CN112266973B

CN112266973B - SNP molecular marker related to wheat ear germination resistance and application thereof

Info

Publication number: CN112266973B
Application number: CN202011088002.2A
Authority: CN
Inventors: 曹佳佳; 常成; 张海萍; 卢杰; 马传喜
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2020-10-13
Filing date: 2020-10-13
Publication date: 2022-03-11
Anticipated expiration: 2040-10-13
Also published as: CN112266973A

Abstract

The invention provides an SNP molecular marker related to wheat ear germination resistance and application thereof, and particularly relates to the technical field of genetic breeding, wherein an SNP locus corresponds to C/T base mutation at 661498776bp of a wheat 4B chromosome of a reference genome IWGSC Refseq v1.0 version; developing a CAPS marker according to the SNP molecular marker, wherein the CAPS marker is named as TaE3, and the TaE3 has a nucleotide sequence shown as SEQ ID NO.5 or a nucleotide sequence shown as SEQ ID NO. 6. The CAPS marker is used for identifying the wheat pre-germination resistant variety. The detection method of the invention is simple and convenient, and is used for the auxiliary breeding of the anti-PHS molecular marker of wheat.

Description

SNP molecular marker related to wheat ear germination resistance and application thereof

Technical Field

The invention belongs to the technical field of wheat genetic breeding, and particularly relates to an SNP molecular marker related to wheat ear germination resistance and application thereof.

Technical Field

Pre-harvest Sprouting (PHS) refers to a phenomenon that seeds directly sprout on wheat ears when wheat is exposed to rainy weather or in a high-humidity environment for a long time before being harvested. The occurrence of PHS not only reduces the wheat yield, but also easily causes the quality to be reduced, and serious people even lose the seed value, thereby causing huge economic loss for agricultural production. For example, storage substances (protein, starch and the like) in endosperm of germinated wheat are degraded, and the nutritional quality is reduced along with the degradation; in particular, bread made with germinated wheat has poor elasticity and is not easily cut, steamed bread has a dark color and increased stickiness, and noodle bite becomes poor and toughness is reduced. In addition, with the increase of the germination degree, the total fat content in the wheat grains is remarkably reduced, the fatty acid content is greatly increased, the rancidity is deteriorated, and the method is extremely unfavorable for seed storage.

Major wheat producing areas of the world, such as canada, the united states, western and northern regions of europe, australia, japan, china, etc., all suffer from PHS hazards to varying degrees, with australia and canada being more serious. It has been reported that PHS disasters caused by summer rainfall affect on average 15% of grain quality annually in northern new south welsh, australia and queensland. In canada, another important wheat export country, where approximately 800 million hectares of spring wheat, 200 million hectares of durum wheat, and 40 million hectares of winter wheat are planted, PHS occurs to some extent each year, causing significant quality degradation and economic loss. In China, the southwest winter wheat area (especially the Sichuan basin), the Huang-Huai-nan winter wheat area, the northeast spring wheat area and the middle and lower reaches of Yangtze river are the main occurrence areas of the PHS disaster. In 1991, the yield reduction of wheat caused by PHS disaster in summer harvest of wheat is reported to be as high as 100 hundred million kilograms nationwide. 1995. In 2001 and 2008, the jingjin Ji area has a serious PHS disaster, which causes huge loss, and leads a large number of farmers in the area to abandon white skin wheat with better processing quality and to improve red skin wheat with relatively low market popularity. In 2008 + 2010, PHS disasters occurred in parts of provinces including Jiangsu, Anhui, Hubei and Henan, and the white-skin wheat is particularly seriously damaged. According to the 'Chinese wheat quality report' (ministry of agriculture) published in 2010-2013, the grain quality parameters of the samples in the southwest wheat area and PHS are lower than those in other PHS-rich areas (Yangtze river downstream, Huang-Huai-wheat area and the like). In 2016, precipitation in most of winter wheat areas in the north, most of south China in the south of the river, parts of the east of the southwest, west of the river, yellow river and north of the west is frequent in wheat harvesting seasons, so that harvesting and airing of wheat are greatly influenced, PHS disasters are serious, the yield is almost halved, the quality is degraded, and farmers and small farmers without dryers lose much disastrous. In 2018, accumulated precipitation of southern Henan, great Anhui, West and southeast Hubei, Central south Jiangsu and Sichuan basins in the harvest period of wheat exceeds 100 milliliters, so that PHS disasters occur in the regions, the yield is reduced seriously, the purchase price of the wheat is reduced to 0.6-0.8 yuan, even to 0.3-0.4 yuan in extreme cases, and the average loss of farmers per mu is about 300 yuan.

Wheat PHS is a quantitative trait belonging to polygene control. The PHS resistance locus or QTL identified by predecessors using linkage analysis and association analysis almost extends over 21 chromosomes of wheat. In addition, by means of map-based cloning or homologous cloning, part of the wheat PHS resistance genes are also excavated successively, and functional markers are developed for screening germplasm resources or breeding materials with strong PHS resistance. If linkage analysis is used for detecting that a major site QPhs exists on the short arm of the 3A chromosome, pseru-3AS, a target gene TaMFT (TaPHS1) is further excavated by a fine positioning and map-based cloning method, and the forward regulation and control effect of the TaMFT on PHS resistance is verified by using an RNA interference mediated gene silencing method. A homologous gene TaSdr of a rice seed dormancy gene OsSdr4 is cloned by adopting a comparative genomics method, is positioned in the 2 nd homologous group of wheat and is respectively named as TaSdr-A1, TaSdr-B1 and TaSdr-D1. SNPs are respectively found at the upstream-11 bp of the initiation codon of the TaSdr-B1 gene and at the +643bp of the TaSdr-A1 gene, and are developed into CAPS markers (cleaned amplified polymorphic sequence marker) Sdr2B and Sdr2A, and the two variations are verified to be obviously related to seed dormancy and germination in a linkage population and a natural population. The gene TaVp-1 of the wheat transcription factor VIVIPAROUS-1 is positioned on the long arm of the 3 rd homologous group by a method combining comparative genomics and linkage map analysis. The expression level of the TaVp-1 gene in the mature embryo in the resting stage and the non-resting stage is measured, and the expression level of the TaVp-1 gene is found to be positively correlated with the sensitivity of the embryo to ABA and the resting stage of the seeds. In addition, TaPM19-A1 and TaPM19-A2 belonging to the ABA-induced wheat plasma membrane 19 gene family, TaMKK3-A encoding mitogen-activated protein kinase 3 and TaQsd1 encoding alanine aminotransferase were also identified and confirmed to be closely related to wheat ear germination resistance and seed dormancy. Although the genetic mechanism of wheat ear germination resistance has been well developed, the detailed genetic basis and molecular control mechanism are still unclear.

Given the large size of the wheat genome (about 17Gb) and the relatively complex nature of wheat (allohexaploids, containing more than about 80% repeats), the genetic mechanism of PHS resistance may differ significantly from one another in different genetic backgrounds. Therefore, more PHS major sites are identified from heterogeneous resources, candidate genes are mined, the molecular mechanism of PHS resistance is further analyzed, and abundant gene resources are provided for PHS-resistant molecular polymerization breeding.

Disclosure of Invention

The invention aims to develop CAPS markers which are easy to detect in large scale and in batch aiming at a candidate gene TaE3-4B of a new major site Qphs.

The technical scheme is as follows:

the invention provides an SNP molecular marker related to wheat ear germination resistance, wherein the SNP locus of the SNP molecular marker corresponds to C/T base mutation at 661498776bp of wheat 4B chromosome of a reference genome IWGSC Refseq v1.0 version.

The invention also provides a CAPS marker which is developed according to the SNP molecular marker and is used for identifying the wheat pre-germination resistant variety, the CAPS marker is named as TaE3, and the TaE3 has a nucleotide sequence shown as SEQ ID NO.5 or a nucleotide sequence shown as SEQ ID NO. 6.

The invention also provides a method for identifying the wheat pre-harvest sprouting resistant variety by using the CAPS marker, which comprises the following specific steps:

s1, designing specific primers according to the sequence SEQ ID NO.5 and the sequence SEQ ID NO.6, and selecting restriction endonucleases according to the difference between the sequence SEQ ID NO.5 and the sequence SEQ ID NO.6, so that the sequence SEQ ID NO.5 and the sequence SEQ ID NO.6 can be cut into fragments with different numbers;

s2, extracting the wheat genome DNA to be detected;

s3, performing PCR amplification by using the DNA extracted in the step S2 as a template and the specific primer designed in the step S1 as a primer to obtain a PCR amplification product;

s4, carrying out enzyme digestion on the PCR amplification product obtained in the step S3 by using the restriction enzyme selected in the step S1, carrying out electrophoresis detection on the enzyme digestion product, and judging the wheat variety according to the number of bands.

Preferably, the upstream primer and the downstream primer in the specific primer designed in step S1 have the nucleotide sequences shown in SEQ ID No.3 and SEQ ID No.4, respectively.

Preferably, the restriction enzyme used in step S3 is AccII, and if one electrophoresis band of the enzyme-cleaved product in step S4 is one, the cut product is a pre-sprouting susceptible variety, and if several cut products are obtained, the cut product is a pre-sprouting resistant variety

The invention has the beneficial effects that:

the present invention develops CAPS marker TaE3 for candidate gene TaE3-4B of a new major site Qphs. ahau-4B for controlling PHS resistance. Compared with SSR molecular markers, the CAPS marker has clear and distinguishable bands after electrophoresis detection, obvious band type difference among different PHS resistant wheat varieties, simple and convenient detection method, and is beneficial to improving the target property and pertinence of molecular marker selection, thereby improving the efficiency of genetic improvement of the PHS resistance of wheat.

Drawings

FIG. 1 is the PHS-resistant new major site Qphs. ahau-4B detected in the Jing 411/Hongmanchun 21 population in example 1;

FIG. 2 shows the differential expression of TaE3-4B gene in the Qphs. ahau-4B segment in example 1 in the two parents and the resistance and susceptibility pools of the Jing 411/Hongmanchun 21 population;

FIG. 3 is the electrophoresis chart of the different varieties of wheat of Jing411 and Hongmangchun 21 with different PHS resistances detected by CAPS marker TaE3 in example 2.

Wherein the reference numerals are: m is a strip of a 2K Marker; j is a Beijing 411 type strip; h is a Hongmanchun 21 type strip.

Detailed Description

Example 1

Identification of candidate genes

The invention utilizes a promoted variety 'Jing 411' which is sensitive to PHS and a highly PHS-resistant 'Hongmanchun spring 21' as parents to construct a recombinant inbred line group (JH-RIL) containing 174 families, adopts a simplified genome sequencing means to construct a high-density genetic linkage map based on a SLAF marker, combines Germination Index (GI) data measured in 2014-plus 2019 after harvesting for carrying out PHS-related major QTL analysis, and discovers a major site Qphs.ahau-4B for controlling PHS on a wheat 4B chromosome. A secondary segregating population was constructed for this site and screened for recombinants, reducing the physical segment from 2.25Mb to 154.7 Kb. Combining transcriptome separation cluster mixed pool sequencing, and screening out the candidate gene which is uniquely and differentially expressed in the segment and encodes E3 ubiquitin ligase, wherein the candidate gene is named TaE 3-4B. Cloning and sequencing are carried out among anti-PHS and anti-PHS varieties, and 18 Single Nucleotide mutations (SNP) are found in total, wherein 5 is positioned in a coding region. The CAPS marker TaE3 was developed using the C/T (sense/anti) base mutation (located at 661498776bp on chromosome 4B, which is referred to the genome IWGSC RefSeq v1.0 version) present on exon 3 of the TaE3-4B gene, the sense material (lacing 411-type) cannot be cut by the restriction enzyme AccII, and the resistant material (HMC21-type) is reversed (FIG. 3). The details are as follows:

(I) measurement of germination-related phenotypic Properties

Germination Index (GI) determination

2014-2019, at different periods (5 days and 15 days after harvesting), taking the complete seeds of 50 embryos of the group parents and each family, repeating 2 times, uniformly placing the seeds in a culture dish with the diameter of 90mm with the ventral sulcus facing downwards, adding 10mL of sterile water, and culturing for 3 days under the conditions of 20 ℃ and 14h (day)/10 h (night). Recording the number of germinated seeds n on the first day₁The number of germinated seeds n in the next day₂And the number of the germinated seeds n on the third day₃And the number n of the remaining ungerminated seeds after 3 days₀. Calculating the germination index GI of the seeds according to the following formula:

GI＝(3*n₁+2*n₂+n₃)/3*(n₁+n₂+n₃+n₀)。

field Natural germination Rate (FS) determination

In the harvest season of wheat, if continuous rainfall occurs, 20 ears of each family are left in the field, after the rainfall is finished, the ears of each family are immediately collected and dried in an oven (105 ℃, 2 hours), and the number n of germinated seeds is recorded after threshing_GAnd number of ungerminated seeds n_RCalculating the field natural germination rate FS according to the following formula:

FS＝n_G/(n_G+n_R)。

determination of seed coat color

In 2015 and 2017, 50 seeds of each JH-RILs family were taken, and the number of the seeds was repeated 2, and the seeds were soaked in sterile water in a culture dish, and the color of the seed coat was observed and recorded, and indicated by GC, as 1 for white skin, 3 for red skin and 2 for medium color.

In 2016-2018, seed coat color was measured for the parent and each family of JH-RILs using a colorimeter (Minolta CR-5). The colorimeter uses the L x a b color system to assess color (CIE 1931). In this color system, the L value measured black is 0 white to 100, when a is a positive value, it represents red, otherwise it represents green, when b is a positive value, it represents yellow, otherwise it represents blue, and the brightness of the seed is also a factor to consider the seed coat color, and this study uses the a/L value to represent the seed coat color measurement value and uses GrainColor to represent it. About 20g of seeds are weighed and placed in a culture dish with the radius of about 25mm of the instrument for 3 times of repetition, and the average value represents the color phenotype value of each variety of seed coats.

The phenotype data consolidation is completed by Excel software, and the correlation analysis between the phenotype data and the marker is completed by SPSS software.

(II) extraction of wheat genome DNA

The test materials for DNA extraction are parent Jing411 and Hongmangchun 21, 174 recombinant inbred line RIL (Jing 411/Hongmangchun 21) groups, 255 wheat promoted varieties and 225 Chinese wheat micro-core germplasm resources. The specific method comprises the following steps:

1. wheat single grains were ground, and 0.1g of 0.7ml of an extract (0.1M Tris-HCl (pH 8.5), 0.1M NaCl, 0.05M EDTA (pH 8.0), 2% SDS) was added thereto to lyse at a constant temperature of 60 ℃ for 45min with shaking.

2. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min.

3. The supernatant was taken and added with equal volume of phenol: chloroform: isopentanol (25:24:1) was rotated upside down until the two phases did not separate quickly.

4. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min.

5. The supernatant was taken and added with equal volume of phenol: chloroform: isopentanol (25:24:1) was rotated upside down several times.

6. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min.

7. Adding equal volume of isopropanol into the supernatant, and standing in a refrigerator at-20 deg.C for 30 min.

8. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min.

9. Washed twice with 70% ethanol.

10. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min.

11. The precipitate is naturally dried in the air and stored at 4 ℃ for later use.

(III) design and sequencing of simplified genome digestion scheme

1. Selection of the enzyme digestion scheme: and finally selecting a wheat A genome as a reference genome for enzyme digestion prediction according to the information such as the size of the wheat genome, GC content and the like. And performing enzyme digestion prediction on the reference genome by enzyme digestion prediction software, and selecting the most appropriate enzyme digestion scheme.

2. The specific experimental process comprises the following steps: the genomic DNA of each sample that was found to be acceptable for the detection was digested with the restriction enzyme RsaI (New England Biolabs, NEB) according to the selected optimal digestion protocol. Subjecting the obtained digested Fragment (SLAF tag) to Klenow Fragment (3 ' → 5 ' exo-) (NEB) and dATP at 37 ℃ to 3 ' end A adding treatment, connecting double-index sequencing adaptor, PCR amplification (PCR amplification primer: the nucleotide sequence of an upstream primer is shown as SEQ ID NO.7, the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 8), purifying, mixing, cutting gel to extract a target Fragment, and using IlluminaHiSeq after the quality of the library is qualified^TM2500 for sequencing. To evaluate the accuracy of the digestion experiment, Nipponbare (Oryza sativa japonica) was used as a control for sequencing.

3. And identifying the original data obtained by sequencing by using the Dual-index to obtain the reads of each sample. The filtered adapters were evaluated for sequencing quality and data size of sequencing reads. And evaluating the enzyme cutting efficiency of the enzyme by the comparison efficiency of Control data, and further verifying the accuracy and the effectiveness of the experimental process. And (3) developing a large number of SLAF labels in parents and filial generations by adopting a method of comparing the reads with the genome, and screening the polymorphic SLAF labels. After the polymorphic SLAF label is subjected to genotype coding, the quality control is carried out according to the following standards:

(1) filtering reads with the sequencing depth of 10 multiplied by less than the parental stock;

(2) filtering tags with SNP number larger than 5;

(3) indicia with filtration integrity less than 70%;

(4) the label was severely biased (P <0.01) by filtration.

And constructing a high-density genetic map by using the controlled SLAF label through a HighMap mapping software, and using the map for QTL analysis after the map is qualified.

(IV) QTL analysis

An additive effect algorithm (ICIM-ADD) for mapping a complete composite interval in QTLICIMapping v4.1 software, wherein the LOD value is set to be 3, and the step interval is 1 cM.

(V) construction of Secondary segregating populations

Through analyzing the molecular marker band types of each family of the Jing 411/Hongmangchun 21 recombinant inbred line group in all PHS resistance-related major and minor QTL sections, JH86 family with stronger PHS resistance is found, 5 marker band types in the Qphs. ahau-4B section are consistent with the resistance parent 'Hongmangchun 21', and the molecular marker band types at all other QTL sites are consistent with the sensitivity parent 'Jing 411'. Based on the results, in the study, JH86 is used as a female parent and Jing411 is used as a male parent to construct a secondary separation population (JH86/J411) in 4 months in 2017. Harvesting F of secondary population JH86/J411 in 5 months of 2018₂Seeds are randomly divided into two sets, sowed in autumn in 2018 in Dayang shop in Hefei city and Huanyou city Huanyuan county Longkang experiment base in 10 months. At the end of 6 months in 2019, respectively completing Qphs_2:3Numbering of 1071 families (JH86/J411-HF) and 1309 families (JH86/J411-LK), leaf sample collection, single grain outcrop, threshing and PHS resistance phenotype evaluation.

(VI) "30 + 30" PHS-resistant extremely pooled transcriptome sequencing BSR-seq

30 PHS resistant and susceptible families are screened in a JH-RIL group according to the relative phenotype characters of multi-year PHS resistance, 30 seeds are taken from each family, 50 seeds are taken from parent Jing411 and Hongmangchun 21 respectively, the ventral furrow is placed on 2 layers of germinating paper in a sterilized culture dish downwards, 800ul of sterile water is added for moistening, and the mixture is placed in an incubator for culturing for 10 hours under the conditions of constant temperature of 20 ℃ and darkness. At this time, most of the seeds which are sensitive to the PHS family and the parents are in the sprouting state that embryo parts are exposed to white, and most of the seeds of the PHS resistant family and the parents are in the sprouting state that the embryo parts are expanded and not exposed to white and sprouting. The PHS material is prepared by mixing 2 seeds in each family to form a sensitive pool, preparing the anti-PHS material into an anti-pool by the same method, respectively preparing 10 seeds from Jing411 and Hongmangchun 21, and respectively filling the prepared PHS resistant and sensitive pools and 2 parent samples into a freezing tube to carry out liquid nitrogen quick freezing. The samples were then stored on dry ice and sent to behcet biosciences, inc for RNA extraction (equal mixing after single pellet extraction of mixed pool material) and RNA sample quality control.

The RNA samples qualified for purity, concentration and integrity detection were subjected to library construction according to the following procedures:

(1) eukaryotic mRNA was enriched with magnetic beads carrying oligo (dT).

(2) Fragmentation buffer was added to randomly break the mRNA.

(3) Using mRNA as template, the first strand was synthesized using hexabasic random primers (random hexamers)

And (3) adding a cDNA strand, then adding DNA polymerase I, dNTPs, a buffer solution and RNase H to synthesize another cDNA strand, and purifying the cDNA by using AMPure XP beads.

(4) And (3) carrying out end repair on the purified double-stranded cDNA, adding A tail and connecting a sequencing joint, and then carrying out fragment size selection by using AMPure XP beads.

(5) And finally, obtaining a cDNA library through PCR enrichment.

After the library is constructed, the concentration and Insert Size of the library are detected by Agilent 2100 and Qubit2.0 respectively, and the effective concentration is quantified by a q-PCR method (the effective concentration of the library is required to be not less than 2nM for ensuring the quality of the library).

After the quality of the library is qualified, sequencing is carried out by using a high-throughput sequencer HiSeq 2500. And performing bioinformatics analysis on the data generated by sequencing to obtain reliable data related to the differentially expressed genes, wherein the differentially expressed situation of the TaE3-4B gene between the parent and the mixed pool of anti-and anti-PHS is shown in FIG. 2.

(VII) preliminary identification and cloning of candidate genes

And screening the annotated genes in the Qphs. ahau-4B segment by using the differential expression analysis result obtained by the BSR-seq to preliminarily determine the candidate genes. The full length of the gene was obtained by referring to the wheat genome sequence, and using this as a template, specific primers were designed using Primer Premier 5.0(https:// www.PremierBiosoft.com) software for fragment amplification.

Gene cloning was performed on the sensitive material Jing411 (J411), Jimai 20(JM20), Zhongyou 9507(ZY9507) and the resistant material Hongmanchun 21(HMC21), Chengninglumong (SNTT) and Triticum aestivum (YXM) as follows.

Fragment amplification

The high fidelity enzyme Fastpfu with high amplification efficiency and high speed is used for PCR amplification according to the following reaction system: 10 μ l of 5 XPCR Buffer (15mM MgCl)₂) Mu.l dNTPmix (2.5mM), 2. mu.l forward primer (10. mu.M), 2. mu.l reverse primer (10. mu.M), 4ul template DNA (50-100ng/ul), 1. mu.l Fastpfu (2.5U/. mu.l), distilled water to make up to 50. mu.l. Reaction procedure: pre-denaturation at 95 ℃ for 2min, denaturation at 95 ℃ for 20s, annealing at the annealing temperature required by each pair of primers for 20s, extension at 72 ℃ (the extension time is calculated according to the fragment length and the amplification efficiency of Fastpfu from 2 kb/min to 4 kb/min), 3 steps of denaturation-annealing-extension are circulated for 35 times, additional extension at 72 ℃ for 5min, and the product is stored at 4 ℃.

Electrophoresis and gel recovery

Mu.l of 6 XDNA loading Buffer was added to the PCR product and electrophoresed on 1.5% agarose gel. After completion of the electrophoresis, the gel containing the desired fragment was excised, and the desired fragment was recovered and purified by the procedure of instructions (https:// www.cwbiotech.com/uploads/websiteddf/216 c4037-3eae-4ac2-b86a-762277a7adc1.pdf) using an agarose gel DNA recovery kit available from Kangji Biotech Co., Ltd.

Sequencing by cloning

Mu.l of plasmid (Blunt-zero, available from Kyoto Kogyo Biotech, Inc., N.t.: http:// www.transgen.com.cn /) and 4. mu.l of PCR-purified product were added to the sample tubes, ligated for 20min at 25 ℃ and cooled at 12 ℃. The ligated sample was removed, 50. mu.l of competent cells (Trans-T, stored at-80 ℃ before use) were added to the UV-sterilized bench, gently mixed, and ice-cooled for 20 min. After completion, the sample was transferred to a water bath pan and heat-shocked at 42 ℃ for 45s, and then immediately placed on ice to stand for 2 min. To the sample was added 950. mu.l of SOC medium (per 100mL of formulation: 2g tryptone, 0.5g yeast extract, 0.06g NaCl, 0.02g KCl, 0.2033g MgCl 2.6H2O, 0.2465g MgSO 4.7H2O, 0.36g glucose, 100mL ultrapure water) on a sterile console, and shaken at 37 ℃ for 1-1.5H. Next, 200. mu.l of the bacterial solution was taken out from the sterile console and uniformly applied to LB solid medium (per 100mL of the formulation: 1g of tryptone, 0.5g of yeast extract, 0.5g of NaCl, 1.5g of agar powder, 100. mu.l of kanamycin solution, 100mL of ultrapure water) to which kanamycin had been added, followed by incubation at 37 ℃ for 15 to 16 hours. After the bacterial plaque grows to a proper size, 6 mul of sterile water is added into a PCR sample hole, the bacteria are picked into the hole by using a pipette gun with the maximum range of 10 mul, after the bacteria are gently sucked and uniformly stirred, 4 mul of bacteria are taken out and added into a centrifuge tube with 1000 mul of LB liquid culture medium (each 100mL of formula: 1g of tryptone, 0.5g of yeast extract, 0.5g of NaCl, 100 mul of kanamycin solution and 100mL of ultrapure water). And (3) placing the centrifugal tube in an environment at 37 ℃ for shaking bacteria for 4-6h, and simultaneously carrying out PCR detection by using M13 as a primer and using 2 mu l of bacteria liquid left in a sample hole as a template. If the PCR reaction result of the bacterial liquid is positive and the target fragment is contained, the bacterial liquid can be sent for sequencing.

(eight) results

The results are shown in table 1 and fig. 1 to 2.

TABLE 1 information of anti-PHS new major site Qphs. ahau-4B detected in Jing 411/Hongmanchun 21 population

Note that 14GI-1, 15GI-1, 17GI-1 and 18GI-1 refer to germination index GI measured 5 days after harvest in 2014, 2015, 2017 and 2018, respectively; 15GI-2 and 16GI-2 refer to germination index GI measured 10 days after harvest in 2015 and 2016, respectively; 15FS and 16FS refer to field natural germination rates FS measured in 2015 and 2016 respectively; 15GC and 17GC respectively refer to GC of the seed coat color observed by naked eyes after seeds are soaked in sterile water in 2015 and 2017; 17 and 18 graincolors refer to the seed coat color measured by the colorimeter in 2017 and 2018, respectively.

FIG. 1 (a) shows the major site of pre-germination resistance Qphs. ahau-4B and associated traits detected by QTL analysis at chromosome 4B 660-662 Mb; (b) shows that the differential SNP is found to be enriched in the 640-680Mb section after the 660K chip scans the anti-cluster germination mixed pool and the cluster infection germination mixed pool. The results of the two can be verified against each other. (nine) sequence analysis and functional marker development of candidate genes

Splicing, alignment and structural analysis of the sequencing results of the candidate genes were performed by DNAMAN software (https:// www.lynnon.com/DNAMAN. html). It can be found that the sequence of the coding region of the candidate gene TaE3-4B gene in the PHS-resistant material is shown as SEQ ID NO.1, and the sequence of the coding region in the PHS-sensitive material is shown as SEQ ID NO. 2. The sequence difference found by comparison in the anti-PHS material and the PHS-sensitive material is designed into a CAPS marker (namely CAPS marker TaE3) by using PrimerPremier 5.0(http:// www.premierbiosoft.com) software, the nucleotide sequence in the anti-PHS material is shown as SEQ5, and the nucleotide sequence in the PHS-sensitive material is shown as SEQ 6. The sequence of the upstream primer in the designed primer pair is shown as SEQ ID NO.3, and the sequence of the downstream primer is shown as SEQ ID NO. 4.

(ten) amplification, cleavage and electrophoresis of CAPS marker

1. And (3) PCR amplification system configuration: mu.l of 2.5mM dNTP, 0.25. mu.l of 10. mu.M primer, 1. mu.l of 10 × EasyTaq Buffer, 0.5U EasyTaq, and 100ng of DNA template were made up to 10. mu.l with double distilled water.

2. PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 62 ℃ and 0.3 ℃ for 30s after dropping every cycle; extension at 72 ℃ for 30s for 40 cycles; extension at 72 ℃ for 8 min.

3. And (3) enzyme digestion system after amplification: mu.l of PCR product, 1. mu.l of 10 XCutSmart Buffer, 0.5UAccII, was made up to 10. mu.l with double distilled water.

4. And (3) enzyme digestion reaction program: 6h at 37 ℃.

After the digestion, 3. mu.l of 6 XDNA Loading Buffer was added to the sample, and 5. mu.l of the digested product was fractionated by 2.5% agarose gel electrophoresis. The results are shown in FIG. 3: the Jing411 (sensitive PHS, T allelic type) band can not be cut by enzyme, and the Hongmanchun spring 21 (anti-PHS, C allelic type) band can be cut. Wherein, the nucleotide sequence of the amplification product of the Hongmanchun 21 (anti-PHS, C allelic type) is shown as SEQ ID NO.5, and the nucleotide sequence of the amplification product of Jing411 (sensitive PHS, T allelic type) is shown as SEQ ID NO. 6.

Verification of (eleven) CAPS signatures in natural populations

Mannich-Whitney test (U test) analysis among different allelic types and PHS resistance related traits in 255 wheat promoted varieties and 225 wheat micro-core germplasm resources is completed by IBM SPSS Statistics 20(www.spss.com) software. The specific names of 255 wheat promoted varieties and 225 wheat micro-core germplasm resources are shown in table 2, and the experimental results are shown in table 3:

TABLE 2 wheat promoted varieties and detailed names of wheat micro-core germplasm resources

TABLE 3 verification of the CAPS marker TaE3 with the PHS trait by using 255 wheat promoted varieties and 225 wheat micro-core germplasm

Note: 12GI-1, 13GI-1, 14GI-1 and 15GI-1 refer to germination index GI measured 5 days after harvest in 2012, 2013, 2014 and 2015, respectively; 14GI-2 and 15GI-2 refer to the germination index GI measured 10 days after harvest in 2014 and 2015, respectively; 13FS and 15FS refer to 2013 and 2015 measurements, respectivelyThe field natural germination rate FS;^**the two different allelic types representing the markers had a very significant correlation with the trait at the 0.01 level.

The results show that in the 2 natural populations mentioned above, the difference between the values of the germination-associated phenotype (GI and FS) reached a very significant level (P <0.01) between wheat varieties carrying two allelic variation types (TaE3-HMC21 type and TaE3-J411 type) of CAPS marker TaE 3.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Sequence listing

<110> agriculture university of Anhui

<120> SNP molecular marker related to wheat ear germination resistance and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4388

<212> DNA

<213> wheat (Triticum aestivum)

<400> 1

gaaattgaca gaagaaagaa aaggaggaac gaaaaatccc gttcaacttt aaaccgctct 60

tgccctcgcc cttctccacc gccgccgccg ccgccgccgc cgtcatgccg ccgcgccggg 120

gaaggcgcgc gtctcctccg gtgccgatcc tggaggacga ggacctcgat ttcgaggtca 180

gctacagcga aggggaggac ggggaagatg gcggaggatc ggattcctcg tcgtcgtcgt 240

cgggggagga gggaacggat gtagaggacg gcgaaagatc ggatgagggg gaggaggagg 300

aggagcagga ggggcaggag gagagcgtcg cggagggatg cgggccggcc gtgcaagcta 360

gggttcccgg cggcgcggcg gcggagaggg ccgccagcgc ccccacctgc cccgtctgta 420

tggagccctg gacctccgaa ggcgagcacc gcatcaggtg aggccgaatc gctgcctcat 480

tcttgcctgg tctgcgtctg ctaggtagta ctagctgtca caggaacagg ggcgccgtgg 540

gtgtttcttt ccaagtaaca atggttactg ggctgcaaga aaatggaaac ttacaatgtt 600

ctaacggtac tactacatga ttgcacatta gcgccatggt cgacgtcgcc ttgaattttg 660

acggaccatg gctggttcct catgtaatcc tggtttttgt ttccaatgct ctgtggattc 720

ggtgtgcaga tggctgttga ctgaaattgt actctatgct gtagttgcat tccttgcggg 780

catgtctacg gcagatcttg cctggagaga tggttaacgc agcgtggtaa cgctagtgcg 840

acggtacgtg tgccgaatgg ttttttcgtt tggaataaca gcgtgtgcat acacatcctt 900

ctgcgcaatc agccaatcac tgtccaatgt gttgtttgct agtgccctca atgtggaaga 960

aggtttaaac ataaggacat tatcaacatc tatgcaccgg aggttgctgt tccaaataac 1020

gatcttgaga aggttgggtt tgtttctcca ctttgtcggt atattttgtt ttggccgcga 1080

tctgaacatc gctgcttgtc tgcagcaatt atggttttgc cggcaaaagc tcgaatccct 1140

tgaggaagtg gtaagcaatt tccttatgct acccgcgagc ttctttagtt gctcaccatg 1200

tgcaaaatgc tgttctcttt ctagtttgta ttagtaagtt cctgctaaca taattttttt 1260

cttgtcttaa ggtcctgaaa caagggaagc tgcttgacga gataatttct gagaaggtat 1320

gctatagatt ttagcaatct ttgctgctta ttgcatgccc cacattctaa acgattaaaa 1380

ttgtgtcaag tgcatagtgt cactagatag tatgtttgca cctgttattc aggtactgta 1440

ataatataca aagaaggtat tagaggttct tcatattgaa ctaatacatc tctcatcttg 1500

ctttattcat cgattgttcc tgcatcagaa aattaatgtc gcaatgatca atagttgtca 1560

cacttgtgag agacttatgc cttcattttc ttatgtgtgt gccttgcaga atcacaggtc 1620

ggcagatgtt ggtgtctcaa aacgacaggt aataccattt aaatttttca atgatcaatg 1680

acaccacatg agtgtatatg ccaaatcttc ttcctctgct ttgtagaaaa tagcagagca 1740

ctcagatgga agaacatatc tggaaccgtc agcctcagcc agtgcagcct cagcttctgg 1800

caacagttgc cgtttcgttt tactggtaaa tatttgttac gaagtgctat tttcagctgt 1860

aaacattcag catatactat ttactatgta ctacatgttt tactgataat gggtgtcttg 1920

tattcttgtc tgatctgaaa catatggtca ttttagtttt agtaaaagca gcatcgaagt 1980

agaaattggg ttgggcaggg aattcatgat tctggcccag cttctgttag atatataaat 2040

cgaaaatgtt gtcaactatg attattttgt ttcaaataac acatataaac cttgttttgt 2100

ttgcatgtta tggtttagga ggttgaacag aatattaaga ctcgtaatac ccatcttatt 2160

tttcgtgatt aaggaagctg aaacttttgg ttgtgttgat tgtcagatct tagaacagtt 2220

ttcacttggt catgaaaatg tgtgatctgc acataggaac ttgatcccac ctcaaccccg 2280

cgaaaaagaa cttggtggaa ctgatggctg tctaattttt tatggcttag tacagaatac 2340

tccagttttg tcccacccca tgtgtaccta ttgtgaataa ttgaaatata ttaaagcaaa 2400

gtggcaccaa atgcttagct agtgtcgaaa gaccttcata atttggactg ctaattctcg 2460

tggattatct gtttgtttga aaccgctgtg cattgcctac atatttcttc caccaatttt 2520

gcagttggat tctggctgga tttatctctt tccaaattta atctagccat ccttgtaatt 2580

tctttaccat gctatacaga aggaattatc ttttgatggt gctcgagtca tgggcataga 2640

tgcatgtaac caaataatac ttgcttctgg gaaggcacct ggtgtgggtg gagaacatgt 2700

tcttagaaag gtatatttag atcatgtttt tctcccaaaa tggatatcac cattaaaatt 2760

ttgaaacacc ttattgtgaa ttttcaatgt caacagatta gcatgctatc cagccatgaa 2820

gcacgtaaaa tacagcttcc tcctgatact aaagttgtca aggacatatg tatcttgcct 2880

ggtggctctg ctctttttgc atcactaggc agaaggctct cactctttag gtttgtcttg 2940

aattttttgt atgacaaatg gtggttcttc tgttttactg tcgcgtctaa gttagtttta 3000

ccattatttc ttttgaacac taatcgttat atttgaatcc agcatgacga ccaacagtgt 3060

tgttcttcaa tgtaatttac cggtaattat ctgaccttcc aatcttcaac acgtaatgtt 3120

ttgtattttg aatcggcaaa tcaggtgaac caatttcaga taatgacatt atgacctaat 3180

tctctatcac actaccttgc aggttcctgc ttggtcatgt tcagcacatg actctgattc 3240

acgtcgcgtt tatgctggct tgcaggttca gcatatcctg atcttaagca tattgaattt 3300

tgataccctt tgcaagattt gcatgaatgt ccttgttgtg ttctgcagga tggcagggtt 3360

ttggtatttg atactcgtca gcattcaaga cccttgcatt ccatggcggg gctatctaaa 3420

catctggtcc atacactcca ctctgtcact gataacagcg gttccagaaa ggttctttca 3480

gcttctgcta ttgggccttg catgtgggat gctgatggca atcaaagcag gtatgtgaca 3540

cagggatggt tcaaaaacat taatactatg tgcttcttat cattactgat gtcgattggt 3600

gttcgcctag gaccgccttt gcaactttgc cgatctgcaa atcggttttg gttctcatgt 3660

atgttgtcgt tttttctcag tccgaagcta ctactagagg atgataacca acacgtctgc 3720

ttctctcttg cgtgtgcccc tccatcgagc gatctgttgg tggcctccta ccggcccaag 3780

gccgattact catcaggaga cgccgctgct ccatctcaag cgtacctatc acagacgtcg 3840

acacagtctg gtgcagggaa actggggcag cacaccgtca tcaggaggac aggcaatgca 3900

tccttcgccg agggcagcac atgccactcc aacgtaagcg aagtgcgcat gtgcaaatca 3960

gcaatcgtac cttgcgggaa cgacgaacat ctcttcgcct acggggacga gtcacaccgc 4020

ggggtcctga cctggcgact accttccctt ggggtccatt ctggcctgat accccaccgc 4080

cagccaatcc tcgacctaag gtatgcggga agtcgagtgg gaggtgggta ccttgggtgc 4140

ctgagcgatg ataagttgca agtttacaga gttgatagat aggtagaatg gcagcaggga 4200

tccaacagtc atcataactt tagttcgcga tgattagccc tcctaattat gcggtatgac 4260

tgtcagtcct tttgctgtgt acaaagcaga acagctagct gtatatgcac cgtgatgttg 4320

tgatgtagta caaattaaag gcagcgatgt ttatttctta ccatggaagt gagaatctgt 4380

tttgagtt 4388

<210> 2

<211> 4388

<212> DNA

<213> wheat (Triticum aestivum)

<400> 2

gaaattgaca gaagaaagaa aaggaggaac gaaaaatccc gttcaacttt aaaccgctct 60

tgccctcgcc cttctccacc gccgccgccg ccgccgccgc cgtcatgccg ccgcgccggg 120

gaaggcgcgc gtctcctccg gtgccgatcc tggaggacga ggacctcgat ttcgaggtca 180

gctacagcga aggggaggac ggggaagatg gcggaggatc ggattcctcg tcgtcgtcgt 240

cgggggagga gggaacggat gtagaggacg gcgaaagatc ggatgagggg gaggaggagg 300

aggagcagga ggggcaggag gagagcgtcg cggagggatg cgggccggcc gtgcaagcta 360

gggttcccgg cggcgcggcg gcggagaggg ccgccagcgc ccccacctgc cccgtctgta 420

tggagccctg gacctccgaa ggcgagcacc gcatcaggtg aggccgaatc gctgcctcat 480

tcttgcctgg tctgcgtctg ctaggtagta ctagctgtca caagaacagg ggcgccgtgg 540

gtgtttcttt ccaagtaaca atggttactg ggctgcaaga aaatggaaac ttacaatgtt 600

ctaacggtac tactacatga ttgcacatta gcgccatggt cgacgtcgcc ttgaattttg 660

acggaccatg gctggttcct catgtaatcc tggtttttgt ttccaatgct ctgtggattc 720

ggtgtgcaga tggctgttga ctgaaattgt actctatgct gtagttgcat tccttgcggg 780

catgtctacg gcagatcttg cctggagaga tggttaacgc agcgtggtaa cgctagtgcg 840

acggtacgtg tgccgaatgg ttttttcgtt tggaataaca gcgtgtgcat acacatcctt 900

ctgcgcaatc agccaatcac tgtccaatgt gttgtttgct agtgccctca atgtggaaga 960

aggtttaaac ataaggacat tatcaacatc tatgcaccgg aggttgctgt tccaaataac 1020

gatcttgaga aggttgggtt tgtttctcca ctttgtcggt atattttgtt ttggccgcga 1080

tcagaacatc gctgcttgtc tgcagcaatt atggttttgc aggcaaaagc tcgaatccct 1140

tgaggaagtg gtaagcaatt tccttatgct acccgcgagc ttctttagtt gctcaccatg 1200

tgcaaaatgc tgttctcttt ctagtttgta ttagtaagtt cctgctaaca taattttttt 1260

cttgtcttaa ggtcctgaaa caagggaagc tgcttgacga gataatttct gagaaggtat 1320

gctatagatt ttagcaatct ttgctgctta ttgcatgccc cacattctaa acgattaaaa 1380

ttgtgtcaag tgcatagtgt cactagatag tatgtttgca cctgttattc aggtactgta 1440

ataatataca aagaaggtat tagaggttct tcatattgaa ctaatacatc tctcatcttg 1500

ctttattcat cgattgttcc tgcatcagaa aattaatgtc gcaatgatca atagttgtca 1560

cacttgtgag agacttatgc cttcattttc ttatgtgtgt gccttgcaga atcacaggtc 1620

ggcagatgtt ggtgtctcaa aacgacaggt aataccattt aactttttca atgatcaatg 1680

acaccacatg agtgtatatg ccaaatcttc ttcctctgct ttgtagaaaa tagcagagca 1740

ctcagatgga agaacatatc tggaaccgtc agcctcagcc agtgcagcct cagcttctgg 1800

caacagttgc cgtttcgttt tactggtaaa tatttgttac gaagtgctat tttcagctgt 1860

aaacattcag catatactat ttactatgta ctacatgttt tactgataat ggatgtcttg 1920

tattcttgtc tgatctgaaa catatggtca tcttagtttt agtaaaagca gcatcgaagt 1980

agaaattggg ttgggcaggg aattcatgat tctggcccag cttctgttag atatataaat 2040

ctaaaatgtt gtcaactatg attattttgt ttcaaataac acatataaac cttgttttgt 2100

ttgcatgtta tggtttagga ggttgaacag aatattaaga ctcgtaatac ccatcttatt 2160

tttcgtgatt aaggaagctg aaacttttgg ttgtgttgat tgtcagatct tagaacagtt 2220

ttcacttggt catgaaaatg tgtgatctgc acataggaac ttgatcccac ctcaaccccg 2280

cgaaaaagaa cttggtggaa ctgatggctg tctaattttt tatggcttag tacagaatac 2340

tccagttttg tcccacccca tgtgtaccta ttgtgaataa ttgaaatata ttaaagcaaa 2400

gtggcaccaa atgcttagct agtgtcgaaa gaccttcata atttggactg ctaattctcg 2460

tggattatct gtttgtttga aaccgctgtg cattgcctac atatttcttc caccaatttt 2520

gcagttggat tctggctgga tttatctctt tccaaattta atctagccat ccttgtaatt 2580

tctttaccat gctatacaga aggaattatc ttttgatggt gctcgagtca tgggcataga 2640

tgcatgtaac ctaataatac ttgcttctgg gaaggcacct ggtgtgggtg gagaacatgt 2700

tcttagaaag gtatatttag atcatgtttt tctcccaaaa tggatatcac cattaaaatt 2760

ttgaaacacc ttattgtgaa ttttcaatgt caacagatta gcatgctatc cagccatgaa 2820

gcacgtacaa tacagcttcc tcctgatact aaagttgtca aggacatatg tatcttgcct 2880

ggtggctctg ctctttttgc atcactaggc agaaggctct cactctttag gtttgtcttg 2940

aattttttgt atgacaaatg gtggttcttc tgttttactg tcgcgtctaa gttagtttta 3000

ccattatttc ttttgaacac taatcgttat atttgaatcc agcatgacga ccaacagtgt 3060

tgttcttcag tgtaatttac cggtaattat ctgaccttcc aatcttcaac acctaatgtt 3120

ttgtattttg aatcggcaaa tcaggtgaac caatttcaga taatgacatt atgacctaat 3180

tctctatcac actaccttgc aggttcctgc ttggtcatgt tcagcacatg actctgattc 3240

acgtcgtgtt tatgctggct gcaaagttca gcatatcctg atcttaagca tattgaattt 3300

tgataccctt tgcaagattt gcatgaatgt ccttgttgtg ttctgcagga tggcagggtt 3360

ttggtatttg atactcgtca gcattcaaga cccttgcatt ccatggcggg gctatctaaa 3420

catctggtcc atacactcca ctctgtcact gataacagcg gttccagaaa ggttctttca 3480

gcttctgcta ttgggccttg catgtgggat gctgatggca atcaaagcag gtatgtgaca 3540

cagggatggt tcaaaagcat taatactatg tgcttcttat cattactgat gtcgattggt 3600

gttcgcctag gaccgccttt gcaactttgc cgatctgcaa atcggttttg gttctcatgt 3660

atgttgtcgt tttttctcag tccgaagcta ctactagagg atgataacca acgtgtctgc 3720

ttctctcttg cgtgtgcccc tccatcgagc gatctgttgg tggcctccta ccggcccaag 3780

gccgattact catcaggaga cgccgctgct ccatctcaag cgtacctatc acagacgtcg 3840

acacagtctg gtgcagggaa actggggcag cacaccgtca tcaggaggac aggcaatgca 3900

tccttcgccg agggcagcac atgccactcc aacgtaagcg aagtgcgcat gtgcaaatca 3960

gcaatcgtac cttgcgggaa cgacgaacat ctcttcgcct acggggacga gtcacaccgc 4020

ggggtcctga cctggcgact accttccctt ggggtccatt ctggcctgat accccaccgc 4080

cagccaatcc ttgacctaag gtatgcggga agtcgagtgg gaggtgggta ccttgggtgc 4140

ctgagcgatg ataagttgca agtttacaga gttgatagat aggtagaatg gcagcaggga 4200

tccaacagtc atcataactt tagttcgcga tgattagccc tcctaattat gcggtatgac 4260

tgtcagtcct tttgctgtgt acaaagcaga acagctagct gtatatgcac cgtgatgttg 4320

tgatgtagta caaattaaag gcagcgatgt ttatttctta ccatggaagt gagaatctgt 4380

tttgagtt 4388

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tgccatcagc atcccaca 18

<210> 4

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cagcatgacg accaaca 17

<210> 5

<211> 481

<212> DNA

<213> wheat (Triticum aestivum)

<400> 5

tgccatcagc atcccacatg caaggcccaa tagcagaagc tgaaagaacc tttctggaac 60

cgctgttatc agtgacagag tggagtgtat ggaccagatg tttagatagc cccgccatgg 120

aatgcaaggg tcttgaatgc tgacgagtat caaataccaa aaccctgcca tcctgcagaa 180

cacaacaagg acattcatgc aaatcttgca aagggtatca aaattcaata tgcttaagat 240

caggatatgc tgaacctgca agccagcata aacgcgacgt gaatcagagt catgtgctga 300

acatgaccaa gcaggaacct gcaaggtagt gtgatagaga attaggtcat aatgtcatta 360

tctgaaattg gttcacctga tttgccgatt caaaatacaa aacattacgt gttgaagatt 420

ggaaggtcag ataattaccg gtaaattaca ttgaagaaca acactgttgg tcgtcatgct 480

g 481

<210> 6

<211> 481

<212> DNA

<213> wheat (Triticum aestivum)

<400> 6

tgccatcagc atcccacatg caaggcccaa tagcagaagc tgaaagaacc tttctggaac 60

cgctgttatc agtgacagag tggagtgtat ggaccagatg tttagatagc cccgccatgg 120

aatgcaaggg tcttgaatgc tgacgagtat caaataccaa aaccctgcca tcctgcagaa 180

cacaacaagg acattcatgc aaatcttgca aagggtatca aaattcaata tgcttaagat 240

caggatatgc tgaactttgc agccagcata aacacgacgt gaatcagagt catgtgctga 300

acatgaccaa gcaggaacct gcaaggtagt gtgatagaga attaggtcat aatgtcatta 360

tctgaaattg gttcacctga tttgccgatt caaaatacaa aacattaggt gttgaagatt 420

ggaaggtcag ataattaccg gtaaattaca ctgaagaaca acactgttgg tcgtcatgct 480

g 481

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aatgatacgg cgaccaccga 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caagcagaag acggcatacg 20

Claims

1. An SNP molecular marker related to wheat ear germination resistance, which is characterized in that the SNP locus corresponds to the C/T base mutation at 661498776bp of wheat 4B chromosome of a reference genome IWGSC Refseq v1.0 version.

2. A CAPS marker developed according to the SNP molecular marker of claim 1 and used for identifying a wheat pre-germination resistant variety, wherein the CAPS marker is named as TaE3, and the nucleotide sequence of TaE3 is shown as SEQ ID NO.5 or as SEQ ID NO. 6.

3. A method for identifying a wheat pre-emergence resistant variety by using the CAPS marker of claim 2, comprising the following steps:

s1, designing specific primers according to the sequence SEQ ID No.5 and the sequence SEQ ID No.6 as described in claim 2, selecting restriction endonucleases according to the difference of the sequence SEQ ID No.5 and the sequence SEQ ID No.6 as described in claim 2, so that the sequence SEQ ID No.5 and the sequence SEQ ID No.6 can be cut into different numbers of fragments;

s2, extracting the wheat genome DNA to be detected;

4. The method for identifying the pre-harvest sprouting variety of wheat according to the CAPS marker of claim 3, wherein the upstream primer and the downstream primer in the specific primer designed in the step S1 are respectively represented by SEQ ID No.3 and SEQ ID No. 4.

5. The method for identifying the wheat pre-harvest sprouting resistant variety according to the CAPS marker of claim 3 or 4, wherein the restriction enzyme used in the step S3 is AccII, the wheat pre-harvest sprouting susceptible variety is obtained if the electrophoresis band of the enzyme digestion product in the step S4 is one, and the wheat pre-harvest sprouting resistant variety is obtained if the electrophoresis band of the enzyme digestion product is several.