CN110894540B - SNP chip for peanut variety identification, preparation method and application thereof - Google Patents
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Abstract
The invention discloses an SNP chip for identifying peanut varieties, which comprises 725 SNP molecular markers, wherein the numbers of the SNP molecular markers are SNP 001-SNP 725, and the nucleotide sequences are respectively shown as SEQ ID NO:1 to SEQ ID NO:725, respectively. The invention also provides a design and preparation method of the SNP chip, namely, the SNP marker contained in the chip detects SNP variation by analyzing peanut cultivar re-sequencing data; screening the SNP markers; designing a probe to manufacture the SNP chip. The invention also provides the application of the SNP chip in peanut variety identification. The 725 SNP markers designed by the invention provide molecular technical means for peanut cultivar variety identification, and further improve the peanut variety identification level in China.
Description
Technical Field
The invention relates to the field of molecular marker chips for plant variety identification, in particular to an SNP chip for peanut variety identification, a preparation method and application thereof.
Background
The peanut is one of the important oil crops in China and is of great importance to guarantee the safety of edible oil in China. The seeds are used as the source of crop production, and the fine varieties play an important role in improving the yield and the quality of crops. The development of variety identification of crops is an important basis for solving the variety disputes in the current seed market and granting the right to new varieties, and is also an effective means for guaranteeing the ordered competition of the seed industry in China.
At present, the peanut seed operation main bodies in China are large in number, the problems of counterfeit and inferior seeds, fake-brand infringement operation and the like exist, the seed market is relatively disordered, and the development of the peanut industry is seriously influenced. The peanut variety identification technology has gradually shown the effects of establishing a seed management system, striking the fake plate operation, judging the new plant variety protection and intellectual property disputes and maintaining the seed market order. At present, the peanut variety identification technology has no unified identification program and related file procedures. The peanut variety identification technology is mainly used for judging by observing some important morphological characters through traditional field planting. The peanuts have different botanical types, such as pearl bean type, common type, multi-grain type, dragon type and the like, belong to varieties of different botanical types, have obvious agronomic characters, but are difficult to distinguish among varieties belonging to the same botanical type. In a book of 'trait descriptors of peanuts' published by the international committee on plant genetic resources and the international research institute on semi-dry crops in 1992, only more than 30 morphological traits are provided for peanut germplasm identification. Therefore, accurate identification of peanut varieties is difficult only according to traditional agronomic traits.
With the development of molecular marker technology, crop variety identification enters the molecular identification level. At present, the most mature molecular marker identification method is SSR detection, and a perfect DNA fingerprint detection technical system and a variety identification database are established on rice. However, the method also has the defects of low detection flux, difficulty in realizing data integration and sharing and the like, and is difficult to adapt to the requirement of rapid identification of varieties in the future. In addition, due to high peanut genome homology, polymorphic SSR screening difficulty is high, coverage rate is low, and distribution is uneven, so that peanut variety identification by utilizing SSR markers is difficult. SNP is sequence polymorphism formed by single nucleotide variation on genome, is a detection technology which is considered to have the most research and development potential in peanut variety identification at present, and has the advantages of high throughput, large quantity, uniform distribution, easy realization of data integration comparison and the like compared with SSR markers.
In recent years, detection techniques based on SNP markers have become the focus of research at home and abroad. The companies such as Illumina, Affymetrix and the like successively provide different SNP genotyping high-throughput detection platforms, and provide good technical support for the research and development of an automatic, high-throughput and low-cost SNP chip detection technology. SNP molecular marker detection technology has also been widely applied to variety identification of crops such as rice, soybean, kidney bean, sweet pepper and the like. Therefore, the method for identifying the peanut varieties based on the SNP chip technology is established on the peanuts, the technical level of identifying the peanut varieties in China is hopeful to be improved, and the yield of excellent peanut varieties is effectively improved.
Disclosure of Invention
The invention aims to provide an SNP chip for identifying peanut varieties and a preparation method thereof.
The invention also aims to provide application of the SNP chip for peanut variety identification.
In order to realize the purpose of the invention, the invention provides an SNP chip for identifying peanut varieties, which comprises a probe for detecting 725 SNP molecular markers, wherein the numbers of the SNP molecular markers are SNP 001-SNP 725 respectively, and the nucleotide sequences are shown as SEQ ID NO 1-SEQ ID NO 725 respectively.
The invention also provides a preparation method of the SNP chip for variety identification, which comprises the following steps:
(1) performing whole genome re-sequencing on the Fuhua to obtain a peanut cultivar reference genome;
(2) 10X resequencing is carried out on 418 peanut cultivars, sequence assembly and comparison are carried out on the peanut cultivars and the reference genome obtained in the step (1), whole genome variation is detected, and 832,949 SNP molecular markers are obtained;
(3) carrying out linkage disequilibrium analysis on the 832,949 SNP molecular markers obtained in the step (2) by utilizing the biological statistical software TASSEL4.0 and Haploview 4.2, and screening 2-4 SNP with marking property in each window by taking the recession distance as a window according to the linkage disequilibrium recession distance of the whole genome of the peanut to obtain 3,700 SNP molecular markers;
(4) 725 SNP molecular markers are screened out from the 3,700 SNP molecular markers obtained in the step (3) according to the marker distribution characteristics, polymorphism, gene distribution, site deletion rate and the like;
(5) designing probes for the 725 SNP molecular markers screened in the step (4), and manufacturing an SNP chip which is named as Peanout 725.
The invention also provides application of the SNP chip in identification of peanut varieties. The SNP chip is utilized to detect the genome DNA of the peanut sample to be detected, the genotypes of 725 SNP markers are obtained, the genetic similarity between the peanut sample to be detected and the known peanut sample is calculated through genetic statistics, and whether the peanut sample is the same variety is judged.
The invention also provides application of the SNP chip in identification of peanut variety authenticity. And detecting the genome DNA of the peanut sample to be detected and the control peanut sample by using the SNP chip, respectively obtaining the genotypes of the 725 SNP markers, calculating the genetic similarity between the peanut sample to be detected and the control peanut sample through genetic statistics, and judging whether the peanut samples are the same variety.
The specific genetic statistical method is as follows: and (3) comparing the 725 SNP marker genotypes of different peanut samples, and calculating the genetic similarity of the different peanut samples according to the formula (1). If the genetic similarity of the two peanut samples is more than 99.0%, the two peanut samples belong to the same variety; and if the genetic similarity of the two samples is less than or equal to 99.0%, judging that the two samples have significant difference and do not belong to the same variety.
Genetic Similarity (GS) ═ 2Nij/(Ni + Nj) equation (1)
Wherein Nij is the number of polymorphic marker bands shared by the two peanut samples, Ni is the number of differential marker bands of one sample, and Nj is the number of differential marker bands of the other sample. For example, the following steps are carried out: if the total number of SNP sites compared between sample 1 and sample 2 is 725 and the number of ectopic sites is 15, the genetic similarity GS between these two samples is 2(725 × 2-15)/(725 × 2+725 × 2) ═ 98.97%.
The invention has the beneficial effects that: the marker used by the SNP chip for identifying the peanut varieties is obtained by whole genome re-sequencing development and design, and has the advantages of high flux, low cost and easy data integration and comparison compared with the traditional molecular marker such as SSR marker. The method is applied to peanut variety identification, can greatly improve the accuracy of peanut variety identification and detection, shortens the detection time, reduces the labor cost and the like.
Drawings
FIG. 1 is a distribution diagram of all SNP markers of a Peanout 725 chip on a Peanut genome, wherein each short line represents 1 SNP site;
FIG. 2 is a diagram of the detection quality effect of the SNP marker of the Peanout 725 chip, A: score >0.85, indicating high confidence; b: score <0.2, indicating low confidence; c: typing of SNP on a Peanout 725 chip to 265 varieties to test the Score effect;
FIG. 3 is a genetic similarity distribution graph between varieties and within varieties of 265 Peanut cultivars detected by Peanout 725 chips.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Modifications and substitutions may be made by those skilled in the art without departing from the spirit of the invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 design and preparation of a Peanout 725 chip.
Carrying out whole genome resequencing on the Volvox to obtain a peanut cultivar reference genome, then carrying out 10X whole genome resequencing on 418 peanut cultivars, carrying out sequence assembly comparison with the peanut cultivar reference genome, and detecting whole genome variation to obtain 832,949 SNP data; performing linkage disequilibrium analysis by using biometric software TASSEL4.0 and Haploview 4.2, and screening 2-4 SNP with markers in each window by taking a fading distance as a window to obtain 3,700 SNP molecular markers; each site was then evaluated for screening according to the following characteristics: (1) the deletion rate is less than or equal to 5 percent; (2) rare allele frequency is less than or equal to 5 percent; (3) single copy in the genome; (4) not in the transposon and repeat region; (5) no other SNP exists within 100bp upstream and downstream of the site; (6) the genome is uniformly distributed; (7) a second allelic polymorphism; (8) the repeatability is more than 99%. And finally, comprehensively considering the genotyping cost of detection and identification, screening out 725 SNP markers, designing probes according to the genotyping requirement customized by Illumina extension Infinium iSelect HD, and manufacturing a chip. The 725 SNP molecular markers are shown as SEQ ID NO 1-SEQ ID NO 725 in the sequence table, and the distribution diagram of all SNP markers of the Peanout 725 chip on the Peanut genome is shown in FIG. 1.
Example 2 use of the Peanout 725 chip
(1) Extraction of peanut genome DNA
265 parts of main cultivars of peanuts from north and south China are selected and shown in Table 2. Taking young and young leaves in the seedling stage, and extracting peanut genome DNA by adopting a biotek DP3111 plant genome DNA extraction kit (centrifugal column type) according to the standard flow of the instruction.
(2) DNA quality detection
The quality of the genomic DNA was checked by 0.5% (w/v) agarose gel electrophoresis, and 1. mu.L of the DNA solution was taken, mixed with 6 Xloading buffer solution, and loaded with 1. mu.L of lambda DNA as a marker. The agarose power showed a single band of no apparent diffuse DNA, indicating better DNA quality. Further, an ultraviolet spectrophotometer is used for determining the OD value of the genomic DNA solution, the OD260/OD280 is between 1.8 and 2.0, and the OD260/OD230 is between 1.8 and 2.0, which indicates that the purity of the DNA is qualified; the DNA concentration is required to be 50 ng/. mu.L or more.
(3) Peanout 725 chip detection
The extracted genomic DNA was diluted to a working concentration of 50 ng/. mu.L, and 4. mu.L of each sample was subjected to loading detection. Genotyping of the samples was performed according to the standard protocol for Illumina Infinium Gene chip detection. And (3) carrying out genotype scanning on the samples by using an Illumina iScan chip scanner to obtain SNP genotype data of each sample. (4) Peanout 725 chip result analysis
Analyzing off-line data by utilizing genome studio software of Illumina to obtain the genotypes of 265 varieties, analyzing all SNP genotypes of each sample, and counting the genotype distribution of all samples at each SNP locus, wherein the genotype distribution is shown in a figure 2.
TABLE 2265 parts of peanut cultivars
Example 3 application of Peanout 725 chip in identification of Peanut variety
The following will illustrate how to use the SNP chip designed and prepared in example 1 to identify whether the sample to be tested is a known peanut sample. Firstly, 265 peanut varieties in example 2 are taken as a fingerprint gallery; secondly, the DNA extraction, quality detection and chip detection of the sample to be detected are the same as those in the embodiment 2; and finally, calculating the genetic similarity between the sample to be detected and each sample in the fingerprint database, and judging. The result shows that the genetic similarity of the sample to be tested and the Zhanjiu oil 75 in the fingerprint gallery is the highest and reaches 99.80 percent. Therefore, the sample was judged to be "oil-spreading 75".
The judgment standard is to detect the 725 SNP genotypes based on the 265 peanut varieties in the example 2 to obtain 725 SNP genotypes of the 265 peanut varieties, and to calculate the Genetic Similarity (GS) between every two varieties by using a formula (1):
genetic Similarity (GS) ═ 2Nij/(Ni + Nj) equation (1)
Wherein Nij is the number of polymorphic marker bands shared by the two peanut samples, Ni is the number of differential marker bands of one sample, and Nj is the number of differential marker bands of the other sample. For example, the following steps are carried out: if the total number of SNP sites compared between sample 1 and sample 2 is 725 and the number of ectopic sites is 15, the genetic similarity GS between these two samples is 2(725 × 2-15)/(725 × 2+725 × 2) ═ 98.97%.
Through the comparison and calculation, the genetic similarity between every two 265 main peanut cultivars popularized and applied in the current production of China is lower than 99.0 percent, as shown in figure 3. Similarly, referring to example 2, 725 SNP genotypes are performed on 50 individuals in the Guangdong oil No. 7 peanut variety, and the genetic similarity between every two individuals in the variety is counted, so that the genetic similarity in the variety is greater than 99.0%. Therefore, in the process of identifying the peanut variety, 99.0 percent is taken as a judgment standard, namely the genetic similarity between the sample to be detected and the contrast sample is more than 99.0 percent, and the two samples are the same variety; otherwise, the genetic similarity between the sample to be detected and the control sample is less than or equal to 99.0 percent, and the two samples are different varieties.
Example 4 application of Peanout 725 chip in Peanut variety authenticity identification
Hereinafter, how to identify whether the sample to be tested is the known control sample "Yueji No. 7" using the SNP chip designed and prepared in example 1 will be exemplified. Selecting 3 single leaves of different strains from a strain garden, selecting 1 single leaf from a reference variety of Yuejie No. 7, taking 4 samples as samples to be detected, randomly disordering, and respectively numbering L1, L2, L3 and L4. Genomic DNA extraction, quality testing and chip testing were as in example 2. The chip typing results show that the number of the differential sites of L1, L2, L3 and L4 and the control 'Yueyou No. 7' are respectively 128, 7, 226 and 56, and according to the genetic similarity calculation formula (1), the specific calculation mode is as described in example 3, and the calculated results show that the genetic similarity GS of the three and the control is respectively 91.17%, 99.51%, 84.41% and 96.13%. According to the judgment criteria in example 3, L2 is "yueji No. 7", and the other three samples are not the control variety "yueji No. 7". After maturation, the L1, L3, and L4 samples were further confirmed to be significantly different from the controls by agronomic phenotypic identification.
The above detailed description is specific to possible embodiments of the present invention, and the embodiments are not intended to limit the scope of the present invention, and all equivalent implementations or modifications that do not depart from the scope of the present invention are intended to be included within the scope of the present invention.
Claims (4)
1. The SNP chip for identifying peanut varieties is characterized by comprising a detection probe, wherein the detection probe consists of probes for detecting 725 SNP molecular markers, the number of the SNP molecular markers is SNP 001-SNP 725, and the nucleotide sequences are respectively shown as SEQ ID NO 1-SEQ ID NO 725.
2. A method for preparing the SNP chip according to claim 1, comprising the steps of:
1) performing whole genome re-sequencing on the Fuhua to obtain a peanut cultivar reference genome;
2) 10X resequencing is carried out on 418 peanut cultivars, sequence assembly and comparison are carried out on the 418 peanut cultivars and the reference genome obtained in the step 1), whole genome variation is detected, and 832,949 SNP molecular markers are obtained;
3) screening 2-4 SNP with markers in each window according to the linkage disequilibrium recession distance of the whole genome of the peanut and the recession distance as windows on the 832,949 SNP molecular markers obtained in the step 2) to obtain 3,700 SNP molecular markers;
4) 725 SNP molecular markers are screened out from the 3,700 SNP molecular markers obtained in the step 3) according to the marker distribution characteristics, polymorphism, gene distribution and site deletion rate;
5) designing probes for the 725 SNP molecular markers screened in the step 4) and manufacturing the SNP chip.
3. Use of the SNP chip according to claim 1 for identification of peanut varieties.
4. Use of the SNP chip according to claim 1 for the identification of the authenticity of a peanut variety.
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