CN107164481B - Powdery mildew resistance related SSR (simple sequence repeat) marker for muskmelon and application of powdery mildew resistance related SSR marker - Google Patents
Powdery mildew resistance related SSR (simple sequence repeat) marker for muskmelon and application of powdery mildew resistance related SSR marker Download PDFInfo
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Abstract
The invention discloses a melon powdery mildew resistance related SSR marker and application thereof, and relates to the field of biotechnology-assisted breeding. SSR markers related to melon powdery mildew resistance have the following sequences: SSR168167-F/SSR168167-R GTCTTCATTGCCTTCCTCGTC/CATTCGTATTCTTACTCCCT. The SSR marker obtained by the method can be used for screening the powdery mildew resistance of the melon material at any stage, has the advantages of high efficiency, accuracy and less limitation, and can accelerate the breeding of powdery mildew resistant melon varieties and shorten the breeding process.
Description
Technical Field
The invention relates to biotechnology-assisted breeding, a melon powdery mildew resistance related SSR marker and application thereof in melon seed and resource selection.
Background
The Cucumis melo (Cucumis melo L.) belongs to cucurbitaceae Cucumis, is an annual viniferous herbaceous plant, and the chromosome 2n =2x =24, so that the economic benefit is good, the planting area is increased year by year, and the sowing area of the Cucumis melo in China is the first in the world. Melon powdery mildew is a worldwide disease in production and has a very serious influence on the yield and quality of melons. Therefore, the cultivation and popularization of disease-resistant varieties are the most economic and effective measures for preventing and treating powdery mildew of melons. The development of molecular markers linked with the powdery mildew resistance gene of the melon is of great significance.
Recent researches show that the powdery mildew of melons in China is caused by P.xanthohii, and dominant races in most areas are P.xanthohii Race1 and P.xanthohii Race 2F. The research of Xuzhihao et al (1999) considers that the main pathogenic bacteria of powdery mildew of melons in Zhejiang area is P.xanthii physiological race 2; the research of Wang Juan et al (2006) considers that P.xanthii Race1 and P.xanthii Race 2F are mainly caused in Beijing area, and the dominant Race is P.xanthii Race 2F; research on baohai et al (2008) showed that the dominant physiological race of melon powdery mildew in the southern hai area is p.xanthii 2F; studies in wayama et al (2010) suggest that the dominant physiological Race in the shanghai region is p.xanthii Race1, which is found only in the new region of purdong; the research of Bingfeng et al (2010) considers that the main pathogenic microspecies of powdery mildew of melons in Shaanxi province are P.xanthii Race 2F; researches on Mahong Yan et al (2011) show that the melon powdery mildew pathogens existing in Heilongjiang province have P.xanthii Race1 and P.xanthii Race6, and the dominant Race is P.xanthii Race1; studies of Zhangli et al (2011) show that pathogenic bacteria of powdery mildew of melons in Gansu province include P.xanthii Race1 and P.xanthii Race 2F, and dominant species are P.xanthii Race1; the studies of zhangbo et al (2011) indicate that p.xanthii Race1 and p.xanthii Race 2F are the mainstream of p.xanthii Race 2F; studies of Surui (2013) consider that the dominant Race of powdery mildew of melons in northern Xinjiang is P.xanthii Race1; the study of plum apple aroma and the like (2015) shows that the dominant species of powdery mildew of melons in Jianghu and Zhejiang Shanghai is P.xanthii Race1.
The research on the resistance gene of the melon powdery mildew starts earlier, the pathogenic races of the melon powdery mildew are different, the corresponding resistance genes are different, and the resistance genes contained in different disease-resistant materials are possibly different. The P merin et al (2002) has the powdery mildew resistance gene Pm-x positioned in the II linkage group, the Pm-W gene positioned in the V linkage group, and the Pm-y gene positioned in the XII linkage group. Perchepied et al (2005) found two genes located in the II th and V th linkage groups, respectively. Dogiment et al (2008) located the powdery mildew resistance gene Pm-W on the V linkage group, and verified the research results of Berin et al (2002); meanwhile, the gene and an aphid-resistant gene Vat allele are found. Fukino et al (2008) found a site of disease resistance in each of the II and XII linkage groups. The Teixeira et al (2008) study showed that the resistance gene Pm-1 contained is located in the IX linkage group. Yuste-Lisbona et al (2011) mapped powdery mildew QTL to the V th linkage group. Wang et al (2011) mapped the powdery mildew resistance gene between the two co-dominant molecular markers AF and CA in the V-th linkage group. Yan Weili (2014) located the powdery mildew resistance gene on melon linkage group II. Ning et al (2014) mapped the melon powdery mildew resistance gene on the II, V linkage group. Chenjing (2015) now that the resistance of the melon WMR-29 line to powdery mildew P.xanthii race1 is controlled by two dominant disease-resistant genes, located on LGII, LGXII linkage groups, respectively. Qizheng (2015) located powdery mildew resistance gene in melon RE-33 at position of chromosome 2, 135976bp-1418829 bp. Blumea et al (2016) mapped 1 melon powdery mildew resistance-related gene site on linkage group VII. Wang et al (2016) mapped the melon powdery mildew resistance gene on chromosome 2.
Disclosure of Invention
The invention provides a melon powdery mildew resistance related SSR marker, and provides an application of the marker in selecting melon germplasm resources with resistance to powdery mildew, lays a foundation for fine positioning and cloning of powdery mildew resistance genes of melons, can be used for screening powdery mildew resistance of melons at any stage of melon materials, has the advantages of high efficiency, accuracy and less limitation, can accelerate breeding of powdery mildew resistance melon varieties, and accelerates a breeding process.
The technical scheme provided by the invention is as follows: a primer pair for detecting a molecular marker for melon powdery mildew resistance comprises the following sequences:
SSR168167-F：GTCTTCATTGCCTTCCTCGTC
SSR168167-R：CATTCGTATTCTTACTCCCT。
the invention also provides application of the primer pair in screening melon powdery mildew resistance germplasm resources, which comprises the following steps:
(1) The primers are adopted to carry out PCR amplification on the genome DNA of the melon variety to be selected respectively,
(2) Carrying out non-denaturing polyacrylamide gel electrophoresis detection on the amplification result;
(3) And selecting a material with amplified anti-powdery mildew characteristic bands from the detection result.
In the above application, the PCR reaction system is 7.5ng of DNA, 50ng of each of the forward and reverse primers, 5. Mu.L of Go
Green Master Mix, add double distilled water to 10ul.
The application, the PCR amplification program is: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 15 seconds, annealing at 55 ℃ for 15 seconds, extension at 72 ℃ for 30 seconds, 35 cycles; keeping the temperature at 72 ℃ for 5 minutes, and storing at 4 ℃.
The application comprises the following steps of: 8% non-denatured polyacrylamide gel is adopted, 1.5ul 750bp Marker (product of Genstar company) is used for spotting, electrophoresis separation is carried out for 75min at constant power of 150V, and finally silver staining is carried out for color development.
The invention also provides a method for identifying powdery mildew resistance of melons by using the primer pair, which comprises the following steps:
(1) The primers are adopted to carry out PCR amplification on the genome DNA of the melon variety to be detected,
(2) Carrying out non-denaturing polyacrylamide gel electrophoresis detection on the amplification result;
(3) And determining the resistance of the melon variety to be detected to the powdery mildew according to the existence of the amplified strips.
The invention aims to cause the disease of '25113' (P) 1 ) "25117" (P) for anti-powdery mildew 2 ) Constructing six generation population for parent and identifying host of melon powdery mildew pathogenic bacteria for phenotype identification, and using powdery mildew susceptible '11016' (P) 1 ) "25117" (P) for anti-powdery mildew 2 ) Construction of F for parents 2 And the population and the RIL population are planted with melon powdery mildew pathogenic bacteria identification hosts at the same time for phenotype identification. DNA of each population was extracted, and polymorphic markers were selected by the BSA method. And applying the screened markers to each group for verification, and comparing the genotype identification result with the field identification result to obtain the linked markers with higher accuracy.
To induce powdery mildew, "25113" (P) 1 ) "25117" (P) for anti-powdery mildew 2 ) Constructing six generation population as material for parent, and marking at F 2 The accuracy in the population was 98.2%, in B 1 The accuracy in the population was 96.7%, in B 2 The accuracy in the population was 99.9%; and "11016" (P) infected with powdery mildew 1 ) "25117" (P) for anti-powdery mildew 2 ) Construction of F for parents 2 The population and RIL population are verified as materials and marked at F 2 The accuracy in the population was 98.1%, and the accuracy in the RIL population was 98.1%. The accuracy of the marking was 82.1% as verified by 28 other materials which had no relationship to the test material.
The invention not only lays a foundation for the fine positioning and cloning of the powdery mildew resistance gene of the melon, but also provides a theoretical basis for the molecular marker-assisted breeding of a new powdery mildew resistance melon variety.
Drawings
FIG. 1 is a schematic representation of a graded field survey of powdery mildew onset of melon;
FIG. 2 shows that SSR marker SSR168167 is '25113' (P) for melon powdery mildew 1 ) "25117" (P) for anti-powdery mildew 2 ) And F 1 The detection result of the generation;
FIG. 3 is "11016" (P ') of SSR marker SSR168167 to powdery mildew of melon' 1 ) "25117" (P) for anti-powdery mildew 2 ) And the detection results of 30 disease-resistant single-plant DNA mixed pools (R) and 30 susceptible single-plant DNA mixed pools (S);
FIG. 4 shows SSR marker SSR168167 on melon "25113" (P) 1 ) "25117" (P) for anti-powdery mildew 2 ) Constructed of F 2 Detecting the individual plants in the population part;
FIG. 5 is SSR marker SSR168167 to melon "11016" (P' 1 ) "25117" (P) for anti-powdery mildew 2 ) Constructed of F 2 Detecting the single plants of the population part;
FIG. 6 shows SSR marker SSR168167 to melon "11016" (P' 1 ) And the detection result of a part of individual plants of the RIL7 group constructed by the anti-powdery mildew 25117 (P2).
Detailed Description
The melon parents of 25113, 25117 and 11016 are stored in the laboratory, and are guaranteed to be released to the public for verification experiments within twenty years from the application date. 20113 is thick-skinned melon with white color, bright skin, yellow color, and orange flesh; "25117" is powdery mildew-resistant muskmelon with thick peel, and its fruit has reticulate pattern, black green peel, and orange red flesh; 11016 is a thick-skinned melon susceptible to powdery mildew, with the fruits having reticulate pattern, grayish green skin, green yellow flesh.
SSR primers from the subject group of the inventor are designed by self according to the EST-SSR sequence of muskmelon by using Primer5.0 software to obtain 61 pairs (the sequences of the primers are detailed in the following table).
PCR experiment Using ShanGhai PromeGa
Green Master Mix; as the gel, a 40% non-denatured polyacrylamide gel from GenStar company was used after diluting it to 8%, and a 750bp Marker (Genstar company) was used for spotting.
Example 1 screening of melon SSR markers associated with powdery mildew resistance
Step 1, field identification of melon powdery mildew resistance
The test was carried out in 2016 at the experimental base of vegetable and flower institute of Chinese academy of agricultural sciences, and the melon was infected with "25113" (P) of powdery mildew 1 ) "25117" (P) for anti-powdery mildew 2 ) Obtaining F for parents 1 、F 2 And B 1 、B 2 A population;
p is planted in 2016 spring in vegetable and flower institute experimental greenhouse of Chinese academy of agricultural science 1 、P 2 And F 1 60 strains each, F 2 320 strains, B 1 100 strains, B 2 100 plants and 30 plants of melon powdery mildew identification hosts respectively.
The powdery mildew pathogenic bacteria are inoculated after two leaves and one heart, and the inoculation method comprises the following steps: the pathogenic bacteria spore suspension is evenly sprayed on the leaf surface. The disease condition of each individual plant of the six-generation population is investigated after about 15 days of inoculation, and the disease grade investigation is divided into 0-5 grades:
(the field disease survey is shown in FIG. 1)
The preparation method of the pathogenic bacteria spore suspension comprises the following steps: brush conidia of pathogenic bacteria on diseased leaves into 0.1% Tween 80 solution to prepare spore suspension, uniformly spray on leaves with spray can, and inoculate with minimum concentration requirement of 10 5 Each/ml.
In the six-generation population planted, the powdery mildew resistance expression is investigated, disease progression in 3 or above is marked as susceptible disease, and segregation ratio is calculated. Statistical data analysis was performed using Microsoft Excel 2012 software and results were carte tested using SAS 9.2.
The results show that: f 1 All appear to be anti-powdery mildew; at F 2 In the population, the separation ratio of disease-resistant plants to disease-susceptible plants is in accordance with 3 by chi-square test, the backcross population taking disease-resistant '25117' as a backcross parent is all expressed as powdery mildew resistance, and in the backcross population obtained by backcrossing with another disease-susceptible parent, the separation ratio of the number of the disease-resistant plants to the disease-susceptible plants is in accordance with 1 by chi-square test. From this, it is clear that the resistance to powdery mildew is controlled by 1 pair of nuclear genes, and the disease resistance is dominant against susceptible diseases.
Step 2. Determination of physiological races of powdery mildew pathogenic bacteria
According to the anti-infection condition of the host, the physiological race of the pathogenic bacteria inoculated in the season is identified by comparing the identification standard of the pathogenic race of the powdery mildew:
calculating the disease index RI:
The germ plasmavirulence disease resistance grade was determined according to the average disease grade and the following instructions.
| 1 | High Resistance (HR) (RI < 1.0) |
| 3 | Anti (R) (RI less than or equal to 1.0 and less than 2.0) |
| 5 | Moderate Resistance (MR) (RI < 3.0 > 2.0 ≤) |
| 7 | Feeling (S) (RI < 4.0 > 3.0 ≤) |
| 9 | High Sensitivity (HS) (RI is more than or equal to 4.0) |
(S means infection, R means disease resistance, ND means currently unknown, H means heterozygosis)
The results show that: 2016 spring inoculated powdery mildew 2016 spring physiological races for powdery mildew P.xanthii race1, autumn physiological races for powdery mildew P.xanthii race 2F.
Step 3.DNA extraction and SSR analysis
The method is characterized in that young leaves of melon plants are taken, and genome DNA of parents and each individual plant of each group is extracted by using an improved CTAB (cetyl trimethyl ammonium bromide) method.
The PCR reaction system was 10. Mu.L of total reaction system, 3. Mu.L of DNA (2.5 ng. Mu.L-1), 1. Mu.L of each of forward and reverse primers (50 ng. Mu.L-1), and 5. Mu.L of Go
Green Master Mix (product of Promega). The PCR amplification procedure was: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 30s,35 cycles; keeping the temperature at 72 ℃ for 5min, and keeping the temperature at 4 ℃ for forever. The amplified products were separated with 8% non-denaturing polyacrylamide gel, 1.5ul 750bp Marker (Genstar) was used for spotting, electrophoresis buffer was 0.5 XTBE, separation was performed for 75min at 150V constant power, silver staining was developed after electrophoresis, and banding patterns were counted.
Step 4.SSR marker screening and data statistics
Mainly comprises 4 steps: (1) Screening at P 1 、P 2 SSR markers which show polymorphism in the world; (2) At F 2 Selecting 30 DNAs of single plants resistant to powdery mildew and susceptible to powdery mildew respectively in the population, constructing 2 near-isogenic pools resistant to powdery mildew and susceptible to powdery mildew according to a BSA (bovine serum albumin) method, and screening polymorphic primers; (3) Analysis of F Using the obtained polymorphic primers 1 Population and F 2 Group B 1 、B 2 The genotype of each individual plant of the population; (4) And comparing the result of the genotype identification with the result of the phenotype identification, and calculating the accuracy of the SSR marker identification. (5) According to the identification result, the molecular marker with high accuracy is obtained.
Statistical methods for dominant markers: individuals with the same maternal banding pattern are designated as a, individuals with the same paternal banding pattern are designated as c, and individuals with no amplification or ambiguity are designated as u.
Using 61 pairs of primers (the sequences of the primers are detailed in table 1), performing polymorphism screening between parent and parent (the screening result of SSR168167 marker is shown in figure 2), and screening 31 markers with polymorphism, wherein the polymorphism rate is 50.8%. And further screening by combining a BSA method to obtain 6 markers (the screening result of the SSR168167 marker is shown in figure 3).
F of the selected combination of polymorphic marker pairs "25113" x "25117 1 Population and F 2 Group B 1 、B 2 And analyzing the genotypes of the individual plants in the population (the identification result of part of the individual plants is shown in figure 4), and combining with the survey character data to obtain the SSR marker SSR168167 with the highest accuracy in each population. SSR168167 primer sequences are as follows:
SSR168167-F：GTCTTCATTGCCTTCCTCGTC；
SSR168167-R：CATTCGTATTCTTACTCCCT。
example 2 validation of SSR markers related to powdery mildew resistance of melon
Another powdery mildew material "11016" (P' 1 ) With "25117" (P) for powdery mildew resistance 2 ) Obtaining F 'from parent' 1 、F’ 2 And each individual plant of the RIL population and the population in which the melon powdery mildew resistance related SSR marker SSR168167 is positioned is verified to determine that the marker is used for molecular marker-assisted selectionThe accuracy of selection: the mark is at F 2 The accuracy in the population was 98.1% (see FIG. 5 for some individual identifications) and 98.1% (see FIG. 6 for some individual identifications) in the RIL population.
Further verification was made using 28 parts of the test material collected in this subject, which showed no relationship with the disease resistance, and 15 parts of the material showing disease susceptibility, among the 28 parts. The calculated accuracy was 82.1% when the phenotypic data reflecting the banding pattern of 23 of the total 28 materials marked as compared to the field phenotype of the selected material was consistent with the field survey results, with 12 of the diseased materials amplifying the disease genotype bands.
The SSR markers related to powdery mildew resistance of the melons are obtained in the research, so that a foundation is laid for fine positioning and cloning of powdery mildew resistance genes of the melons, and a theoretical basis is provided for the molecular marker-assisted breeding of new powdery mildew resistance melon varieties.
Claims (6)
1. The primer pair for detecting the molecular marker for the melon powdery mildew resistance is characterized by comprising the following sequences:
SSR168167-F ：GTCTTCATTGCCTTCCTCGTC
SSR168167-R ：CATTCGTATTCTTACTCCCT。
2. the application of the primer pair of claim 1 in screening powdery mildew resistant germplasm resources of melons, which comprises the following steps:
(1) The primers are adopted to carry out PCR amplification on the genome DNA of the melon variety to be selected respectively,
(2) Carrying out non-denaturing polyacrylamide gel electrophoresis detection on the amplification result;
(3) And selecting a material for amplifying the characteristic strip of the powdery mildew resistance from the detection result.
3. The use of claim 2, wherein the PCR reaction system is 7.5ng DNA, 50ng each of the forward and reverse primers, 5. Mu.L of Go Taq Green Master Mix, and 10. Mu.L of double distilled water.
4. The use according to claim 2, wherein the PCR amplification procedure is: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 15 seconds, annealing at 55 ℃ for 15 seconds, extension at 72 ℃ for 30 seconds, 35 cycles; keeping the temperature at 72 ℃ for 5 minutes, and storing at 4 ℃.
5. Use according to any one of claims 2 to 4, characterized in that: and (3) detecting by gel electrophoresis: 8% non-denatured polyacrylamide gel is adopted, 1.5ul 750bp Marker is used for spotting, electrophoresis separation is carried out for 75min at 150V constant power, and finally silver staining is carried out for color development.
6. The method for identifying the powdery mildew resistance of the melon by using the primer pair as claimed in claim 1, which comprises the following steps:
(1) The primers are adopted to carry out PCR amplification on the genome DNA of the melon variety to be detected,
(2) Carrying out non-denaturing polyacrylamide gel electrophoresis detection on the amplification result;
(3) And determining the resistance of the melon variety to be detected to the powdery mildew according to the existence of the amplified strips.
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