CN113684300A

CN113684300A - SNP loci obviously associated with wheat ear number and application thereof in wheat genetic breeding

Info

Publication number: CN113684300A
Application number: CN202111030823.5A
Authority: CN
Inventors: 刘树兵; 庞昀龙; 朱华强; 梁云龙; 燕强
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2021-09-03
Filing date: 2021-09-03
Publication date: 2021-11-23

Abstract

The invention discloses a group of (50) SNP loci obviously associated with the number of grains per spike of wheat and an application method thereof in heredity and breeding. These SNPs were identified by performing simplified genome sequencing (GBS) on 768 wheat varieties and excellent lines from the major producing areas of wheat in China, searching for Single Nucleotide Polymorphisms (SNPs) by comparing with reference genome sequences, and then performing whole genome association analysis. The SNP locus has high accuracy, can be converted into KASP markers and SNP chips, and can be widely applied to positioning of wheat ear grain number related genes, fine mapping and candidate gene identification, marker-assisted selective breeding and whole genome selective breeding of wheat ear grain number, breeding of new wheat germplasm and new wheat variety with increased ear grain number, and improving wheat yield.

Description

SNP loci obviously associated with wheat ear number and application thereof in wheat genetic breeding

Technical Field

The invention relates to the technical field of plant molecular markers, in particular to a group of SNP (single nucleotide polymorphism) which are obviously associated with wheat ear grain number related gene loci and application thereof in inheritance and molecular breeding of wheat ear grain number.

Background

Wheat is one of important grain crops, and continuous breeding of high-yield new varieties and continuous improvement of the wheat yield are important targets of wheat breeding at home and abroad. The wheat yield mainly comprises three factors of the number of ears per unit area, the number of grains per ear and the grain weight, and is influenced by variety genetic characteristics and environmental conditions, and the variation range of the number of grains per ear is larger than that of the number of ears per unit area and the grain weight, so that the selection of the number of grains per ear is enhanced on the basis of coordinating the three factors of the number of ears per unit area, the number of grains per ear and the grain weight, and the yield increasing potential is large.

The grain number per spike is an important selection index for high-yield breeding of wheat. However, the grain number per ear is a complex quantitative trait, is controlled by multiple genes and is susceptible to environmental conditions, and the effect of improving the grain number per ear through phenotypic selective breeding is limited. The basic process is to utilize molecular markers covering the whole genome, construct a prediction model by training the marker genotype and phenotype of a population, and then utilize the marker information carried by individuals in a breeding population to predict unknown phenotypes, namely, estimate the genome breeding value and select. The whole genome selection has the advantage of high accuracy of early generation individual selection, and has important application value in improving the breeding efficiency and genetic progress of wheat ear grain number improvement.

The genetic research of the grain number of wheat per ear plays an important role in understanding the genetic development, gene mapping and cloning of the grain number of wheat per ear and the selective breeding and utilization of the whole genome. Quantitative Trait Locus (QTL) location is a common method for profiling the genetic basis of complex Quantitative traits. At present, linkage analysis is carried out by various types of genetic segregation populations derived from parental hybridization or whole genome association analysis is carried out by natural populations, and QTL related to solidity and spike grain number are reported on 21 chromosomes of wheat. Yu Xin et al (2020) performed meta-analysis on QTL controlling panicle number and identified 35 stable and consistent QTL on 10 chromosomes. However, the QTL positioning interval is often large, the linkage between the marker and the target QTL is not tight enough, the effect of applying the QTL positioning to marker-assisted selective breeding is poor, most of the QTL positioning is the traditional molecular marker, the limitations of low flux, small quantity, complicated operation process and the like exist, and the QTL positioning method is difficult to be widely applied to molecular marker-assisted selective breeding of the wheat ear grain number QTL.

Therefore, identifying molecular markers which are closely linked with the panicle number QTL and are easy to carry out high-throughput detection is the key for improving the breeding efficiency of marker-assisted selection. Compared with the traditional molecular marker, the SNP marker has the characteristics of rich variation, two-state property and easy high-throughput automatic detection, and is the molecular marker technology with the most application prospect in the research of functional genome and genetic breeding. At present, the common high-throughput SNP site identification technology mainly comprises two types, namely genome sequencing and SNP chip. High-density SNP sites can be obtained by second-generation or third-generation sequencing, but the cost is higher for wheat (16G) with a huge genome, and the requirements of the processes of sequencing data processing, sequence comparison, genotyping and the like on data analysis are higher, and the processes can be completed by personnel with professional bioinformatics backgrounds.

Disclosure of Invention

The SNP chip is used for genotyping, the data processing is simpler, and the SNP chip is easier to master and utilize for breeders. The KASP (Kompetitive Allele Specific PCR) marker, namely competitive Allele Specific PCR technology, which is formed based on the transformation of a single SNP locus can carry out high-flux accurate genotyping on the single SNP locus, has the characteristics of high stability and accuracy, low cost and high flux, and can be widely applied to gene positioning, fine mapping, cloning and high-flux screening of large-scale breeding materials, so that the method has important application value in genetic research, molecular marker assisted breeding and genome selective breeding.

Aiming at the defects in the prior art, the invention utilizes the mature simplified-by-sequencing (GBS) technology in wheat to carry out large-scale simplified genome sequencing on 768 wheat materials, and carries out sequence comparison with a wheat reference genome to obtain a high-density SNP locus of a whole genome; through multi-point and multi-year phenotypic character identification and whole genome association analysis, the SNP locus obviously associated with the number of grains per spike is identified, and the method can be widely applied to SNP chip preparation, KASP marker development, gene positioning, marker-assisted selection and whole genome selection, and is used for genetic research and molecular breeding of the number of grains per spike of wheat.

The invention provides a set of SNP loci obviously related to the grain number of wheat ears, can be applied to marker-assisted selection and whole genome selective breeding of the grain number of wheat ears, can be used for developing a single KASP marker and an SNP chip, and is convenient to use in inheritance and breeding groups. Can be widely applied to the positioning of wheat ear grain number related genes, fine mapping and cloning, and the marker-assisted selection, aggregation and whole genome selection of single or multiple QTL (quantitative trait loci) for controlling ear grain number in breeding.

The technical scheme adopted by the invention is as follows:

the invention provides a group (50) of Single Nucleotide Polymorphism (SNP) sites which are obviously related to the number of grains per spike of wheat, comprising SNP flanking sequences, SNP site information and base mutation information, wherein the SNPs are positioned on 17 different chromosomes of common wheat.

The group of SNP loci obviously related to the wheat ear grain number provided by the invention comprises 50 SNP loci, the numbers of which are respectively SNP 01-SNP 50, and the information of the SNP loci is as follows:

the physical position in the table takes the Chinese spring genome IWGSC reference genome v1.1(IWGSC,2018) as a reference sequence;

the sequences listed in the table are shown in sequence tables SEQ ID NO. 1-SEQ ID NO. 100. SNPs 01 to SNPs 50 each have 2 sequences, i.e., one sequence for each of the original sequence and the mutation site.

The SNP loci obviously associated with the wheat ear grain number and the base mutation types thereof provided by the invention can be applied to the identification of the wheat ear grain number QTL.

The SNP sites and the base mutation types thereof which are obviously related to the wheat ear grain number can be applied to the preparation of single detectable KASP markers or SNP gene chips.

The SNP loci obviously associated with wheat and the base mutation types thereof provided by the invention can be applied to preparation of KASP markers K4B28740074 linked with spike grain number gene loci qGN4B.1.

The SNP loci obviously associated with the wheat ear grain number provided by the invention can be applied to molecular marker-assisted selection and whole genome selection detection methods of the wheat ear grain number.

In the application of the detection method, KASP primers can be designed according to SNP sites, and are designed according to DNA short sequences which comprise 50bp of the upstream and downstream of the SNP sites. Specifically, using website PolyMarker (http:// www.polymarker.info/) And (4) designing primers, and setting default parameters of a website. The primer is added with a joint, the FAM sequence is GAAGGTGACCAAGTTCATGCT, and the HEX sequence is GAAGGTCGGAGTCAACGGATT.

After the primers are designed and synthesized, the effectiveness detection can be carried out by utilizing a separation population or a natural population, and whether the QTL identified by GWAS exists or not is verified, and the using methods are respectively as follows:

(1) wheat DNA is used as a PCR amplification template, and a synthesized KASP primer is designed to carry out PCR amplification, wherein the reaction system is 6 mu L. The reaction system specifically comprises: 20-50 ng/. mu.L of DNA 3. mu.L, 2 XKASP Master mix 3. mu.L, and KASP Assay mix (upstream and downstream primer mix) 0.0825. mu.L. Amplified in 384-well PCR instrument.

(2) The PCR amplification program is pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, renaturation at 65-57 ℃ for 60s (0.8 ℃ per cycle), 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 57 ℃ for 60s, 30 cycles; storing at 10 deg.C;

(3) after the PCR is finished, placing the sample in an Omega SNP typing instrument to detect the PCR typing result;

(4) analyzing and identifying, and analyzing the genotype according to the typing result.

Compared with the prior art, the research has the following advantages:

(1) the invention identifies a group of (50) obvious associated SNPs closely linked with grain number per ear, and basically covers grain number per ear related quantitative trait genetic loci in wheat germplasm in China at present.

(2) The SNP can be further converted into a single detectable SNP marker (such as KASP marker), and is used for positioning of the panicle number related gene, fine mapping, cloning and high-throughput molecular marker-assisted selection applied to breeding materials in a large scale, so that the efficiency of molecular breeding is improved.

(3) The SNPs can also be made into gene chips, and can be applied to molecular marker-assisted selection and whole genome selection of the number of grains per ear of breeding materials, so that the efficiency and the accuracy of molecular breeding are further improved.

(4) The KASP marker K4B28740074 linked with the panicle grain number gene locus qGN4B.1 is developed, so that the SNP identified by the inventor is proved to be very effective, and an efficient detection marker is provided for marker-assisted selection of qGN4B.1.

Drawings

FIG. 1 is the accuracy of prediction of spike size using markers. The left side shows the result of prediction by using SNP markers associated with QTL, and the right side shows the result of prediction by randomly screening the same number of SNP sites in the whole genome. GZ17, LY17, TA19, TA16, TA17, TA18, YT17 represent different environments, respectively.

Detailed Description

Example 1 identification of SNPs by simplified genomic sequencing

1.1 sequencing materials

768 wheat varieties (lines) were selected for simplified genome sequencing and SNP identification. The materials are mainly wheat varieties and excellent strains from main wheat producing areas in China, including Huang-Huai wheat areas, northern winter wheat areas, middle and lower Yangtze river wheat areas and southwest wheat areas.

1.2 methods of investigation

1.2.1 extraction of wheat genomic DNA

Seedling leaves were used to provide genomic DNA. The DNA extraction was carried out by the modified CTAB (butyl trimethyl ammonium bromide) method (Stewart and Via, 1993). The method comprises the following specific operations: taking young and tender wheat leaves in a 2mL centrifuge tube, freezing by using liquid nitrogen, and grinding into powder on a tissue grinder; (b) adding 800 μ L CTAB into 2mL tube, placing in 65 deg.C water bath for 90min, and shaking gently for 5-8 times during the water bath period to fully crack DNA; (c) adding 800 μ L chloroform isoamyl alcohol (volume ratio 24:1) and shaking for 10 min; (d) centrifuging at 12000rpm for 10min, and placing 600 μ L of supernatant in a new 2mL tube (note corresponding number); (e) mu.L of 3M sodium acetate (pH 5.2) and 600. mu.L of isopropanol (frozen at-20 ℃ C. in advance) were added thereto, and the mixture was gently shaken and mixed to see the generation of white DNA flocs, which were then put in a refrigerator at-20 ℃ for 1 hour to increase the DNA yield. (f) Centrifuging at 12000rpm for 10min, pouring out supernatant, washing the precipitate with 70% ethanol (freezing in a refrigerator at-20 deg.C in advance) for 2-3 times, standing in a fume hood, and air drying; (g) add 200. mu.L of ddH₂O dissolves the DNA.

1.2.2 DNA sample quality detection

And detecting by using agarose gel electrophoresis with the mass fraction of 1%, and checking an electrophoresis result by using a gel imaging system to ensure the integrity of the genome DNA. The ratio of A260/280 of the genomic DNA should be between 1.8 and 2.0, and the ratio of A260/230 should be between 1.8 and 2.2. The DNA was diluted to a working concentration of 20 ng/ul. Storing at-20 deg.C for use.

1.2.3 DNA library construction and GBS sequencing

Construction of GBS DNA libraries was performed with reference to Poland et al (2012 b). Genomic DNA was digested with two restriction enzymes PstI and MspI (New England BioLabs, Inc., Ipswich, MA, United States). The barcode sequence was ligated to the digested DNA fragment using T4(New England BioLabs, Inc., Ipswich, MA, United States) ligase. All products from each plate were mixed and purified using QIAq rapid PCR purification kit (Qiagen, inc., Valencia, CA, United States). PCR amplification was performed using primers complementary to the barcode sequence. The PCR product was again purified using QIAquick PCR purification kit and using a Qubit^TMDouble-stranded DNA high sensitivity fluorescent quantitation kit (Life technology)ies, inc., Grand Island, NY, United States). Screening of 200-300 size DNA fragments, the Qubit 2.0 fluorescent agent and the Qubit using agarose gel electrophoresis (Life Technologies, Inc., Grand Island, NY, United States)^TMThe double-stranded DNA high-sensitivity fluorescent quantitation kit estimates the concentration of each DNA library. Fragment size-screened DNA libraries were loaded onto P1v3 chips using an Ion CHEF instrument (Ion PI Hi-Q CHEF Kit) and sequenced using an Ion Proton sequencer (Life Technologies, Inc., Grand Island, NY, United States, software version 5.10.1). This Ion Torrent system can produce sequences of various read lengths.

1.2.4 SNP site identification

The sequencing sequence was sequenced by adding 80 poly-A bases to its 3' end and then using TASSEL 5.0, so that it was possible to process sequences shorter than 64 bases by the train Analysis by Association, Evolution and Linkage (TASSEL) pipeline 5.0(TASSEL 5.0) (Bradbury et al, 2007) rather than just discarding these short sequences. Sequence alignment is carried out by taking the IWGSC reference gene v1.1(IWGSC,2018) of the Chinese spring genome as a reference sequence and using TASSEL 5.0(Bradbury et al, 2007) to identify SNP sites. All parameters are set to default settings of TASSEL 5.0. A total of 432,588 SNP sites were obtained covering approximately 14Gb of the whole genome, with an average distance between markers of 34.0 kb. Wherein 150784 sites on the A genome are spaced at an average distance of 32.9 kb; 182192 loci on the B genome with an average spacing of 28.9 kb; the D genome has 99612 sites and the average distance is 40.3 kb. The number of SNP markers on each chromosome is 10177 to 31149, and the variation range of the marker interval is 26.1-50.1 kb. These SNPs were mainly located in the intergenic region, 364203, accounting for 84.1, followed by the CDS region, 39901 (9.2%), the intron region 22215 (5.1%), the 5 'UTR region 3543 (0.8%), and the 3' UTR region 3300 (0.8%).

TABLE 1 distribution of SNP sites identified by GBS in wheat genome

Example 2 Whole genome identification of spike number quantitative trait loci

2.1 materials

768 wheat materials for genotyping by GBS technique, as in example 1.

2.2 methods

2.2.1 identification of grain count per ear

To identify the number of grains per spike (GN) of these materials, 768 wheat materials were planted at the tai an shandong agriculture university laboratory in 2017-. Planting 1 row of each material, wherein the row length is 3m, the row spacing is 25 cm, sowing 50 full seeds in each row, repeating the steps twice, and performing conventional field management.

2.2.2 Whole genome Association analysis

And (3) further screening the SNP sites with the Minimum Allele Frequency (MAF) of more than 0.01 and the deletion rate of less than 80% of all the identified 432588 SNP sites to obtain 327609 SNPs. And performing whole genome association analysis on the number of grains per ear. The GAPIT v.3 package was used, which uses EMMA, a Compressed Mixed Linear Model (CMLM) and a population parameters previous determined (P3D) to improve the efficiency of GWAS operation. Kinship was analyzed using EMMA algorithm, using the first 3 principal components to control population structure. Significance threshold was set at 1.0 × 10^-5。

2.3 results

2.3.1 Whole genome Association analysis of ear grain number

By performing GWAS on the spike grain numbers measured under a plurality of environments, 99 correlated SNP Sites (MTAs) were identified in total, and combined into 49 spike grain number QTLs according to LD. 38 of these QTLs were located within the 1.0Mb interval, each containing less than 10 annotated genes. The method shows that the QTL can be positioned in a narrower physical interval by using the high-density SNP marker identified by GBS to carry out GWAS, and great convenience is brought to the fine positioning, gene cloning and molecular marker-assisted breeding of the subsequent QTL.

2.3.2 identified SNPs associated with spike size number

For the SNPs identified by GWAS, one SNP most significantly associated in each environment was selected for each site, and 50 SNP sites significantly associated with the number of grains per ear were obtained in total, and the information of these sites is shown in table 2. In the table, "QTL" column, the QTL name linked to SNP is indicated; in the table, a list of 'chromosomes and physical positions of SNPs' indicates the chromosomes and the physical positions of the SNPs, and the physical positions refer to IWGSC reference gene v1.1(IWGSC,2018) of the Chinese spring genome; in the table, "sequence and SNP variation site" is listed, and "base" in "[ ]" indicates a variation site, and some sites have only one base, indicating that the base is deleted after variation.

TABLE 2 SNP site information significantly associated with wheat ear number of grains

Example 3 application of wheat grain number per ear QTL in genome prediction

3.1 materials

The same as in example 1.

3.2 methods

3.2.1 spike size phenotype identification

The same as in example 1.

3.2.2 predictive model construction

And respectively constructing a whole genome selection model by using a rrBLUP method in R software. And (3) constructing a prediction model by using the identified 50 significant SNP markers so as to evaluate the prediction capability of the QTL. Meanwhile, random SNP sites with the same number are extracted from the whole genome to construct a prediction model, and the prediction effect of the random marker is evaluated.

3.2.3 Cross-validation of predictive models

The prediction accuracy of the model was analyzed using 5-fold cross-validation. The whole population is randomly divided into 5 parts, 1 part of the 5 parts is taken as a breeding population, and the other 4 parts are taken as a training population. And (3) estimating a marker effect value by using the phenotype and the genotype of the training population, predicting the phenotype of the breeding population according to the genotype of the breeding population, calculating the correlation between the predicted phenotype and the actually measured phenotype to be used as the accuracy of model prediction, repeating the steps for 100 times, and taking an average value.

3.3 results

Data under 7 environments or years, such as GZ17, LY17, TA19, TA16, TA17, TA18, YT17 and the like, are respectively utilized to construct a QTL prediction model and a random SNP site prediction model. The identified QTL was used for prediction, and the prediction accuracy in each environment was-0.60, which was significantly higher than that of the random SNP site (the accuracy in each environment was only-0.2) (fig. 1). The identified QTLs and the linked SNP markers thereof have higher prediction capability on the number of panicle grains, and can be applied to marker-assisted selection and whole genome selective breeding of the panicle grains.

FIG. 1 accuracy of prediction of spike size using markers. The left side shows the result of prediction by using SNP markers associated with QTL, and the right side shows the result of prediction by randomly screening the same number of SNP sites in the whole genome. GZ17, LY17, TA19, TA16, TA17, TA18, YT17 represent different environments, respectively.

Example 4 verification and mapping of grain number per wheat ear gene qGN4B.1

4.1 materials

The research material comprises a florid and fructification parent nicotiana 19, a parent sunshine No. 5 and F constructed by hybridization and hybridization of the two₂Segregating population, total 156F₂And (4) single plants.

4.2 methods

4.2.1 segregating population spike number phenotype identification

The same as in example 1.

4.2.2 DNA extraction

The same as in example 1.

4.2.3 KASP primer design, dilution:

according to the GWAS analysis result, a group of (three) KASP primers K4B28740074 is designed for one GBS-SNP (S4B28740074) which is obviously associated with the spike grain number QTL qGN4B.1 according to the SNP locus information. FAM-R primer sequence CACGTACAGTACGCGCAATGGT, HEX-S primer sequence ACGTACAGTACGCGCAATGGC, reverse primer sequence CCCGATCGTACAGGCGCAGGTA. After the three primers are diluted to 100 mu M with ultrapure water, the volume ratio of the forward primer-FAM-R: forward primer-HEX-S: reverse primer: ultrapure water 6: 6: 15: 23, and storing the mixture to-20 ℃ for later use.

4.2.4 KASP PCR amplification System and procedure are as follows:

the KASP reaction system used a 6 μ L system:

the system was prepared on ice according to the following table, 3. mu.L (about 20 ng/. mu.L) of template DNA, 2 × Master mix 3. mu.L (LGC Group UK), and 0.0825. mu.L of KASP assay primer (synthesized by Shanghai Biotech).

TABLE 3 KASP reaction System

The PCR procedure was as follows:

pre-denaturation at 1.94 ℃ for 5 min;

denaturation at 2.94 ℃ for 20 s;

3.65 ℃ for 30s (0.8 ℃ per cycle), and steps 2-3 are cycled 10 times.

Denaturation at 4.94 ℃ for 20 s;

annealing at 5.57 deg.C for 30s, and circulating step 4-5 for 35 times.

Storing at 6.10 deg.C. And (5) signal detection.

4.3 results

4.3.1 variation in spike size in segregating populations

GWAS analysis results show that qGN4B.1 is a QTL for controlling the number of grains per wheat ear and is positioned on 4 BS. Polymorphism exists between sunlight No. 5 and tobacco grower 19 at the two sites, and the grain number of the tobacco grower 19 is obviously higher than that of sunlight No. 5. In order to further verify the QTL and develop the KASP marker linked with the QTL, F is constructed by hybridizing sunshine No. 5 with the Nicotiana 19₂Segregating the population and selfing to F₄。

Spike number survey of this population revealed that F₄The number of grains per spike of the generation plants has wide variation and obvious super-parent separationSeparation of (4).

4.3.2 correlation of ear size to marker genotype in segregating populations

After a GBS-SNP (S4B28740074) which is most obviously associated with the QTL is converted into a KASP marker (K4B28740074), the group is genotyped, and the result shows that the KASP marker can convert F into the protein₂And (5) carrying out group typing. ANOVA analysis is carried out on the genotype and the grain number per ear expression, and the result P value<0.001, indicating that the marker is extremely obviously related to the number of grains per ear, and the SNP marker has the same genotype as F of Nicotiana tabacum 19₄The grain number per ear of the strain is obviously more than that of the strain with the same sunshine No. 5, and the grain number per ear of the heterozygous strain is between the two, which indicates that a QTL for controlling the grain number per ear of wheat exists in the interval.

The foregoing is only a preferred embodiment of this patent, and it should be noted that, for those skilled in the art, various modifications and substitutions can be made without departing from the technical principle of this patent, and these modifications and substitutions should also be regarded as the protection scope of this patent.

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<210> 21

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 21

atagatgttc tacttctttc attcagtcag gttctactga aactttttgc caaaaaaaaa 60

agttctcctg gaactgtcta aaggcacaaa tataaacgtt t 101

<210> 22

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 22

atagatgttc tacttctttc attcagtcag gttctactga aactttttgc aaaaaaaaaa 60

agttctcctg gaactgtcta aaggcacaaa tataaacgtt t 101

<210> 23

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 23

cagaaatcaa atgtacctaa aaaggacgga tctatcatcc tactgcagaa tgagtagcca 60

ctcatgattc atgactcatc taatagtggc aagcagcaac a 101

<210> 24

<211> 102

<212> DNA

<213> wheat (Triticum aestivum)

<400> 24

cagaaatcaa atgtacctaa aaaggacgga tctatcatcc tactgcagaa atgagtagcc 60

actcatgatt catgactcat ctaatagtgg caagcagcaa ca 102

<210> 25

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 25

tctttgtgtt gatttgtttc acctctgcag gataatccta gagccagggg atgtacggta 60

ttccatttcc gcatttcgat gtgtttgatg cggtgtatgg c 101

<210> 26

<211> 102

<212> DNA

<213> wheat (Triticum aestivum)

<400> 26

tctttgtgtt gatttgtttc acctctgcag gataatccta gagccagggg gatgtacggt 60

attccatttc cgcatttcga tgtgtttgat gcggtgtatg gc 102

<210> 27

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 27

cgctggtctc gcgcctccct cagccgccgt cgcctatgcc tgtcccgcgc ttccctccgc 60

cgctcgcgcc gccgcttccc cgtcgagctt cacgtcctcg g 101

<210> 28

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 28

cgctggtctc gcgcctccct cagccgccgt cgcctatgcc tgtcccgcgc ctccctccgc 60

cgctcgcgcc gccgcttccc cgtcgagctt cacgtcctcg g 101

<210> 29

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 29

tcttctccaa tggaggctcc ccatggcgca ccatcttctt tagacctgtc actgacgctg 60

gcccccatgc caccacctcc atcctcctct gtttgggctg c 101

<210> 30

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 30

tcttctccaa tggaggctcc ccatggcgca ccatcttctt tagacctgtc gctgacgctg 60

gcccccatgc caccacctcc atcctcctct gtttgggctg c 101

<210> 31

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 31

tctcccacga gatcgacctg ttgaaggaga agaacaccgg ctggaagaag ttgcatgaga 60

tagtagttcc tcgatgaaga tgctgcaggc cctcgccatg g 101

<210> 32

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 32

tctcccacga gatcgacctg ttgaaggaga agaacaccgg ctggaagaag ctgcatgaga 60

tagtagttcc tcgatgaaga tgctgcaggc cctcgccatg g 101

<210> 33

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 33

ggggcgcggc cacgcaagag gttgccgtag tacttgttgt cgaagacgtc tggggtgatt 60

gtgtcgagat tcgccagcgt ctcctcgtcc tgcccagcgc t 101

<210> 34

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 34

ggggcgcggc cacgcaagag gttgccgtag tacttgttgt cgaagacgtc cggggtgatt 60

gtgtcgagat tcgccagcgt ctcctcgtcc tgcccagcgc t 101

<210> 35

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 35

ggcctctagc ttcaggagga gggttgctgc agcaggtctt catgggtcgc actggaggtc 60

tccgccagcg gaggcttggc cttcacccgt ggctggcagg c 101

<210> 36

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 36

ggcctctagc ttcaggagga gggttgctgc agcaggtctt catgggtcgc gctggaggtc 60

tccgccagcg gaggcttggc cttcacccgt ggctggcagg c 101

<210> 37

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 37

tcagtgccgc tctcactctc acgacagaac atagtcttat cccgctagcc agaggagcag 60

gacgacaccg tggacgggcc tcgcgcgtga tctggcagca g 101

<210> 38

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 38

tcagtgccgc tctcactctc acgacagaac atagtcttat cccgctagcc ggaggagcag 60

gacgacaccg tggacgggcc tcgcgcgtga tctggcagca g 101

<210> 39

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 39

ctgcatcaca agcgcccact ccatgtagtt ggtgcgcgtg agcatcatgg gggcgcccgg 60

ctcctcgcgc acgatgctct ccaccaccct gatctcgccg c 101

<210> 40

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 40

ctgcatcaca agcgcccact ccatgtagtt ggtgcgcgtg agcatcatgg aggcgcccgg 60

ctcctcgcgc acgatgctct ccaccaccct gatctcgccg c 101

<210> 41

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 41

cgcctctcac tcaggggctt agctgcagtc cagtactcgc ctaagtgata taaatcctac 60

cgagtggtga gtccgcctct cactcggggg cttagctgca g 101

<210> 42

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 42

cgcctctcac tcaggggctt agctgcagtc cagtactcgc ctaagtgata aaaatcctac 60

cgagtggtga gtccgcctct cactcggggg cttagctgca g 101

<210> 43

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 43

tcgacggata cgcctcccag gaaatcaaac ctgcagcaca gcaggacacg ttttcacgct 60

tgatctattt cagggcaaaa aggagggttg gggtttgggc g 101

<210> 44

<211> 102

<212> DNA

<213> wheat (Triticum aestivum)

<400> 44

tcgacggata cgcctcccag gaaatcaaac ctgcagcaca gcaggacacg tttttcacgc 60

ttgatctatt tcagggcaaa aaggagggtt ggggtttggg cg 102

<210> 45

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 45

tcctggtggc ctctggcttt aagcggacgg ttgctgcagc agggcctcct aggcttcact 60

ggagatctcc gtcgtcggga acgtggccct gacccgcaca c 101

<210> 46

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 46

tcctggtggc ctctggcttt aagcggacgg ttgctgcagc agggcctcct gggcttcact 60

ggagatctcc gtcgtcggga acgtggccct gacccgcaca c 101

<210> 47

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 47

tccagcatct tcttctcttc ggcgatcacg agggactcgg ccagcggact gctggctgca 60

gcacactcag aagacttctt gtagtctccg gctatggtga t 101

<210> 48

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 48

tccagcatct tcttctcttc ggcgatcacg agggactcgg ccagcggact tctggctgca 60

gcacactcag aagacttctt gtagtctccg gctatggtga t 101

<210> 49

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 49

aggcagcatc tttggcagct cgaaaagctg ctgtcggttg tgctgcaaaa ggtcgccgtg 60

ctgtagcagc agcttcagca gctggaaacg ctgctaccat c 101

<210> 50

<211> 100

<212> DNA

<213> wheat (Triticum aestivum)

<400> 50

aggcagcatc tttggcagct cgaaaagctg ctgtcggttg tgctgcaaaa gtcgccgtgc 60

tgtagcagca gcttcagcag ctggaaacgc tgctaccatc 100

<210> 51

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 51

tgcagttgca ggcctgcagc acacgatcga ctcggcgagc cagccaacga ttacggaccc 60

aacggctgta gctatggggt atacaggagc ctactgtggt a 101

<210> 52

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 52

tgcagttgca ggcctgcagc acacgatcga ctcggcgagc cagccaacga ctacggaccc 60

aacggctgta gctatggggt atacaggagc ctactgtggt a 101

<210> 53

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 53

gagagctgca gcgcgagcaa gagcaagagc aatagcaaga gcagaccaag taggggaccg 60

agagcaagag cagagcagca gcgcgagcaa gagctaaagc a 101

<210> 54

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 54

gagagctgca gcgcgagcaa gagcaagagc aatagcaaga gcagaccaag caggggaccg 60

agagcaagag cagagcagca gcgcgagcaa gagctaaagc a 101

<210> 55

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 55

ctcgaccacc tcatcatcct ggcacgcggg cagctcatgt acagcggcgg cccaaggagg 60

tgaccgcgca cctcggccgc atgggccgca aggtgcccaa a 101

<210> 56

<211> 102

<212> DNA

<213> wheat (Triticum aestivum)

<400> 56

ctcgaccacc tcatcatcct ggcacgcggg cagctcatgt acagcggcgg gcccaaggag 60

gtgaccgcgc acctcggccg catgggccgc aaggtgccca aa 102

<210> 57

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 57

gtggtgagca cggtggccat cttcaccgac ggcaacgccc tgcggacgcg gcgcttcaag 60

gtgttcgccg tcgacgagcg cgaggacagg tggcgcgagc t 101

<210> 58

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 58

gtggtgagca cggtggccat cttcaccgac ggcaacgccc tgcggacgcg ccgcttcaag 60

gtgttcgccg tcgacgagcg cgaggacagg tggcgcgagc t 101

<210> 59

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 59

atctggaggg gaaaggggct ggcggaagga gggcgacagg gggtggcggg ggagggaaga 60

ggaggctggc ggaaggaggg cgacagggga aaggggccgc a 101

<210> 60

<211> 102

<212> DNA

<213> wheat (Triticum aestivum)

<400> 60

atctggaggg gaaaggggct ggcggaagga gggcgacagg gggtggcggg aggagggaag 60

aggaggctgg cggaaggagg gcgacagggg aaaggggccg ca 102

<210> 61

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 61

gagtgttgcc gtgacagcgt gctgcagtca ctgaccaacg tttcgggcga tgcgtggatg 60

gcagcccaga ctctgccatc cagtgcgacc atttgctcca g 101

<210> 62

<211> 102

<212> DNA

<213> wheat (Triticum aestivum)

<400> 62

gagtgttgcc gtgacagcgt gctgcagtca ctgaccaacg tttcgggcga atgcgtggat 60

ggcagcccag actctgccat ccagtgcgac catttgctcc ag 102

<210> 63

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 63

gcgatccgcg cgagactgca gcaggagtcc acgtacagta cgcgcaatgg cgacgggcgg 60

gcctacctgc gcctgtacga tcggggatcg gattgtacca c 101

<210> 64

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 64

gcgatccgcg cgagactgca gcaggagtcc acgtacagta cgcgcaatgg tgacgggcgg 60

gcctacctgc gcctgtacga tcggggatcg gattgtacca c 101

<210> 65

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 65

cgtatcctct atactactcg taacacgtga accatcttgc gaagaagata cactaccact 60

gcagtctaca gtagtatagg agtatcaagg atttgctgag c 101

<210> 66

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 66

cgtatcctct atactactcg taacacgtga accatcttgc gaagaagata tactaccact 60

gcagtctaca gtagtatagg agtatcaagg atttgctgag c 101

<210> 67

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 67

ctgcagcagg gtcatgggcg ccaggctcga caggttgcta cccccgctgc gcatcccggc 60

gctccgacgt tcaaaacgcc ctccgccacc ccgtccccat c 101

<210> 68

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 68

ctgcagcagg gtcatgggcg ccaggctcga caggttgcta cccccgctgc ccatcccggc 60

gctccgacgt tcaaaacgcc ctccgccacc ccgtccccat c 101

<210> 69

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 69

cagcgtatcg ccactaggac ggaagaggct gcggcagcag ctaccacggg tgagttcggg 60

ttcaccgtgg aggaggcaga ggcagcagag gcggcaagcc t 101

<210> 70

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 70

cagcgtatcg ccactaggac ggaagaggct gcggcagcag ctaccacggg cgagttcggg 60

ttcaccgtgg aggaggcaga ggcagcagag gcggcaagcc t 101

<210> 71

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 71

atttggaatt gcagaaatga tttggctttt aacagaacaa caaatattca ctttttcagg 60

ttttattccg agctactgcg ctgatccgta tgtggtcgtt a 101

<210> 72

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 72

atttggaatt gcagaaatga tttggctttt aacagaacaa caaatattca ttttttcagg 60

ttttattccg agctactgcg ctgatccgta tgtggtcgtt a 101

<210> 73

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 73

gacagaagca actatcgggt accactcatc cctcaggagc aagcttacgc caaccatggg 60

tcgcaggagg tacaggttgc cgcagcggat ggctgcccca a 101

<210> 74

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 74

gacagaagca actatcgggt accactcatc cctcaggagc aagcttacgc taaccatggg 60

tcgcaggagg tacaggttgc cgcagcggat ggctgcccca a 101

<210> 75

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 75

aacgtgtcgc cgagctgttg gatgctgcag ccaggggtac gctgcctttc tgcgccctac 60

ttacgtctca agttttggcc aggtcgcacg acggccctcg t 101

<210> 76

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 76

aacgtgtcgc cgagctgttg gatgctgcag ccaggggtac gctgcctttc cgcgccctac 60

ttacgtctca agttttggcc aggtcgcacg acggccctcg t 101

<210> 77

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 77

aactagctgc gctcgttctt gcactatgta gtatagtcct ttgtgctgct cagaaacgac 60

gtcttctcac ttgtatgcgg aatgcagctt ggcatgtcga c 101

<210> 78

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 78

aactagctgc gctcgttctt gcactatgta gtatagtcct ttgtgctgct tagaaacgac 60

gtcttctcac ttgtatgcgg aatgcagctt ggcatgtcga c 101

<210> 79

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 79

tgctggtgat gttcctcgtg gcctccgccg ctgcaggatt gcctttcccc cttttttttg 60

caggtttttc gggctgacaa tgatcagcag tacggccttt a 101

<210> 80

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 80

tgctggtgat gttcctcgtg gcctccgccg ctgcaggatt gcctttcccc tttttttttg 60

caggtttttc gggctgacaa tgatcagcag tacggccttt a 101

<210> 81

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 81

gccggccatg gccacaatct ggtcaaggat ctttttctcg gcagcaatca tgagggactc 60

ggccagctgg ctgctggctg cagcgcaggc agaggatttc t 101

<210> 82

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 82

gccggccatg gccacaatct ggtcaaggat ctttttctcg gcagcaatca cgagggactc 60

ggccagctgg ctgctggctg cagcgcaggc agaggatttc t 101

<210> 83

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 83

atcgcgcgga gacgactcag ctcctcggag atataggtga agaaggagac taggtcagga 60

agctgcagct cgttgaagtc gaccgggtgg tcgaacgggt t 101

<210> 84

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 84

atcgcgcgga gacgactcag ctcctcggag atataggtga agaaggagac caggtcagga 60

agctgcagct cgttgaagtc gaccgggtgg tcgaacgggt t 101

<210> 85

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 85

ctacgaacgg tagcgagcaa tgtaggtttg ggtgggggtg ggggtgcagt tgctagccca 60

aaaaaaacag atcaaccagg ggagatttag agggatggtc g 101

<210> 86

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 86

ctacgaacgg tagcgagcaa tgtaggtttg ggtgggggtg ggggtgcagt ggctagccca 60

aaaaaaacag atcaaccagg ggagatttag agggatggtc g 101

<210> 87

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 87

aggggtcgtg caagtcctat atttcctcga ggccgtgggc cagggacgaa ggttgtcaac 60

ctccaactca aacccaaaga ccaaggggtc gagcataggc t 101

<210> 88

<211> 102

<212> DNA

<213> wheat (Triticum aestivum)

<400> 88

aggggtcgtg caagtcctat atttcctcga ggccgtgggc cagggacgaa aggttgtcaa 60

cctccaactc aaacccaaag accaaggggt cgagcatagg ct 102

<210> 89

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 89

tactagggcg gaagaggctg cggccgcagc tgctgcgggt gtgtttggga ataatgtgga 60

ggaggcggag gctgcagagg cgggaaacct tgccgaacgc g 101

<210> 90

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 90

tactagggcg gaagaggctg cggccgcagc tgctgcgggt gtgtttggga ttaatgtgga 60

ggaggcggag gctgcagagg cgggaaacct tgccgaacgc g 101

<210> 91

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 91

gacattaaca aattcccatc tgccataacc gcgggcacgg ctttcgaaag attataccct 60

gcaggggtga cccaacttag cccatgataa gctctcgcga t 101

<210> 92

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 92

gacattaaca aattcccatc tgccataacc gcgggcacgg ctttcgaaag tttataccct 60

gcaggggtga cccaacttag cccatgataa gctctcgcga t 101

<210> 93

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 93

ctgtgcactg ttgggctagc ttgcagattc agcccgcatg tgtttttcct cttctggacg 60

ttgtgctaat tatccaggga actgcagctt tgcagaaaac c 101

<210> 94

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 94

ctgtgcactg ttgggctagc ttgcagattc agcccgcatg tgtttttcct gttctggacg 60

ttgtgctaat tatccaggga actgcagctt tgcagaaaac c 101

<210> 95

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 95

tgatcgatgg tgtgcctcgg atttattcta acaattgata tcatgagcaa cgtaacggag 60

aggctgcagg ttattgatct agaggaagta tcaagtttga a 101

<210> 96

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 96

tgatcgatgg tgtgcctcgg atttattcta acaattgata tcatgagcaa tgtaacggag 60

aggctgcagg ttattgatct agaggaagta tcaagtttga a 101

<210> 97

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 97

ttggtgaact taccatctac tctcttctac atgctgcaag atggaggtgg tcagaagcgt 60

agtcttcgac aggattagct atcccccctc ttattctggc a 101

<210> 98

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 98

ttggtgaact taccatctac tctcttctac atgctgcaag atggaggtgg ccagaagcgt 60

agtcttcgac aggattagct atcccccctc ttattctggc a 101

<210> 99

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 99

gaaatttaaa cctttcgatc agctaatctt actaaagaac acaaatgtgt atttttataa 60

acttgaattg tcctggtaaa agtacggatt ttcactttca c 101

<210> 100

<211> 101

<212> DNA

<213> wheat (Triticum aestivum)

<400> 100

gaaatttaaa cctttcgatc agctaatctt actaaagaac acaaatgtgt ctttttataa 60

acttgaattg tcctggtaaa agtacggatt ttcactttca c 101

Claims

1. A group of SNP sites and base mutation types which are obviously related to the number of grains of the wheat ear is characterized in that: the SNP sites comprise 50 SNP sites and base mutation types, the numbers of the SNP sites are respectively 01-50, and the information of the SNP sites is as follows:

the sequences listed in the table are shown in sequence tables SEQ ID NO. 1-SEQ ID NO. 100.

2. The use of the set of SNP sites and the base mutation types thereof in significant association with the grain number of wheat ear according to claim 1 in the identification of QTL for controlling the grain number of wheat ear.

3. The use of the set of SNP sites and the base mutation types thereof significantly associated with the number of grains of wheat ear according to claim 1 in the preparation of a single detectable SNP marker or gene chip.

4. The use of the set of SNP sites and base mutation types thereof significantly associated with ear number of wheat according to claim 1 in the preparation of KASP marker S4B _28740074 linked to ear number gene site qGN4B.1.

5. The use of the set of SNP sites in significant association with wheat ear grain number according to claim 1 in wheat ear grain number marker-assisted selection and genome selection breeding.

6. Use in a detection method according to claim 6, characterized in that the detection method comprises the steps of:

designing KASP primers according to the SNP sites, and designing KASP primers according to DNA short sequences containing 50bp of upstream and downstream of the SNP sites; specifically, a website http:// www.polymarker.info/is used for primer design, and default parameter setting of the website is adopted; adding a joint in front of the primer, wherein the FAM sequence is GAAGGTGACCAAGTTCATGCT, and the HEX sequence is GAAGGTCGGAGTCAACGGATT;

after the primers are designed and synthesized, the effectiveness detection can be carried out by utilizing a separation population or a natural population, whether the QTL identified by GWAS exists or not can be verified, and the method can also be used for gene mapping, marker-assisted selection and the like, and the using methods are respectively as follows:

(1) using wheat DNA as a PCR amplification template, designing a synthesized KASP primer, and carrying out PCR amplification with a reaction system of 6 mu L; the reaction system specifically comprises: 20-50 ng/. mu.L of DNA 3. mu.L, 2 XKASP Master mix 3. mu.L, and KASP Assay mix upstream and downstream primer mixture 0.0825. mu.L; amplifying in a 384-well PCR instrument;

(2) the PCR amplification procedure was: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 deg.C for 20s, and renaturation at 65-57 deg.C for 60 s; the reduction per cycle is 0.8 ℃; 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 57 ℃ for 60s, 30 cycles; storing at 10 deg.C;