CN113403423A

CN113403423A - Non-heading Chinese cabbage core SNP molecular marker set developed based on KASP technology and application thereof

Info

Publication number: CN113403423A
Application number: CN202110913496.1A
Authority: CN
Inventors: 张艳; 陈汉才; 黎庭耀; 沈卓; 杨易; 周轩; 吴增祥
Original assignee: Vegetable Research Institute of Guangdong Academy of Agriculture Sciences
Current assignee: Vegetable Research Institute of Guangdong Academy of Agriculture Sciences
Priority date: 2021-08-10
Filing date: 2021-08-10
Publication date: 2021-09-17
Anticipated expiration: 2041-08-10
Also published as: CN113403423B

Abstract

The invention belongs to the technical field of molecular biology and plant molecular breeding, and particularly relates to a core SNP molecular marker set of a non-heading Chinese cabbage developed based on a KASP technology and application thereof, in order to develop a rapid, accurate and effective identification and evaluation method for germplasm and variety of the non-heading Chinese cabbage.

Description

Non-heading Chinese cabbage core SNP molecular marker set developed based on KASP technology and application thereof

Technical Field

The invention belongs to the technical field of molecular biology and plant molecular breeding, and particularly relates to a non-heading Chinese cabbage core SNP molecular marker set developed based on a KASP technology and application thereof.

Background

Non-heading Chinese cabbage (Brassica rapa ssp. chinensis Makino) is a Brassica seed Brassica rapa subspecies of Brassicaceae, and comprises various varieties such as pakchoi, Brassica rapa, Brassica oleracea, Wuta-tsai, tillered vegetable and the like. The non-heading Chinese cabbage has the characteristics of short growth cycle, rich nutrition, strong adaptability and the like, is widely cultivated all over the world, and is a leaf vegetable with important economic value.

Molecular markers (RAPD, SSR, Indel, SNP and the like) are widely applied to aspects of genetic diversity evaluation of genetic resources of the non-heading Chinese cabbage, genetic map construction, gene positioning, molecular marker assisted breeding and the like. Among them, the RAPD technique is susceptible to various factors. Regardless of the quality and concentration of the template, short primer sequences, the number of PCR cycles, the complexity of the genomic DNA, the technical equipment, etc., may lead to unstable and difficult to repeat RAPD results. SSR markers have been widely used in genetic breeding of vegetable crops in China due to the advantages of high polymorphism, simple operation and the like. But it does not meet the needs for large-scale, high-throughput, automated testing. The SNP marker as a third generation molecular marker has the following advantages: firstly, the density is high and the distribution is more uniform in the genome; secondly, the flux is high, and the number of detection sites can reach millions; and thirdly, automation of data statistics and data integration comparison are easy to realize. At present, SNP has been applied to variety identification and seed purity detection of vegetable crops such as hot pepper, melon, watermelon and cucumber. With the development of high-throughput genotyping technology, Competitive allele-specific PCR (KASP) technology with higher SNP use efficiency and assay throughput has emerged. KASP is a genotyping technology based on fluorescence detection, namely SNP is typed based on specific matching of base at the tail end of a primer, and a plurality of SNP sites are detected by two site specific probes and 2 fluorescent probes, so that the target SNP is precisely typed by double alleles, and the method has high stability and accuracy. The technology is mainly applied to SNP or Indel gene typing research at present and is gradually becoming a main technical means for genetic map construction, gene positioning and molecular assisted breeding technology germplasm resource assessment.

The traditional identification and evaluation of the germplasm and variety of the non-heading Chinese cabbage mainly depends on phenotype. With the continuous promotion of the collection and protection work of the germplasm resources of the Chinese cabbage and the annual increase of the number of breeding hybrid varieties, the traditional phenotype identification method becomes the bottleneck of the efficient utilization of the germplasm resources due to time and labor consumption, long period and low accuracy.

Therefore, there is a need to develop a rapid, accurate and effective identification and evaluation method for the germplasm and variety of non-heading Chinese cabbage.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a core SNP molecular marker set of the non-heading Chinese cabbage developed based on the KASP technology, and the identification and evaluation of the germplasm and variety of the non-heading Chinese cabbage can be realized based on the SNP molecular marker set.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a core SNP molecular marker set of a non-heading Chinese cabbage, which is developed based on a KASP technology, and comprises 42 SNP markers, wherein the number of the 42 SNP markers is SNP 1-42 (SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, SNP12, SNP 13);

the SNP1 is marked as a basic group C/T, is positioned on the chromosome 1 of the non-heading Chinese cabbage and is positioned at the 21612761 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP2 is marked as a basic group C/T, is positioned on the chromosome 1 of the non-heading Chinese cabbage and is positioned at the 21624075 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP3 is marked as a base G/A, is positioned on the chromosome 1 of the non-heading Chinese cabbage and is positioned at the 27291001 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP4 is marked as a basic group C/T, is positioned on the chromosome 1 of the non-heading Chinese cabbage and is positioned at the 29042081 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP5 is marked as a basic group C/T, is positioned on the No. 2 chromosome of the non-heading Chinese cabbage and is positioned at the 12579271 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP6 is marked as a base T/A, is positioned on the No. 2 chromosome of the non-heading Chinese cabbage and is positioned at the 14670199 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP7 is marked as a base T/C, is positioned on the No. 2 chromosome of the non-heading Chinese cabbage and is positioned at the 6287090 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP8 is marked as a base G/C, is positioned on the No. 2 chromosome of the non-heading Chinese cabbage and is positioned at the 17101169 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP9 is marked as a basic group C/A, is positioned on the chromosome 3 of the non-heading Chinese cabbage and is positioned at the 7872365 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP10 is marked as a base T/G, is positioned on the chromosome 3 of the non-heading Chinese cabbage and is positioned at the 27336042 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP11 is marked as a base T/C, is positioned on the chromosome 3 of the non-heading Chinese cabbage and is positioned at the 10862382 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP12 is marked as a base T/A, is positioned on the chromosome 3 of the non-heading Chinese cabbage and is positioned at the 10870310 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP13 is marked as a basic group C/T, is positioned on the chromosome 3 of the non-heading Chinese cabbage and is positioned at the 11407563 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP14 is marked as a base T/C, is positioned on the chromosome 3 of the non-heading Chinese cabbage and is positioned at the 14544571 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP15 is marked as a basic group C/T, is positioned on the chromosome 4 of the non-heading Chinese cabbage and is positioned at the 10563214 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP16 is marked as a basic group C/G, is positioned on the chromosome 4 of the non-heading Chinese cabbage and is positioned at the 19380396 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP17 is marked as a basic group C/T, is positioned on the chromosome 4 of the non-heading Chinese cabbage and is positioned at the 11760387 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP18 is marked as a basic group C/G, is positioned on the chromosome 4 of the non-heading Chinese cabbage and is positioned at the 21624754 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP19 is marked as a base G/C, is positioned on a No. 5 chromosome of the non-heading Chinese cabbage and is positioned at the 1066885 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP20 is marked as a base G/T, is positioned on the chromosome 5 of the non-heading Chinese cabbage and is positioned at the 17807854 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP21 is marked as a basic group C/A, is positioned on a No. 5 chromosome of the non-heading Chinese cabbage and is positioned at the 1889510 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP22 is marked as a basic group C/T, is positioned on a No. 5 chromosome of the non-heading Chinese cabbage and is positioned at the 1581635 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP23 is marked as a base G/A, is positioned on the No. 6 chromosome of the non-heading Chinese cabbage and is positioned at the 23758128 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP24 is marked as a base G/C, is positioned on the No. 6 chromosome of the non-heading Chinese cabbage and is positioned at the 25457983 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP25 is marked as a basic group T/C, is positioned on the No. 6 chromosome of the non-heading Chinese cabbage and is positioned at the 18142570 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP26 is marked as a base G/C, is positioned on the No. 6 chromosome of the non-heading Chinese cabbage and is positioned at the 19301511 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP27 is marked as a base G/C, is positioned on the No. 7 chromosome of the non-heading Chinese cabbage and is positioned at the 15969630 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP28 is marked as a base T/C, is positioned on the No. 7 chromosome of the non-heading Chinese cabbage and is positioned at the 14610710 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP29 is marked as a base G/T, is positioned on the No. 7 chromosome of the non-heading Chinese cabbage and is positioned at the 15631302 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP30 is marked as a base A/G, is positioned on the No. 7 chromosome of the non-heading Chinese cabbage and is positioned at the 1750463 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP31 is marked as a base G/C, is positioned on a No. 8 chromosome of the non-heading Chinese cabbage and is positioned at the 18036348 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP32 is marked as a basic group T/C, is positioned on a No. 8 chromosome of the non-heading Chinese cabbage and is positioned at the 19882040 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP33 is marked as a basic group C/T, is positioned on a No. 8 chromosome of the non-heading Chinese cabbage and is positioned at the 1929087 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP34 is marked as a base T/A, is positioned on a No. 8 chromosome of the non-heading Chinese cabbage and is positioned at the 14605882 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP35 is marked as a basic group C/G, is positioned on the chromosome 9 of the non-heading Chinese cabbage and is positioned at the 37635371 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP36 is marked as a basic group C/A, is positioned on the chromosome 9 of the non-heading Chinese cabbage and is positioned at the 3011899 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP37 is marked as a basic group C/A, is positioned on the chromosome 9 of the non-heading Chinese cabbage and is positioned at the 34173525 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP38 is marked as a base G/T, is positioned on the chromosome 9 of the non-heading Chinese cabbage and is positioned at the 39079259 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP39 is marked as a base G/A, is positioned on a 10 th chromosome of the non-heading Chinese cabbage and is positioned at the 9999223 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP40 is marked as a basic group C/A, is positioned on a 10 th chromosome of the non-heading Chinese cabbage and is positioned at the 11493584 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP41 is marked as a base A/T, is positioned on the 10 th chromosome of the non-heading Chinese cabbage and is positioned at the 16824105 th site of the whole genome sequence of the non-heading Chinese cabbage;

the SNP42 is marked as a base T/A, is positioned on a 10 th chromosome of the non-heading Chinese cabbage and is positioned at the 19172885 th site of the whole genome sequence of the non-heading Chinese cabbage.

The invention also provides KASP primers for amplifying the core SNP molecular marker set of the non-heading Chinese cabbage, wherein the KASP primers comprise 42 KASP primer combinations, and each KASP primer combination is used for amplifying a corresponding SNP marker.

Further, each KASP primer combination consists of two allele forward primers F1 and F2 differing in terminal base and one reverse primer R.

Furthermore, a FAM fluorescent tag sequence is added to the 5' end of the primer F1, and the FAM fluorescent tag sequence is shown as SEQ ID NO. 127; the 5' end of the primer F2 is added with a HEX fluorescent tag sequence, and the HEX fluorescent tag sequence is shown as SEQ ID NO: 128.

Specifically, the KASP primer consists of a nucleotide sequence shown in SEQ ID NO. 1-126. Specifically, KASP primer combinations corresponding to 42 SNP markers (SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, SNP12, SNP13, SNP14, SNP15, SNP16, SNP17, SNP18, SNP19, SNP20, SNP21, SNP22, SNP23, SNP24, SNP25, SNP26, SNP27, 28, SNP29, SNP30, SNP31, SNP32, SNP33, SNP34, SNP35, SNP36, SNP37, SNP38, SNP39, SNP40, SNP41, SNP42) are: 1-3 parts of SEQ ID NO, 4-6 parts of SEQ ID NO, 7-9 parts of SEQ ID NO, 10-12 parts of SEQ ID NO, 13-15 parts of SEQ ID NO, 16-18 parts of SEQ ID NO, 19-21 parts of SEQ ID NO, 22-24 parts of SEQ ID NO, 25-27 parts of SEQ ID NO, 28-30 parts of SEQ ID NO, 31-33 parts of SEQ ID NO, 34-36 parts of SEQ ID NO, 37-39 parts of SEQ ID NO, 40-42 parts of SEQ ID NO, 43-45 parts of SEQ ID NO, 46-48 parts of SEQ ID NO, 49-51 parts of SEQ ID NO, 52-54 parts of SEQ ID NO, 55-57 parts of SEQ ID NO, 58-60 parts of SEQ ID NO, 61-63 parts of SEQ ID NO, 64-66 parts of SEQ ID NO, 67-69 parts of SEQ ID NO, 70-72 parts of SEQ ID NO, 73-75 parts of SEQ ID NO, 76-78 parts of SEQ ID NO, SEQ ID NO, 79-81 parts of SEQ ID NO, 82-84 parts of SEQ ID NO, 85-87 parts of SEQ ID NO, 88-90 parts of SEQ ID NO, 91-93 parts of SEQ ID NO, 94-96 parts of SEQ ID NO, 97-99 parts of SEQ ID NO, 100-102 parts of SEQ ID NO, 103-105 parts of SEQ ID NO, 105-108 parts of SEQ ID NO, 109-111 parts of SEQ ID NO, 112-114 parts of SEQ ID NO, 115-1117 parts of SEQ ID NO, 118-120 parts of SEQ ID NO, 121-123 parts of SEQ ID NO and 124-126 parts of SEQ ID NO.

The invention also provides a detection product containing the KASP primer, and the detection product comprises a detection reagent, a kit and a chip.

The invention also provides the application of the non-heading Chinese cabbage core SNP molecular marker set or the KASP primer or the detection product in any one of the following aspects:

(1) identifying germplasm resources or varieties of the non-heading Chinese cabbages;

(2) constructing a non-heading Chinese cabbage SNP fingerprint gallery;

(3) analyzing genetic diversity of the non-heading Chinese cabbage;

(4) constructing a genetic map of the non-heading Chinese cabbage and genotyping;

(5) molecular marker assisted breeding of non-heading Chinese cabbage;

(6) detecting the purity of the non-heading Chinese cabbage germplasm resources or varieties;

(7) molecular gene mapping of non-heading Chinese cabbage.

Preferably, the specific method of the above application comprises the steps of:

s1, extracting genome DNA of a non-heading Chinese cabbage sample;

s2, using the DNA in the step S1 as a template, adopting the KASP primer of any one of claims 2-5 to perform PCR amplification, and placing the PCR amplification product on a Bio-Rad CFX Connect real-time fluorescence quantitative PCR instrument to obtain a corresponding product fluorescence signal value to complete genotyping;

s3, analyzing the experimental result of the step S2 by using data analysis software Bio-Rad CFX Manager 3.1.

Further, the reaction system for PCR amplification was 10. mu.L, including 2. mu.L of DNA at 20 ng/. mu.L to 30 ng/. mu.L, 0.28. mu.L of each KASP primer combination (0.07. mu.L of each of the two F primers, 0.14. mu.L of the R primer), and 5. mu.L of KASP Master mix.

Further, the reaction conditions for PCR amplification are: 15min at 94 ℃; 94 ℃,20sec, 63-55 ℃,1min, reducing the temperature by 0.8 ℃ in each cycle, and taking 10 cycles; 26 cycles of 94 ℃,20sec, 55 ℃,1 min; the reading conditions of the fluorescence signal values were 16 ℃ for 20 s.

Compared with the prior art, the invention has the beneficial effects that:

the core SNP molecular marker set of the non-heading Chinese cabbage is developed based on the KASP technology, can quickly, accurately and effectively identify and evaluate germplasm resource materials or breed varieties, and can solve the problems of resource redundancy, inconvenient storage and management, intellectual property disputes and the like caused by mass germplasm accumulation. Provides molecular markers for genetic map construction, gene positioning, molecular assisted breeding and the like.

Drawings

FIG. 1 is a polymorphism map of A06:19301511 marker in 82 resources;

FIG. 2 is a polymorphism map of A03:7876365 marker in 82 resources;

FIG. 3 is a KaSP tag based genetic distance clustering chart for non-heading Chinese cabbage;

FIG. 4 is a finger print of 25 local germplasm resources of Chinese cabbage of Dongdong province.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

Example 1 development of core SNP molecular markers of non-heading Chinese cabbage based on KASP technology

1. KASP primer design

Extracting genome DNA of the leaf mustard heart for 70 days (Guangdong province crop germplasm resource protection library GD II 2D00187) and the leaf mustard heart for C40 (Guangdong province crop germplasm resource protection library GD II 2D 00114).

The genetic re-sequencing is carried out on genome DNA of 70 days and C40 by utilizing an illumina HiSeqTM technology, the re-sequencing result and the Chinese cabbage genome (http:// fibrous db. cn /) are compared and analyzed, 150 SNP loci with polymorphism and uniform distribution on chromosomes are selected, and 150 SNP loci without other variation exist in 50bp before and after the loci, and then primers are designed to convert the 150 SNP loci into KASP markers.

The method for designing the primer comprises the following steps: KASP marker primers were designed for differential sites based on SNP sites and flanking sequences using the software Primer Premier 5.0 to develop KASP molecular markers by the Primer design method described by Awais et al (Awais R, Wen W E, Gao F M, ZHai S N, Jin H, Liu J D, Guo Q, Zhang Y J, Dreisidentifier S, Xia X C, He Z H.development and evaluation of KASP assays for genes interfacing with electronic primers in branched from molecular and tagged Genetics,2016,129(10):1843 1860). 2 SNP specific primers (F1/F2) and one universal primer (R) are designed for each marker, wherein a specific sequence 5'-GAAGGTGACCAAGTTCATGCT-3' capable of binding with FAM fluorescence is added to the tail of F1, and a specific sequence 5'-GAAGGTCGGAGTCAACGGATT-3' capable of binding with HEX fluorescence is added to the tail of F2. The primers were synthesized by Biotechnology engineering (Shanghai) Inc.

2. KASP primer screening

(1) And (2) collecting 82 parts of germplasm resources of the non-heading Chinese cabbage to form a natural population of the non-heading Chinese cabbage, wherein the natural population comprises 53 parts of flowering cabbage germplasm resources and 29 parts of Chinese cabbage germplasm resources (shown in table 1).

Table 182 details of germplasm resources of non-heading Chinese cabbage

(2) Extracting DNA of the non-heading Chinese cabbage:

firstly, taking about 2g of tender leaves of the non-heading Chinese cabbage, grinding the tender leaves by using liquid nitrogen, quickly adding 1000 mu L of 2% CTAB extraction buffer solution when the liquid nitrogen is quickly evaporated to be dry, uniformly mixing, placing the mixture in a water bath at 65 ℃ for warm bath for 50min, and shaking the mixture once every 10 min;

② standing to room temperature, centrifuging at 12000rpm at 4 ℃ for 10min, transferring about 800 microliter of supernatant to a new 2mL centrifuge tube;

③ adding chloroform/isoamyl alcohol with the same volume (24: 1), reversing and mixing evenly, standing for 3-5 minutes, centrifuging at 12000rpm at 4 ℃ for 10min, transferring about 600 mu L of supernatant into a new centrifugal tube of 1.5 mL;

adding equal volume of isopropanol precooled at-20 ℃, slowly mixing uniformly, slowly reversing for 20 times, and culturing at-20 ℃ for 30 min;

centrifuging at 12000rpm at 4 deg.C for 10min to obtain white precipitate at the bottom, discarding the supernatant, washing with 800 μ L of 75% and 95% ethanol for twice, discarding the supernatant, and air drying at room temperature in a fume hood;

sixthly, adding 100 mu L of sterile water to dissolve, and obtaining the DNA of the non-heading Chinese cabbage germplasm resource.

The KASP primers designed for development were screened using "70 days" and "C40" as experimental materials. Using the "70 day" and "C40" DNAs as templates, KASP primers and a general-purpose KASPMaster mix (available from Guangzhou, Mass., Biotechnology, Inc.) were added for PCR (polymerase chain reaction) amplification. The PCR reaction system is as follows: 10 u L including 20 ng/L ~ 30 ng/L DNA2 u L, the molecular marker primer total 0.28 u L, two F primers 0.07 u L, R primer 0.14 u L, and 5 u L2 x KASPMaster mix. The reaction conditions were: 15min at 94 ℃; 94 ℃,20sec, 63-55 ℃,1min, reducing the temperature by 0.8 ℃ in each cycle, and taking 10 cycles; 94 ℃,20sec, 55 ℃,1min, for 26 cycles. After the reaction is finished, placing the PCR amplification product on a Bio-Rad CFX Connect real-time fluorescence quantitative PCR instrument to obtain a corresponding product fluorescence signal value, and finishing genotyping. The reading conditions of the fluorescence signal values were 16 ℃ for 20 s.

And carrying out genotype detection on 82 parts of non-heading Chinese cabbage germplasm resources by using the successfully-typed KASP markers to finally obtain 42 clearly-typed high-quality KASP markers, wherein the 42 KASP markers are uniformly distributed on 10 chromosomes of the non-heading Chinese cabbage to form the non-heading Chinese cabbage core SNP molecular marker set. The name and nucleotide sequence of each primer pair are shown in Table 2.

Calculating the allele frequency and the polymorphism information content of each MARKER by using POWER MARKER V3.25 software, wherein the variation range of the Polymorphism Information Content (PIC) of 42 KASP MARKERs in 82 parts of non-heading Chinese cabbage germplasm resources is 0.1700-0.4944, the average value is 0.4032, and sites with PIC values larger than 0.4032 account for 61.9% of all sites; major Allele Frequency (MAF) varied from 0.4817 to 0.9024 with an average of 0.6289 (polymorphism maps for partial markers are shown in FIGS. 1 and 2).

TABLE 2 primer information of non-heading Chinese cabbage core SNP molecular marker developed based on KASP technique

Example 2 application of KASP molecular marker of non-heading Chinese cabbage in genetic diversity analysis of non-heading Chinese cabbage

Genotyping data for the 42 KASP-labeled natural populations of Brassica campestris in example 1 were used to form an A-B matrix, and the Power MARKER V3.25 software was used to calculate the Nei's genetic distance between various germplasm resources. The result shows that the genetic distance of the Nei's of 82 germplasm resources is 0.2352-0.9594, and the average value is 0.5025. Meanwhile, a phylogenetic tree is constructed according to the distance matrix, Neighbor-Joining clustering analysis is carried out, and the generated result is shown in figure 3, so that 82 parts of non-heading Chinese cabbage germplasm resources can be divided into three groups.

Example 3 development of core SNP molecular markers of non-heading Chinese cabbage based on KASP technology and application of core SNP molecular markers to construction of germplasm resource fingerprint of non-heading Chinese cabbage

Screening the markers with PIC values larger than the average value of 0.4032 and deletion rate smaller than 0.1 and evenly distributed on 10 chromosomes from the 42 KASP markers in the embodiment 1 as core markers for constructing the germplasm resource fingerprint of the non-heading Chinese cabbage, and finally obtaining 21 KASP markers for constructing the fingerprint of the germplasm resource of the non-heading Chinese cabbage.

And (3) carrying out genotype detection on 26 local germplasm resources (shown in table 3) of the non-heading Chinese cabbage in Guangdong province by using 21 core KASP markers to construct a DNA fingerprint of the germplasm resources of the non-heading Chinese cabbage. The core locus typing results of 26 genetic resources of the Guangdong non-heading Chinese cabbage are converted into binary coded data, and the fingerprint spectrum of the genetic resources of the Guangdong non-heading Chinese cabbage is obtained (figure 4). The DNA fingerprint spectrum can be used for effectively distinguishing local germplasm resources of the Chinese cabbage of the Guangdong province.

Table 326 details table of germplasm resources in ungrounded places of Guangdong province

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Sequence listing

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<213> SNP7-F2(Artificial Sequence)

<400> 20

tgaaggtcgg agtcaacgga ttccaaaatc tcagatttca agattc 46

<210> 21

<211> 25

<212> DNA

<213> SNP7-R(Artificial Sequence)

<400> 21

cgcttcatgg gataaacaag aagtg 25

<210> 22

<211> 44

<212> DNA

<213> SNP8-F1(Artificial Sequence)

<400> 22

tgaaggtgac caagttcatg cttccccaag aagtccaaac tgtg 44

<210> 23

<211> 44

<212> DNA

<213> SNP8-F2(Artificial Sequence)

<400> 23

tgaaggtcgg agtcaacgga tttccccaag aagtccaaac tgtc 44

<210> 24

<211> 25

<212> DNA

<213> SNP8-R(Artificial Sequence)

<400> 24

gtagctcgga agtagcagaa tctcc 25

<210> 25

<211> 44

<212> DNA

<213> SNP9-F1(Artificial Sequence)

<400> 25

tgaaggtgac caagttcatg ctacctcaaa accgtttctt ttgc 44

<210> 26

<211> 44

<212> DNA

<213> SNP9-F2(Artificial Sequence)

<400> 26

tgaaggtcgg agtcaacgga ttacctcaaa accgtttctt ttga 44

<210> 27

<211> 20

<212> DNA

<213> SNP9-R(Artificial Sequence)

<400> 27

ttccggcgtc tctgcttctg 20

<210> 28

<211> 44

<212> DNA

<213> SNP10-F1(Artificial Sequence)

<400> 28

tgaaggtgac caagttcatg ctggtgtttg aagatgctcc ttct 44

<210> 29

<211> 44

<212> DNA

<213> SNP10-F2(Artificial Sequence)

<400> 29

tgaaggtcgg agtcaacgga ttggtgtttg aagatgctcc ttcg 44

<210> 30

<211> 23

<212> DNA

<213> SNP10-R(Artificial Sequence)

<400> 30

cactaactgc tcatccgctg agg 23

<210> 31

<211> 44

<212> DNA

<213> SNP11-F1(Artificial Sequence)

<400> 31

gaaggtgacc aagttcatgc ttctgctttg agatctttgg aact 44

<210> 32

<211> 44

<212> DNA

<213> SNP11-F2(Artificial Sequence)

<400> 32

gaaggtcgga gtcaacggat ttctgctttg agatctttgg aacc 44

<210> 33

<211> 22

<212> DNA

<213> SNP11-R(Artificial Sequence)

<400> 33

caaactcaca acggtacctc ag 22

<210> 34

<211> 43

<212> DNA

<213> SNP12-F1(Artificial Sequence)

<400> 34

gaaggtgacc aagttcatgc tgccgtcgat gaccatgctc tgt 43

<210> 35

<211> 43

<212> DNA

<213> SNP12-F2(Artificial Sequence)

<400> 35

gaaggtcgga gtcaacggat tgccgtcgat gaccatgctc tga 43

<210> 36

<211> 22

<212> DNA

<213> SNP12-R(Artificial Sequence)

<400> 36

caccataggc gtcgagtttc ag 22

<210> 37

<211> 44

<212> DNA

<213> SNP13-F1(Artificial Sequence)

<400> 37

gaaggtgacc aagttcatgc tataagcatc ggaagatgac tagc 44

<210> 38

<211> 44

<212> DNA

<213> SNP13-F2(Artificial Sequence)

<400> 38

gaaggtcgga gtcaacggat tataagcatc ggaagatgac tagt 44

<210> 39

<211> 23

<212> DNA

<213> SNP13-R(Artificial Sequence)

<400> 39

tgctcagatc atatagtagc atc 23

<210> 40

<211> 44

<212> DNA

<213> SNP14-F1(Artificial Sequence)

<400> 40

gaaggtgacc aagttcatgc taatgaagca acctcactag atct 44

<210> 41

<211> 44

<212> DNA

<213> SNP14-F2(Artificial Sequence)

<400> 41

gaaggtcgga gtcaacggat taatgaagca acctcactag atcc 44

<210> 42

<211> 23

<212> DNA

<213> SNP14-R(Artificial Sequence)

<400> 42

gactcaactg aacctaagcc atg 23

<210> 43

<211> 43

<212> DNA

<213> SNP15-F1(Artificial Sequence)

<400> 43

gaaggtgacc aagttcatgc ttgtgaattc ctatttgcgt ctc 43

<210> 44

<211> 43

<212> DNA

<213> SNP15-F2(Artificial Sequence)

<400> 44

gaaggtcgga gtcaacggat ttgtgaattc ctatttgcgt ctt 43

<210> 45

<211> 25

<212> DNA

<213> SNP15-R(Artificial Sequence)

<400> 45

ggagaggaag tagaagccga cattg 25

<210> 46

<211> 43

<212> DNA

<213> SNP16-F1(Artificial Sequence)

<400> 46

gaaggtgacc aagttcatgc tcttggagtt tcaggtctct ttc 43

<210> 47

<211> 43

<212> DNA

<213> SNP16-F2(Artificial Sequence)

<400> 47

gaaggtcgga gtcaacggat tcttggagtt tcaggtctct ttg 43

<210> 48

<211> 25

<212> DNA

<213> SNP16-R(Artificial Sequence)

<400> 48

ccgaagtcag agagctttgc gttcc 25

<210> 49

<211> 43

<212> DNA

<213> SNP17-F1(Artificial Sequence)

<400> 49

gaaggtagcc aagttcatgc tacgaaaggc taagtgctta tcc 43

<210> 50

<211> 43

<212> DNA

<213> SNP17-F2(Artificial Sequence)

<400> 50

gaaggtcgga gtcaacggat tacgaaaggc taagtgctta tct 43

<210> 51

<211> 25

<212> DNA

<213> SNP17-R(Artificial Sequence)

<400> 51

atgaggaagg gcatcaagac cagtg 25

<210> 52

<211> 43

<212> DNA

<213> SNP18-F1(Artificial Sequence)

<400> 52

gaaggtgacc aagttcatgc tagagaatgt aataaacctt ctc 43

<210> 53

<211> 43

<212> DNA

<213> SNP18-F2(Artificial Sequence)

<400> 53

gaaggtcgga gtcaacggat tagagaatgt aataaacctt ctg 43

<210> 54

<211> 25

<212> DNA

<213> SNP18-R(Artificial Sequence)

<400> 54

gaacgataga tgctgtttct tcctg 25

<210> 55

<211> 43

<212> DNA

<213> SNP19-F1(Artificial Sequence)

<400> 55

gaaggtgacc aagttcatgc tgtaagagag aaaacggcag gag 43

<210> 56

<211> 43

<212> DNA

<213> SNP19-F2(Artificial Sequence)

<400> 56

gaaggtcgga gtcaacggat tgtaagagag aaaacggcag gac 43

<210> 57

<211> 25

<212> DNA

<213> SNP19-R(Artificial Sequence)

<400> 57

acagggtcaa cggagaagtg tttac 25

<210> 58

<211> 44

<212> DNA

<213> SNP20-F1(Artificial Sequence)

<400> 58

gaaggtgacc aagttcatgc tctggtaacg aagacctata ctcg 44

<210> 59

<211> 44

<212> DNA

<213> SNP20-F2(Artificial Sequence)

<400> 59

gaaggtcgga gtcaacggat tctggtaacg aagacctata ctct 44

<210> 60

<211> 22

<212> DNA

<213> SNP20-R(Artificial Sequence)

<400> 60

ctggttgatt ccttggcgag ac 22

<210> 61

<211> 44

<212> DNA

<213> SNP21-F1(Artificial Sequence)

<400> 61

gaaggtgacc aagttcatgc tgagaaggag ttttctcgaa tccc 44

<210> 62

<211> 44

<212> DNA

<213> SNP21-F2(Artificial Sequence)

<400> 62

gaaggtcgga gtcaacggat tgagaaggag ttttctcgaa tcca 44

<210> 63

<211> 22

<212> DNA

<213> SNP21-R(Artificial Sequence)

<400> 63

ctttaagcgc gctagcgaaa gc 22

<210> 64

<211> 44

<212> DNA

<213> SNP22-F1(Artificial Sequence)

<400> 64

gaaggtgacc aagttcatgc taggaagccc catgatgaat cctc 44

<210> 65

<211> 44

<212> DNA

<213> SNP22-F2(Artificial Sequence)

<400> 65

gaaggtcgga gtcaacggat taggaagccc catgatgaat cctt 44

<210> 66

<211> 22

<212> DNA

<213> SNP22-R(Artificial Sequence)

<400> 66

tgtggaaagc aaagtgttgg ac 22

<210> 67

<211> 46

<212> DNA

<213> SNP23-F1(Artificial Sequence)

<400> 67

gaaggtgacc aagttcatgc tagtttattt gcagaatgat atgggg 46

<210> 68

<211> 47

<212> DNA

<213> SNP23-F2(Artificial Sequence)

<400> 68

gaaggtcgga gtcaacggat taagtttatt tgcagaatga tatggga 47

<210> 69

<211> 29

<212> DNA

<213> SNP23-R(Artificial Sequence)

<400> 69

taaatataaa gtatcaccct tcactcact 29

<210> 70

<211> 46

<212> DNA

<213> SNP24-F1(Artificial Sequence)

<400> 70

gaaggtgacc aagttcatgc tgaaggtcag agtcttccta aacccg 46

<210> 71

<211> 46

<212> DNA

<213> SNP24-F2(Artificial Sequence)

<400> 71

gaaggtcgga gtcaacggat tgaaggtcag agtcttccta aacccc 46

<210> 72

<211> 29

<212> DNA

<213> SNP24-R(Artificial Sequence)

<400> 72

aggaggcgag accgtcgatt taacggact 29

<210> 73

<211> 43

<212> DNA

<213> SNP25-F1(Artificial Sequence)

<400> 73

gaaggtgacc aagttcatgc tggtatgtca ctcggatctc ttt 43

<210> 74

<211> 43

<212> DNA

<213> SNP25-F2(Artificial Sequence)

<400> 74

gaaggtcgga gtcaacggat tggtatgtca ctcggatctc ttc 43

<210> 75

<211> 25

<212> DNA

<213> SNP25-R(Artificial Sequence)

<400> 75

acaaacgcag agcaccagaa agtag 25

<210> 76

<211> 43

<212> DNA

<213> SNP26-F1(Artificial Sequence)

<400> 76

gaaggtgacc aagttcatgc ttggagagat cgagcaaccc tag 43

<210> 77

<211> 43

<212> DNA

<213> SNP26-F2(Artificial Sequence)

<400> 77

gaaggtcgga gtcaacggat ttggagagat cgagcaaccc tac 43

<210> 78

<211> 25

<212> DNA

<213> SNP26-R(Artificial Sequence)

<400> 78

tcacgatcac gtcgatccac ttgtc 25

<210> 79

<211> 38

<212> DNA

<213> SNP27-F1(Artificial Sequence)

<400> 79

gaaggtgacc aagttcatgc tcagagcgac ccgcgtcg 38

<210> 80

<211> 38

<212> DNA

<213> SNP27-F2(Artificial Sequence)

<400> 80

gaaggtcgga gtcaacggat tcagagcgac ccgcgtcc 38

<210> 81

<211> 25

<212> DNA

<213> SNP27-R(Artificial Sequence)

<400> 81

caaatggtcc gtgttggaag gtagc 25

<210> 82

<211> 46

<212> DNA

<213> SNP28-F1(Artificial Sequence)

<400> 82

gaaggtgacc aagttcatgc tttcgcttct caacgcgacg gagctt 46

<210> 83

<211> 45

<212> DNA

<213> SNP28-F2(Artificial Sequence)

<400> 83

gaaggtcgga gtcaacggat ttcgcttctc aacgcgacgg agctc 45

<210> 84

<211> 29

<212> DNA

<213> SNP28-R(Artificial Sequence)

<400> 84

tcgccggaga tgtggttacg cgacagatc 29

<210> 85

<211> 46

<212> DNA

<213> SNP29-F1(Artificial Sequence)

<400> 85

gaaggtgacc aagttcatgc tgtttggaat gtcagttact gatcag 46

<210> 86

<211> 47

<212> DNA

<213> SNP29-F2(Artificial Sequence)

<400> 86

gaaggtcgga gtcaacggat tagtttggaa tgtcagttac tgatcat 47

<210> 87

<211> 28

<212> DNA

<213> SNP29-R(Artificial Sequence)

<400> 87

aggtaggacc cgagcctcaa ctgtggct 28

<210> 88

<211> 47

<212> DNA

<213> SNP30-F1(Artificial Sequence)

<400> 88

gaaggtgacc aagttcatgc ttctccgtcg cgccgcaaac gcgccta 47

<210> 89

<211> 46

<212> DNA

<213> SNP30-F2(Artificial Sequence)

<400> 89

gaaggtcgga gtcaacggat tctccgtcgc gccgcaaacg cgcctg 46

<210> 90

<211> 29

<212> DNA

<213> SNP30-R(Artificial Sequence)

<400> 90

aacctagctt cctggagaag gtgtttgag 29

<210> 91

<211> 42

<212> DNA

<213> SNP31-F1(Artificial Sequence)

<400> 91

gaaggtgacc aagttcatgc tgacgcaaca acaacgctgg ag 42

<210> 92

<211> 42

<212> DNA

<213> SNP31-F2(Artificial Sequence)

<400> 92

gaaggtcgga gtcaacggat tgacgcaaca acaacgctgg ac 42

<210> 93

<211> 25

<212> DNA

<213> SNP31-R(Artificial Sequence)

<400> 93

tgcaaagctg aagatgagcc gtgtc 25

<210> 94

<211> 43

<212> DNA

<213> SNP32-F1(Artificial Sequence)

<400> 94

gaaggtgacc aagttcatgc tcaatgaagc agcacctgat tct 43

<210> 95

<211> 43

<212> DNA

<213> SNP32-F2(Artificial Sequence)

<400> 95

gaaggtcgga gtcaacggat tcaatgaagc agcacctgat tcc 43

<210> 96

<211> 25

<212> DNA

<213> SNP32-R(Artificial Sequence)

<400> 96

cacatcctcc ttctcctcag ccatc 25

<210> 97

<211> 41

<212> DNA

<213> SNP33-F1(Artificial Sequence)

<400> 97

gaaggtgacc aagttcatgc tagctagacc ttccctcttg c 41

<210> 98

<211> 41

<212> DNA

<213> SNP33-F2(Artificial Sequence)

<400> 98

gaaggtcgga gtcaacggat tagctagacc ttccctcttg t 41

<210> 99

<211> 25

<212> DNA

<213> SNP33-R(Artificial Sequence)

<400> 99

gccagatgct gtgaattgtt gcttg 25

<210> 100

<211> 47

<212> DNA

<213> SNP34-F1(Artificial Sequence)

<400> 100

gaaggtgacc aagttcatgc tatacgtcgc attctcttgc cagtcct 47

<210> 101

<211> 47

<212> DNA

<213> SNP34-F2(Artificial Sequence)

<400> 101

gaaggtcgga gtcaacggat tatacgtcgc attctcttgc cagtcca 47

<210> 102

<211> 29

<212> DNA

<213> SNP34-R(Artificial Sequence)

<400> 102

agcgtagact ttagaatatg gtcgatggt 29

<210> 103

<211> 40

<212> DNA

<213> SNP35-F1(Artificial Sequence)

<400> 103

gaaggtgacc aagttcatgc tggcgtacca gaccgagtcc 40

<210> 104

<211> 40

<212> DNA

<213> SNP35-F2(Artificial Sequence)

<400> 104

gaaggtcgga gtcaacggat tggcgtacca gaccgagtcg 40

<210> 105

<211> 24

<212> DNA

<213> SNP35-R(Artificial Sequence)

<400> 105

cggtagagcg tgagggtcgt agtg 24

<210> 106

<211> 46

<212> DNA

<213> SNP36-F1(Artificial Sequence)

<400> 106

gaaggtgacc aagttcatgc taagtcccgc catggctgta gcgatc 46

<210> 107

<211> 47

<212> DNA

<213> SNP36-F2(Artificial Sequence)

<400> 107

gaaggtcgga gtcaacggat ttaagtcccg ccatggctgt agcgata 47

<210> 108

<211> 29

<212> DNA

<213> SNP36-R(Artificial Sequence)

<400> 108

cccatgaaga agagaatagc gatcacgac 29

<210> 109

<211> 45

<212> DNA

<213> SNP37-F1(Artificial Sequence)

<400> 109

gaaggtgacc aagttcatgc tacggatgta ctgagacctc ttgtc 45

<210> 110

<211> 46

<212> DNA

<213> SNP37-F2(Artificial Sequence)

<400> 110

gaaggtcgga gtcaacggat taacggatgt actgagacct cttgta 46

<210> 111

<211> 29

<212> DNA

<213> SNP37-R(Artificial Sequence)

<400> 111

ctgttgtaca attatacagc ctgtttctg 29

<210> 112

<211> 45

<212> DNA

<213> SNP38-F1(Artificial Sequence)

<400> 112

gaaggtgacc aagttcatgc tgcagagagg aaacgctcct caacg 45

<210> 113

<211> 46

<212> DNA

<213> SNP38-F2(Artificial Sequence)

<400> 113

gaaggtcgga gtcaacggat tagcagagag gaaacgctcc tcaact 46

<210> 114

<211> 29

<212> DNA

<213> SNP38-R(Artificial Sequence)

<400> 114

cggtgtggac gggttagtgg gagtggttg 29

<210> 115

<211> 47

<212> DNA

<213> SNP39-F1(Artificial Sequence)

<400> 115

gaaggtgacc aagttcatgc tcgcttccgg tgtcgtttcc atctccg 47

<210> 116

<211> 48

<212> DNA

<213> SNP39-F2(Artificial Sequence)

<400> 116

gaaggtcgga gtcaacggat tacgcttccg gtgtcgtttc catctcca 48

<210> 117

<211> 29

<212> DNA

<213> SNP39-R(Artificial Sequence)

<400> 117

cgaccatcgg taaccacgag gtttccgtc 29

<210> 118

<211> 47

<212> DNA

<213> SNP40-F1(Artificial Sequence)

<400> 118

gaaggtgacc aagttcatgc ttagctataa cttgtatact ccacacc 47

<210> 119

<211> 47

<212> DNA

<213> SNP40-F2(Artificial Sequence)

<400> 119

gaaggtcgga gtcaacggat ttagctataa cttgtatact ccacaca 47

<210> 120

<211> 29

<212> DNA

<213> SNP40-R(Artificial Sequence)

<400> 120

gtgattattg ttctttgtgg ctagagatg 29

<210> 121

<211> 47

<212> DNA

<213> SNP41-F1(Artificial Sequence)

<400> 121

gaaggtgacc aagttcatgc tatcgatctt aatctgttgg gccctca 47

<210> 122

<211> 47

<212> DNA

<213> SNP41-F2(Artificial Sequence)

<400> 122

gaaggtcgga gtcaacggat tatcgatctt aatctgttgg gccctct 47

<210> 123

<211> 29

<212> DNA

<213> SNP41-R(Artificial Sequence)

<400> 123

tttttacgct ctctcctctt ggtctggtg 29

<210> 124

<211> 46

<212> DNA

<213> SNP42-F1(Artificial Sequence)

<400> 124

gaaggtgacc aagttcatgc ttgtcttata aagattacta acgagt 46

<210> 125

<211> 46

<212> DNA

<213> SNP42-F2(Artificial Sequence)

<400> 125

gaaggtcgga gtcaacggat ttgtcttata aagattacta acgaga 46

<210> 126

<211> 29

<212> DNA

<213> SNP42-R(Artificial Sequence)

<400> 126

gatctgaacg catgctacca ataaggtgc 29

<210> 127

<211> 21

<212> DNA

<213> FAM(Artificial Sequence)

<400> 127

gaaggtgacc aagttcatgc t 21

<210> 128

<211> 21

<212> DNA

<213> HEX(Artificial Sequence)

<400> 128

gaaggtcgga gtcaacggat t 21

Claims

1. The core SNP molecular marker set of the non-heading Chinese cabbage developed based on the KASP technology is characterized by comprising 42 SNP markers, wherein the 42 SNP markers are numbered as SNP 1-42;

2. KASP primers for amplifying the core SNP molecular marker set of non-heading Chinese cabbage according to claim 1, wherein the KASP primers comprise 42 KASP primer combinations, and each KASP primer combination is used for amplifying a corresponding SNP marker.

3. The KASP primer set of claim 2, wherein each KASP primer combination is comprised of two allele forward primers F1 and F2 differing in terminal base and one reverse primer R.

4. The KASP primer of claim 3, wherein the 5' end of primer F1 is added with FAM fluorescent tag sequence as shown in SEQ ID NO: 127; the 5' end of the primer F2 is added with a HEX fluorescent tag sequence, and the HEX fluorescent tag sequence is shown as SEQ ID NO: 128.

5. The KASP primer of claim 3, consisting of the nucleotide sequence set forth in SEQ ID NO 1-126.

6. The test product comprising the KASP primer of any of claims 2-5, wherein the test product comprises a test reagent, a kit, and a chip.

7. The use of the non-heading cabbage core SNP molecular marker set of claim 1, or the KASP primer of any one of claims 2 to 5, or the test product of claim 6 in any one of the following aspects:

(2) constructing a non-heading Chinese cabbage SNP fingerprint gallery;

(3) analyzing genetic diversity of the non-heading Chinese cabbage;

(5) molecular marker assisted breeding of non-heading Chinese cabbage;

(7) molecular gene mapping of non-heading Chinese cabbage.

8. Use according to claim 7, characterized in that it comprises the following steps:

s1, extracting genome DNA of a non-heading Chinese cabbage sample;

9. The use of claim 8, wherein the reaction system for PCR amplification is 10 μ L, comprising 2 μ L of DNA from 20ng/μ L to 30ng/μ L, 0.28 μ L of each KASP primer combination, and 5 μ L of KASP Master mix.

10. The use according to claim 8, wherein the PCR amplification is performed under the following conditions: 15min at 94 ℃; 94 ℃,20sec, 63-55 ℃,1min, reducing the temperature by 0.8 ℃ in each cycle, and taking 10 cycles; 26 cycles of 94 ℃,20sec, 55 ℃,1 min; the reading conditions of the fluorescence signal values were 16 ℃ for 20 s.