CN104630215A - Series molecular markers closely linked with muskmelon powdery mildew resistant gene Pm-2Fand acquisition method of series molecular markers - Google Patents
Series molecular markers closely linked with muskmelon powdery mildew resistant gene Pm-2Fand acquisition method of series molecular markers Download PDFInfo
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Abstract
The invention belongs to the field of genetic engineering and provides series molecular markers closely linked with a muskmelon powdery mildew resistant gene Pm-2F and an acquisition method of the series molecular markers. Each molecular marker contains at least one nucleotide base sequences shown in SEQ No:1-8 in a sequence table. The acquisition method of the series molecular markers comprises the following steps: selecting a test material, primarily positioning the muskmelon powdery mildew resistant gene Pm-2F, acquiring a site SNP/Indel linked with target characters, and acquiring molecular markers closely linked with the muskmelon powdery mildew resistant gene Pm-2F, so that the molecular markers closely linked with the muskmelon powdery mildew resistant gene Pm-2F are obtained. When the molecular markers are used for early screening and identification of a powdery mildew resistant variety, the breeding period can be greatly shortened, and the breeding efficiency is improved, so that the series molecular markers closely linked with the muskmelon powdery mildew resistant gene Pm-2F have important theory and practice significance.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a series of molecular markers closely linked with a melon powdery mildew resistance gene Pm-2F and an acquisition method thereof.
Background
The melon (Cucumis melo L.) is an important fruit-type vegetable crop of the Cucumis genus in the cucurbitaceae family, and is generally cultivated at home and abroad; the muskmelon is a dominant crop in high-end agriculture in China, and has important significance for increasing the income of farmers and adjusting agricultural structures. Powdery mildew is a worldwide disease seriously harming melon production and can occur from a seedling stage to a harvest stage. Powdery mildew mainly harms leaves of the melons and also infects stems and stems, which can cause the reduction of the photosynthetic capacity of plants, cause premature senility and even death, and seriously affect the yield and quality of the melons. In recent years, with the increase of scale and commercialization degree of production, powdery mildew rapidly spreads, and has become a main obstacle for green production of melon crops such as melons at home and abroad.
The pathogenic bacteria causing melon powdery mildew are mainly ascochyta (podosphaea xanthorrhii) and erysiphe cichoracearum (Golovinomyces cichororaceae), wherein ascochyta (p. The physiological races of melon powdery mildew are multiple and are fast to differentiate, only P.xanthonti common physiological races are as many as 7, including 0, 1, 2US, 2F, 3, 4 and 5, wherein P.xanthonti 2F is the dominant physiological race in most areas in China (Chenghong, the research progress of melon powdery mildew and the preliminary identification of physiological races in China, Changjiang vegetables 2011(18):1-5), so that the development of the disease-resistant gene research aiming at the physiological race of melon powdery mildew P.xanthonti 2F is the first breakthrough for solving the powdery mildew of melons. In Chinese patent application 'specific fragment closely linked with melon powdery mildew resistance gene Pm-2F and obtaining method thereof' (application number: 200710178022.7), a specific fragment closely linked with melon powdery mildew resistance gene PM-2F is disclosed, which has a DNA sequence represented by SEQ ID NO. 1-4 in a sequence table; the obtained SSR specific fragment has the advantages of good repeatability, stable and reliable marking, convenient statistics and the like. In 2008, Zhang Hai Ying and the like construct RILs population containing 106 strains by taking Japanese netted melon inbred line K7-1 with high powdery mildew resistance P.xanthii 2F and susceptible Hami melon inbred line K7-2 as parents, genetic analysis finds that the resistance of K7-1 to P.xanthii 2F is controlled by a single dominant gene Pm-2F, and molecular markers CMBR8 (corresponding to SSR509 in patent application 200710178022.7) and CMBR120 (corresponding to SSR510 in patent application 200710178022.7) linked with Pm-2F are obtained by screening, wherein the linkage distances are respectively 1cm and 3cm (Zhang Hai Ying, genetic characteristics of melon powdery mildew resistance gene Pm-2F and specific fragments thereof closely linked, gardening reports 2008, 35(12)1773 and 1780). However, due to the problems that the population used by marker linkage analysis is small, the genetic distance between the marker and the target gene is relatively long, and the like, the application of the two markers to the powdery mildew resistant assisted breeding of the melon has certain limitations.
Therefore, in the current scientific research and practice, the development of the molecular marker which is more closely linked with the Pm-2F is beneficial to applying the molecular marker to assist in breeding the melon variety of the powdery mildew resistant physiological race P.xanthothii 2F, and meanwhile, the method lays a foundation for cloning the Pm-2F gene.
Disclosure of Invention
The invention aims to provide a series of closely linked markers of a melon powdery mildew resistance gene Pm-2F and an obtaining method thereof.
The purpose of the invention is realized by the following technical scheme:
the series of molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F have at least one nucleotide base sequence of SEQ ID NO. 1-8 in a sequence table.
The purpose of the invention is realized by another technical scheme as follows:
the method for obtaining the series of molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F comprises the following steps:
selecting test materials: selecting a test material;
preliminary positioning of the muskmelon powdery mildew resistance gene Pm-2F:
performing seedling-stage disease-resistant inoculation identification on the test material to obtain a resistance and sensitivity material; extracting the genome DNA of the anti-influenza pool material by using a group mixed analysis method to construct an anti-influenza DNA pool;
screening SSR markers in the cucumber high-density genetic map through PCR amplification reaction to obtain SSR markers linked with target characters;
performing genetic linkage analysis on the SSR marker linked with the target character, and preliminarily positioning a target gene Pm-2F;
obtaining SNP/Indel sites linked with target characters:
designing a primer in the Pm-2F gene primary positioning interval, carrying out PCR amplification reaction on a muskmelon anti-infection parent and the anti-infection DNA pool, and obtaining the SNP/Indel locus linked with the target character through sequence comparison after sequencing;
the method comprises the following steps of (1) obtaining a molecular marker closely linked with a melon powdery mildew resistance gene Pm-2F:
and respectively designing CAPS/Indel markers aiming at the SNP/Indel sites linked with the target traits, and verifying the candidate SNP/Indel sites in a verification material to obtain molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F.
Further, in the preliminary positioning step of the melon powdery mildew resistance gene Pm-2F, the SSR marker linked with the target character has a sequence shown in SEQ ID NO: 9-14, wherein:
SEQ ID NO. 9 in the sequence table is a nucleotide base sequence of primer SSR02733 for specific amplification of a disease-resistant material;
SEQ ID NO. 10 in the sequence table is a nucleotide base sequence of primer SSR02733 for specific amplification of a susceptible material;
SEQ ID NO. 11 in the sequence table is a nucleotide base sequence of primer SSR02734 for specific amplification of a disease-resistant material;
SEQ ID NO. 12 in the sequence table is a nucleotide base sequence of the primer SSR02734 for specific amplification of the susceptible material.
SEQ ID NO. 13 in the sequence table is a nucleotide base sequence of a primer CS27 for specific amplification of a disease-resistant material;
SEQ ID NO. 14 in the sequence list is a nucleotide base sequence of the primer CS27 for the specific amplification of the susceptible material.
Preferably, in the preliminary positioning step of the melon powdery mildew resistance gene Pm-2F, the nucleotide base sequence of the primer for PCR amplification of the SSR marker is as follows:
SSR primer SSR 02733:
the sequence of the upstream primer is as follows: 5'-TTGTTAGGTAAGCCATGCCC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TTTGCCTGAGGAAGAATCTGA-3', respectively;
SSR primer SSR 02734:
the sequence of the upstream primer is as follows: 5'-TGTTGTTGGACCCCTTCAAT-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TGTCAAAGGAGGAGGTGGAG-3', respectively;
SSR primer CS 27:
the sequence of the upstream primer is as follows: 5'-GCTGAGTTATGGGGAAAGCA-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-ATGTTGTTGGACCCCTTCAA-3' are provided.
Preferably, in the preliminary localization step of the melon powdery mildew resistance gene Pm-2F, the PCR amplification reaction program for the SSR marker is as follows:
stage 1: pre-denaturation at 94 ℃ for 3 min; and (2) stage: circulating for 30 times at 94 ℃ for 20s, 55 ℃ for 20s and 72 ℃ for 30 s; and (3) stage: extending for 10min at 72 ℃; and (4) stage: keeping at 4 ℃.
Further, in the step of obtaining the SNP/Indel site linked with the target trait, the sequence specifically amplified by the disease-resistant material and the disease-sensitive material obtained by the PCR amplification reaction has at least one nucleotide base sequence of SEQ ID NO. 15-22 in the sequence table, wherein:
15 in the sequence table is a nucleotide base sequence of a primer Cum-1 for specific amplification of the disease-resistant material;
16 in the sequence table is a nucleotide base sequence of a primer Cum-1 for specific amplification of the susceptible material;
SEQ ID NO. 17 in the sequence table is a nucleotide base sequence of the primer Cum-2 for specific amplification of the disease-resistant material;
18 in the sequence table is a nucleotide base sequence of a primer Cum-2 for specific amplification of the susceptible material;
SEQ ID NO. 19 in the sequence table is a nucleotide base sequence of the primer Mel-1 for specific amplification of the disease-resistant material;
20 in the sequence table is a nucleotide base sequence of a primer Mel-1 for specific amplification of the susceptible material;
21 in the sequence table is a nucleotide base sequence of a primer Cum-3 for specific amplification of the disease-resistant material;
SEQ ID NO. 22 in the sequence table is a nucleotide base sequence of the primer Cum-3 for the specific amplification of the susceptible material.
Preferably, in the step of obtaining the SNP/Indel site linked to the target trait, the nucleotide base sequence of the primer for amplifying the sequence specifically amplified by the disease-resistant and disease-sensitive material is as follows:
cum-1 primer:
the sequence of the upstream primer is as follows: 5'-GTACAAGAGTTCTGCTGTGAAGGA-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TTGAATCTCATTTTTCTGTTGCAT-3', respectively;
cum-2 primer:
the sequence of the upstream primer is as follows: 5'-ATCTTCGAACATCATCTTTGAC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-ATCCCAAATCTCCAATATCG-3', respectively;
mel-1 primer:
the sequence of the upstream primer is as follows: 5'-CGAGTCTTCTTCTTCCAAATATCC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-GAAAGATTCATAGGAGAACTCGTCC-3', respectively;
cum-3 primer:
the sequence of the upstream primer is as follows: 5'-CACACAGTGGACAACAGTACAT-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-AAGATTGATTGACGATTGATTG-3' are provided.
Further, in the step of obtaining the series of markers closely linked with the melon powdery mildew resistance gene Pm-2F, the nucleotide base sequence of the primers for PCR amplification of the CAPS markers is as follows:
CAPS primer CAPS-Dde I:
the sequence of the upstream primer is as follows: 5'-GCCCAACCTTCAACTCGATA-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TTGAATCTCATTTTTCTGTTGCAT-3', respectively;
CAPS primer CAPS-Hph I:
the sequence of the upstream primer is as follows: 5'-CAGTAGGGACACACAACC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TATGCCATACGCTAATGT-3', respectively;
further, in the step of obtaining the molecular marker closely linked with the melon powdery mildew resistance gene Pm-2F, the PCR amplification reaction program used for the CAPS marker is as follows:
stage 1: pre-denaturation at 94 ℃ for 3 min; and (2) stage: circulating 30 times at 94 ℃ for 20s, Tm value for 20s and 72 ℃ for 30 s; and (3) stage: extending for 10min at 72 ℃; and (4) stage: keeping at 4 ℃; wherein CAPS marker Tm values are: CAPS-Dde I: 57 ℃, CAPS-Hph I: 55 ℃;
further, in the step of obtaining the molecular marker closely linked with the melon powdery mildew resistance gene Pm-2F, the endonuclease of the marker CAPS-Dde I is Dde I restriction endonuclease, and the endonuclease of the marker CAPS-Hph I is Hph I restriction endonuclease;
in the step of obtaining the molecular marker closely linked with the melon powdery mildew resistance gene Pm-2F, the enzyme cutting site of the marker CAPS-Dde I is as follows: GCACT, CAPS-Hph I site: GGTGA;
in the step of obtaining the series markers closely linked with the melon powdery mildew resistance gene Pm-2F, the enzyme digestion reaction conditions are as follows:
stage 1: carrying out enzyme digestion at 37 ℃ for 12-16 h; and (2) stage: enzyme denaturation at 65 deg.C for 20 min; and (3) stage: keeping at 4 ℃.
Further, in the step of obtaining the molecular marker tightly linked with the melon powdery mildew resistance gene Pm-2F, the nucleotide base sequence for PCR amplification of the Indel marker primer is as follows:
indel primers Indel 01:
the sequence of the upstream primer is as follows: 5'-GTCCACAGCAATCCAAGCTC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-CGAGTCTTCTTCTTCCAAATATCC-3' are provided.
Indel primers Indel 02:
the sequence of the upstream primer is as follows: 5'-GTGTGTGTGTGCAGAAATGGA-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-ATGGAACAACCTCCATCTGC-3' are provided.
Further, in the step of obtaining the molecular marker closely linked with the melon powdery mildew resistance gene Pm-2F, the PCR amplification reaction program for the Indel marker is as follows:
stage 1: pre-denaturation at 94 ℃ for 3 min; and (2) stage: circulating 30 times at 94 ℃ for 20s, Tm value for 20s and 72 ℃ for 30 s; and (3) stage: extending for 10min at 72 ℃; and (4) stage: keeping at 4 ℃; wherein, the Tm value of the molecular marker is as follows: indel 01: 58 ℃, Indel 02: and 57 ℃.
Further, in the test material selecting step, the test material includes:
the anti-influenza parent: male parent K7-1, a typical japanese type melon material of high powdery mildew resistant physiological races p.xanthothii 2F; the female parent K7-2 is a typical high-susceptibility powdery mildew physiological race P.xanthii 2F Sinkiang Hami melon material;
f obtained by hybridization of the resistant parent1Generation group;
with said F1F obtained by inbreeding of generations2And (4) separating the populations.
The invention has the beneficial effects that:
1. the invention adopts a BSA method, utilizes a muskmelon-cucumber comparative genome to combine with a cucumber high-density genetic linkage map, screens molecular markers linked with target characters, and initially positions a muskmelon powdery mildew resistance gene Pm-2F; designing primers according to genome information and melon EST information of cucumber homologous sections corresponding to the primary positioning interval, amplifying melon resistance and induction parents and resistance and induction pool sequences, and comparing to obtain candidate SNP/Indel sites completely conforming to target characters; respectively designing CAPS/Indel markers according to candidate SNP/Indel sites linked with the traits, obtaining the markers closely linked with the target traits by combining F2 generation segregation population verification analysis and natural population verification analysis, and confirming the close linkage degree of the series of SNP/Indel sites and the target traits; the molecular marker is used for early screening and identifying powdery mildew resistant varieties, so that the breeding period can be greatly shortened, the breeding efficiency is improved, and the molecular marker has important theoretical and practical significance.
2. Most of the work in the present invention was done in the case where the melon reference genome was not yet published (the melon genome was published in 2012). According to the invention, a series of molecular markers which are closely linked with the Pm-2F gene are developed by a melon-cucumber comparative genome method and cucumber genome sequence information, so that a new thought and method are provided for developing molecular markers of related species by using comparative genomes and plant genomes in a sequenced mode.
Drawings
FIG. 1 shows primers SSR02734, SSR02733 and CS27 for resistant parent strain F1PCR amplification result of generation, resistance and sensing DNA pool.
FIG. 2 is a diagram of the preliminary positioning result of the melon powdery mildew resistance gene Pm-2F.
FIG. 3 shows the primer CAPS-Dde I pair F2Separation ofPCR amplification and Dde I enzyme cutting result diagram of the individual strains of the population part.
FIG. 4 shows primer CAPS-Hph I pair F2Separating PCR amplification and Hph I enzyme cutting result graph of partial individual strains of the population.
FIG. 5 shows the primer pair F of Indel012And (3) separating a PCR amplification result graph of a population part single strain.
FIG. 6 shows the primer pair F of Indel022And (3) separating a PCR amplification result graph of a population part single strain.
FIG. 7 is a fine positioning result diagram of the melon powdery mildew resistance gene Pm-2F.
Detailed Description
The following describes in detail specific embodiments of the present invention.
According to the first aspect of the invention, the invention provides a series of CAPS/Indel markers closely linked with a melon powdery mildew resistance gene Pm-2F, wherein the CAPS/Indel markers have at least one nucleotide base sequence of SEQ ID NO. 1-8 in a sequence table.
Wherein,
SEQ ID NO. 1 in the sequence table is a nucleotide base sequence of the specific amplification of the primer CAPS-Dde I on the disease-resistant material;
SEQ ID NO. 2 in the sequence table is a nucleotide base sequence of the primer CAPS-Dde I for the specific amplification of the susceptible material;
SEQ ID NO. 3 in the sequence table is a nucleotide base sequence of the specific amplification of the primer CAPS-Hph I on the disease-resistant material;
SEQ ID NO. 4 in the sequence table is a nucleotide base sequence of the primer CAPS-Hph I for the specific amplification of the susceptible material;
SEQ ID NO. 5 in the sequence table is a nucleotide base sequence of a primer Indel01 for specific amplification of a disease-resistant material;
SEQ ID NO. 6 in the sequence table is a nucleotide base sequence of a primer Indel01 for specific amplification of a susceptible material;
SEQ ID NO. 7 in the sequence table is a nucleotide base sequence of a primer Indel02 for specific amplification of a disease-resistant material;
SEQ ID NO. 8 in the sequence list is a nucleotide base sequence of the primer Indel02 for specific amplification of the susceptible material.
According to the second aspect of the invention, the invention provides a method for obtaining the series of molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F,
the acquisition method comprises the following steps:
(1) selecting a test material:
the test material comprises male parent, female parent and F1Generation group, RILs group, F2Separating the population;
the male parent is: k7-1, a typical japanese type melon material of high powdery mildew resistant physiological races p.xanthonthii 2F;
the female parent is: k7-2, which is a typical high-susceptibility powdery mildew physiological race P.xanthii 2F Sinkiang Hami melon material;
F1the generation is as follows: f obtained by hybridization by taking the male parent and the female parent as parents1Generation group;
the RILs population was: the F1The RILs population obtained by self-crossing 8 generations comprises 58 disease-resistant strains and 48 susceptible strains.
F2The population is as follows: with F12200F strains obtained by generation selfing2Separating the population;
the tested materials are all from West melon germplasm resource library of vegetable research center of agriculture and forestry academy of sciences of Beijing.
(2) And preliminary positioning of the muskmelon powdery mildew resistance gene Pm-2F:
performing seedling-stage disease-resistant inoculation identification on a test material to obtain an anti-infection pool material, and extracting genome DNA by using a group mixing Analysis method (BSA) to construct an anti-infection pool; then, screening SSR markers in the cucumber high-density genetic map through PCR amplification reaction to obtain markers linked with target characters; and combining with the RILs group phenotype identification result and the linkage marker analysis result, performing genetic linkage analysis by using JoinmapP 4.0 software, and preliminarily positioning a target gene-powdery mildew resistance Pm-2F gene.
The method comprises the following specific steps:
firstly, performing disease-resistant inoculation identification on a test material at a seedling stage;
(a) the male parent, the female parent and F in the test material are used1The generation group is sown by taking 30 seeds respectively, each strain of RILs group is sown by taking 15 seeds, F22200 seeds are taken for the generation separation group to be sown and planted in a sunlight greenhouse of a vegetable research center of agriculture and forestry academy of sciences of Beijing;
(b) separating and purifying powdery mildew pathogenic bacteria and preparing an inoculation bacterial source:
powdery mildew collected from Sijiqing farms at the vegetable research center of the academy of agriculture and forestry, Beijing is inoculated on 13 identification hosts (PI 124112, PMR45, PMR 6, PI 124111, Top mark, Vedrantais, WMR 29, Nantais blong, Edison 47, PI 414723, PMR 5, Iran H and MR 1) identified by physiological races of powdery mildew by adopting a spore suspension spraying method, the physiological races of the powdery mildew pathogenic bacteria are determined to be P.xanthii 2F according to the anti-infection reaction of the identification hosts, the powdery mildew P.xanthii 2F is separated and purified by utilizing a monospore culture technology, and the purified powdery mildew pathogenic bacteria are inoculated on Cucurbita pepo seedlings for propagation;
brushing spores on leaves of the powdery mildew pumpkin with a sterilized writing brush in a beaker filled with sterile distilled water, filtering all spore suspensions with clean gauze to remove impurities, sucking a small amount of spore suspension with a suction tube, and dripping the spore suspension on a blood counting chamberCounting up, adjusting the concentration of inoculated bacteria liquid to 2.0 × 105spore/mL;
(c) and identifying disease-resistant inoculation at seedling stage: 13 parts of physiological races of male parent, female parent and melon powdery mildew differential hosts and F1The generation group respectively takes 30 seeds, each strain of RILs group takes 15 seeds, F22200 seeds are taken from the generation separation population; soaking and sterilizing the seeds, putting the seeds in a constant-temperature incubator at 28 ℃ for accelerating germination for 18 hours, and then sowing the seeds in a plug tray filled with sterilized nutrient soil; after cotyledon flattening, spraying spore suspension to inoculate P.xanthii 2F, and after 12-15 days of full disease, investigating male parent, female parent and F1RILs and F2The anti-infection reaction of the individual plants of the population;
(d) and grading standard of seedling stage disease-resistant inoculation identification: the disease resistance identification adopts 6-grade grading standard of melon seedling stage disease identification; the whole plant without any lesion is 0 grade; only cotyledons had a very small number of lesions (less than 20%) of grade 1; only cotyledons have more scabs (21-50 percent) or cotyledons have scabs, and the number of the scabs on stems is 2 grade; the cotyledon has a plurality of disease spots (51% -70%), and the stem has a small amount of disease spots (21% -50%) which are 3 grades; the cotyledon and stem are covered with disease spots or the cotyledon and stem are covered with disease spots (more than 71 percent), and the true leaf has disease spots (less than 20 percent) which are 4 grades; the whole plant is uniformly distributed with white powder or the plant dies to 5 grades due to infection; resistance determination disease index (PDI) was used to determine (Zhanghaiying, Sources of resistance to trace 2WF powder mile in US water plant expression, HortSci 46(10): 1349-: 0< PDI < 40; intermediate type: 40< PDI < 60; the infection: PDI is more than or equal to 60; wherein, PDI is calculated according to the following formula:
PDI＝[Σ(s×n)/(N×S)]×100
in the above formula, Σ is a sum formula; s is a value representing each disease level; n is disease strain of each disease level; n is the total number of the investigated plants; s is the highest disease level representative value;
F2the resistance of the individual plant of the group is determined according to disease classification, wherein 0, 1 and 2 are disease resistance, and 3, 4 and 5 are infection;
secondly, extracting the genome DNA of the anti-infection and infection pool material by using a group mixed Analysis method (BSA), and constructing an anti-infection and infection DNA pool;
finding out cucumber homologous sections corresponding to the Pm-2F gene linkage markers CMBR8 and CMBR120 by utilizing melon-cucumber comparative genomics, screening SSR markers in high-density genetic maps of corresponding cucumber chromosome sections, and obtaining SSR markers linked with target characters;
the specific operation is as follows:
(a) and extracting genome DNA of the antibiotic and sensitive pool material:
DNA extraction was improved based on the method of Murray et al (1980) (Murray M, Thompson W.Rapidation of high molecular weight plant DNA [ J ]. Nucl Acid Res,1980,8: 668-673.); the method comprises the following specific steps:
taking 1.5 g of leaf of an anti-influenza cell material, grinding the leaf into powder in liquid nitrogen, adding 9ml of 2% CTAB extracting solution (2% CTAB, 1.4mM NaCl, 100mM Tris-HCl pH8.0, 20mM EDTApH8.0, 1% PVP-40, 0.2% beta-mercaptoethanol), uniformly mixing, and carrying out water bath at 65 ℃ for 1 hour to obtain a mixture A;
stopping the water bath of the mixture A, adding 1/3 volumes of 5M potassium acetate solution, mixing uniformly, and carrying out ice bath for 20 minutes; adding chloroform/isoamyl alcohol (24: 1) with the same volume for extraction twice to obtain supernatant A;
adding 2/3 volumes of isopropanol to supernatant A to precipitate the DNA; washing with washing buffer (76% ethanol, 10mM ammonium acetate), blow-drying, and dissolving with TE buffer (10mM Tris-HCl, 1mM EDTA, pH7.4) to obtain solution A;
iv, adding the RNase A into the solution A to enable the final concentration to reach 100 mu g/ml, and uniformly mixing in a water bath at 37 ℃ for 1 hour; extracting with chloroform/isoamyl alcohol (24: 1) to obtain supernatant B;
v, adding 1/2 volumes of 7.5M ammonium acetate and 2 volumes of absolute ethanol into the supernatant B to obtain DNA precipitate;
vi, washing DNA precipitate with 70% ethanol, blowing to dry, adding appropriate amount of ddH2Dissolving DNA to obtain genome DNA of the antibiotic and sensitive cell material;
then, the OD was measured using an ultraviolet spectrophotometer (Shimadzu UV-1201, Japan)260Measuring the concentration of the test material genome DNA, and detecting the extraction quality of the genome DNA of the antigen-sensitive cell material by 0.8% agarose gel electrophoresis;
(b) and (3) constructing an anti-infection DNA pool:
the anti-infection DNA pool is a disease-resistant DNA pool formed by mixing 10 homozygous disease-resistant (disease index is 0 level) single DNA strains, and the infection DNA pool formed by mixing 10 homozygous powdery mildew (disease index is 5 level) single DNA strains;
(c) utilizing a muskmelon-cucumber comparative genome to find out cucumber genome homologous regions corresponding to molecular markers CMBR8 and CMBR120 linked with a Pm-2F gene, screening SSR markers in a high-density genetic map of a corresponding cucumber chromosome segment, and obtaining the SSR molecular markers linked with target characters:
the sequence of SSR primer SSR02733 is as follows:
the sequence of the upstream primer is as follows: 5'-TTGTTAGGTAAGCCATGCCC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TTTGCCTGAGGAAGAATCTGA-3', respectively;
the SSR primer SSR02734 has the following sequence:
the sequence of the upstream primer is as follows: 5'-TGTTGTTGGACCCCTTCAAT-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TGTCAAAGGAGGAGGTGGAG-3', respectively;
the sequence of the SSR primer CS27 is as follows:
the sequence of the upstream primer is as follows: 5'-GCTGAGTTATGGGGAAAGCA-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-ATGTTGTTGGACCCCTTCAA-3', respectively;
the primer selected by the SSR marker is synthesized by Beijing synthesis part of Shanghai bio-engineering company;
(d) screening the parent muskmelon and the resistance and sensitivity DNA pool by PCR amplification reaction by using SSR markers:
the PCR reaction system (15. mu.L) contained: 1.5 μ L of MgCl containing 15mM210 × Buffer; 0.5. mu.L of dNTPs at a concentration of 2.0 mM; 0.5U Taq DNA polymerase; 1.0 μ L of 10uM PCR upstream and downstream mixed primers; 20ng of template DNA; ddH2O to 15 μ L; taq DNA polymerase and reaction buffer were purchased from TaKaRa; dNTPs were purchased from Beijing Quanji Biotech Ltd;
the PCR amplification reaction program is as follows: stage 1: pre-denaturation at 94 ℃ for 3 min; and (2) stage: circulating for 30 times at 94 ℃ for 20s, 55 ℃ for 20s and 72 ℃ for 30 s; and (3) stage: extending for 10min at 72 ℃; and (4) stage: keeping at 4 ℃; wherein the PCR instrument is a Veriti 96 well Thermal Cycler available from Applied Biosystems;
detection of PCR amplification product: adding 15 ul of amplification product into 3 ul of loading buffer (6 XLoadingBuffer), mixing evenly by a pipettor, and then taking 3 ul of amplification product and dropping the 3 ul of amplification product into 8% polyacrylamide gel; 140V/500mA electrophoresis for 90 min; the electrophoresis results are shown in FIG. 1;
wherein, the 8% polyacrylamide gel formula is as follows:
dd H2O：10mL；10×TBE：2mL；20％Acr-Bis：8mL；Temed：20μL；10％APS：200μL；
the buffer solution is as follows: 1 × TBE electrode buffer; the electrophoresis apparatus is: purchased from JY600 electrophoresis apparatus (JINGYI corporation);
after the amplified product is visualized by silver staining, analyzing an electrophoresis band; homozygous banding pattern from the male parent K7-1 is designated "a", homozygous banding pattern from the female parent K7-2 is designated "b", heterozygous banding pattern is designated "h", ambiguous or missing banding is designated "-";
the series SSR markers linked with the melon powdery mildew resistance gene Pm-2F have nucleotide base sequences of SEQ ID NO. 9-14 in a sequence table, wherein:
SEQ ID NO. 9 in the sequence table is a nucleotide base sequence of primer SSR02733 for specific amplification of a disease-resistant material;
SEQ ID NO. 10 in the sequence table is a nucleotide base sequence of primer SSR02733 for specific amplification of a susceptible material;
SEQ ID NO. 11 in the sequence table is a nucleotide base sequence of primer SSR02734 for specific amplification of a disease-resistant material;
SEQ ID NO. 12 in the sequence table is a nucleotide base sequence of the primer SSR02734 for specific amplification of a susceptible material;
SEQ ID NO. 13 in the sequence table is a nucleotide base sequence of a primer CS27 for specific amplification of a disease-resistant material;
SEQ ID NO. 14 in the sequence list is a nucleotide base sequence of the primer CS27 for the specific amplification of the susceptible material;
thirdly, genetic linkage analysis is carried out on the phenotype and the linkage marker of the RILs group strain by using Joinmap4.0 software, and the Pm-2F gene of powdery mildew resistance is preliminarily positioned.
(3) And obtaining SNP/Indel sites linked with the target characters: utilizing a melon-cucumber comparative genome to find a cucumber genome region corresponding to a preliminary positioning powdery mildew resistance Pm-2F gene interval, designing primers according to cucumber genome and melon EST information, and carrying out amplification and sequencing in a melon anti-susceptible parent and an anti-susceptible DNA pool to obtain a specific amplification sequence of a disease-resistant and disease-susceptible material, wherein the specific amplification sequence has a nucleotide base sequence of SEQ ID NO. 15-22 in a sequence table, and the nucleotide base sequence comprises the following components:
15 in the sequence table is a nucleotide base sequence of a primer Cum-1 for specific amplification of the disease-resistant material;
16 in the sequence table is a nucleotide base sequence of a primer Cum-1 for specific amplification of the susceptible material;
SEQ ID NO. 17 in the sequence table is a nucleotide base sequence of the primer Cum-2 for specific amplification of the disease-resistant material;
18 in the sequence table is a nucleotide base sequence of a primer Cum-2 for specific amplification of the susceptible material;
SEQ ID NO. 19 in the sequence table is a nucleotide base sequence of the primer Mel-1 for specific amplification of the disease-resistant material;
20 in the sequence table is a nucleotide base sequence of a primer Mel-1 for specific amplification of the susceptible material;
21 in the sequence table is a nucleotide base sequence of a primer Cum-3 for specific amplification of the disease-resistant material;
SEQ ID NO. 22 in the sequence table is a nucleotide base sequence of a primer Cum-3 for specific amplification of the susceptible material;
and obtaining SNP/Indel sites (namely candidate sites) linked with the target traits through sequence comparison.
The method comprises the following specific steps:
taking the genomic DNA of the parent muskmelon and an anti-infection DNA pool as templates, and carrying out PCR amplification reaction to obtain a PCR amplification product; extracting the genome DNA of the parent muskmelon and the resistant and sensitive DNA pool in the step (2); the primers selected for PCR amplification reaction are designed according to cucumber homologous region sequence information and melon EST information and synthesized by Beijing synthesis part of Shanghai's worker company; wherein, the primer sequences selected by the PCR amplification reaction are as follows:
cum-1 primer (corresponding to CAPS-Dde I marker long fragment sequence amplification):
the sequence of the upstream primer is as follows: 5'-GTACAAGAGTTCTGCTGTGAAGGA-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TTGAATCTCATTTTTCTGTTGCAT-3', respectively;
cum-2 primer (corresponding to CAPS-Hph I-tagged long fragment sequence amplification):
the sequence of the upstream primer is as follows: 5'-ATCTTCGAACATCATCTTTGAC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-ATCCCAAATCTCCAATATCG-3', respectively;
mel-1 primer (corresponding to Indel 01-labeled long fragment sequence amplification):
the sequence of the upstream primer is as follows: 5'-CGAGTCTTCTTCTTCCAAATATCC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-GAAAGATTCATAGGAGAACTCGTCC-3', respectively;
cum-3 primer (corresponding to Indel 02-labeled long fragment sequence amplification):
the sequence of the upstream primer is as follows: 5'-CACACAGTGGACAACAGTACAT-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-AAGATTGATTGACGATTGATTG-3', respectively;
the reaction system (25. mu.L) in which the above primers were used for PCR amplification contained: 2.5 μ L of MgCl containing 15mM210 × Buffer; 1.0. mu.L of dNTPs at a concentration of 2.5 mM; 1U Taq DNA polymerase; 2.0. mu.L of PCR upstream and downstream mixed primers with the concentration of 10 uM; 50ng of template DNA; ddH2O to 25 μ L; taq DNA polymerase and reaction buffer were purchased from TaKaRa; dNTPs were purchased from Beijing Quanji Biotech Ltd;
the PCR amplification reaction program is as follows: stage 1: pre-denaturation at 94 ℃ for 3 min; and (2) stage: circulating for 30 times at 94 ℃ for 20s, Tm value for 30s and 72 ℃ for 1 min; and (3) stage: extending for 10min at 72 ℃; and (4) stage: keeping at 4 ℃; wherein the series of labeled Tm values: cum-1: 53 ℃ and Cum-2: 54 ℃ and Mel-1-3: 58 ℃, Cum-3: 52 ℃; wherein the PCR instrument is Veriti 96 well thermal cycler available from Applied Biosystems;
then, 25. mu.L of the PCR amplification product is mixed with 4. mu.L of a sample Loading Buffer solution (6 XLoading Buffer, Beijing all-purpose gold biotechnology, Ltd.), added into 1.0% agarose gel, injected with 180V/90A and subjected to 90V/100A electrophoresis; wherein, the agarose is: agarose RA (available from amresco corporation); the buffer solution is as follows: 1 XTAE buffer solution; the electrophoresis apparatus is a JY600 electrophoresis apparatus purchased from JINGYI company;
secondly, detection and recovery of PCR products and acquisition of specific amplification sequences:
purifying the PCR amplification product, and then carrying out a connection reaction with a pEASY-T1 vector to obtain a recombinant plasmid; then transferring the recombinant plasmid into competent cells, and then carrying out screening, detection and sequencing to obtain a specific amplification sequence;
the method comprises the following specific steps:
(a) cutting off the specific amplification strips by using a blade, respectively putting the cut specific amplification strips into a marked 2.0ml centrifuge tube, and recovering and purifying the product by using a gel cutting recovery and purification Kit Easypure Quick gel extraction Kit purchased from Beijing all-type gold biotechnology limited company, wherein the operation method is detailed in a product specification to obtain a purified amplification product;
(b) connecting the purified amplification product with a pEASY-T1 vector for reaction to obtain a recombinant plasmid; the ligation reaction adopts a pEASY-T1Cloning Kit of Beijing holotype gold biotechnology limited;
(c) transferring the recombinant plasmid into competent cells, and then carrying out screening, detection and sequencing to obtain a specific amplification sequence;
the specific operation is as follows:
the recombinant plasmid was transferred to Trans-T1 competent cells purchased from Beijing Quanyujin Biotechnology Ltd, and then cultured in SOC liquid medium at 37 ℃ and 200rpm/min for 1h, and then the bacterial liquid was uniformly applied to LB/Amp previously applied with IPTG (8. mu.L, 500mM) and X-gal (40. mu.L, 20mg/ml)+(100ug/ml) in solid medium, and cultured overnight at 37 ℃; then, a single white colony was picked up with a 10. mu.L pipette tip and 1ml LB/Amp was added+Culturing in (100ug/ml) liquid culture medium at 37 deg.C and 200rpm/min for 8 hr, collecting 1 μ L of the culture solution, performing PCR detection with universal primer M13, and if positive clone is detected, transferring the corresponding culture solution to Beijing Nuosai genome researchSequencing by research center company Limited; wherein M13 primer used for PCR of bacterial liquid is synthesized by Beijing synthesis part of Shanghai's worker company, and DNA polymerase is Easytaq DNApolymerase;
obtaining SNP/Indel sites linked with the target characters:
and (3) analyzing and comparing the specific amplification sequence obtained by sequencing by using DNAMAN software, and combining a phenotype identification result to obtain the SNP/Indel locus (namely a candidate locus) linked with the target character.
(4) And respectively designing CAPS/Indel markers aiming at the SNP/Indel sites linked with the target traits, verifying the SNP/Indel sites linked with the target traits in a verification material, and obtaining the molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F.
The verification material comprises: above F2A generation segregating population (including 2200 individuals) and a natural population; the natural population is: 120 parts of melon germplasm resources, wherein the melon germplasm resources comprise 96 parts of local melon germplasm resources in Xinjiang, 20 parts of typical western Asia-European and American type melon resources introduced from abroad and 4 parts of other control materials; the natural population comprises 13 disease-resistant materials such as Perlia, Mainstream, TAMUvalde, Saticoy, PMR honeydew, Ogen-op, Piel de sapo and Munger's 339 and 107 disease-sensitive materials such as Hale's Best Jumbo, Crenshaw, Juan Canavy, Turkey variety, Im16 puredeo, Lorta and Mirela RZ; the verification materials are all from West melon germplasm resource bank of vegetable research center of Beijing agriculture and forestry academy of sciences.
The verification steps are as follows:
firstly, taking the genome DNA of a verification material as a template, and carrying out PCR amplification reaction to obtain a PCR amplification product;
the extraction method of the verification material genome DNA is shown in the step (2);
the CAPS/Indel labeled primer is synthesized by Beijing synthesis part of Shanghai bio-chemical company;
the sequence of the CAPS/Indel marker primers was as follows:
CAPS primer CAPS-Dde I:
the sequence of the upstream primer is as follows: 5'-GCCCAACCTTCAACTCGATA-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TTGAATCTCATTTTTCTGTTGCAT-3', respectively;
CAPS primer CAPS-Hph I:
the sequence of the upstream primer is as follows: 5'-CAGTAGGGACACACAACC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TATGCCATACGCTAATGT-3', respectively;
indel primers Indel 01:
the sequence of the upstream primer is as follows: 5'-GTCCACAGCAATCCAAGCTC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-CGAGTCTTCTTCTTCCAAATATCC-3', respectively;
indel primers Indel 02:
the sequence of the upstream primer is as follows: 5'-GTGTGTGTGTGCAGAAATGGA-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-ATGGAACAACCTCCATCTGC-3', respectively;
the PCR amplification reaction system (15. mu.L) with the CAPS/Indel primers contained: 1.5. mu.L of 15mM MgCl210 × Buffer; 0.5. mu.L of dNTPs at a concentration of 2.5 mM; 0.5U Taq DNA polymerase; 1.0 μ L of 10uM PCR upstream and downstream mixed primers; 20ng of template DNA; ddH2O to 15 μ L; TransStart Taq DNA Polymerase was purchased from Beijing Quanji Biotech Ltd, dNTPs was purchased from TaKaRa;
the PCR amplification reaction program is as follows: stage 1: pre-denaturation at 94 ℃ for 3 min; and (2) stage: circulating 30 times at 94 ℃ for 20s, Tm value for 20s and 72 ℃ for 30 s; and (3) stage: extending for 10min at 72 ℃; and (4) stage: keeping at 4 ℃; wherein the Tm value of the primer is as follows: CAPS-Dde I: 57 ℃, CAPS-Hph I: 55 ℃ Indel 01: 58 ℃ Indel 02: 57 ℃; wherein the PCR instrument is Veriti 96 well thermal cycler of Applied Biosystems company;
secondly, carrying out enzyme digestion reaction on the PCR amplification product to obtain an enzyme digestion product;
the reaction system 10. mu.L of the enzyme digestion reaction contained: 1.0. mu.L of 10 XBuffer, 0.5. mu.L of restriction enzyme, 3. mu.L of LPCR product, 5.5. mu.L of L H2O; dde I and Hph I restriction endonucleases were purchased from Fermantas;
the enzyme digestion reaction program is as follows: carrying out enzyme digestion at 37 ℃ for 12-16 h, then carrying out denaturation at 65 ℃ for 20min, and storing at 4 ℃, wherein the PCR instrument is a Veriti 96 well Thermal Cycler purchased from Applied Biosystems, and the labeled CAPS-Dde I enzyme digestion site is as follows: GCACT, CAPS-Hph I site: GGTGA.
The verification results and analysis in the above steps are as follows:
1. genetic rule analysis of melon powdery mildew resistance gene Pm-2F
In the step (2), a disease-resistant parent K7-1 (male parent), a disease-susceptible parent K7-2 (female parent) and F1Individual plants, RILs and F of the population2And (4) carrying out seedling stage disease-resistant inoculation identification on the generation segregating population, and investigating the disease progression of each single plant according to the grading standard of the powdery mildew disease of the melons. The results showed that K7-1, K7-2 and F1Disease indexes of generation groups are respectively expressed as disease resistance, infection resistance and disease resistance, which indicates that the resistance of the melon K7-1 to the white powdery mildew physiological race P.xanthii 2F is controlled by dominant single genes. F of K7-2 XK 7-12The disease-resistant identification result of the generation segregation population shows that 1673 disease-resistant individuals and 527 susceptible individuals exist in 2200 individuals, and the chi-square proportion suitability test F2 population meets the segregation rule that the infection-resistant proportion is 3:1 (chi)21.28, P0.26). Synthetic parents, F1And F2As a result of the seedling-stage disease-resistant identification of 2200 individuals in a population, the resistance of the melon K7-1 to the powdery mildew physiological race P.xanthonthii 2F is controlled by a dominant single gene Pm-2F. The identification result of each strain in the RILs group is consistent with that of RILs group of Zhang Hai Ying et al (2008).
2. Preliminary positioning analysis of muskmelon powdery mildew resistance gene Pm-2F
In the second step of the step (2), F is determined according to K7-2 XK 7-12Separating the disease-resistant identification results of the population at the seedling stage, and mixing 10 homozygous resistant and susceptible single plant DNAs to construct an anti-susceptible gene pool. And (2) carrying out polymorphism screening on the SSR markers by adopting a clustered mixed Analysis method (BSA) and utilizing 136 on the chromosome 1 of the cucumber high-density genetic map to obtain the SSR markers linked with target characters by utilizing the homologous segments of the cucumber chromosomes corresponding to CMBR8 and CMBR 120.
The analysis result shows that the SSR primers SSR02733, SSR02734 and CS27 amplified bands are linked with the target traits. As shown in FIG. 1, FIG. 1 shows SSR02733, SSR02734 and CS27 resistant parent, F1PCR amplification results of Generation and influenza pools (in FIG. 1, P1：K7-1；P2：K7-2；F1：K7-2×K7-1；BR: a disease-resistant gene pool; bs: the pool of susceptibility genes).
In the third step of the step (2), genetic linkage analysis is performed on the marker linked with the target trait by using Joinmap4.0 software, the SSR02733, the SSR02734 and the CS27 are positioned on the same side of the target gene, and the genetic distances to the target gene are respectively 0.5, 0.5 and 1.3cM, as shown in FIG. 2, and FIG. 2 is a preliminary positioning genetic map of the melon powdery mildew resistance gene Pm-2F.
3. SNP/Indel site acquisition linked to target traits
In the step (3), primer design is carried out according to cucumber genome interval sequence information and melon EST information corresponding to wing marks SSR02734 and CMBR8 on two sides of a Pm-2F gene of a primary positioning result, PCR amplification and sequencing are carried out in a neutralizing-resistant and sensing pool of a melon parent, and sequence comparison shows that an AC-GT base mutation site exists in a resistant and sensing material in a Cum1 amplification sequence; T-C base mutation sites exist in the anti-sensitive material of the Cum2 amplification sequence, ACT … A4 base insertion deletion mutation exists in the anti-sensitive material of the Mel-1 amplification sequence, GCGGCG 6 base insertion deletion mutation exists in the anti-sensitive material of the Cum3 amplification sequence, and CAPS/Indel markers are designed aiming at the mutation sites in the next step.
4、F2Genotype verification analysis of generation segregation population and natural population material
(1)、F2Generation segregation population genotype verification analysis
In the step (4), the parents and the parents are paired by the primers CAPS-Dde I and CAPS-Hph I22200 individuals of the segregating population are subjected to PCR amplification, and then the DNA restriction enzymes Dde I and Hph I are used for enzyme digestion, and the results show that:
after the primer CAPS-Dde I is subjected to PCR amplification, restriction enzyme Dde I is used for enzyme digestion, and in 1673 single plants with disease-resistant phenotype identification results, the genotype detection results of 1665 single plants are all shown as disease-resistant banding types, and the detection results of 8 disease-resistant plants are shown to be inconsistent with the phenotype results, so that the disease-resistant single plants are shown as exchange plants; among 527 individuals with disease-sensitive phenotype identification results, 521 individuals have disease-sensitive genotype identification results, and 6 individuals with disease-sensitive genotype do not accord with the phenotype detection results, so that the disease-sensitive individuals are cross-over plants; thus, 2200F2There were 14 crossover individuals in the population. Referring to FIG. 3, lanes P1: k7-1; lane P2: k7-2; lanes 1-16: f of K7-2 XK 7-12Part of the individual plants, lanes 4, 5, 6, 11, 13, 14, 16 are homozygous disease-resistant banding patterns, and lanes 1, 2, 3, 7, 8, 9, 10, 12, 15 are homozygous disease-resistant banding patterns.
After the primer CAPS-Hph I PCR amplification, carrying out enzyme digestion by using restriction endonuclease Hph I, wherein in 1673 single plants with disease-resistant phenotype identification results, the genotype detection results of 1668 single plants are all shown as disease-resistant banding types, and the detection results of 5 disease-resistant plants are shown to be inconsistent with the phenotype results, so that the disease-resistant single plants are shown as exchange plants; among 527 individuals with the disease-sensitive phenotype identification results, 522 individuals have the disease-sensitive genotype identification results, and the detection results of 5 disease-sensitive strains show that the disease-sensitive individuals are the cross-over strains; thus, 2200F2There were 10 crossover individuals in total among the population individuals. Referring to FIG. 4, lanes P1: k7-1; lane P2: k7-2; lanes 1-16: f2 partial individual of K7-2 XK 7-1, wherein 4, 5, 6, 14 and 16 are homozygous disease-resistant type, 1-3, 7-13 and 15 is heterozygous disease-resistant banding pattern.
The pair of parents F is formed by primers Indel01 and Indel022The progeny segregating population (this population included 2200 individuals) was PCR amplified and the results showed:
in the PCR amplification of the primer Indel01, the identification results of the genotypes of 1673 disease-resistant single strains are disease-resistant banding types; the genotype identification results of 527 susceptible individual strains are all susceptible banding patterns; thus, 2200F2The genotype analysis result of the single plant completely accords with the phenotype identification result. See FIG. 5, lanes P1: k7-1; lane P2: k7-2; lanes 1-16: f of K7-2 XK 7-12Part of the single plants, wherein 1, 2, 4, 5 and 13 are homozygous disease-resistant banding patterns, 3, 6, 9, 10, 11, 12, 14 and 15 are heterozygous disease-resistant banding patterns, and 7, 8 and 16 are homozygous disease-resistant banding patterns.
In the PCR amplification of the primer Indel02, in 1673 single plants with disease-resistant phenotype identification results, the genotype detection results of 1670 single plants are all shown as disease-resistant banding types, and the detection results of 3 disease-resistant plants are shown to be inconsistent with the phenotype results, which indicates that the disease-resistant single plants are cross-over plants; among 527 individuals with disease-sensitive phenotype identification results, 526 individuals with disease-sensitive genotype identification results are disease-sensitive banding types, and 1 individual-susceptible strain detection result shows that the disease-sensitive individuals are cross-over strains; thus, 2200F2There were 4 crossover individuals in the population. Referring to FIG. 6, lanes P1: k7-1; lane P2: k7-2; lanes 1-16: f of K7-2 XK 7-12Part of the single plants are heterozygous disease-resistant types 1, 2, 3, 5, 7-13, and homozygous disease-resistant types 4, 6, 14, 15, 16.
The linkage relationship between the markers and the target traits is analyzed by using Joinmap4.0 software, and the results are shown in FIG. 7, wherein the markers CAPS-Hph I, Indel02, CAPS-Dde I and the melon powdery mildew resistance gene Pm-2F are in linkage relationship, and the linkage distances are 0.26cM, 0.12cM and 0.35cM respectively; the Indel01 marker appeared to co-segregate with the Pm-2F gene, and the flanking markers Indel02 and CAPS-Dde I mapped the Pm-2F gene to within 0.47 cM.
(2) Natural population genotype verification analysis
In order to further verify the linkage relationship between the marker and the melon powdery mildew resistance gene Pm-2F, 120 melon germplasm resource materials in the natural population are verified, wherein 13 disease-resistant varieties and 107 susceptible varieties exist.
After the primer CAPS-Dde I is subjected to PCR amplification, the restriction enzyme Dde I is used for enzyme digestion, and in 120 parts of melon germplasm resource materials, 10 parts of material results do not accord with the field disease-resistant phenotype, and the coincidence rate is 91.6%.
After the primer CAPS-Hph I is subjected to PCR amplification, the restriction enzyme Hph I is used for enzyme digestion, and in 120 parts of melon germplasm resource materials, 8 parts of material results do not accord with the field disease-resistant phenotype, and the coincidence rate is 93.3%.
In the PCR amplification of the primer Indel01, the molecular marker detection genotype in 120 parts of melon germplasm resource materials is completely consistent with the field phenotype.
In the PCR amplification of the primer Indel02, the results of 3 parts of the melon germplasm resource materials are not consistent with the field disease-resistant phenotype in 120 parts of melon germplasm resource materials, and the coincidence rate is 97.5%.
The verification result further proves the close linkage relationship of the markers CAPS-Hph I, Indel02, CAPS-Dde I and the melon powdery mildew resistance gene Pm-2F, and the marker Indel01 and the melon powdery mildew resistance gene Pm-2F are coseparated and are gene-specific molecular markers; the marker can be effectively applied to molecular assisted breeding of the powdery mildew resistance of the melon, and has higher utilization value in the aspect of identifying the powdery mildew resistance of the melon.
In the invention, the adopted reagents and instruments are all conventional products in the market.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. Not all embodiments are exhaustive. All obvious changes and modifications which are obvious to the technical scheme of the invention are covered by the protection scope of the invention.
Claims (10)
1. A series of molecular markers closely linked with a melon powdery mildew resistance gene Pm-2F is characterized in that: the molecular marker has at least one nucleotide base sequence of SEQ ID NO 1-8 in a sequence table.
2. The method for acquiring the series of molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F as claimed in claim 1, which is characterized in that: the acquisition method comprises the following steps:
selecting test materials: selecting a test material;
preliminary positioning of the muskmelon powdery mildew resistance gene Pm-2F:
performing seedling-stage disease-resistant inoculation identification on the test material to obtain an infection-resistant pond material; extracting the genome DNA of the anti-influenza pool material by using a group mixed analysis method to construct an anti-influenza DNA pool;
screening SSR markers in the cucumber high-density genetic map through PCR amplification reaction to obtain SSR markers linked with target characters;
performing genetic linkage analysis on the SSR marker linked with the target character, and preliminarily positioning a target gene Pm-2F;
obtaining SNP/Indel sites linked with target characters:
designing a primer in the Pm-2F gene primary positioning interval, carrying out PCR amplification reaction on a muskmelon anti-infection parent and the anti-infection DNA pool, and obtaining the SNP/Indel locus linked with the target character through sequence comparison after sequencing;
the method comprises the following steps of (1) obtaining a molecular marker closely linked with a melon powdery mildew resistance gene Pm-2F:
and respectively designing CAPS/Indel markers aiming at the SNP/Indel sites linked with the target traits, and verifying the candidate SNP/Indel sites in a verification material to obtain molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F.
3. The method for acquiring the series of molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F as claimed in claim 2, which is characterized in that:
in the preliminary positioning step of the melon powdery mildew resistance gene Pm-2F, the SSR marker linked with the target character has a sequence shown in SEQ ID NO: 9-14, wherein:
SEQ ID NO. 9 in the sequence table is a nucleotide base sequence of primer SSR02733 for specific amplification of a disease-resistant material;
SEQ ID NO. 10 in the sequence table is a nucleotide base sequence of primer SSR02733 for specific amplification of a susceptible material;
SEQ ID NO. 11 in the sequence table is a nucleotide base sequence of primer SSR02734 for specific amplification of a disease-resistant material;
SEQ ID NO. 12 in the sequence table is a nucleotide base sequence of the primer SSR02734 for specific amplification of the susceptible material.
SEQ ID NO. 13 in the sequence table is a nucleotide base sequence of a primer CS27 for specific amplification of a disease-resistant material;
SEQ ID NO. 14 in the sequence list is a nucleotide base sequence of the primer CS27 for the specific amplification of the susceptible material.
Preferably, in the preliminary positioning step of the melon powdery mildew resistance gene Pm-2F, the nucleotide base sequence of the primer for PCR amplification of the SSR marker is as follows:
SSR primer SSR 02733:
the sequence of the upstream primer is as follows: 5'-TTGTTAGGTAAGCCATGCCC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TTTGCCTGAGGAAGAATCTGA-3', respectively;
SSR primer SSR 02734:
the sequence of the upstream primer is as follows: 5'-TGTTGTTGGACCCCTTCAAT-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TGTCAAAGGAGGAGGTGGAG-3', respectively;
SSR primer CS 27:
the sequence of the upstream primer is as follows: 5'-GCTGAGTTATGGGGAAAGCA-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-ATGTTGTTGGACCCCTTCAA-3' are provided.
Preferably, in the preliminary localization step of the melon powdery mildew resistance gene Pm-2F, the PCR amplification reaction program for the SSR marker is as follows:
stage 1: pre-denaturation at 94 ℃ for 3 min; and (2) stage: circulating for 30 times at 94 ℃ for 20s, 55 ℃ for 20s and 72 ℃ for 30 s; and (3) stage: extending for 10min at 72 ℃; and (4) stage: keeping at 4 ℃.
4. The method for acquiring the series of molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F as claimed in claim 2, which is characterized in that:
in the step of obtaining the SNP/Indel site linked with the target character, the sequence specifically amplified by the disease-resistant material and the disease-sensitive material obtained by the PCR amplification reaction has at least one nucleotide base sequence in SEQ ID NO. 15-22 in a sequence table, wherein:
15 in the sequence table is a nucleotide base sequence of a primer Cum-1 for specific amplification of the disease-resistant material;
16 in the sequence table is a nucleotide base sequence of a primer Cum-1 for specific amplification of the susceptible material;
SEQ ID NO. 17 in the sequence table is a nucleotide base sequence of the primer Cum-2 for specific amplification of the disease-resistant material;
18 in the sequence table is a nucleotide base sequence of a primer Cum-2 for specific amplification of the susceptible material;
SEQ ID NO. 19 in the sequence table is a nucleotide base sequence of the primer Mel-1 for specific amplification of the disease-resistant material;
20 in the sequence table is a nucleotide base sequence of a primer Mel-1 for specific amplification of the susceptible material;
21 in the sequence table is a nucleotide base sequence of a primer Cum-3 for specific amplification of the disease-resistant material;
SEQ ID NO. 22 in the sequence table is a nucleotide base sequence of the primer Cum-3 for the specific amplification of the susceptible material.
Preferably, in the step of obtaining the SNP/Indel site linked to the target trait, the nucleotide base sequence of the primer for amplifying the sequence specifically amplified by the disease-resistant and disease-sensitive material is as follows:
cum-1 primer:
the sequence of the upstream primer is as follows: 5'-GTACAAGAGTTCTGCTGTGAAGGA-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TTGAATCTCATTTTTCTGTTGCAT-3', respectively;
cum-2 primer:
the sequence of the upstream primer is as follows: 5'-ATCTTCGAACATCATCTTTGAC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-ATCCCAAATCTCCAATATCG-3', respectively;
mel-1 primer:
the sequence of the upstream primer is as follows: 5'-CGAGTCTTCTTCTTCCAAATATCC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-GAAAGATTCATAGGAGAACTCGTCC-3', respectively;
cum-3 primer:
the sequence of the upstream primer is as follows: 5'-CACACAGTGGACAACAGTACAT-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-AAGATTGATTGACGATTGATTG-3' are provided.
5. The method for acquiring the series of molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F according to claim 2, which is characterized in that:
in the step of obtaining the series of markers closely linked with the melon powdery mildew resistance gene Pm-2F, the nucleotide base sequence of a primer for PCR amplification of the CAPS marker is as follows:
CAPS primer CAPS-Dde I:
the sequence of the upstream primer is as follows: 5'-GCCCAACCTTCAACTCGATA-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TTGAATCTCATTTTTCTGTTGCAT-3', respectively;
CAPS primer CAPS-Hph I:
the sequence of the upstream primer is as follows: 5'-CAGTAGGGACACACAACC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-TATGCCATACGCTAATGT-3' are provided.
6. The method for acquiring the series of molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F according to claim 5, which is characterized in that:
in the step of obtaining the molecular marker closely linked with the melon powdery mildew resistance gene Pm-2F, the PCR amplification reaction program used for the CAPS marker is as follows:
stage 1: pre-denaturation at 94 ℃ for 3 min; and (2) stage: circulating 30 times at 94 ℃ for 20s, Tm value for 20s and 72 ℃ for 30 s; and (3) stage: extending for 10min at 72 ℃; and (4) stage: keeping at 4 ℃; wherein CAPS marker Tm values are: CAPS-Dde I: 57 ℃, CAPS-Hph I: at 55 ℃.
7. The method for acquiring the series of molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F as claimed in claim 6, which is characterized in that:
in the step of obtaining the molecular marker closely linked with the melon powdery mildew resistance gene Pm-2F, the endonuclease marked with CAPS-Dde I is Dde I restriction endonuclease, and the endonuclease marked with CAPS-Hph I is Hph I restriction endonuclease;
in the step of obtaining the molecular marker closely linked with the melon powdery mildew resistance gene Pm-2F, the cleavage sites of the marker CAPS-Dde I are as follows: GCACT, the labeled CAPS-Hph I restriction site is: GGTGA;
in the step of obtaining the series markers closely linked with the melon powdery mildew resistance gene Pm-2F, the enzyme digestion reaction conditions are as follows:
stage 1: carrying out enzyme digestion at 37 ℃ for 12-16 h; and (2) stage: enzyme denaturation at 65 deg.C for 20 min; and (3) stage: keeping at 4 ℃.
8. The method for acquiring the molecular marker tightly linked with the melon powdery mildew resistance gene Pm-2F according to claim 2, which is characterized in that:
in the step of obtaining the molecular marker tightly linked with the melon powdery mildew resistance gene Pm-2F, the nucleotide base sequence of the Indel marker primer for PCR amplification is as follows:
indel primers Indel 01:
the sequence of the upstream primer is as follows: 5'-GTCCACAGCAATCCAAGCTC-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-CGAGTCTTCTTCTTCCAAATATCC-3' are provided.
Indel primers Indel 02:
the sequence of the upstream primer is as follows: 5'-GTGTGTGTGTGCAGAAATGGA-3' the flow of the air in the air conditioner,
the sequence of the downstream primer is as follows: 5'-ATGGAACAACCTCCATCTGC-3' are provided.
9. The method for acquiring the series of molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F according to claim 8, which is characterized in that:
in the step of obtaining the molecular marker closely linked with the melon powdery mildew resistance gene Pm-2F, the PCR amplification reaction program used for the Indel marker is as follows:
stage 1: pre-denaturation at 94 ℃ for 3 min; and (2) stage: circulating 30 times at 94 ℃ for 20s, Tm value for 20s and 72 ℃ for 30 s; and (3) stage: extending for 10min at 72 ℃; and (4) stage: keeping at 4 ℃; wherein, the Tm value of the molecular marker is as follows: indel 01: 58 ℃, Indel 02: and 57 ℃.
10. The method for acquiring the series of molecular markers closely linked with the melon powdery mildew resistance gene Pm-2F according to claim 2, which is characterized in that:
in the test material selecting step, the test material includes:
the anti-influenza parent: male parent K7-1, a typical japanese type melon material of high powdery mildew resistant physiological races p.xanthothii 2F; the female parent K7-2 is a typical high-susceptibility powdery mildew physiological race P.xanthii 2F Sinkiang Hami melon material;
f obtained by hybridization of the resistant parent1Generation group;
with said F1F obtained by inbreeding of generations2And (4) separating the populations.
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| CN113881801A (en) * | 2021-10-29 | 2022-01-04 | 东北农业大学 | Molecular marker composition with tightly linked dark green stem characters of American pumpkin and application thereof |
| CN113881801B (en) * | 2021-10-29 | 2023-08-11 | 东北农业大学 | Molecular marker composition closely linked with deep green stem traits of cucurbita moschata and application of molecular marker composition |
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