidence investigation result is taken as the disease condition of the pepper seedlings of the variety for statistics; the disease rate A = (number of infected plants of the variety/total number of plants of the variety). times.100%.
3. The method for screening tomato spotted wilt virus resistant hot pepper plants as claimed in claim 2, characterized in that: the disease investigation adopts a method of serological detection or molecular biological detection; during the serological detection, collecting leaves of a pepper seedling plant to be detected with disease resistance, and performing ELISA detection by using a tomato spotted wilt virus test strip or a tomato spotted wilt virus antibody, wherein if the leaves are positive, the pepper seedling plant is indicated as a disease-sensitive plant, and if the leaves are negative, the pepper seedling plant is indicated as a disease-resistant plant; and during the molecular biological detection, total RNA of plants is extracted, and the tomato spotted wilt virus specific primers are used for RT-PCR detection, wherein if the total RNA is positive, the plant is indicated as a susceptible plant, and if the total RNA is negative, the plant is indicated as a disease-resistant plant.
4. The method for screening tomato spotted wilt virus resistant hot pepper plants as claimed in claim 3, wherein the method comprises the following steps: the method adopted by the disease investigation also comprises symptom observation; in the observation of symptoms, the symptoms of the affected plants include: the systemic leaves have necrotic spots, shrinkage, yellowing, short plants and necrosis of the whole plant at the later stage of disease.
5. The method for screening tomato spotted wilt virus resistant hot pepper plants as claimed in claim 2, characterized in that: the relation between the polymorphic site and the strength of anti-TSWV is divided into three types through morbidity, wherein the morbidity A =0 is an immune variety, the morbidity a is more than 0 and less than or equal to 20 percent and is a disease-resistant variety, and the morbidity a is more than 20 percent and less than or equal to 100 percent and is a disease-susceptible variety; finally, the disease-resistant variety is used as a molecular marker of PCR.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4221419A (en) * | 1977-02-19 | 1980-09-09 | Keith Riley | Gripping devices |
| CN105907755A (en) * | 2016-06-10 | 2016-08-31 | 中国农业科学院蔬菜花卉研究所 | Molecular marker closely linked with resistance of hot peppers to cucumber mosaic virus and application of molecular marker |
| CN109182575A (en) * | 2018-09-10 | 2019-01-11 | 广东省农业科学院作物研究所 | A kind of method of the anti-southern rust inbred line of sweet corn of molecular marking supplementary breeding |
| CN110777197A (en) * | 2019-08-02 | 2020-02-11 | 中国农业科学院棉花研究所 | Major QTL method for rapidly identifying cotton-related traits through compound BSA-seq |
-
2020
- 2020-10-28 CN CN202011169270.7A patent/CN112216343B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4221419A (en) * | 1977-02-19 | 1980-09-09 | Keith Riley | Gripping devices |
| CN105907755A (en) * | 2016-06-10 | 2016-08-31 | 中国农业科学院蔬菜花卉研究所 | Molecular marker closely linked with resistance of hot peppers to cucumber mosaic virus and application of molecular marker |
| CN109182575A (en) * | 2018-09-10 | 2019-01-11 | 广东省农业科学院作物研究所 | A kind of method of the anti-southern rust inbred line of sweet corn of molecular marking supplementary breeding |
| CN110777197A (en) * | 2019-08-02 | 2020-02-11 | 中国农业科学院棉花研究所 | Major QTL method for rapidly identifying cotton-related traits through compound BSA-seq |
Non-Patent Citations (3)
| Title |
|---|
| 华明艳等: "辣椒中可用于辅助选择的分子标记及其研究进展", 长江蔬菜, no. 06, pages 14 - 17 * |
| 王立浩等: "甜椒抗番茄斑点萎蔫病毒的种质创新", 中国蔬菜, no. 02, pages 19 - 23 * |
| 陈小翠等: "辣椒遗传图谱构建和重要性状QTL定位研究进展", 辣椒杂志, no. 03, pages 1 - 12 * |
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