CN114231657B

CN114231657B - SNP locus for detecting corn variety purity and application thereof

Info

Publication number: CN114231657B
Application number: CN202111678747.9A
Authority: CN
Inventors: 李乐; 彭佩; 田冰川; 蒋友如; 唐顺学
Original assignee: Huazhi Biotechnology Co ltd
Current assignee: Huazhi Biotechnology Co ltd
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2023-06-23
Anticipated expiration: 2041-12-31
Also published as: CN114231657A

Abstract

The invention discloses an SNP locus for detecting the purity of a corn variety and application thereof. The invention selects a set of 40 SNP markers with high quality and high polymorphism through screening, can be used for accurately detecting the purity of corn varieties with different sources, and has wide application universality; the marker is a co-dominant marker, and has high specificity, sensitivity and resolution; the mark is not influenced by environmental conditions, seeds or any type of plant tissues can be used, and the detection result is accurate and has good repeatability and stability.

Description

SNP locus for detecting corn variety purity and application thereof

Technical Field

The invention belongs to the field of agricultural molecular biology, and particularly relates to a SNP locus for detecting the purity of corn varieties and application thereof.

Background

Corn is the primary food crop; the main production of corn is from hybrid seeds. With the continuous development of corn breeding technology and the large-area popularization and application of hybrid varieties, the purity detection of parent varieties and genetic materials for seed production becomes an essential quality assurance for corn breeding research and development, seed production and seed transaction.

Today corn variety purity identification is mainly dependent on the traditional field plot planting identification method (Grow-out Test). The field planting identification is to plant the variety in a field test plot, and the purity of the corn variety is identified by observing the difference of plant morphological characteristics (such as height and size of the plant, tillering, leaf color, leaf shape, seed size, seed coat color and the like) and biological characteristics (such as growth period, photoperiod, disease resistance, drought resistance, seed shatter property and the like) of the plant in different growth periods (seedling period, growth period, flowering period, mature period and seed). The method depends on visual identification of morphological characteristics and biological characteristics of plants in fields, and the judgment standard is difficult to precisely quantify, so that the method is high in subjectivity and low in detection sensitivity and resolution; is easily influenced by environment and cultivation conditions, and has poor accuracy and stability; long time consumption and poor timeliness; a great deal of manpower and material resources are needed to be input, and the cost is high.

SSR has become one of the main methods for detecting purity and variety authenticity at present, has the advantages of simple laboratory operation, low cost, better repeatability, true and reliable results and the like when used in large scale in the national standard of SSR for various crops such as rice, corn, wheat, soybean and the like. Compared with an SSR labeling method, the SNP labeling method has simpler technology, easy automation, high detection flux and high speed; the unit data point detection cost is low; the data results of different detection laboratories can be compared and verified, and the data has universal comparability; is the most common method for rapidly, simply, sensitively, accurately, stably and with low cost for identifying the purity of the varieties. At present, a corn purity detection method based on SNP markers is rarely reported.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a SNP locus for detecting the purity of corn.

The invention also provides a primer group for detecting the SNP locus.

The invention also provides a kit.

The invention also provides a gene chip.

The invention also provides application of the SNP locus, the primer set, the kit and/or the gene chip.

The invention also provides a detection method of the SNP locus.

According to an embodiment of the first aspect of the present invention, the SNP site is selected from at least one of the following first SNP site to forty SNP site:

a first SNP locus which is positioned at a corresponding locus on a 82587178 nucleotide of a 1 st chromosome of a corn reference genome or an inter-variety homologous genome fragment thereof, and the nucleotide base of the locus is G/T;

a second SNP locus which is positioned at a corresponding locus on the 291348623 th nucleotide of the 1 st chromosome of the corn reference genome or the homologous genome fragment between varieties thereof, and the nucleotide base of the locus is G/A;

a third SNP locus which is positioned at a corresponding locus on the 157283336 th nucleotide of the 1 st chromosome of the corn reference genome or the homologous genome fragment between varieties thereof, and the nucleotide base of the locus is G/A;

a fourth SNP locus which is positioned at a corresponding locus on the 213137297 th nucleotide of the 1 st chromosome of the corn reference genome or the homologous genome fragment between varieties thereof, and the nucleotide base of the locus is A/G;

a fifth SNP locus located at a corresponding locus on the 141204710 nucleotide of chromosome 2 of the maize reference genome or on an inter-variety homologous genome fragment thereof, the nucleotide base of the locus being C/T;

A sixth SNP locus which is positioned at a corresponding locus on a 28211592 nucleotide of a 2 nd chromosome of a corn reference genome or an inter-variety homologous genome fragment thereof, and the nucleotide base of the locus is C/G;

a seventh SNP locus which is positioned at a corresponding locus on a 207814666 nucleotide of a 2 nd chromosome of a corn reference genome or an inter-variety homologous genome fragment thereof, and the nucleotide base of the locus is G/A;

an eighth SNP locus located at the corresponding locus on the 237488672 nd nucleotide of the 2 nd chromosome of the maize reference genome or on the inter-variety homologous genome fragment thereof, the nucleotide base of the locus being T/G;

a ninth SNP locus located at a corresponding locus on the 129484896 nucleotide of chromosome 3 of the maize reference genome or on an inter-variety homologous genomic fragment thereof, the nucleotide base of the locus being G/A;

a tenth SNP locus which is positioned at a corresponding locus on a 225786467 nucleotide of a 3 rd chromosome of a corn reference genome or an inter-variety homologous genome fragment thereof, and the nucleotide base of the locus is A/C;

an eleventh SNP locus which is positioned at a corresponding locus on the 184720677 nucleotide of the 3 rd chromosome of the corn reference genome or the homologous genome fragment between varieties thereof, and the nucleotide base of the locus is C/T;

A twelfth SNP locus which is positioned at a corresponding locus on the 22035145 nucleotide of the 3 rd chromosome of the corn reference genome or the inter-variety homologous genome fragment thereof, and the nucleotide base of the locus is A/C;

a thirteenth SNP locus located at a corresponding locus on the 157469658 th nucleotide of the 4 th chromosome of the maize reference genome or on an inter-variety homologous genomic fragment thereof, the nucleotide base of the locus being T/C;

a fourteenth SNP locus located at a corresponding locus on the 84668766 th nucleotide of the 4 th chromosome of the maize reference genome or on an inter-variety homologous genome fragment thereof, the nucleotide base of the locus being T/G;

a fifteenth SNP locus located at a corresponding locus on the 243991142 th nucleotide of the 4 th chromosome of the maize reference genome or on an inter-variety homologous genome fragment thereof, the nucleotide base of the locus being C/T;

a sixteenth SNP locus located at a corresponding locus on the 236715436 th nucleotide of the 4 th chromosome of the maize reference genome or on an inter-variety homologous genome fragment thereof, the nucleotide base of the locus being G/A;

A seventeenth SNP locus located at a corresponding locus on the 7486959 th nucleotide of the 5 th chromosome of the maize reference genome or on an inter-variety homologous genome fragment thereof, the nucleotide base of the locus being T/C;

an eighteenth SNP locus which is positioned at a corresponding locus on a 135108514 th nucleotide of a 5 th chromosome of a corn reference genome or an inter-variety homologous genome fragment thereof, wherein the nucleotide base of the locus is C/T;

a nineteenth SNP locus located at a corresponding locus on the 199452705 th nucleotide of the 5 th chromosome of the maize reference genome or on an inter-variety homologous genome fragment thereof, the nucleotide base of the locus being G/T;

a twentieth SNP locus which is positioned at a corresponding locus on a 215650073 th nucleotide of a 5 th chromosome of a corn reference genome or an inter-variety homologous genome fragment thereof, wherein the nucleotide base of the locus is C/T;

a twenty-first SNP locus which is positioned at a corresponding locus on a 13727981 th nucleotide of a 6 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is C/A;

A twenty-second SNP locus which is positioned at a corresponding locus on a 132514637 th nucleotide of a 6 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is C/T;

a twenty-third SNP locus which is positioned at a corresponding locus on a 95771968 th nucleotide of a 6 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is A/G;

a twenty-fourth SNP locus which is positioned at a corresponding locus on a 160230101 th nucleotide of a 6 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, and the nucleotide base of the locus is G/A;

a twenty-fifth SNP locus which is positioned at a corresponding locus on a 8195312 th nucleotide of a 7 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is C/T;

a twenty-sixth SNP locus which is positioned at a corresponding locus on a 74052945 nucleotide of a 7 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is G/A;

A twenty-seventh SNP locus which is positioned at a corresponding locus on a 169955912 th nucleotide of a 7 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is A/C;

a twenty-eighth SNP locus which is positioned at a corresponding locus on a 136144222 th nucleotide of a 7 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is G/A;

a twenty-ninth SNP locus which is positioned at a corresponding locus on a 4978106 th nucleotide of a 8 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is T/G;

a thirty-first SNP locus located at a corresponding locus on the 126914854 th nucleotide of the 8 th chromosome of the maize reference genome or on an inter-variety homologous genome fragment thereof, the nucleotide base of the locus being T/C;

a thirty-first SNP locus which is positioned at a corresponding locus on a 173505727 th nucleotide of a 8 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is G/A;

A thirty-second SNP locus which is positioned at a corresponding locus on a 75160235 th nucleotide of a 8 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is T/C;

a thirty-third SNP locus which is positioned at a corresponding locus on a 11442148 th nucleotide of a 9 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is G/A;

a thirty-fourth SNP locus which is positioned at a corresponding locus on a 150460620 th nucleotide of a 9 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is C/T;

a thirty-fifth SNP locus which is positioned at a corresponding locus on a 89076495 th nucleotide of a 9 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is G/A;

a thirty-sixth SNP locus located at a corresponding locus on the 121462317 th nucleotide of the 9 th chromosome of the maize reference genome or on an inter-variety homologous genome segment thereof, the nucleotide base of the locus being C/A;

A thirty-seventh SNP locus which is positioned at a corresponding locus on a 15090132 th nucleotide of a 10 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is G/A;

a thirty-eighth SNP locus which is positioned at a corresponding locus on a 41610224 th nucleotide of a 10 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is G/A;

a thirty-ninth SNP locus which is positioned at a corresponding locus on a 113193861 th nucleotide of a 10 th chromosome of a corn reference genome or an inter-variety homologous genome segment thereof, wherein the nucleotide base of the locus is G/C;

a forty SNP locus located at the corresponding locus on the 119856037 th nucleotide of the 10 th chromosome of the maize reference genome or on the inter-variety homologous genome segment thereof, the nucleotide base of the locus being C/T;

wherein the maize reference genome is a maize B73V 3 version reference genome.

According to a second aspect of the present invention, there is provided a primer set for amplifying the above SNP site.

In some embodiments of the invention, the primer sets each independently comprise two specific primers and 1 universal primer.

In some embodiments of the invention, the primer set comprises:

the first SNP primer set is used for amplifying the first SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.1 and SEQ ID NO.2 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 3;

the second SNP primer set is used for amplifying the second SNP locus and comprises specific primer nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO.5 and universal primer nucleotide sequences shown as SEQ ID NO. 6;

the third SNP primer set is used for amplifying the third SNP locus and comprises specific primer nucleotide sequences shown as SEQ ID NO.7 and SEQ ID NO.8 and also comprises a universal primer nucleotide sequence shown as SEQ ID NO. 9;

a fourth SNP primer set for amplifying the fourth SNP locus, comprising a specific primer nucleotide sequence shown as SEQ ID NO.10 and SEQ ID NO.11 and a universal primer nucleotide sequence shown as SEQ ID NO. 12;

a fifth SNP primer set for amplifying the fifth SNP locus, comprising a specific primer nucleotide sequence shown as SEQ ID NO.13 and SEQ ID NO.14 and a universal primer nucleotide sequence shown as SEQ ID NO. 15;

A sixth SNP primer set for amplifying the sixth SNP locus, comprising a specific primer nucleotide sequence as shown in SEQ ID NO.16 and SEQ ID NO.17 and a universal primer nucleotide sequence as shown in SEQ ID NO. 18;

a seventh SNP primer set for amplifying the seventh SNP locus, comprising a specific primer nucleotide sequence shown as SEQ ID NO.19 and SEQ ID NO.20 and a universal primer nucleotide sequence shown as SEQ ID NO. 21;

an eighth SNP primer set for amplifying the eighth SNP locus, comprising a specific primer nucleotide sequence as shown in SEQ ID NO.22 and SEQ ID NO.23 and a universal primer nucleotide sequence as shown in SEQ ID NO. 24;

a ninth SNP primer set for amplifying the ninth SNP locus, comprising a specific primer nucleotide sequence as shown in SEQ ID NO.25 and SEQ ID NO.26, and further comprising a universal primer nucleotide sequence as shown in SEQ ID NO. 27;

a tenth SNP primer set for amplifying the tenth SNP locus, comprising a specific primer nucleotide sequence as shown in SEQ ID NO.28 and SEQ ID NO.29 and a universal primer nucleotide sequence as shown in SEQ ID NO. 30;

an eleventh SNP primer set for amplifying the eleventh SNP locus, comprising a specific primer nucleotide sequence as shown in SEQ ID NO.31 and SEQ ID NO.32, and further comprising a universal primer nucleotide sequence as shown in SEQ ID NO. 33;

A twelfth SNP primer set for amplifying the twelfth SNP locus, comprising a specific primer nucleotide sequence as shown in SEQ ID NO.34 and SEQ ID NO.35 and a universal primer nucleotide sequence as shown in SEQ ID NO. 36;

a thirteenth SNP primer set for amplifying the thirteenth SNP locus, comprising a specific primer nucleotide sequence as set forth in SEQ ID NO.37 and SEQ ID NO.38, and further comprising a universal primer nucleotide sequence as set forth in SEQ ID NO. 39;

a fourteenth SNP primer set for amplifying the fourteenth SNP locus, comprising a specific primer nucleotide sequence as shown in SEQ ID NO.40 and SEQ ID NO.41 and a universal primer nucleotide sequence as shown in SEQ ID NO. 42;

a fifteenth SNP primer set for amplifying the fifteenth SNP locus, comprising a specific primer nucleotide sequence as shown in SEQ ID NO.43 and SEQ ID NO.44 and further comprising a universal primer nucleotide sequence as shown in SEQ ID NO. 45;

a sixteenth SNP primer set for amplifying the sixteenth SNP locus, comprising a specific primer nucleotide sequence shown as SEQ ID NO.46 and SEQ ID NO.47 and further comprising a universal primer nucleotide sequence shown as SEQ ID NO. 48;

A seventeenth SNP primer set for amplifying the seventeenth SNP locus, wherein the seventeenth SNP locus comprises a specific primer nucleotide sequence shown as SEQ ID NO.49 and SEQ ID NO.50 and further comprises a universal primer nucleotide sequence shown as SEQ ID NO. 51;

an eighteenth SNP primer set for amplifying the eighteenth SNP locus, wherein the eighteenth SNP locus comprises a specific primer nucleotide sequence shown as SEQ ID NO.52 and SEQ ID NO.53 and further comprises a universal primer nucleotide sequence shown as SEQ ID NO. 54;

a nineteenth SNP primer set for amplifying the nineteenth SNP locus, comprising a specific primer nucleotide sequence as shown in SEQ ID NO.55 and SEQ ID NO.56 and further comprising a universal primer nucleotide sequence as shown in SEQ ID NO. 57;

the twentieth SNP primer set is used for amplifying the twentieth SNP locus and comprises specific primer nucleotide sequences shown as SEQ ID NO.58 and SEQ ID NO.59 and also comprises a universal primer nucleotide sequence shown as SEQ ID NO. 60;

and the twenty-first SNP primer set is used for amplifying the twenty-first SNP locus and comprises specific primer nucleotide sequences shown as SEQ ID NO.61 and SEQ ID NO.62 and also comprises a universal primer nucleotide sequence shown as SEQ ID NO. 63.

A twenty-second SNP primer set for amplifying the twenty-second SNP locus, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.64 and SEQ ID NO.65 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 66;

a twenty-third SNP primer set for amplifying the twenty-third SNP locus, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.67 and SEQ ID NO.68 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 69;

a twenty-four SNP primer set for amplifying the twenty-four SNP locus, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.70 and SEQ ID NO.71 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 72;

a twenty-five SNP primer set for amplifying the twenty-five SNP loci, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.73 and SEQ ID NO.74 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 75;

a twenty-first SNP primer set for amplifying the twenty-first SNP locus, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.76 and SEQ ID NO.77 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 78;

A twenty-seventh SNP primer set for amplifying the twenty-seventh SNP locus, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.79 and SEQ ID NO.80 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 81;

the twenty-eighth SNP primer set is used for amplifying the twenty-eighth SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.82 and SEQ ID NO.83 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 84;

a second nineteenth SNP primer set for amplifying the second nineteenth SNP locus, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.85 and SEQ ID NO.86 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 87;

a thirty-first SNP primer set for amplifying the thirty-first SNP locus, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.88 and SEQ ID NO.89 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 90;

a thirty-one SNP primer set for amplifying the thirty-one SNP locus, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.91 and SEQ ID NO.92 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 93;

A thirty-two SNP primer set for amplifying the thirty-two SNP loci, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.94 and SEQ ID NO.95 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 96;

a thirteenth SNP primer set for amplifying the thirteenth SNP locus, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.97 and SEQ ID NO.98 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 99;

the thirty-fourth SNP primer set is used for amplifying the thirty-fourth SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.100 and SEQ ID NO.101 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 102;

a thirty-fifth SNP primer set for amplifying the thirty-fifth SNP locus, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.103 and SEQ ID NO.104 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 105;

a thirty-sixth SNP primer set for amplifying the thirty-sixth SNP locus, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.106 and SEQ ID NO.107 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 108;

A seventeenth SNP primer set for amplifying the seventeenth SNP locus, comprising a specific primer with a nucleotide sequence shown as SEQ ID NO.109 and SEQ ID NO.110 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 111;

the thirty-eighth SNP primer set is used for amplifying the thirty-eighth SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.112 and SEQ ID NO.113 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 114;

a third nineteenth SNP primer set for amplifying the third nineteenth SNP locus, wherein the third nineteenth SNP locus comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.115 and SEQ ID NO.116 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 117;

and the forty SNP primer set is used for amplifying the forty SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.118 and SEQ ID NO.119 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 120.

According to some embodiments of the invention, the two specific primers have different fluorophore tag sequences attached, preferably the fluorophore tag sequences are selected from FAM, HEX.

According to an embodiment of the third aspect of the present invention, a kit for identifying the purity of maize varieties is provided, comprising the above-mentioned primer set.

According to a fourth aspect of the present invention, there is provided a gene chip for identifying the purity of maize varieties, comprising the above primer set.

A method of detecting the purity of a maize variety according to an embodiment of the fifth aspect of the present invention comprises the steps of:

s1, extracting genome DNA from a corn sample to be detected;

s2, carrying out polymorphism detection on the SNP loci in the genome DNA extracted in the step S1 to obtain genotypes of the SNP loci corresponding to the corn samples to be detected;

s3, carrying out statistical analysis on the genotype of the sample individuals to be detected obtained in the step S2, identifying the hybrid strains, and calculating the purity of the varieties according to the number of the individual hybrid strains and the total detection number.

In some embodiments of the invention, in step S1, genomic DNA is extracted from corn using a simplified CTAB process (cetyl trimethylammonium bromide process).

In some embodiments of the invention, in step S2, SNP sites are detected using the KASP (competitive allele-specific PCR) technique.

In some embodiments of the present invention, in step S3, the number of the corn samples to be tested is more than 100, preferably 200.

In some embodiments of the present invention, in step S3, if the genotype of a certain corn individual to be tested is different from the genotype of other most individuals to be tested in 2 or more SNP loci, the corn individual to be tested is determined to be a hybrid plant.

In some embodiments of the invention, the method of calculating the purity of the cultivar is: degree of homozygosity = (total number of assays-number of hybrid plants)/total number of assays x 100%.

The use of the above-described SNP sites, primer sets, kits and/or gene chips according to the sixth aspect of the embodiments of the invention is:

(1) The application in identifying or assisting in identifying the purity of the corn varieties;

(2) Application in corn molecular marker assisted breeding;

(3) The application of the corn seed breeding product is prepared.

A method of maize breeding comprising the steps of: by using the corn purity detection method, corn with the required purity is selected for subsequent breeding.

The detection of corn SNP loci according to the embodiment of the invention has at least the following beneficial effects: a set of (4 SNP/chromosome) SNP markers with 40 high quality and high polymorphism are selected through screening, can be used for accurately detecting the purity of corn varieties (lines) with different sources, and has wide application universality; the marker is a co-dominant marker, and has high specificity, sensitivity and resolution; the mark is not affected by environmental conditions, seeds or any type of plant tissues can be used, and the detection result is accurate and has good repeatability and stability; different detection laboratories and different data results can be compared and verified, and the data has universal comparability; the SNP marker developed by the scheme of the invention is subjected to genotyping by utilizing the KASP technology based on Douglas Array Tape platform, the genotyping is accurate, the technology is simple, the automation is easy, the speed is high, the automation degree reaches 90%, and the manpower and human errors in a laboratory are greatly reduced; the unit data point detection cost is low, the detection flux is high, and the breeding process of corn varieties is accelerated.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a site development flow chart of an embodiment of the present invention;

FIG. 2 is a genotyping schematic diagram of SNP locus ZM_K_10000630 according to an embodiment of the invention.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

In order to describe the technical contents, the achieved objects and effects of the present invention in detail, the following description will be made with reference to the embodiments in conjunction with the accompanying drawings. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

The embodiment of the invention comprises the following steps: SNP locus for detecting purity of corn varieties

In the site design process of the embodiment of the invention, as shown in fig. 1, 56110 corn SNP site information is collected, SNP sites for corn variety purity detection are obtained through comparison and screening, SNP sites and flanking sequences are extracted, and a primer sequence of a marker is designed and synthesized, and then the marker is verified and detected, specifically as follows:

screening of 1.40 SNP loci for corn variety purity detection

Based on 156 parts of whole genome detection data of corn, according to screening conditions: MAF >0.1, deletion rate <0.02, even distribution of chromosome, good PCA clustering effect, high discrimination degree and conservation of 25bp sequences of two wings, at least 2 site differences exist in genotypes of database samples corresponding to the selected SNP set, a plurality of high-quality SNP sites are selected, and finally, the high-quality SNP combination with the least proposal of the invention is selected as 40 SNP sites. The basic information of the 40 SNP loci is shown in Table 1. Wherein the position of the SNP site on the chromosome is determined based on alignment of the maize reference genome B73 sequences.

TABLE 1 physical location of SNP loci for corn variety purity detection

2 primer design

Design of the KASP marker: KASP primer design was performed on the 40 SNP sites screened using Batchprimer3 (http:// probes. Pw. Usda. Gov/Batchprimer3 /). Each KASP marker consists of three primers, including two Allele-specific primers X (rule-Specific Primer X; primer_X) and Y (rule-Specific Primer Y; primer_Y) and one universal Primer C (Common Primer; primer_C). Two allele-specific primers were ligated to FAM and HEX (or VIC) fluorophore sequence tags, respectively. If only FAM fluorescence is detected in the sample, the genotype of the sample is homozygous Allele X (Allelle_X); if only HEX fluorescence is detected, the genotype of the sample is homozygous Allele Y (allele_Y); if both FAM and HEX fluorescence are detected, the genotype of the sample is heterozygous (with both alleles X and Y). The genotypes and primer sequences of the 40 KASP markers used for corn variety purity detection are shown in table 2.

TABLE 2 primer sequence listing

3 KASP detection

DNA extraction: genomic DNA was extracted from maize using a simplified CTAB method.

KASP label detection procedure: the verification and detection of the KASP markers was performed with the Array Tape system of Douglas Scientific. The Array Tape genotyping platform included NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescent signal scanning, and INTELLICS for data analysis.

PCR reaction system: automatic assembly of the PCR reaction system was performed using NEXAR, and the PCR reaction system is shown in Table 3 below.

And (3) PCR amplification: PCR amplification was performed using SOELLEX under the following conditions: pre-denaturation at 94 ℃ for 15 min; the first step of amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 65-57 ℃ and extension for 60 seconds, 10 cycles, wherein the annealing and extension temperature of each cycle is reduced by 0.8 ℃; the second amplification step was performed by denaturation at 94℃for 20 seconds, annealing at 57℃and extension for 60 seconds, 30 cycles.

Signal scanning and genotyping: after the PCR reaction is completed, ARAYA is used for scanning fluorescent signals of the reaction system; genotyping and data analysis were then performed using INTELLICS. In the KASP marker genotyping assay, the genotypes of the samples were divided into 3 clusters, an X cluster, a Y cluster, and a heterozygous genotype cluster (see fig. 2). As can be seen in FIG. 2, the red color in the figure, the X cluster in the upper left hand corner of the figure, indicates that the sample contains homozygous X alleles at this KASP marker locus, the blue color in the figure, the Y cluster in the lower right hand corner of the figure, indicates that the sample contains homozygous Y alleles at this KASP marker locus, and the purple color in the figure, indicates that the sample contains X and Y heterozygous alleles at this KASP marker locus.

TABLE 3 PCR reaction System for KASP detection

4.40 SNP locus verification for corn variety purity detection

Genotyping quality verification of 40 KASP markers for corn variety purity detection: 40 KASP markers were validated using 376 maize diversity materials according to the assay described above. Through verification, two homozygous and heterozygous clusters of each KASP mark are good and compact in typing, the sites are single copies, the detection rate is higher than 99.5%, the detection rate is consistent with the sequencing result, the result shows that 40 KASP marks can be accurately typed, and the genotyping quality can completely meet the accurate detection of the purity of corn varieties. The KASP marker genotyping map of the ZM_K_10000630 locus is shown in FIG. 2, and the results of other loci are identical to those of ZM_K_ 10000630.

Calculation of purity of maize variety

The calculation of the purity of the corn varieties comprises the following steps:

(1) The genotypes of the 40 SNP loci of 184 corns (corn B73) of the sample to be tested are analyzed statistically. If more than 2 loci are different from the other most individuals to be tested in a certain individual to be tested, judging the wheat individual to be tested as a hybrid plant; single plants with the difference number less than or equal to 1 with control sites or colony consistent sites (genotypes with the genotype frequency ratio more than 50% are consistent sites) in the individuals to be detected are pure plants; and if the SNP purity detection deletion site is lower than 5% of the total detection site number, judging that the single plant is effective, and incorporating purity judgment statistics.

As shown in Table 4 (Table 4 includes tables 4-1, 4-2, 4-3), the genotypes of 184 individuals at 40 SNP loci are identical, and the individuals are all effective individuals (the number of effective individuals is the total number of detection); wherein, the individual with the number of 6 is different from other 183 individuals in 10 SNP loci such as ZM_K_10000154, ZM_K_10000225 and the like, and is judged as a hybrid strain; the individual with the number 14 is different from other 183 individuals in 8 SNP loci such as ZM_K_10000154, ZM_K_10000225 and the like, and is judged as a hybrid strain; the individual No. 15 was judged as a hybrid strain, differing from the other 183 individuals in 9 SNP sites such as zm_k_10000154, zm_k_10000225, etc.; the individual with the number of 27 is different from other 183 individuals in 10 SNP loci such as ZM_K_10000225, ZM_K_10000154 and the like, and is judged to be a hybrid plant; the individual with the number 63 is different from other 183 individuals in 9 SNP loci such as ZM_K_10000154, ZM_K_10000312 and the like, and is judged as a hybrid; the individual with the number 72 is different from other 183 individuals at 8 SNP loci such as ZM_K_10000154, ZM_K_10000225, ZM_K_10000312 and the like, and is judged as a hybrid; the individual with the number of 107 is different from other 183 individuals in 9 SNP loci such as ZM_K_10000154, ZM_K_10000225, ZM_K_10000312 and the like, and is judged as a hybrid; the individual with the number 164 is different from other 183 individuals at 8 SNP loci such as ZM_K_10000154, ZM_K_10000225, ZM_K_10000312 and the like, and is judged as a hybrid; the 169 individual was judged as a hybrid strain, differing from the other 183 individuals in 8 SNP loci such as ZM_K_10000154, ZM_K_10000225, etc.; the individual numbered 176 was determined to be a hybrid strain, differing from the other 183 individuals in 8 SNP loci such as ZM_K_10000154, ZM_K_10000225, ZM_K_10000312, etc.

(2) And (3) calculating the purity of the varieties according to the individual number of the hybrid strains and the total detection number in the step (1).

The calculation of the degree of homozygosity is: degree of homozygosity = (total number of assays-number of hybrid plants)/total number of assays x 100%.

The variety purity of the corn variety B73 to be detected is as follows: (184-174)/184×100% = 94.57%.

TABLE 4-1

TABLE 4-2

TABLE 4-3

In summary, the invention provides a set of 40 SNP loci which can be used for accurately detecting the purity of corn varieties and genetic materials with different sources, has wide application universality and can trace and infer pollution sources of impure samples; has co-dominant mark, high specificity, sensitivity and resolution; the mark is not affected by environmental conditions, seeds or any type of plant tissues can be used, and the detection result is accurate and has good repeatability and stability; the technology is simple, the automation is easy, the detection flux is high, and the speed is high; the unit data point detection cost is low; the data results of different detection laboratories can be compared and verified, and the data has universal comparability.

The Douglas Array Tap genotyping platform used in the invention comprises NEXAR for assembling a PCR amplification system, SOELLEX for PCR amplification, ARAYA for fluorescent signal scanning and INTELLICS for data analysis, and reagent consumables matched with the NEXAR, SOELLEX and ARAYA are all purchased from LGC company in England.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

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Claims

1. A primer group for detecting SNP locus of corn variety purity is characterized in that: the primer set includes:

a twenty-first SNP primer set for amplifying the twenty-first SNP locus, comprising a specific primer nucleotide sequence shown as SEQ ID NO.61 and SEQ ID NO.62 and a universal primer nucleotide sequence shown as SEQ ID NO. 63;

2. The primer set of claim 1, wherein the specific primer is linked to FAM and HEX fluorophore tag sequences, respectively.

3. A kit comprising the primer set of claim 1 or 2.

4. A gene chip comprising the primer set according to claim 1 or 2.

5. Use of the primer set of claim 1 or 2, the kit of claim 3 or the gene chip of claim 4 for identifying the purity of maize varieties.

6. A method for detecting the purity of maize varieties using the primer set of SNP locus as set forth in claim 1, characterized in that it comprises the steps of:

s1, extracting genome DNA from a corn sample to be detected;

s2, carrying out polymorphism detection on the SNP locus in the genomic DNA extracted in the step S1 by using the primer set of the SNP locus to obtain the genotype of the SNP locus corresponding to the corn sample to be detected;

S3, carrying out statistical analysis on genotypes of individuals of the sample to be detected obtained in the step S2, identifying hybrid strains, and calculating variety purity according to the number of the individuals of the hybrid strains and the total detection number; if the genotype of a certain corn individual to be detected is different from the genotype of other most individuals to be detected in more than 2 SNP loci, judging that the corn individual to be detected is a hybrid plant.

7. A method of maize breeding comprising the steps of: the method for detecting the purity of corn varieties according to claim 6, wherein corn of a desired purity is selected for subsequent breeding.