CN113151549B - Molecular marker closely linked with peanut aspergillus flavus resistance major site qAFTA07 and application thereof - Google Patents
Molecular marker closely linked with peanut aspergillus flavus resistance major site qAFTA07 and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular markers, in particular to a molecular marker closely linked with a peanut aspergillus flavus resistance major effective site qAFTA07 and application thereof. The molecular marker of the invention is AFT-1; the molecular marker AFT-1 is linked with a QTL locus qAFTA07, and the molecular marker AFT-1 can be obtained by amplifying a primer pair shown as SEQ ID NO. 1-2. The molecular marker AFT-1 linked with the major QTL site qAFTA07 for the resistance of peanut aspergillus flavus, which is obtained by the invention, can assist in selecting materials with high resistance to aspergillus flavus in peanut breeding, is favorable for promoting the breeding of peanut varieties resistant to aspergillus flavus and improving the breeding efficiency.
Description
Technical Field
The invention relates to the technical field of molecular markers, in particular to a molecular marker closely linked with a peanut aspergillus flavus resistance major effective site qAFTA07 and application thereof.
Background
Peanuts (Arachis Hypogaea L.) play an important role in the aspects of oil supply guarantee, increase of income of growers, adjustment of planting structures and the like as important oil crops and economic crops. With the rapid development of the peanut industry, the aflatoxin pollution problem is increasingly prominent. Not only restricts the development of the peanut industry, but also poses serious threat to the health of consumers. Aflatoxins are a class of microbial toxins produced by aspergillus flavus and aspergillus parasiticus that have very strong toxicity and carcinogenicity. A plurality of factors inducing aflatoxin pollution exist in the peanuts in various links of planting, harvesting, processing, transporting and storing. The method for cultivating and planting the disease-resistant peanut variety is the most direct and effective method for relieving the aflatoxin pollution problem of the peanuts.
Aspergillus flavus resistance is a complex quantitative genetic trait whose phenotype is greatly influenced by environmental factors. In order to eliminate the influence of environmental factors, the identification of the resistance of the peanut aspergillus flavus is mostly carried out by adopting a laboratory artificial inoculation method, but the artificial inoculation identification process is very complicated and has higher requirements on experimental conditions. The current Aspergillus flavus resistance breeding work progresses very slowly due to the difficulty of phenotype identification and screening. With the development of molecular biology and genetics, a genetic linkage map is drawn by developing molecular markers at the whole genome level, and a QTL (quantitative trait locus) for controlling a target agronomic trait can be positioned on a genome by utilizing a linkage analysis method in combination with phenotypic data. Phenotypic traits are selected by markers closely linked to QTLs, which can improve the breeding efficiency of target traits. However, the related QTL of the peanut aspergillus flavus resistance is reported recently, and the progress of molecular marker-assisted selection of the aspergillus flavus resistant peanut variety is limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the QTL locus qAFTA07 with stable main effect of peanut aspergillus flavus resistance and a linked marker thereof, which are used for molecular marker-assisted selection of an anti-aspergillus flavus peanut material.
Specifically, the invention provides the following technical scheme:
in a first aspect, the invention provides a molecular marker closely linked with a peanut aspergillus flavus resistance major effective site, which is characterized in that the molecular marker is AFT-1; the molecular marker AFT-1 is linked with a QTL locus qAFTA07, and the molecular marker AFT-1 can be obtained by amplifying a primer pair shown as SEQ ID NO. 1-2.
In a second aspect, the present invention provides primers for amplifying the above molecular markers.
The primer comprises a sequence shown as SEQ ID NO. 1-2.
In a third aspect, the present invention provides a reagent or a kit comprising the above primer.
In a fourth aspect, the present invention provides any one of the following uses of the above molecular marker or primer or reagent or kit:
(1) the application in identifying the resistance trait phenotype of peanut aspergillus flavus;
(2) the application in peanut germplasm resource identification, improvement or molecular marker-assisted breeding;
(3) the application in early prediction of peanut aspergillus flavus resistance characters;
(4) the application in screening or creating peanut materials with different Aspergillus flavus resistance traits;
(5) application in peanut aspergillus flavus resistance genotyping.
In a fifth aspect, the present invention provides a method for identifying a peanut aspergillus flavus resistance trait phenotype, comprising:
(1) extracting the genomic DNA of the peanut to be identified;
(2) taking genome DNA as a template, and carrying out PCR amplification by using a primer shown in SEQ ID NO. 1-2;
(3) and judging the resistance trait phenotype of the peanut aspergillus flavus to be identified according to the size of the DNA fragment in the PCR amplification product.
The method for judging the resistance trait phenotype of the peanut aspergillus flavus to be identified in the step (3) comprises the following steps:
and after PCR amplification is carried out by using the primer shown in SEQ ID NO.1-2, judging that the peanut to be identified has high aspergillus flavus resistance when the size of the DNA fragment in the product is 381 bp.
The invention has the beneficial effects that:
the molecular marker AFT-1 linked with the major QTL site qAFTA07 for the resistance of peanut aspergillus flavus, which is obtained by the invention, can assist in selecting materials with high resistance to aspergillus flavus in peanut breeding, is favorable for promoting the breeding of peanut varieties resistant to aspergillus flavus and improving the breeding efficiency.
Drawings
FIG. 1 shows the position and effect value of major QTL site qAFTA07 for resistance to Aspergillus flavus of peanut, the abscissa represents the position of linkage group A07 of genetic linkage map, the ordinate represents LOD effect value of QTL, 2016ENV, 2017ENV and 2018ENV represent the environment of 2016, 2017 and 2018 respectively.
FIG. 2 shows the sequence information obtained by amplifying Xuhua No. 13 and Zhonghua No. 6 with the primer set shown in SEQ ID NO. 1-2.
FIG. 3 is a histogram of aflatoxin content of Xuhua No. 13 XZhonghua No. 6 RIL population extreme resistant and Susceptible materials, the abscissa is the material number, the left light color column in the graph is extreme resistant material (Resistance bulk), the right dark color column is extreme Susceptible material (Susceptible bulk), the last two columns are parent materials (Parents), and the ordinate represents aflatoxin concentration.
FIG. 4 is an agarose gel electrophoresis of the amplification products of the parent and extreme resistance and sensitivity materials using the primer set shown in SEQ ID NO.1-2, lanes 1-11 are Xuhua 13 and the extreme sensitivity materials, lanes 12 are DL2000 Marker, and lanes 13-23 are Zhonghua No. 6 and the extreme resistance materials.
FIG. 5 is an agarose gel electrophoresis of the amplification product of 99 shares of Chinese peanut micro-core germplasm materials using the primer pair shown in SEQ ID NO.1-2, and the numbers above the amplification bands are the numbers of the materials corresponding to the respective lanes.
Fig. 6 shows the aflatoxin content phenotypic distribution of 99 peanut micro-core germplasm materials in a 3-year environment, wherein horizontal coordinates ENV2016, ENV2017 and ENV2018 represent the environments of 2016, 2017 and 2018 respectively, and the vertical coordinate is the aflatoxin concentration.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Hybridizing the disease-susceptible slowly-flowering peanut 13 serving as a female parent and the disease-resistant medium-flowering 6 serving as a male parent, and constructing a Recombinant Inbred Line (RIL) population F by a single-seed-transmission method7(2016) -F9Generation (2018). Previous laboratories have constructed a high-density genetic linkage map containing 817 loci and 1756.48cM total length based on SSR markers, and the average distance between markers of the map is 2.1 cM. The parents and RIL groups (187 parts) were planted in the Yanghuo test base of the oil crop research institute of Chinese academy of agricultural sciences in 2016-2018. Two replicates were designed using a fully randomized block experiment. Each strain is repeatedly planted with 12 single plants in 1 row, the row spacing is 30 cm, and the plant spacing is 20 cm. The normal 8 single-plant mature pods were harvested by standard field management. Approximately 60 full mature non-diseased-spot seeds were selected for each line. After artificially inoculating an aspergillus flavus strain AF2202 to peanut seeds, drying and grinding the peanut seeds, extracting aflatoxin by using a 55% methanol solution, and detecting the aflatoxin content in the seeds by adopting an HPLC method. After the phenotype data are obtained, the independent sample T test is utilized to perform differential analysis on the aflatoxin content of the parents, and the significant difference of the aflatoxin content between the parents is found. The toxin content of No. 6 flower in the disease-resistant parent is always lower than that of Xuhua 13 of the susceptible parent. The phenotype of the RIL population is analyzed, and the aflatoxin content is found to be distributed in the population, which shows that the aflatoxin resistance of the peanut seeds is a quantitative character controlled by multiple genes. Performing linkage analysis of Aspergillus flavus resistance by using composite interval mapping method of Windows QTL Cartographer 2.5 software to identify a peanut seedAspergillus flavus resistance QTL qAFTA07, which is repeatedly detected under 2016 (see FIG. 1), 2017 (see FIG. 7) and 2018 (see FIG. 8), has LOD values of 12.31, 7.81 and 6.00 respectively, and contribution rates of 25.74%, 15.88% and 11.88% respectively. The QTL contribution rate detected in 3 environments is more than 10%, and the QTL qAFTA07 is considered as a main QTL site for controlling the resistance of peanut seeds to aspergillus flavus. The main effect QTL interval contains 5 SSR markers, the genetic distance is 10.32cM, and the corresponding physical interval distance is 3.6 Mb. Combining the QTL positioning result, designing a pair of molecular marker primers AFT-1F according to a reference genome sequence: 5'-CTGCGGATGCCAGATCTTGTA-3' (SEQ ID NO. 1); AFT-1R: 5'-AGGAGTTAACCTTGCAGAGGAT-3' (SEQ ID NO. 2). The fragments amplified by the pair of primers are closely linked with a target QTL, the PCR product of the primers is 435bp in Xuhua 13 of a susceptible parent and 381bp in No. 6 of a disease-resistant parent, and the sequence of the product amplified by the two parents is shown in figure 2. The PCR amplification conditions were: denaturation at 94 ℃ for 3 min; 30 seconds at 94 ℃, 30 seconds at 60 ℃, 60 seconds at 72 ℃ and 32 cycles; 5 minutes at 72 ℃.
Example 2
All 187 materials were ranked according to their aflatoxin content phenotype (3 replicates per material) for the xuhua 13 × zhonghua 6 RIL population material of example 1, and 10 extremely high-sensory and extremely high-resistant materials were found, with the distribution of the toxin content phenotype shown in fig. 3, and the T-test results showed significant differences in the mean values of the toxin content between the two groups of materials. Amplifying the 20 extreme materials by using a primer pair shown in SEQ ID NO.1-2, wherein the result is shown in figure 4, the genotypes of all the susceptible materials are consistent with those of the susceptible parents Xuhua 13 to obtain a 435bp product fragment, and the genotypes of the disease-resistant materials are consistent with those of the disease-resistant parents Mihua 6 to obtain a 381bp product fragment. The molecular marker of the invention can effectively screen the Aspergillus flavus resistant and susceptible peanut material.
Example 3
PCR amplification is carried out on 99 parts of Chinese peanut micro-core germplasm materials by using a primer pair shown in SEQ ID NO.1-2, the result is shown in figure 5, the genotypes of the other 96 parts of materials without amplification bands of No. 51 and No. 76 in the 99 parts of materials are consistent with that of diseased parent Xuhua 13, and a 435bp product fragment is obtained; only lane 9 material genotype is consistent with that of the disease-resistant parent, No. 6, and 381bp product fragment is obtained. As shown in FIG. 6, the aflatoxin content (67.32 +/-9.12 mu g/g) of the No. 9 material is remarkably lower than the average value (180.60 +/-109.90 mu g/g) of the rest 98 materials by the aflatoxin resistance phenotype of 2016 (3 replicates per material) of 99 materials among 3 environments in 2018, which indicates that the aflatoxin resistance is better.
The specific 99 core germplasm names and corresponding lanes are shown in table 1.
TABLE 199 accession numbers for micronucleus materials and their corresponding aflatoxin concentrations
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> institute of oil crop of academy of agricultural sciences of China
<120> molecular marker closely linked with peanut aspergillus flavus resistance major effective site qAFTA07 and application thereof
<130> KHP211112612.7
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<170> SIPOSequenceListing 1.0
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ctgcggatgc cagatcttgt a 21
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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aggagttaac cttgcagagg at 22
Claims (6)
1. The molecular marker is closely linked with the peanut aspergillus flavus resistance major effective site and is characterized in that the molecular marker is AFT-1; molecular marker AFT-1 and QTL locusqAFTA07Linkage, wherein the molecular marker AFT-1 can be obtained by amplifying a primer pair shown as SEQ ID NO.1-2, and the reference genome is peanut; and after PCR amplification is carried out by using the primer shown in SEQ ID NO.1-2, judging that the peanut to be identified has high aspergillus flavus resistance when the size of the DNA fragment in the product is 381 bp.
2. The primer for amplifying the molecular marker of claim 1, which comprises a sequence shown as SEQ ID No. 1-2.
3. A reagent or kit comprising the primer of claim 2.
4. Use of any one of the molecular marker of claim 1 or the primer of claim 2 or the reagent or kit of claim 3:
(1) the application in identifying the resistance trait phenotype of peanut aspergillus flavus;
(2) the application in peanut germplasm resource identification, improvement or molecular marker-assisted breeding;
(3) the application in early prediction of peanut aspergillus flavus resistance characters;
(4) the application in screening or creating peanut materials with different Aspergillus flavus resistance traits;
(5) application in peanut aspergillus flavus resistance genotyping.
5. The method for identifying the resistance trait phenotype of peanut aspergillus flavus is characterized by comprising the following steps:
(1) extracting the genomic DNA of the peanut to be identified;
(2) taking genome DNA as a template, and carrying out PCR amplification by using a primer shown in SEQ ID NO. 1-2;
(3) and judging the resistance trait phenotype of the peanut aspergillus flavus to be identified according to the size of the DNA fragment in the PCR amplification product.
6. The method as claimed in claim 5, wherein the method for judging the peanut aspergillus flavus resistance trait phenotype to be identified in the step (3) is as follows:
and after PCR amplification is carried out by using the primer shown in SEQ ID NO.1-2, judging that the peanut to be identified has high aspergillus flavus resistance when the size of the DNA fragment in the product is 381 bp.
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| US7449617B2 (en) * | 2004-01-05 | 2008-11-11 | The Regents Of The University Of California | Plants with elevated levels of gallic acid/polyphenol oxidase and methods of generating such plants |
| CN104328198A (en) * | 2014-11-14 | 2015-02-04 | 河南省农业科学院 | Primer for detecting mutant genotypes of FAD2A and FAD2B genetic loci of high oleic acid peanuts and PCR detection method of primer |
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| High‑density SNP map facilitates fine mapping of QTLs and candidate genes discovery for Aspergillus flavus resistance in peanut (Arachis hypogaea);Shahid Ali Khan等;《Theoretical and Applied Genetics》;20200413;全文 * |
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