CN112575101B - Molecular marker related to resistance of Cucurbita pepo PRSV-W virus disease and application thereof - Google Patents
Molecular marker related to resistance of Cucurbita pepo PRSV-W virus disease and application thereof Download PDFInfo
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Abstract
The invention relates to a molecular marker related to resistance of Cucurbita pepo PRSV-W virus disease and application thereof. The Cucurbita pepo PRSV-W virus molecular marker provided by the invention is a DNA molecule obtained by taking genome DNA of the Cucurbita pepo as a template and adopting a primer to amplify A1; the A1 consists of single-stranded DNA named as P1 and P2, the P1 is the single-stranded DNA specifically combined with the 39 th upstream of the SEQ ID No.1, and the P2 is the single-stranded DNA specifically combined with the 54 th downstream of the SEQ ID No.1. Experiments prove that the primer pair A1 for identifying or assisting in identifying the resistance traits of the Cucurbita pepo PRSV-W virus disease and the molecular markers of the Cucurbita pepo PRSV-W virus disease can be used for identifying the resistance conditions of the PRSV-W virus disease of the Cucurbita pepo.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a molecular marker related to resistance of Cucurbita pepo PRSV-W virus and application thereof.
Background
The Cucurbita pepo (Cucurbita pepo) is an important melon vegetable in China, the cultivation area tends to expand year by year, and particularly, the cultivation area of a protected area is only second to that of cucumbers at present and becomes an important planting crop for increasing the income of farmers and adjusting the agricultural structure.
The papaya ringspot virus watermelon strain (PRSV-W) is one of the main virus diseases damaging the production of the pumpkin, and the field morbidity is common and the harm is serious. After the zucchini plants are infected with PRSV-W virus, leaves show symptoms of mosaic, shrinkage, curling and the like, the later period is serious, the symptoms of plant dwarfing, uneven surface tumor, stiff pulp, bitter taste and the like can be caused, the fruits lose commodity value, more than 40 percent of yield loss can be caused, and the safety production of the zucchini is seriously threatened.
At present, no effective reagent and measure for preventing and treating PRSV-W virus diseases exist, and the spread and the harm of the virus diseases are controlled and reduced mainly by removing and burying diseased plants in time for preventing and treating aphids in field production. The most economical, safe and effective measure for reducing the harm of virus diseases is to cultivate and popularize disease-resistant varieties. However, at present, the resistance identification through artificial inoculation has the problems of complicated operation, poor character identification accuracy, easy cross-infection of other viruses, failure in selection during breeding and the like, and the problems of difficult transformation of resistance characters, low disease-resistant breeding efficiency, long period, easy failure and the like are caused. The molecular marker closely linked with the disease-resistant gene can improve the accuracy of selection and accelerate the disease-resistant breeding process, and is a necessary condition for molecular marker-assisted selection breeding. However, at present, no research report of molecular markers which are closely linked with the Cucurbita pepo PRSV-W disease-resistant gene and can be practically used exists at home and abroad. Therefore, the development of the molecular marker tightly linked with the cucurbit PRSV-W disease-resistant gene has important practical application value for improving the disease-resistant breeding efficiency of the cucurbit pepo, expanding the application range of the disease-resistant gene, cultivating new disease-resistant varieties and ensuring the healthy development of the cucurbit pepo industry.
Disclosure of Invention
The invention aims to solve the technical problem of how to identify the PRSV-W virus disease resistance character of the cucurbita pepo.
In order to solve the technical problems, the invention firstly provides a Cucurbita pepo PRSV-W virus disease molecular marker.
The molecular marker related to the resistance of the Cucurbita pepo PRSV-W virus disease is a DNA molecule obtained by amplifying A1 by using genome DNA of the Cucurbita pepo as a template and adopting a primer; the A1 consists of single-stranded DNA named as P1 and P2, the P1 is the single-stranded DNA specifically combined with the 39 th upstream of the double-stranded DNA shown in SEQ ID No.1, and the P2 is the single-stranded DNA specifically combined with the 54 th downstream of the double-stranded DNA shown in SEQ ID No.1.
The polymorphism of the cucurbita pepo molecular marker can be that the 39 th to 54 th positions corresponding to SEQ ID No.1 in the genome of the cucurbita pepo are the 39 th to 54 th positions of SEQ ID No.1 or the 39 th to 54 th positions of the SEQ ID No.1 are deleted.
In the above Cucurbita pepo molecule marker, P1 can be single-stranded DNA shown in 1 st-22 nd position of SEQ ID No.1, and P2 can be single-stranded DNA reverse complementary with 144 nd-162 nd position of SEQ ID No.1.
In order to solve the technical problems, the invention also provides a method for identifying the genotype of the zucchini.
The method for identifying the genotype of the pumpkin comprises the following steps of I or II:
i, including the following K1) and K2):
k1 Taking the genome DNA of the Cucurbita pepo to be detected as a template, and performing PCR amplification by adopting the A1 to obtain a PCR product;
k2 Detecting the PCR product obtained in the step K1), and determining the genotype of the pumpkin according to the PCR product:
the genotype of the pumpkin to be detected, which corresponds to the SEQ ID No.1 and does not contain the DNA fragment shown in the 39 th-54 th sites of the SEQ ID No.1, of the PCR product is the RR genotype; the PCR product contains a DNA fragment which does not contain the 39 th to 54 th sites of the SEQ ID No.1 and a genotype of the pumpkin to be detected which contains the 39 th to 54 th sites of the SEQ ID No.1, and the genotype is the RS genotype; the genotype of the pumpkin to be detected, which corresponds to the SEQ ID No.1 and contains the DNA fragment shown in the 39 th-54 th sites of the SEQ ID No.1, of the PCR product is an SS genotype;
II, including the following L1) and L2):
l1) carrying out PCR amplification by using the A1 with genome DNA of the Cucurbita pepo to be detected as a template to obtain a PCR product;
l2) the following L21) or L22):
l21) detecting the size of the PCR product obtained in the step L1), and determining the genotype of the cucurbita pepo according to the size of the PCR product:
the genotype of the pumpkin to be detected, of which the PCR product contains DNA fragments of 162bp and 146bp, is an RS genotype; the PCR product contains a 162bp DNA fragment, and the genotype of the pumpkin to be detected, which does not contain a 146bp DNA fragment, is an SS genotype; the PCR product does not contain a 162bp DNA fragment, and the genotype of the pumpkin to be detected containing a 146bp DNA fragment is an RR genotype;
l22) detecting the sequence of the PCR product obtained in the step L1), and determining the genotype of the cucurbita pepo according to the PCR product:
the genotype of the pumpkin to be detected, of which the PCR product contains DNA fragments shown by SEQ ID No.1 and SEQ ID No.2, is an RS genotype; the PCR product contains a DNA fragment shown by SEQ ID No.2 and does not contain a DNA fragment shown by SEQ ID No.1, and the genotype of the Cucurbita pepo to be detected is RR genotype; the PCR product contains a DNA fragment shown in SEQ ID No.1 and does not contain a DNA fragment shown in SEQ ID No.2, and the genotype of the Cucurbita pepo to be detected is SS genotype.
In the above method for identifying the genotype of cucurbita pepo, the system for performing the PCR amplification may comprise: dNTPs with the concentration of 2.5mM for 10 XBuffer, dATP, dTTP, dCTP and dGTP, taq DNA polymerase, the P1, the P2, and the Cucurbita pepo genome DNA to be detected. In the system, the concentrations of dATP, dTTP, dCTP and dGTP are all 0.05mM, the final concentrations of single-stranded DNA shown in SEQ ID No.1 and SEQ ID No.2 are all 0.2 mu M, and the concentration of the genome DNA of the Cucurbita pepo to be detected can be 30 ng/mu L.
The reaction conditions for performing the PCR amplification may be: pre-denaturation at 95 ℃ for 3min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 25s, and circulation for 36 times; extension at 72 ℃ for 10min.
In the method for identifying the genotype of cucurbita pepo, the dNTPs may be a product of tiangen biochemical technology (beijing) ltd, and the product number is CD111. The Taq DNA polymerase and the 10 XBuffer can be products of Beijing Quanjin Biotechnology Limited, and the product number is AP101-01.
In the method for identifying the genotype of the zucchini, the PRSV-W virus resistance of the RR genotype zucchini and the RS genotype zucchini is higher than that of the SS genotype zucchini.
In order to solve the technical problems, the invention also provides a method for identifying or assisting in identifying the resistance traits of the Cucurbita pepo PRSV-W virus.
The method for identifying or assisting in identifying the resistance traits of the Cucurbita pepo PRSV-W virus disease provided by the invention is 1) or 2) as follows:
1) Including the following M1) and M2):
m1) carrying out PCR amplification by using the A1 with genome DNA of the Cucurbita pepo to be detected as a template to obtain a PCR product;
m2) detecting the PCR product obtained in the step M1), and if the PCR product contains a DNA fragment which does not contain the DNA fragment shown in the 39 th-54 th position of SEQ ID No.1 and a DNA fragment which contains the DNA fragment shown in the 39 th-54 th position of SEQ ID No.1, the to-be-detected Cucurbita pepo is or is selected as a Cucurbita pepo for resisting PRSV-W virus disease; if the PCR product corresponds to SEQ ID No.1 and does not contain a DNA fragment shown in 39 th-54 th positions of SEQ ID No.1, the to-be-detected zucchini is or is selected as the zucchini for resisting PRSV-W virus disease; if the PCR product corresponds to SEQ ID No.1 and is a DNA fragment containing the 39 th-54 th sites of SEQ ID No.1, the Cucurbita pepo to be detected is or is selected as a non-PRSV-W virus disease resistant Cucurbita pepo;
2) Including the following N1) and N2):
n1) taking genome DNA of the Cucurbita pepo to be detected as a template, and performing PCR amplification by adopting the A1 to obtain a PCR product;
n2) the following N21) or N22):
n21) detecting the size of the PCR product obtained in the step N1), wherein if the PCR product contains DNA fragments of 162bp and 146bp, the Cucurbita pepo to be detected is or is selected as a Cucurbita pepo for resisting PRSV-W virus disease; if the PCR product contains a 146bp DNA fragment and does not contain a 162bp DNA fragment, the Cucurbita pepo to be detected is or is selected as a Cucurbita pepo for resisting PRSV-W virus disease; if the PCR product does not contain the 146bp DNA fragment and contains the 162bp DNA fragment, the Cucurbita pepo to be detected is or is selected as a non-PRSV-W virus disease resistant Cucurbita pepo;
n22) detecting the sequence of the PCR product obtained in the step N1), wherein if the PCR product contains DNA fragments shown in SEQ ID No.1 and SEQ ID No.2, the to-be-detected Cucurbita pepo is or is candidate to be a Cucurbita pepo for resisting PRSV-W virus disease; if the PCR product contains the DNA segment shown in SEQ ID No.2 and does not contain the DNA segment shown in SEQ ID No.1, the to-be-detected zucchini is or is candidate to be the zucchini with the PRSV-W virus disease resistance; and if the PCR product contains the DNA segment shown in SEQ ID No.1 and does not contain the DNA segment shown in SEQ ID No.2, the Cucurbita pepo to be detected is or is selected as a non-PRSV-W virus disease resistant Cucurbita pepo L.
The system for performing the PCR amplification in the method for identifying or assisting in identifying the resistance trait of the Cucurbita pepo PRSV-W virus disease may be the system for performing the PCR amplification in the method for identifying the genotype of the Cucurbita pepo, and the reaction conditions for performing the PCR amplification may be the reaction conditions for performing the PCR amplification in the method for identifying the genotype of the Cucurbita pepo.
In order to solve the technical problems, the invention also provides a method for identifying or assisting in identifying the stable genetic Cucurbita pepo of PRSV-W resistant virus disease characters.
The method for identifying or assisting in identifying the genetic zucchini with stable PRSV-W virus disease resistance traits provided by the invention is as follows:
I. including the following M1) and M2):
m1) carrying out PCR amplification by using the A1 with genome DNA of the Cucurbita pepo to be detected as a template to obtain a PCR product;
m2) detecting the PCR product obtained in the step M1), wherein if the PCR product corresponding to SEQ ID No.1 is a DNA fragment which does not contain the DNA fragment shown in the 39 th to 54 th sites of SEQ ID No.1, the Cucurbita pepo to be detected is or is selected as a hereditary Cucurbita pepo with stable anti-PRSV-W virus disease character;
II. Including the following N1) and N2):
n1) taking genome DNA of the Cucurbita pepo to be detected as a template, and performing PCR amplification by adopting the A1 to obtain a PCR product;
n2) the following N21) or N22):
n21) detecting the size of the PCR product obtained in the step N1), wherein if the PCR product contains a DNA fragment of 146bp and does not contain a DNA fragment of 162bp, the Cucurbita pepo to be detected is or is selected as a hereditary Cucurbita pepo with stable anti-PRSV-W virus disease character;
n22) detecting the sequence of the PCR product obtained in the step N1), wherein if the PCR product contains the DNA segment shown by SEQ ID No.2 and does not contain the DNA segment shown by SEQ ID No.1, the to-be-detected Cucurbita pepo is or is a candidate of the genetic Cucurbita pepo with the stable anti-PRSV-W virus disease character.
The PCR amplification system in the method for identifying or assisting in identifying the genetic zucchini with stable anti-PRSV-W virus disease character can be the PCR amplification system in the method for identifying the genotype of the zucchini, and the reaction conditions for carrying out the PCR amplification can be the reaction conditions for carrying out the PCR amplification in the method for identifying the genotype of the zucchini.
In order to solve the technical problems, the invention also provides a primer pair for identifying or assisting in identifying the resistance traits of the Cucurbita pepo PRSV-W virus.
The primer pair for identifying or assisting in identifying the resistance traits of the Cucurbita pepo PRSV-W virus disease is A1.
In order to solve the technical problems, the invention also provides a system for identifying or assisting in identifying the resistance traits of the Cucurbita pepo PRSV-W virus disease.
The system for identifying or assisting in identifying the resistance traits of the Cucurbita pepo PRSV-W virus disease consists of X1 and X2; the X1 is the A1, and the X2 is a reagent and/or an instrument required for PCR amplification.
In the above system, the reagent for PCR amplification may comprise dNTPs for dATP, dTTP, dCTP and dGTP, taqDNA polymerase and/or PCR reaction buffer, or the dNTP mixture, taqDNA polymerase and/or PCR reaction buffer alone; the apparatus required for performing PCR amplification may be a PCR apparatus. The dNTPs may be a product of Tiangen Biochemical technology (Beijing) Ltd, with a product number of CD111. The TaqDNA polymerase and the 10 XBuffer can be products of Beijing Quanyujin Biotechnology Limited, and the product number is AP101-01. The PCR instrument can be specifically a product of Tovicon Innovative Biotechnology Limited company of Beijing, and the model is ETC-811.
In the above system, both the A1 and the reagents required for PCR amplification can be packaged separately. The two single-stranded DNAs of each primer pair in A1 can be independently packaged. Each reagent required for PCR amplification can be packaged independently.
The system for identifying or assisting in identifying the zucchini PRSV-W virus disease resistance trait can also contain only the A1 and the reagents or kits required for performing PCR amplification.
In order to solve the technical problem, the invention also provides any one of the following applications H1-H10:
h1, application of the Cucurbita pepo PRSV-W virus disease molecular marker in identification or auxiliary identification of the resistance trait of the Cucurbita pepo PRSV-W virus disease;
h2, application of the molecular marker of the Cucurbita pepo PRSV-W virus disease in identification or auxiliary identification of the stable genetic Cucurbita pepo of PRSV-W virus disease resistance traits;
h3, application of the Cucurbita pepo PRSV-W virus molecular marker in Cucurbita pepo breeding;
h4, application of the method for identifying the genotype of the zucchini or the method for identifying or assisting in identifying the PRSV-W virus disease resistance trait in genetic zucchini;
h5, the application of the method for identifying the genotype of the cucurbita pepo or the method for identifying or assisting in identifying the PRSV-W virus disease resistance traits of the cucurbita pepo in the breeding of the cucurbita pepo;
h6, application of the method for identifying the genotype of the cucurbita pepo to identification or auxiliary identification of the resistance trait of the cucurbita pepo PRSV-W virus disease;
h7, A1 or the system is applied to preparation of a reagent or a kit for identifying or assisting in identifying the resistance traits of the Cucurbita pepo PRSV-W virus disease;
h8, the A1 or the system are applied to identification or auxiliary identification of the zucchini PRSV-W virus disease resistance traits;
the application of H9, A1 or the system in identification or auxiliary identification of PRSV-W virus disease resistance trait stable genetic cucurbita pepo;
the application of H10, A1 or the system in summer squash breeding.
In the application, the PRSV-W virus resistance of the RR genotype Cucurbita pepo and the RS genotype Cucurbita pepo is higher than that of the SS genotype Cucurbita pepo.
In order to solve the technical problems, the invention also provides a summer squash breeding method.
According to the zucchini breeding method provided by the invention, the genotype of the zucchini is identified according to the method for identifying the genotype of the zucchini, and the zucchini with the RR or RS genotype is selected as a parent to carry out breeding.
In the invention, the anti-PRSV-W virus disease zucchini refers to a zucchini with no disease symptoms or slight mosaic expression after being artificially inoculated with PRSV-W virus disease, and the non-anti-PRSV-W virus disease zucchini refers to a zucchini with high mosaic or crimped leaf after being artificially inoculated with PRSV-W virus disease.
In the present invention, the number of moles of the two single-stranded DNAs of A1 may be 1.
In the invention, when the size of the PCR product is detected by electrophoresis, the PCR product contains 162bp and 146bp DNA fragments and can show that two bands exist between 100bp and 200bp, the PCR product contains 146bp DNA fragments and does not contain 162bp DNA fragments and can show that a band close to 100bp exists between 100bp and 200bp, and the PCR product does not contain 146bp DNA fragments and contains 162bp DNA fragments and can show that a band close to 200bp exists between 100bp and 200 bp.
Experiments prove that the resistance property of the PRSV-W virus disease of the cucurbita pepo can be identified by using the molecular marker of the cucurbita pepo PRSV-W virus disease, three PCR products obtained by using a primer pair for identifying or assisting in identifying the resistance property of the PRSV-W virus disease of the cucurbita pepo as a template correspond to different resistance properties of the PRSV-W virus disease of the cucurbita pepo, when the PCR product of the cucurbita pepo contains the DNA fragment (162 bp) shown by the SEQ ID No.1 and does not contain the DNA fragment (146 bp) shown by the SEQ ID No.2, the cucurbita pepo is expressed as the PRSV-W virus disease, when the PCR product of the cucurbita pepo contains the DNA fragment (146 bp) shown by the SEQ ID No.2 and does not contain the DNA fragment (162 bp) shown by the SEQ ID No.1, the cucurbita pepo is expressed as the PRSV-W virus disease resistance, and when the PCR product of the cucurbita pepo contains the DNA fragment (162 bp) shown by the SEQ ID No.1 and the SEQ ID No.2, the PCR product of the cucurbita pepo is expressed as the PRSV-W virus disease resistance property. The primer pair A1 for identifying or assisting in identifying the resistance traits of the Cucurbita pepo PRSV-W virus disease and the molecular marker of the Cucurbita pepo PRSV-W virus disease are used for identifying the resistance conditions of the PRSV-W virus disease of the Cucurbita pepo.
According to the invention, a PRSV-W virus disease dominant disease-resistant gene CpPRSV-W is found in a Cucurbita pepo inbred line 08-13, and the excellent disease resistance of the gene CpPRSV-W has important utilization value in the Cucurbita pepo breeding for disease resistance. The invention obtains the molecular marker Cp126 closely linked with the disease-resistant gene. The obtained molecular marker is used for auxiliary selection, and the disease-resistant gene is successfully introduced into the cucurbita pepo excellent inbred line fiber-white through hybridization and backcross transformation, so that PRSV-W virus disease resistance is obtained.
Drawings
FIG. 1 is a genetic linkage diagram of a molecular marker Cp126 and a cucurbita pepo disease-resistant gene CpPRSV-W, and the left side is a graph distance between the markers.
FIG. 2 shows an amplification band pattern of the molecular marker Cp126. Wherein P is 1 Is a susceptible parent fibraurea, P 2 Is a disease-resistant parent 08-13,F 1 Is a first filial generation; 1-13 are F 2 Carrying out single plant cultivation; m: trans DNA marker II, arrow indicates 146bp specific amplification band.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples, the cucurbita pepo inbred line 08-13 and the cucurbita pepo inbred line fibro-leu, (the cucurbita pepo PRSV _ CMV resistance genetic analysis and resistance gene preliminary location, zhang dong, 2016) are publicly available from the vegetable center of the beijing academy of agricultural sciences, and the biomaterial is only used for repeating the related experiments of the present invention, and is not used for other purposes.
PRSV-W virus, (Cucurbita pepo anti-PRSV _ CMV genetic analysis and resistance gene preliminary location, zhang Mei, 2016) is publicly available from vegetable center of Beijing Nongkovictory institute, and the biomaterial is only used for repeating the related experiments of the present invention, but not used for other purposes.
The dNTPs in the following examples are available from Tiangen Biochemical technology (Beijing) Ltd, under the trade name CD111. The TaqDNA polymerase and the 10 XBuffer can be products of Beijing Quanji Biotechnology Limited, with the product number of AP101-01. The PCR instrument is a product of Beijing Dongsheng Innovation Biotechnology Limited company, and the model is ETC-811.
Example 1 identification of resistance trait to Cucurbita pepo PRSV-W Virus disease
In the embodiment, the primer pair A1 for identifying or assisting in identifying the resistance traits of the Cucurbita pepo PRSV-W virus disease is used for identifying the resistance traits of the PRSV-W virus disease of the Cucurbita pepo, wherein A1 is composed of single-stranded DNAs (namely P1 and P2), P1 is the single-stranded DNA shown in the 1 st to 22 th positions of SEQ ID No.1, and P2 is the single-stranded DNA reversely complementary to the 162 th to 144 th positions of SEQ ID No.1.
1. The cucurbita pepo to be identified is as follows, and 50 cucurbita pepo plants are selected for each cucurbita pepo to be tested:
the cucurbita pepo selfing line is fibraurea leucocyte, and the phenotype of the cucurbita pepo selfing line is PRSV-W virus disease;
the cucurbita pepo inbred line 08-13 has a phenotype of resisting PRSV-W virus disease;
episy No.2, which is a product of Peking Episy agricultural science and technology Limited, and has a phenotype of susceptibility to PRSV-W virus;
yuhu No. two F1, which has a phenotype of susceptibility to PRSV-W virus disease and is a product of Beijing Zhongnong Lvqiao science and technology Limited;
spring charm II, the phenotype of which is the PRSV-W virus infection, is a product of Oriental big-seed Limited company;
406 Cucurbita pepo, the phenotype of which is PRSV-W virus disease resistance, and is a product of China seed group Limited company;
DSOT6B, the phenotype of which is PRSV-W virus infection, is a product of Pompe agriculture product Co;
summer green, the phenotype of which is PRSV-W virus, is a product of Eggerzert Limited company in Changji city;
the jadeite has the phenotype of resisting PRSV-W virus disease and is a product of Eggerzert Limited company in Changji city;
zhenyu 369, which is resistant to PRSV-W virus disease and is a product of science and technology development Limited company of Henan Yu skill;
lufeng 89, which is a product of Shouguangsheng breed limited company, and has a phenotype of feeling PRSV-W virus;
jade eight, the phenotype of which is PRSV-W virus infection, and is a product of Beijing Beinong Henli seed Limited company;
the French green Bember 9810, the phenotype of which is PRSV-W virus disease resistance, is a product of Beijing Lifengxin sprout Limited company;
ruifeng Jiu F1, the phenotype of which is PRSV-W virus resistance, is a product of Beijing Zhongnong Henheng seed science and technology Limited;
jinghu No. 8, which is a product of Jingyangyong (Beijing) science and technology cultivars limited company, has a phenotype of susceptibility to PRSV-W virus disease.
2. Detection of resistance traits of Cucurbita pepo PRSV-W virus disease
Extracting the genome DNA of each young leaf of Cucurbita pepo by CTAB method (SuePsorebskil, 1997), and performing PCR amplification by using the genome DNA as a template and A1 as a primer pair, wherein A1 is the primer pair consisting of 5-.
The 15. Mu.L reaction system for PCR amplification using primer pair A1 was: 10 xbuffer 1.5 microliter, dATP, dTTP, dCTP and dGTP concentrations are 2.5mM dNTPs0.3 microliter, taqDNA polymerase 0.2 microliter, P1, P2, pumpkin genome DNA30ng, sterile ultrapure water to make up for 15 microliter, make P1 and P2 final concentration is 0.2. Mu.M. The reaction conditions for PCR amplification in the PCR instrument are as follows: pre-denaturation at 95 ℃ for 3min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 25s, and circulation for 36 times; extension at 72 ℃ for 10min. Sequencing a part of the reaction product, carrying out 160V electrophoresis on 8% non-denaturing polyacrylamide gel for 70min, carrying out silver staining, observing under a lamp box, and recording.
The results of performing non-denaturing polyacrylamide gel electrophoresis and sequencing on the PCR products of each zucchini are shown in Table 1, the PCR product of 162bp in Table 1 represents the DNA fragment shown in SEQ ID No.1, and the PCR product of 146bp represents the DNA fragment shown in SEQ ID No.2. Then, the PRSV-W virus resistance of each Cucurbita pepo was identified, and the relationship between the PCR product and the phenotype is shown in Table 1.
When different cucurbita pepo genome DNAs are used as templates and A1 is used as a primer pair for PCR amplification, PCR products have three conditions: the first case is that the PCR product contains the DNA fragment (162 bp) shown by SEQ ID No.1 and does not contain the DNA fragment (146 bp) shown by SEQ ID No.2, the second case is that the PCR product contains the DNA fragment (146 bp) shown by SEQ ID No.2 and does not contain the DNA fragment (162 bp) shown by SEQ ID No.1, and the third case is that the PCR product contains the DNA fragment (162 bp) shown by SEQ ID No.1 and the DNA fragment (146 bp) shown by SEQ ID No.2. And defining the genotype of the zucchini according to the PCR amplification product, wherein the RR genotype represents that the regions of two homologous chromosomes of the zucchini, which correspond to SEQ ID No.1, are both SEQ ID No.2, the RS genotype represents that the region of one homologous chromosome of the zucchini, which corresponds to SEQ ID No.1, is SEQ ID No.1, the region of the other homologous chromosome, which corresponds to SEQ ID No.1, is SEQ ID No.2, and the SS genotype represents that the regions of the two homologous chromosomes of the zucchini, which correspond to SEQ ID No.1, are both SEQ ID No.1. Wherein, both RR genotype and SS genotype can be stably inherited.
The DNA molecule obtained by taking the genome DNA of the cucurbita pepo as a template and carrying out PCR amplification on the A1 by adopting a primer is named as a cucurbita pepo PRSV-W virus molecular marker, and the polymorphism of the cucurbita pepo PRSV-W virus molecular marker is that SEQ ID No.1 or SEQ ID No.2 corresponding to SEQ ID No.1 in the genome of the cucurbita pepo.
3. The method for judging the resistance and the infection of the cucurbita pepo PRSV-W virus variety comprises the following steps:
drying the tested seeds at 40 ℃ for 2h, performing high-temperature treatment at 65 ℃ for 2h, soaking the seeds for 4h, then accelerating germination in a constant-temperature incubator at 30 ℃, sowing the seeds in a sterilized nutrition pot after 24h, placing the seeds in an insect-proof net chamber for cultivation at 25-30 ℃, planting 7 plants in each material, and repeating for 3 times. After the cotyledon is flattened, a small amount of 600-mesh carborundum is scattered on the cotyledon, the cotyledon is frictionally inoculated by virus inoculation liquid with the mass concentration of 0.5mg/mL (0.02 moL/L of phosphate buffer solution is used for grinding leaf tissues carrying PRSV-W virus), and 2 blank controls are set. After 15-25 days of inoculation, the plants were observed for 1-4 true leaf morbidity and scored according to the following grading criteria.
Grade 0, no symptom of the plant; grade 1, few leaves are local and slight in flower and leaf and have normal shapes; 2-stage, most leaf flowers and leaves are normal in shape, and individual leaves are malformed; grade 3, most of the leaves have serious flower and leaf, blister spot and new leaf deformity; grade 4, almost all the leaves are severely mosaic and misshapen. Disease Index (DI) =100 × Σ (number of diseased plants at each stage × representative value at each stage)/(number of investigated total plants × representative value at highest stage). Wherein DI is less than or equal to 5 is highly disease-resistant (HR), DI is more than 5 and less than or equal to 25 is disease-resistant (R), DI is more than 25 and less than or equal to 70 is susceptible (S), and DI is more than 70 is Highly Susceptible (HS). The above cucurbita pepo varieties were tested according to the above method, and the test results are shown in table 1.
TABLE 1 results of analysis of the PCR products of different Cucurbita pepo and the PRSV-W virus disease resistance phenotype
The results show that the zucchini is manifested as PRSV-W virus disease when the PCR product of the zucchini is in the first case (i.e., the genotype of the zucchini is SS genotype), resistant to PRSV-W virus disease when the PCR product of the zucchini is in the second case (i.e., the genotype of the zucchini is RR genotype), and resistant to PRSV-W virus disease when the PCR product of the zucchini is in the third case (i.e., the genotype of the RS). The three conditions (or SS, RR and RS genotypes of the cucurbita pepo) of the PCR product are completely consistent with the resistance condition of the PRSV-W virus disease of the cucurbita pepo, and the condition that the resistance condition of the PRSV-W virus disease of the cucurbita pepo can be identified by utilizing a primer pair A1 for identifying or assisting in identifying the resistance property of the PRSV-W virus disease of the cucurbita pepo.
Example 2 method for obtaining SSR molecular marker closely linked with dominant disease-resistant gene CpPRSV-W of Cucurbita pepo PRSV-W virus disease
The method for obtaining the SSR molecular marker Cp126 tightly linked to the cucurbita pepo PRSV-W virus disease dominant disease-resistant gene CpPRSV-W is specifically described by the embodiment, which is characterized by naming the cucurbita pepo PRSV-W virus disease molecular marker in example 1 as the molecular marker Cp126 and comprises the following steps:
first, selfing line of pumpkin, fibre, white and 08-13F 2 Generation creation and phenotypic identification:
(1) The selfing line of pumpkin, named as Figbai (female parent, P1), and 08-13 (male parent, P2) are hybridized to obtain hybrid F 1 ，F 1 Selfing to produce F 2 And (4) generation groups.
(2)F 2 Planting the generation group single plants in a greenhouse nutrition pot, covering an insect-proof net, inoculating PRSV-W virus after cotyledons are completely unfolded, and investigating disease resistance after inoculating for 15 days. The results are shown in Table 2.
TABLE 2 Fine-white X08-13F after inoculation of PRSV-W Virus 2 Generation population genetic analysis
R and S respectively represent a disease-resistant single plant and an infection single plant. Chi shape 2 0.05,1 ＝3.84。
(3)F 2 Transplanting the infected plant to greenhouse for planting after character investigation of the generation individual plant, and selfing to obtain F 3 Family members. Each F 3 10 seeds from the pedigree were selected for progeny disease resistance testing to verify F 2 Whether the generation infected single plant carries homozygous infected gene or not.
(4) By making a pair F 2 Genetic analysis is carried out on individual generation plants, and the pumpkin inbred line 08-13 is found to carry a dominant disease-resistant gene.
(II) screening of polymorphic molecular markers
(5) 10 plants of disease-resistant F 2 The leaves of the single plant are mixed into a resistance pool in equal amount, 10 susceptible plants F 2 The leaves of the single plant are mixed into a sensing pool in equal amount. Extraction of disease-resistant 08-13 parent and susceptible parent fiber-white by CTAB method (Sue Porebski L, 1997)DNA of anti-pool and sensing pool, and screening polymorphism molecular marker by using simple repeat sequence marker SSR and BSA (Bulk segegant Analysis) combination method.
(6) Firstly, 843 pairs of SSR primers which are designed and synthesized on 20 chromosomes of the zucchini in the subject group are utilized to carry out polymorphism screening on 08-13, fiebyibai, resistance pool and sensing pool;
the PCR reaction volume was 15. Mu.L, of which 10 XBuffer 1.5. Mu.L, 2.5 mMdNTPs0.3. Mu.L, tag enzyme (5 units/. Mu.L) 0.2. Mu.L, 10. Mu.M primers (F/R) each 0.3. Mu.L, template DNA30ng, and ddH were added 2 O to 15 μ L;
the SSR reaction conditions comprise DNA pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 25s, circulation for 36 times, and final extension at 72 ℃ for 10min;
performing PCR amplification on a PCR amplification instrument, performing electrophoretic separation on an amplification product on 8% non-denaturing polyacrylamide gel, and photographing after silver staining to record a result;
selection between parents and F 2 Amplifying primers with the same polymorphism between resistant and sensitive pools of the generation group, wherein the number of the primers is 3, and the primers are linked with a disease-resistant gene CpPRSV-W;
(III) obtaining closely-linked molecular markers
(7) According to the linkage exchange rule, constructing a linkage map of the molecular marker and the CpPRSV-W gene by using genotype data and resistance data of 3 candidate molecular markers among homozygous susceptible individuals, wherein the used software is Mapmaker/EXP3.0, obtaining the molecular marker Cp126 tightly linked with the disease-resistant gene CpPRSV-W, and the genetic linkage distance is 0.4cM (figure 1);
(IV) transfer of disease-resistant gene CpPRSV-W to susceptible Cucurbita pepo inbred line
(8) The obtained molecular marker CpPRSV-W is used for introducing the disease-resistant gene into a cucurbita pepo excellent susceptible inbred line (shown in figure 2) through hybridization, backcross transformation and selfing purification. By utilizing the strategy, the fine susceptible inbred line fiber-white of the cucurbita pepo is successfully improved, so that the cucurbita pepo carrying CpPRSV-W disease-resistant gene has PRSV-W virus disease resistance.
The cucurbita pepo germplasm resources contain rich gene resources, and the efficient and full utilization of the gene resources has an important role in breeding novel cucurbita pepo varieties. The invention establishes a genetic linkage map of a disease-resistant gene CpPRSV-W and a molecular marker Cp126 by hybridizing the disease-resistant germplasm 08-13 with an infected selfing line, determining that a dominant disease-resistant gene is carried in a disease-resistant material through genetic analysis, establishing a disease-resistant character segregation population and screening polymorphic molecular markers. The disease-resistant gene CpPRSV-W is successfully transferred into the fine disease-susceptible inbred line fiber-white of Cucurbita pepo by molecular marker-assisted selection and hybridization and backcross transfer, so that PRSV-W virus disease resistance is obtained. In the traditional breeding method, due to the lack of molecular markers linked with PRSV-W virus disease resistance genes, resistance inoculation identification is required for the transfer of the disease resistance genes of each generation, time and labor are consumed, and breeding failure is often caused by the problem of poor identification accuracy. Therefore, the development of the molecular marker Cp126 can simply, conveniently and accurately screen the single plant containing the disease-resistant gene CpPRSV-W in the seedling stage, greatly save the screening time and the breeding labor amount of disease-resistant materials, improve the selection accuracy and accelerate the cultivation speed of new varieties of PRSV-W resistant virus diseases.
The invention obtains the codominant molecular marker Cp126 which is tightly linked with the disease-resistant gene CpPRSV-W of the Cucurbita pepo PRSV-W virus disease. The genetic distance of Cp126 from the disease resistance gene is 0.4cM. By using the marker, the disease-resistant gene can be successfully introduced into other cucurbita pepo susceptible excellent inbred lines through auxiliary selection identification, so that the cucurbita pepo susceptible excellent inbred lines can obtain the disease-resistant character. The molecular marker has the advantages of stable amplification, convenient detection, rapidness, accuracy and the like. The marker Cp126 is used for detecting the Cucurbita pepo disease-resistant gene CpPRSV-W, so that whether the disease-resistant gene exists or not and the existence state can be determined, the resistance of the plant to PRSV-W virus diseases can be predicted, and the utilization of the disease-resistant gene is accelerated. The invention provides a method for utilizing disease-resistant genes in Cucurbita pepo PRSV-W virus disease-resistant germplasm resources 08-13: the molecular marker screening is utilized, the disease-resistant gene is introduced into the excellent susceptible inbred line of the cucurbita pepo through hybridization and backcross transformation, and the method is utilized to successfully introduce the disease-resistant gene CpPRSV-W into the excellent inbred line fiber-white of the cucurbita pepo to obtain PRSV-W virus disease resistance.
Sequence listing
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Claims (4)
1. A method for identifying cucurbita pepo genotypes, the genotypes being an RR genotype, an RS genotype and an SS genotype, the method comprising L1) and L2) as follows:
l1) carrying out PCR amplification by using genome DNA of the cucurbita pepo to be detected as a template and adopting A1 to obtain a PCR product;
l2) the following L21) or L22):
l21) detecting the size of the PCR product obtained in the step L1), and determining the genotype of the cucurbita pepo according to the size of the PCR product:
the genotype of the pumpkin to be detected, which contains 162bp and 146bp DNA fragments of the PCR product, is an RS genotype; the PCR product contains 162bp DNA fragment, and the genotype of the pumpkin to be detected, which does not contain 146bp DNA fragment, is SS genotype; the PCR product does not contain a 162bp DNA fragment, and the genotype of the pumpkin to be detected containing a 146bp DNA fragment is an RR genotype;
l22) detecting the sequence of the PCR product obtained in the step L1), and determining the genotype of the cucurbita pepo according to the PCR product:
the genotype of the pumpkin to be detected, of which the PCR product contains DNA fragments shown by SEQ ID No.1 and SEQ ID No.2, is an RS genotype; the PCR product contains a DNA fragment shown in SEQ ID No.2 and does not contain the DNA fragment shown in SEQ ID No.1, and the genotype of the Cucurbita pepo to be detected is RR genotype; the PCR product contains a DNA fragment shown in SEQ ID No.1 and does not contain a DNA fragment shown in SEQ ID No.2, and the genotype of the Cucurbita pepo to be detected is SS genotype;
a1 consists of single-stranded DNA named P1 and P2, P1 is the single-stranded DNA shown in the 1 st to 22 nd positions of SEQ ID No.1, P2 is the single-stranded DNA reverse complementary to the 162 nd to 144 th positions of SEQ ID No.1,
the zucchini with the genotype of SS is a PRSV-W virus disease-resistant zucchini, the zucchini with the genotype of RS is a PRSV-W virus disease-resistant zucchini, and the zucchini with the genotype of RR is a PRSV-W virus disease-resistant zucchini.
2. The method for identifying or assisting in identifying the zucchini PRSV-W virus disease resistance traits comprises the following N1) and N2):
n1) taking genome DNA of a pumpkin to be detected as a template, and performing PCR amplification by adopting A1 to obtain a PCR product, wherein the A1 consists of single-stranded DNA (deoxyribonucleic acid) named P1 and P2, the P1 is the single-stranded DNA shown in the 1 st-22 th positions of SEQ ID No.1, and the P2 is the single-stranded DNA reversely complemented with the 162 nd-144 th positions of SEQ ID No. 1;
n2) the following N21) or N22):
n21) detecting the size of the PCR product obtained in the step N1), wherein if the PCR product contains DNA fragments of 162bp and 146bp, the Cucurbita pepo to be detected is or is selected as a Cucurbita pepo for resisting PRSV-W virus disease; if the PCR product contains a 146bp DNA fragment and does not contain a 162bp DNA fragment, the Cucurbita pepo to be detected is or is selected as a Cucurbita pepo for resisting PRSV-W virus disease; if the PCR product does not contain the 146bp DNA fragment and contains the 162bp DNA fragment, the Cucurbita pepo to be detected is or is selected as a non-PRSV-W virus disease resistant Cucurbita pepo;
n22) detecting the sequence of the PCR product obtained in the step N1), wherein if the PCR product contains DNA fragments shown in SEQ ID No.1 and SEQ ID No.2, the to-be-detected Cucurbita pepo is or is candidate to be a Cucurbita pepo for resisting PRSV-W virus disease; if the PCR product contains the DNA segment shown in SEQ ID No.2 and does not contain the DNA segment shown in SEQ ID No.1, the to-be-detected zucchini is or is candidate to be the zucchini for resisting the PRSV-W virus disease; and if the PCR product contains the DNA segment shown by SEQ ID No.1 and does not contain the DNA segment shown by SEQ ID No.2, the Cucurbita pepo to be detected is or is selected as a non-PRSV-W virus disease resistant Cucurbita pepo.
3. Any one of the following H1-H3 is used:
use of H1, the method of claim 1 or 2 for identifying or aiding in identifying stable genetic zucchini for the resistance trait of a PRSV-W virus;
use of H2, the method of claim 1 or 2, in the breeding of cucurbita pepo;
h3, the use of the method for identifying the genotype of cucurbita pepo as claimed in claim 1 in identifying or assisting in identifying the resistance trait of the cucurbita pepo PRSV-W virus disease.
4. A method for breeding Cucurbita pepo L.according to claim 1, identifying genotype of Cucurbita pepo L.and selecting Cucurbita pepo L.with RR or RS genotype as parent.
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Non-Patent Citations (5)
| Title |
|---|
| Combining ability of summer-squash lines with different degrees of parthenocarpy and PRSV-W resistance;Douglas Willian Nogueira et al.;《Genetics and Molecular Biology》;20111231;616-623 * |
| 南瓜属EST-SSR标记的的开发及在杂种纯度鉴定中的应用;张国裕 等;《华北农学报》;20111231;98-100 * |
| 王冬杰.美洲南瓜（Cucurbit pepo）WMV-2抗性遗传及分子标记研究.《中国优秀硕士学位论文全文数据库（农业科技辑）》.2013,I,56-57. * |
| 美洲南瓜（Cucurbit pepo）WMV-2抗性遗传及分子标记研究;王冬杰;《中国优秀硕士学位论文全文数据库（农业科技辑）》;20131015;I，56-57 * |
| 西葫芦抗PRSV、CMV遗传分析和抗性基因初步定位;张栋;《中国优秀硕士学位论文全文数据库（农业科技辑）》;20160615;I,20-24 * |
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