CN114231657A

CN114231657A - SNP (Single nucleotide polymorphism) locus for detecting purity of corn variety and application thereof

Info

Publication number: CN114231657A
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: 2022-03-25
Anticipated expiration: 2041-12-31
Also published as: CN114231657B

Abstract

The invention discloses an SNP locus for detecting the purity of a corn variety and application thereof. The method can be used for accurately detecting the purity of corn varieties from different sources by screening a set of 40 SNP markers with high quality and high polymorphism, and has wide application universality; the marker is a co-dominant marker, and has high specificity, sensitivity and resolution; the marker 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 (Single nucleotide polymorphism) locus for detecting purity of corn variety and application thereof

Technical Field

The invention belongs to the field of agricultural molecular biology, and particularly relates to an SNP (single nucleotide polymorphism) locus for detecting the purity of a corn variety and application thereof.

Background

Corn is a major food crop; maize is mainly produced from hybrids. 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 the essential quality assurance for corn breeding research and development, seed production and seed transaction.

The purity identification of corn varieties is mainly dependent on the traditional field plot planting identification method (Grow-out Test) at present. The method comprises the steps of planting a variety in a field test cell, and identifying the purity of the corn variety by observing the difference of the vegetative morphological characteristics (such as height and size of the plant, tillering, leaf color, leaf shape, seed size, seed coat color and the like) and the biological characteristics (such as growth period, photoperiod, disease resistance, drought resistance, seed shattering property and the like) of the plant in different growth periods (seedling stage, growth, flowering stage, mature stage and seed). The method depends on visual identification of morphological characteristics and biological characteristics of plants in the field, the judgment standard is difficult to accurately quantify, the subjectivity is strong, and the detection sensitivity and the resolution are low; the method is easily influenced by environment and cultivation conditions, and has poor accuracy and stability; long time consumption and poor timeliness; large amount of manpower and material resources are needed, and the cost is high.

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

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides an 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 the application of the SNP locus, the primer group, the kit and/or the gene chip.

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

A SNP site according to an embodiment of the first aspect of the invention selected from at least one of the following first to fortieth SNP sites:

a first SNP locus, wherein the first SNP locus is located at 82587178 th nucleotide of 1 st chromosome of a maize reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is G/T;

a second SNP site, wherein the second SNP site is located at 291348623 th nucleotide of 1 st chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is G/A;

a third SNP locus, wherein the third SNP locus is located at 157283336 th nucleotide of 1 st chromosome of a maize reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is G/A;

a fourth SNP locus, wherein the fourth SNP locus is located at 213137297 th nucleotide of 1 st chromosome of a maize reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is A/G;

a fifth SNP locus, wherein the fifth SNP locus is located at 141204710 th nucleotide of 2 nd chromosome of a maize reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is C/T;

a sixth SNP locus, wherein the sixth SNP locus is located at 28211592 th nucleotide of 2 nd chromosome of a maize reference genome or at a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is C/G;

a seventh SNP locus, wherein the seventh SNP locus is located at 207814666 th nucleotide of 2 nd chromosome of a maize reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is G/A;

an eighth SNP locus, wherein the eighth SNP locus is located at 237488672 th nucleotide of 2 nd chromosome of a maize reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is T/G;

a ninth SNP site, wherein the ninth SNP site is located at 129484896 th nucleotide of 3 rd chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is G/A;

a tenth SNP site, wherein the tenth SNP site is located at 225786467 th nucleotide of 3 rd chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is A/C;

an eleventh SNP site, wherein the eleventh SNP site is located at 184720677 rd nucleotide of 3 rd chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is C/T;

a twelfth SNP site, wherein the twelfth SNP site is located at 22035145 th nucleotide of 3 rd chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is A/C;

a thirteenth SNP site, wherein the thirteenth SNP site is located at 157469658 th nucleotide of 4 th chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is T/C;

a fourteenth SNP site, which is located at 84668766 th nucleotide of the 4 th chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is T/G;

a fifteenth SNP site, wherein the fifteenth SNP site is located at 243991142 th nucleotide of the 4 th chromosome of a maize reference genome or at a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is C/T;

a sixteenth SNP locus, wherein the sixteenth SNP locus is located at 236715436 th nucleotide of the 4 th chromosome of a maize reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is G/A;

a seventeenth SNP site, wherein the seventeenth SNP site is located at 7486959 th nucleotide of the 5 th chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is T/C;

an eighteenth SNP locus, wherein the eighteenth SNP locus is located at 135108514 th nucleotide of the 5 th chromosome of a maize reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is C/T;

a nineteenth SNP site, wherein the nineteenth SNP site is located at 199452705 th nucleotide of 5 th chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is G/T;

a twentieth SNP site, wherein the twentieth SNP site is located at 215650073 th nucleotide of 5 th chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the twentieth SNP site is C/T;

a twenty-first SNP site, wherein the twenty-first SNP site is located at 13727981 th nucleotide of 6 th chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is C/A;

a twenty-second SNP site, wherein the twenty-second SNP site is located at 132514637 th nucleotide of 6 th chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is C/T;

a twenty-third SNP site, wherein the twenty-third SNP site is located at 95771968 th nucleotide of the 6 th chromosome of a maize reference genome or at a corresponding site on an interspecies homologous genomic fragment thereof, and the nucleotide base of the site is A/G;

a twenty-fourth SNP locus, wherein the twenty-fourth SNP locus is located at 160230101 th nucleotide of the 6 th chromosome of a maize reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is G/A;

a twenty-fifth SNP locus, wherein the twenty-fifth SNP locus is located at 8195312 th nucleotide of 7 th chromosome of a maize reference genome or a corresponding locus on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the locus is C/T;

a twenty-sixth SNP locus, wherein the twenty-sixth SNP locus is located at 74052945 th nucleotide of 7 th chromosome of a maize reference genome or a corresponding locus on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the locus is G/A;

a twenty-seventh SNP locus, wherein the twenty-seventh SNP locus is located at 169955912 th nucleotide of 7 th chromosome of a maize reference genome or a corresponding locus on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the locus is A/C;

a twenty-eighth SNP locus, wherein the twenty-eighth SNP locus is located at 136144222 th nucleotide of the 7 th chromosome of a maize reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is G/A;

a twenty-ninth SNP locus, wherein the twenty-ninth SNP locus is located at 4978106 th nucleotide of 8 th chromosome of a maize reference genome or a corresponding locus on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the locus is T/G;

a thirtieth SNP site, wherein the thirtieth SNP site is positioned at 126914854 th nucleotide of 8 th chromosome of a maize reference genome or a corresponding site on an interspecies homologous genome fragment of the maize reference genome, and the nucleotide base of the site is T/C;

a thirty-first SNP locus, wherein the thirty-first SNP locus is located at 173505727 th nucleotide of 8 th chromosome of a maize reference genome or a corresponding locus on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the locus is G/A;

a thirty-second SNP site, wherein the thirty-second SNP site is located at 75160235 th nucleotide of 8 th chromosome of a maize reference genome or a corresponding site on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the site is T/C;

a thirty-third SNP site, wherein the thirty-third SNP site is located at 11442148 th nucleotide of 9 th chromosome of a maize reference genome or a corresponding site on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the site is G/A;

a thirty-fourth SNP locus, wherein the thirty-fourth SNP locus is located at 150460620 th nucleotide of 9 th chromosome of a maize reference genome or a corresponding locus on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the locus is C/T;

a thirty-fifth SNP locus, wherein the thirty-fifth SNP locus is located at 89076495 th nucleotide of 9 th chromosome of a maize reference genome or a corresponding locus on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the locus is G/A;

a thirty-sixth SNP locus, wherein the thirty-sixth SNP locus is located at 121462317 th nucleotide of 9 th chromosome of a maize reference genome or a corresponding locus on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the locus is C/A;

a thirty-seventh SNP locus, wherein the thirty-seventh SNP locus is located at 15090132 th nucleotide of 10 th chromosome of a maize reference genome or a corresponding locus on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the locus is G/A;

a thirty-eighth SNP locus, wherein the thirty-eighth SNP locus is located at 41610224 th nucleotide of 10 th chromosome of a maize reference genome or a corresponding locus on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the locus is G/A;

a thirty-ninth SNP locus, wherein the thirty-ninth SNP locus is located at 113193861 th nucleotide of 10 th chromosome of a maize reference genome or a corresponding locus on homologous genome fragments among varieties of the maize reference genome, and the nucleotide base of the locus is G/C;

a fortieth SNP locus, wherein the fortieth SNP locus is located at 119856037 th nucleotide of 10 th chromosome of a maize reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is C/T;

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

The primer set for amplifying the SNP site according to the second aspect embodiment of the present invention.

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 group 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 general primer with a nucleotide sequence shown as SEQ ID NO. 3;

the second SNP primer group 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 also comprises a general primer nucleotide sequence shown as SEQ ID NO. 6;

a third SNP primer group, which is used for amplifying the third SNP locus and comprises a specific primer nucleotide sequence shown as SEQ ID NO.7 and SEQ ID NO.8 and a general primer nucleotide sequence shown as SEQ ID NO. 9;

the fourth SNP primer group is used for amplifying the fourth SNP locus and comprises specific primer nucleotide sequences shown as SEQ ID NO.10 and SEQ ID NO.11 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 12;

a fifth SNP primer group, which is used for amplifying the fifth SNP locus and comprises specific primer nucleotide sequences shown as SEQ ID NO.13 and SEQ ID NO.14 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 15;

a sixth SNP primer set used for amplifying the sixth SNP locus, which comprises specific primer nucleotide sequences shown as SEQ ID NO.16 and SEQ ID NO.17 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 18;

a seventh SNP primer set used for amplifying the seventh SNP locus, which comprises specific primer nucleotide sequences shown as SEQ ID NO.19 and SEQ ID NO.20 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 21;

the eighth SNP primer group is used for amplifying the eighth SNP locus and comprises specific primer nucleotide sequences shown as SEQ ID NO.22 and SEQ ID NO.23 and a general primer nucleotide sequence shown as SEQ ID NO. 24;

a ninth SNP primer set for amplifying the ninth SNP locus, which comprises specific primer nucleotide sequences shown as SEQ ID NO.25 and SEQ ID NO.26 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 27;

a tenth SNP primer set used for amplifying the tenth SNP locus, which comprises specific primer nucleotide sequences shown as SEQ ID NO.28 and SEQ ID NO.29 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 30;

an eleventh SNP primer set used for amplifying the eleventh SNP locus, which comprises a specific primer nucleotide sequence shown as SEQ ID NO.31 and SEQ ID NO.32 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 33;

a twelfth SNP primer set used for amplifying the twelfth SNP locus, which comprises specific primer nucleotide sequences shown as SEQ ID NO.34 and SEQ ID NO.35 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 36;

a thirteenth SNP primer set used for amplifying the thirteenth SNP locus, which comprises a specific primer nucleotide sequence shown as SEQ ID NO.37 and SEQ ID NO.38 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 39;

a fourteenth SNP primer group, which is used for amplifying the fourteenth SNP locus and comprises a specific primer nucleotide sequence shown as SEQ ID NO.40 and SEQ ID NO.41 and a general primer nucleotide sequence shown as SEQ ID NO. 42;

a fifteenth SNP primer set used for amplifying the fifteenth SNP locus, which comprises specific primer nucleotide sequences shown as SEQ ID NO.43 and SEQ ID NO.44 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 45;

a sixteenth SNP primer group, which is used for amplifying the sixteenth SNP locus and comprises a specific primer nucleotide sequence shown as SEQ ID NO.46 and SEQ ID NO.47 and a general primer nucleotide sequence shown as SEQ ID NO. 48;

a seventeenth SNP primer set used for amplifying the seventeenth SNP locus, which comprises a specific primer nucleotide sequence shown as SEQ ID NO.49 and SEQ ID NO.50 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 51;

the eighteenth SNP primer group is used for amplifying the eighteenth SNP locus and comprises specific primer nucleotide sequences shown as SEQ ID NO.52 and SEQ ID NO.53 and a universal primer nucleotide sequence shown as SEQ ID NO. 54;

a nineteenth SNP primer set, which is used for amplifying the nineteenth SNP locus and comprises specific primer nucleotide sequences shown as SEQ ID NO.55 and SEQ ID NO.56 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 57;

a twentieth SNP primer set which 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 a universal primer nucleotide sequence shown as SEQ ID NO. 60;

the twenty-first SNP primer group 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 a universal primer nucleotide sequence shown as SEQ ID NO. 63.

A second twelve SNP primer group, which is used for amplifying the second twelve SNP loci and comprises specific primers with nucleotide sequences shown as SEQ ID NO.64 and SEQ ID NO.65 and also comprises a universal primer with nucleotide sequence shown as SEQ ID NO. 66;

a twenty-third SNP primer group which is used for amplifying the twenty-third SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.67 and SEQ ID NO.68 and a general primer with a nucleotide sequence shown as SEQ ID NO. 69;

the twenty-fourth SNP primer group is used for amplifying the twenty-fourth SNP locus and comprises specific primers with nucleotide sequences shown as SEQ ID NO.70 and SEQ ID NO.71 and also comprises a universal primer with nucleotide sequence shown as SEQ ID NO. 72;

a twenty-fifth SNP primer group which is used for amplifying the twenty-fifth SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.73 and SEQ ID NO.74 and a general primer with a nucleotide sequence shown as SEQ ID NO. 75;

a twenty-sixth SNP primer group which is used for amplifying the twenty-sixth SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.76 and SEQ ID NO.77 and a general primer with a nucleotide sequence shown as SEQ ID NO. 78;

a twenty-seventh SNP primer group which is used for amplifying the twenty-seventh SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.79 and SEQ ID NO.80 and a general primer with a nucleotide sequence shown as SEQ ID NO. 81;

a twenty-eighth SNP primer group which 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 general primer with a nucleotide sequence shown as SEQ ID NO. 84;

a twenty-ninth SNP primer group, which is used for amplifying the twenty-ninth SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.85 and SEQ ID NO.86 and a general primer with a nucleotide sequence shown as SEQ ID NO. 87;

the thirtieth SNP primer set is used for amplifying the thirtieth SNP locus and comprises specific primers with nucleotide sequences shown as SEQ ID NO.88 and SEQ ID NO.89 and a universal primer with nucleotide sequence shown as SEQ ID NO. 90;

the thirty-first SNP primer group is used for amplifying the thirty-first SNP locus and comprises specific primers with nucleotide sequences shown as SEQ ID NO.91 and SEQ ID NO.92 and a universal primer with nucleotide sequence shown as SEQ ID NO. 93;

a third twelve SNP primer group, which is used for amplifying the third twelve SNP loci and comprises specific primers with nucleotide sequences shown as SEQ ID NO.94 and SEQ ID NO.95 and also comprises a universal primer with nucleotide sequence shown as SEQ ID NO. 96;

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

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

a fifteenth SNP primer set for amplifying the fifteenth SNP locus, which comprises specific primers with nucleotide sequences shown as SEQ ID NO.103 and SEQ ID NO.104, and also comprises a universal primer with nucleotide sequence shown as SEQ ID NO. 105;

a sixteenth SNP primer group, which is used for amplifying the sixteenth SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.106 and SEQ ID NO.107 and a general primer with a nucleotide sequence shown as SEQ ID NO. 108;

a seventeenth SNP primer group which is used for amplifying the seventeenth SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.109 and SEQ ID NO.110 and a general primer with a nucleotide sequence shown as SEQ ID NO. 111;

a thirty-eighth SNP primer group which 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 general primer with a nucleotide sequence shown as SEQ ID NO. 114;

a nineteenth SNP primer group, which is used for amplifying the nineteenth SNP locus and comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.115 and SEQ ID NO.116 and a general primer with a nucleotide sequence shown as SEQ ID NO. 117;

a fortieth SNP primer set used for amplifying the fortieth SNP locus, which comprises a specific primer with a nucleotide sequence shown as SEQ ID NO.118 and SEQ ID NO.119, and also comprises a general primer with a nucleotide sequence shown as SEQ ID NO. 120.

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

According to the third aspect of the invention, a kit for identifying the purity of a corn variety is provided, and the kit comprises the primer group.

According to the fourth aspect of the invention, a gene chip for identifying the purity of a corn variety is provided, and the gene chip comprises the primer group.

According to a fifth aspect embodiment of the present invention, the method for detecting the purity of the corn variety comprises the following steps:

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

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

s3, statistically analyzing the genotype of the individual to-be-detected sample 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, the simplified CTAB method (cetyl trimethyl ammonium bromide method) is used for extracting genomic DNA from corn.

In some embodiments of the present invention, in step S2, the SNP sites are detected using 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 a genotype of a corn individual to be tested is different from that of a plurality of other corn individuals to be tested at 2 or more SNP sites, the corn individual to be tested is determined to be a hybrid.

In some embodiments of the invention, the method of calculating breed purity is: percent of purity ═ total number of detected-number of hybrid plants)/total number of detected × 100%.

The application of the SNP site, the primer set, the kit and/or the gene chip according to the sixth embodiment of the invention is as follows:

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

(2) the application in the corn molecular marker assisted breeding;

(3) application in preparing corn breeding products.

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

The method for detecting the corn SNP locus has at least the following beneficial effects: the method can be used for accurately detecting the purity of corn varieties (lines) from different sources by screening a set of (4 SNP/chromosome) SNP markers with high quality and high polymorphism, and has wide application universality; the marker is a co-dominant marker, and has high specificity, sensitivity and resolution; the marker is not influenced by environmental conditions, seeds or any type of plant tissues can be used, the detection result is accurate, and the repeatability and the stability are good; different detection laboratories and different data results can be compared and verified with each other, and the data has universal comparability; the invention carries out genotyping on the SNP marker developed by the scheme of the invention by using the KASP technology based on the 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 percent, 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 the corn variety 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 flow chart of site development according to an embodiment of the present invention;

FIG. 2 is a diagram showing genotyping of SNP site ZM _ K _10000630 according to an embodiment of the present invention.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

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

The embodiment of the invention is as follows: SNP locus for detecting purity of corn variety

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 the markers are verified and detected by designing and synthesizing the primer sequences of the markers, which specifically comprises the following steps:

screening of 140 SNP loci for corn variety purity detection

Based on 156 parts of corn whole genome detection data, according to screening conditions: MAF is more than 0.1, the deletion rate is less than 0.02, chromosomes are uniformly distributed, PCA clustering effect is good, discrimination is high, two wing 25bp sequences are conserved, the genotype of a database sample corresponding to the selected SNP set at least has 2 site differences, a plurality of high-quality SNP sites are selected, and finally, the high-quality SNP sites with the least high-quality SNP combination of the scheme of the invention are selected to be 40 SNP sites. The basic information of the above 40 SNP sites is detailed in Table 1. Wherein the location of the SNP site on the chromosome is determined based on alignment of maize reference genome B73 sequences.

TABLE 1 physical location of SNP sites for purity determination of maize varieties

2 primer design

Design of KASP marker: KASP primer design was performed on the 40 selected SNP sites using BatchPrimer3(http:// probes. pw. usda. gov/BatchPrimer3 /). Each KASP marker consists of three primers, including two Allele-Specific primers X (Allle-Specific Primer X; Primer _ X) and Y (Allle-Specific Primer Y; Primer _ Y) and one Common Primer C (Common Primer; Primer _ C). Two allele-specific primers were attached 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 (AlleX); if only HEX fluorescence is detected, the genotype of the sample is homozygous Allele Y (Alley _ Y); if FAM and HEX fluorescence is detected simultaneously, the genotype of the sample is heterozygous (with both alleles X and Y). The allele types and primer sequences of 40 KASP markers for corn variety purity testing 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 tag detection procedure: the validation and detection of KASP markers was performed using the Array Tape system of Douglas Scientific. The Array Tape genotyping platform comprises NEXAR for PCR amplification system assembly, sollex for PCR amplification, ARAYA for fluorescent signal scanning, and INTELLICS for data analysis.

And (3) 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; performing a first-step amplification reaction, namely performing denaturation at 94 ℃ for 20 seconds, annealing at 65-57 ℃ and extending for 60 seconds for 10 cycles, wherein the annealing and extending temperature of each cycle is reduced by 0.8 ℃; the second amplification reaction, 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 finished, carrying out fluorescent signal scanning on the reaction system by using ARAYA; genotyping and data analysis were then performed with INTELLICS. In the KASP marker genotyping assay, the genotype of the sample is divided into 3 clusters, X, Y and heterozygous genotype clusters (see fig. 2). As shown in FIG. 2, it can be seen that the red color indicated in the graph, the X cluster located in the upper left corner of the graph indicates that the sample contains a homozygous X allele at this KASP marker locus, the blue color indicated in the graph, the Y cluster located in the lower right corner of the graph indicates that the sample contains a homozygous Y allele at this KASP marker locus, and the purple color indicated in the graph indicates that the sample contains both X and Y heterozygous alleles at this KASP marker locus.

TABLE 3 PCR reaction System for KASP detection

440 SNP locus verifications for corn variety purity detection

Genotyping quality verification of 40 KASP markers for corn variety purity testing: the verification of 40 KASP markers was performed with 376 corn diversity material according to the detection method described above. The verification shows that the two homozygous and heterozygous clusters of each KASP marker are well and compactly typed, the loci are single copy, the detection rate is higher than 99.5 percent, and the result is consistent with the sequencing result, and the result shows that 40 KASP markers can be accurately typed, and the genotyping quality can completely meet the accurate detection of the purity of corn varieties. Wherein, the KASP marker genotyping map of the position ZM _ K _10000630 is shown in FIG. 2, and the results of other positions are the same as those of ZM _ K _ 10000630.

5 calculation of maize variety purity

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

(1) and statistically analyzing the genotypes of the 40 SNP loci of 184 corns (corn B73) of the sample to be detected. If 2 or more sites in a certain individual to be detected are different from those of other individuals to be detected, judging the individual wheat to be detected to be a mixed plant; the single plant of which the difference number with the consistent locus of the control locus or the group (the genotype with the genotype frequency proportion of more than 50 percent is the consistent locus) in the individual to be detected is less than or equal to 1 is a pure plant; and if the SNP purity detection deletion site is less than 5 percent of the total detection site, judging the single plant as the effective single plant, and taking purity judgment statistics.

As shown in Table 4 (Table 4 includes tables 4-1, 4-2, and 4-3), the genotypes of the 184 individuals at the 40 SNP sites are consistent, and all the individuals are valid individuals (the number of valid individuals is the total number of detections); wherein, the individual with the number of 6 is different from other 183 individuals in 10 SNP sites such as ZM _ K _10000154, ZM _ K _10000225 and the like, and is judged as a hybrid; the individual with the number of 14 is different from the other 183 individuals in 8 SNP sites such as ZM _ K _10000154, ZM _ K _10000225 and the like, and is judged as a hybrid; the individual with the number of 15 is different from the other 183 individuals in 9 SNP sites such as ZM _ K _10000154, ZM _ K _10000225 and the like, and is judged as a hybrid; the individual with the number 27 is judged to be a hybrid when 10 SNP sites such as ZM _ K _10000225 and ZM _ K _10000154 are different from other 183 individuals; the individual with the number 63 is different from the other 183 individuals in 9 SNP sites 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 the other 183 individuals in 8 SNP sites 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 107 is different from the other 183 individuals in 9 SNP sites 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 164 is judged to be a hybrid when the individual is different from the other 183 individuals in 8 SNP sites such as ZM _ K _10000154, ZM _ K _10000225, ZM _ K _ 10000312; the individual with the number 169 is different from other 183 individuals in 8 SNP sites such as ZM _ K _10000154, ZM _ K _10000225 and the like, and is judged to be a hybrid; the individual with the number 176 was judged as a hybrid, differing from the other 183 individuals in 8 SNP sites such as ZM _ K _10000154, ZM _ K _10000225 and ZM _ K _ 10000312.

(2) And (4) calculating the purity of the variety according to the number of the individual hybrid strains and the total detection number in the step (1).

The degree of purity was calculated as: percent of purity ═ total number of detected-number of hybrid plants)/total number of detected × 100%.

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

TABLE 4-1

TABLE 4-2

Tables 4 to 3

In conclusion, 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 conjecture the pollution source of impure samples; the kit has a co-dominant marker and high specificity, sensitivity and resolution; the marker is not influenced by environmental conditions, seeds or any type of plant tissues can be used, the detection result is accurate, and the repeatability and the stability are good; the technology is simple, the automation is easy, the detection flux is high, and the speed is high; the detection cost of unit data points is low; the data results of different detection laboratories can be compared and verified with each other, and the data has universal comparability.

The Douglas Array Tap genotyping platform used in the present invention comprises NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescence signal scanning and INTELLICS for data analysis, and the consumables for its kit are all purchased from LGC company, 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 those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

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Claims

1. An SNP locus for detecting the purity of a corn variety, which is characterized in that: the site is selected from at least one of the following first SNP site to fortieth SNP site:

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

2. The primer group for amplifying the SNP sites for detecting the purity of the corn variety as claimed in claim 1.

3. The primer set according to claim 2, wherein the primer set comprises two specific primers and 1 universal primer, wherein the two specific primers are preferably connected with different fluorophore tag sequences; more preferably, the fluorophore tag sequence is selected from FAM, HEX.

4. The primer set according to claim 2, wherein the primer set comprises:

5. A kit comprising the primer set according to any one of claims 2 to 4.

6. A gene chip comprising the primer set according to any one of claims 2 to 4.

7. Any one of the following uses of the SNP site according to claim 1, the primer set according to any one of claims 2 to 4, the kit according to claim 5, or the gene chip according to claim 6:

(2) the application in the corn molecular marker assisted breeding;

(3) application in preparing corn breeding products.

8. The method for detecting the purity of the maize variety by using the SNP locus according to claim 1, which comprises the following steps:

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

9. The method of claim 8, wherein the individual maize is determined to be a hybrid if the genotype of the individual maize is different from that of the majority of the individuals to be tested at 2 or more SNP sites.

10. A method for breeding corn, comprising the following steps: the method for detecting the purity of the corn variety as claimed in claim 8 or 9, wherein the corn with the required purity is selected for subsequent breeding.