CN111575400A

CN111575400A - Wheat stripe rust resistant QTL molecular marker IWB12253 and application thereof

Info

Publication number: CN111575400A
Application number: CN202010596459.8A
Authority: CN
Inventors: 王文学; 贾梦洁; 李君辉; 刘易科; 佟汉文; 陈泠; 张宇庆; 何伟杰; 邹娟; 朱展望; 高春保
Original assignee: Hubei Academy Of Agricultural Sciences Institute Of Food Crops
Current assignee: Hubei Academy Of Agricultural Sciences Institute Of Food Crops
Priority date: 2020-06-28
Filing date: 2020-06-28
Publication date: 2020-08-25
Anticipated expiration: 2040-06-28
Also published as: CN111575400B

Abstract

The invention discloses a wheat stripe rust resistance QTL molecular marker IWB12253 and application thereof. The wheat stripe rust resistance QTL molecular marker IWB12253 is a nucleotide corresponding to the 51 th position of a sequence 4 in a sequence table in a wheat genome, and the wheat stripe rust resistance molecular marker is T or C.

Description

Wheat stripe rust resistant QTL molecular marker IWB12253 and application thereof

Technical Field

The invention relates to a wheat stripe rust resistance QTL molecular marker IWB12253 and application thereof in the field of biological agriculture.

Background

Wheat stripe rust is a fungal disease that occurs widely worldwide and is caused by wheat stripe rust (puccinitia striata west.f. Stripe rust mainly damages wheat leaves, affects photosynthetic renting and further causes yield reduction. The cultivation and planting of disease-resistant varieties is the most economic, effective and environment-friendly method for preventing and controlling stripe rust. The genetic research of disease resistance is an important basis for breeding disease resistance. Because wheat stripe rust fungus physiological race is easy to generate mutation, the existing disease-resistant gene can lose resistance, and the important significance of exploring new disease-resistant gene and developing its linkage marker for enriching stripe rust resistance source and better performing disease-resistant breeding is achieved.

With the development of Single Nucleotide Polymorphism (SNP) marker chip technology and cost reduction, genome-wide association analysis (GWAS) is applied to genetic research of a plurality of characters of wheat. The related remarkable SNP locus can be converted into a PCR-based molecular marker, so that the PCR-based molecular marker can be better applied to breeding for disease resistance. The PARMS (Penta-primer amplification mutation system) detection technology is a detection technology based on amplification hindered mutation system PCR (ARMS PCR), and is different from the conventional ARMS PCR in that two detection primers with different fluorescence are added in the PARMS detection technology, so that complementary sequences at 5' ends of forward primers of two alleles can be respectively detected, and after the PCR amplification is carried out on the forward primers and the reverse primers, the polymorphism of a site to be detected can be detected through different fluorescence signals. PARMS has been applied to work such as molecular assisted breeding, target character gene location, seed purity and authenticity identification in succession, has advantages such as with low costs, flux height, experiment operation safety and fluorescence signal acquisition data accuracy.

Disclosure of Invention

The invention aims to solve the technical problem of how to detect the wheat stripe rust resistance.

In order to solve the technical problems, the invention firstly provides application of a wheat disease-resistant molecular marker or a substance for detecting the wheat disease-resistant molecular marker in detection or auxiliary detection of wheat stripe rust resistance; the wheat disease-resistant molecule marker is SNP named IWB12253 and is a nucleotide corresponding to the 51 st position of a sequence 4 in a sequence table in a wheat genome, and the wheat disease-resistant molecule marker is T or C.

The wheat disease-resistant molecular marker is located on a wheat chromosome 3BS, and the physical position of the wheat disease-resistant molecular marker is a 9.1Mb locus.

In the above application, the substance for detecting the wheat disease-resistant molecular marker can be a PARMS _ IWB12253 primer set, and the PARMS _ IWB12253 primer set consists of single-stranded DNAs with the names of PARMS _ IWB12253A, PARMS _ IWB12253B and PARMS _ IWB 12253C;

the PARMS _ IWB12253A is (b1) or (b 2):

(b1) single-stranded DNA shown in 22 th to 41 th positions of a sequence 1 in a sequence table;

(b2) single-stranded DNA obtained by substituting and/or deleting and/or adding one or more nucleotides at the 22 nd to the 41 th positions of the sequence 1;

the PARMS _ IWB12253B is (b3) or (b 4):

(b3) single-stranded DNA shown in 22 th to 41 th positions of a sequence 2 in a sequence table;

(b4) single-stranded DNA obtained by substituting and/or deleting and/or adding one or more nucleotides from the 22 nd position to the 41 th position of the sequence 2;

the PARMS _ IWB12253C is a single-stranded DNA shown in a sequence 3 of a sequence table.

In the application, (b2) can be single-stranded DNA shown in a sequence 1 in a sequence table; (b4) can be single-stranded DNA shown in a sequence 2 in a sequence table.

The invention also provides a method for detecting the wheat genotype, wherein the genotype comprises a TT genotype, a TC genotype and a CC genotype, and the method comprises the following steps: detecting the 51 st nucleotide corresponding to the sequence 4 in the sequence table in the chromosome of the wheat to be detected, wherein if the two chromosomes of the wheat to be detected are g1) chromosomes, the wheat to be detected is TT genotype wheat; if the two chromosomes of the wheat to be detected are g2) chromosomes, the wheat to be detected is CC genotype wheat; if one of the two chromosomes of the wheat to be detected is the chromosome of g1) below and the other chromosome of g2) below, the wheat to be detected is TC genotype wheat;

g1) the nucleotide corresponding to the 51 st site of the sequence 4 in the sequence table is T;

g2) the nucleotide corresponding to the 51 st position of the sequence 4 in the sequence table is C.

The method can adopt a 90K SNP chip to analyze and determine the genotype of the wheat to be detected.

In the method, the detection of the nucleotide corresponding to the 51 st position of the sequence 4 in the sequence table in the wheat chromosome to be detected can be carried out by using the PARMS _ IWB12253 primer group.

The method may specifically include: and (2) carrying out PARMS reaction by adopting the primer group PARMS _ IWB12253 to obtain a reaction product, detecting a fluorescent signal of the reaction system, wherein the wheat to be detected only with FAM fluorescent signals is TT genotype wheat (namely, the IWB12253 is marked as homozygous type of T), the wheat to be detected only with HEX fluorescent signals is CC genotype wheat (namely, the IWB12253 is marked as homozygous type of C), and the wheat to be detected with FAM and HEX fluorescent signals is TC genotype wheat (namely, the IWB12253 is marked as heterozygous type of T and C).

The invention also provides a method for detecting wheat stripe rust resistance, which comprises the following steps: detecting the genotype of the wheat to be detected according to the detection method of the wheat genotype, wherein the stripe rust resistance of the wheat to be detected with the TT genotype is higher than or candidate higher than that of the wheat with the CC genotype.

In the method, the wheat to be detected can be homozygous wheat. The wheat to be detected can be TT genotype wheat or CC genotype wheat.

The invention also provides a wheat breeding method, which comprises the following steps: detecting the genotype of the wheat according to the detection method of the wheat genotype, and selecting TT genotype wheat as a parent to breed.

The wheat breeding method can also comprise the step of selecting the wheat with the TT genotype as the target wheat for resisting the stripe rust to realize the wheat breeding.

The wheat disease-resistant molecular marker also belongs to the protection scope of the invention.

The invention also provides a substance with any one of the following uses Y1) -Y4), wherein the substance comprises the PARMS _ IWB12253 primer set:

y1) detecting the disease-resistant molecular marker of wheat;

y2) preparing a product for detecting the disease-resistant molecular marker of wheat;

y3) or assisting in detecting the wheat stripe rust resistance;

y4) preparing a product for detecting or assisting in detecting the wheat stripe rust resistance.

The substance may also include other reagents required to carry out the PARMS reaction, such as 2 × PARMS master mix (product of peptide Biotech, Inc., city of Wuhan, under the trade designation E001-2.

The substance may be a kit. The substance may be only the PARMS _ IWB12253 primer set, or may be a kit of reagents consisting of the PARMS _ IWB12253 primer set and the other reagents required for performing a PARMS reaction.

The invention also provides any of the following applications:

H1) the application of the wheat disease-resistant molecular marker in wheat breeding;

H2) detecting the application of the wheat disease-resistant molecular marker substance in wheat breeding;

H3) the application of the substance for detecting the wheat disease-resistant molecular marker in preparing a product for detecting or assisting in detecting the wheat stripe rust resistance;

H4) the detection method of the wheat genotype is applied to detection or auxiliary detection of wheat stripe rust resistance.

The wheat in the present invention may be any one or more of 240 parts of wheat in table 1, but is not limited to 240 parts of wheat in table 1.

In an embodiment of the invention, the stripe rust disease is caused by the rust stripe fungus race CYR32, CYR33 and/or CYR 34.

The invention discovers a stripe rust resistant locus QYr.hbaas-3BS positioned on the short arm of a wheat 3B chromosome through genome-wide association analysis (GWAS), explains phenotypic variation of 4.1-5.5%, discovers that IWB12253 is related to wheat stripe rust through detecting related SNP IWB12253, can be used for detecting wheat stripe rust resistance and is used for stripe rust resistant molecular breeding.

Drawings

FIG. 1 shows the result of detection of the primer set PARMS _ IWB12253 on the genotype of wheat to be detected. The upper left part is wheat with IWB12253 genotype CC, and the lower right part is wheat with IWB12253 genotype TT.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

Mingxian 169 in the following examples is described in "Huangliang, Liu Tai, Xiaoxing Zhi, etc.. in the field of evaluation of stripe rust resistance and gene molecule detection of the 79 triticale species (line) [ J ]. in Chinese agricultural science, 2017,50(16): 3122-3134", the public can obtain the biological material from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

The physiological races of the yellow rust fungi CYR32, CYR33 and CYR34 are described in the text "Zhang Hua Zhi, Xie Hua Cheng Yong xing, Liu Xue, Wang Yong, Yan Suhong, Yang Meng, Zhao hong, Wang Xicheng, Jia Union, Cao Tingjie, Liu Yong, positioning the yellow rust resistance gene YRZM103[ J ] of the wheat variety Zheng wheat 103 by BSR-Seq. the crop academic newspaper, 2017,43(11): 1643-1649", the public can be obtained from the plant protection institute of Chinese academy of agricultural sciences, and the biological material is used only for repeating the relevant experiments of the present invention and cannot be used for other purposes.

Example 1, the IWB12253 marker can be used to detect wheat stripe rust

Test materials: the GWAS group constructed by 240 parts of wheat varieties (lines) at home and abroad is shown in Table 1. Materials used are described in the literature (Zhu Z, Chen L, Zhang W, Yang L, Li J, Liu Y, Tong H, Fu L, Liu J, Rasheet A, Xia X, He Z, Hao Y, Gao C,2020 Genome-wide association analysis of Fusarium height resistance in core else thin places lines, frontiers in Plant Science,11:206), and are publicly available from the Applicant. Wherein, 14FHBSN6402 is CROC _1/AE.SQUARROSA (205)// KAUZ/3/SASIA/4/TROST in the literature; 14FHBSN6404 is MONARCA F2007/KRONSTAD F2004 in the literature; 14FHBSN6405 is PBW343 x 2/KUKUNA// PBW343 x 2/KUKUNA/3/PBW 343; 14FHBSN6408 is KS82W418/SPN// WBLL1/3/BERKUT in the literature; 14FHBSN6409 is CNDO/R143// ENTE/MEXI75/3/AE.SQ/4/2 FCT/5/KAUZ 2/YACO// KAUZ/6/BERKUT; 14FHBSN6411 is T.DICOCCONPI94625/AE.SQUARROSA (372)// TUI/CLMS/3/2 PASTORR/4/EXCALIBUR; 14FHBSN6418 is NG8675/CBRD// MILAN/3/SAUAL/6/CNDO/R143// ENTE/MEXI _2/3/AEGILOPS SQUARROSA (TAUS)/4/WEAVER/5/2 PASTOR.

TABLE 1 GWAS population varieties (lines) and their sources

In the context of table 1, the following,^athe detection result is the detection result of the primer group PARMS _ IWB 12253;^bis a typing result of the 90K SNP chip;^cfor the strip rust MDS BLUP value (Best linear approximated prediction) in 5 environments, "NN" is data missing.

Discovery of the first, IWB12253 marker

1. Stripe rust resistance identification

The GWAS group stripe rust resistance is inoculated and identified in the fields of Pi county and New Sichuan in 2015-2016, in 2013-2014, in 2016-2017 and in 2018-2019 in Wuhan Hubei. The experiment was performed in a completely randomized block design, repeated twice. Each cell has 2 rows, the length of each row is 1m, and the distance between the rows is 25 cm. The high-susceptibility variety mingxian 169 of the stripe rust is planted around the plot as an induced line, the mingxian 169 is inoculated by the physiological race CYR32 and CYR33 of the stripe rust in the year 2013-2014 in a mixed way, and the mingxian 169 is inoculated by the physiological race CYR32 and the mixed race CYR34 in the rest years. When the severity of the stripe rust disease of Mingxian 169 reaches the highest, the maximum severity (MDS) of the stripe rust disease of each cell is investigated, namely the percentage of the area of the stripe rust disease spore heap on the leaf to the total leaf area when the stripe rust disease is the most serious, and then 5 environmental phenotype BLUP values are calculated by using an R package lme4 as an average value, and the results are shown in Table 1.

2. Genotyping analysis

The GWAS group is subjected to genotype analysis by using a 90K SNP chip, 22922 SNPs with good typing results are selected for subsequent analysis, markers with deletion rate of more than 20% and minimum allele frequency of less than 5% are removed, and 14577 SNPs in total are used for GWAS.

3. GWAS analysis

Association analysis was performed using a mixed linear model of the Tassel v5.2.53 and the GAPIT software kinship (K) + PCA method. When P is less than or equal to 0.001, the marker is considered to be significantly associated with the trait.

4. QYr. hbaas-3BS and linked SNP marker thereof

Correlation analysis finds that the stripe rust resistant locus located on 3BS, BLUP values under two environments of Pi county 2015-2016 and New 2015-2016 and 5 environments are remarkably related to stripe rust resistance, the explanation phenotypic variation is 4.1% -5.5%, a representative correlation marker is IWB12253, the IWB12253 marker is a biallelic polymorphic SNP locus which is T or C, and the flanking sequence is as follows: 5 '-TCTTACCTGGCCTACTAGCTGCGAAAGCTATTGCAGCACGCCATCAGATC [ T/C ] GTGAGGTCATTGATGGCGTAGCACAGTTTGTGACCGAAGTCGGGCTTCGG-3' (SEQ ID NO: 4, SNP site in parentheses, sequence 4 in the sequence listing y represents T or C). The physical position on the Chinese spring reference genome sequence (IWGSC, http:// www.wheatgenome.org) of the wheat variety was 9.1Mb (Table 2).

^aA representative SNP marker is a marker for a protein,^bthe disease-resistant alleles are shown underlined,^cchinese spring reference genome physical location (IWGSC, http:// www.wheatgenome.org),^daccounting for phenotypic variation.

Second, the special primer for detecting the IWB12253 marker is used for detecting the IWB12253 marker of wheat and stripe rust

1. Genome specific primer design

IWB 12253-labeled chromosome-specific primers PARMS _ IWB12253 (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3) were designed using Polymarker (www.polymarker.tgac.ac.uk) and synthesized by Bioengineering Ltd.

The PARMS primer for identifying the SNP site IWB12253 is a PARMS _ IWB12253 primer group, and the specific steps are as follows:

PARMS_IWB12253A：

5′-GAAGGTGACCAAGTTCATGCTACGCCATCAATGACCTCACA-3' (SEQ ID NO: 1);

PARMS_IWB12253B：

5′-GAAGGTCGGAGTCAACGGATTACGCCATCAATGACCTCACG-3' (SEQ ID NO: 2);

PARMS _ IWB 12253C: 5'-TGGCCTACTAGCTGCGAAAG-3' (SEQ ID NO: 3).

The underlined sequence in PARMS _ IWB12253A is the FAM binding sequence; the underlined sequence in PARMS _ IWB12253B is the HEX binding sequence.

The single-stranded DNA molecules shown in the sequence 1 and the sequence 3 can amplify a DNA fragment including an SNP site IWB12253, the nucleotide at the SNP site IWB12253 in the PCR product is T, and a fluorescence signal of a fluorescent group FAM combined with the FAM binding sequence in PARMS master mix can be read by a microplate reader or a fluorescent quantitative PCR instrument;

the single-stranded DNA molecules shown in the sequence 2 and the sequence 3 can amplify a DNA fragment including the SNP site IWB12253, the nucleotide at the SNP site IWB12253 in the PCR product is C, and a fluorescence signal of a fluorescent group HEX combined with the HEX binding sequence in PARMS master mix can be read by a microplate reader or a fluorescent quantitative PCR instrument.

2. Detection of genotype

Extracting genome DNA of wheat to be detected, adding ddH₂O lysis was used as template, and PARMS reaction was performed using primer set PARMS _ IWB12253 to detect the nucleotides at SNP site IWB 12253.

The PARMS reaction system is shown in Table 3;

table 3 PARMS reaction System of primer set PARMS _ IWB12253

Wherein the 2 xPARMS master mix is a product of peptide Biotechnology Limited in the city of Wuhan, and the product number is E001-2.

The PARMS reaction program is shown in table 4:

TABLE 4 PARMS reaction program for primer set PARMS _ IWB12253

After the PARMS reaction is finished, the obtained product is subjected to fluorescence data reading by using a microplate reader or a fluorescence quantitative PCR (polymerase chain reaction) instrument, fluorescence signal processing is carried out by using an online software SNP decoder (http:// www.snpway.com/snpdecoder01/), and the genotype of the wheat SNP site IWB12253 to be detected is determined: the wheat to be detected with only FAM fluorescence signals is TT genotype wheat (namely, the homozygous type marked by IWB12253 as T), the wheat to be detected with only HEX fluorescence signals is CC genotype wheat (namely, the homozygous type marked by IWB12253 as C), and the wheat to be detected with FAM and HEX fluorescence signals is TC genotype wheat (namely, the heterozygous type marked by IWB12253 as T and C).

The results of the genotype tests on wheat to be tested are shown in Table 1 and FIG. 1, which indicate that the above-described method can be used to test the IWB12253 marker of wheat.

3. Genotype and phenotype analysis

Further, from the genotyping results of step 2, BLUP mean values of the respective genotypes of stripe rust MDS were calculated, see table 5.

TABLE 5 BLUP averages of wheat stripe rust MDS of different genotypes

PARMS _ IWB12253 genotype	Number of varieties	Stripe rust MDS BLUP (%)
			TT	21	29.97
CC	204	51.16

From the results in table 5, it can be seen that the BLUP average value of wheat stripe rust MDS to be detected at SNP site IWB12253 with the TT genotype is significantly lower than that of wheat to be detected at SNP site IWB12253 with the CC genotype, which indicates that stripe rust resistance of wheat to be detected with the TT genotype at SNP site IWB12253 is significantly higher than that of wheat to be detected with the CC genotype at SNP site IWB12253, and stripe rust resistance of wheat to be detected with T at SNP site IWB12253 is significantly higher than that of wheat to be detected with C at SNP site IWB 12253.

Therefore, the SNP locus IWB12253 can be used for auxiliary detection of the stripe rust resistance of wheat to be detected and for stripe rust resistance molecular breeding.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

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Claims

1. The application of the wheat disease-resistant molecular marker or the substance for detecting the wheat disease-resistant molecular marker in detecting or assisting in detecting the wheat stripe rust resistance; the wheat disease-resistant molecular marker is a nucleotide corresponding to the 51 th site of a sequence 4 in a sequence table in a wheat genome, and the wheat disease-resistant molecular marker is T or C.

2. Use according to claim 1, characterized in that: the substance for detecting the wheat disease-resistant molecular marker is PARMS _ IWB12253 primer group, and the PARMS _ IWB12253 primer group consists of single-stranded DNA with the names of PARMS _ IWB12253A, PARMS _ IWB12253B and PARMS _ IWB12253C respectively;

the PARMS _ IWB12253A is (b1) or (b 2):

the PARMS _ IWB12253B is (b3) or (b 4):

3. Use according to claim 2, characterized in that: (b2) is single-stranded DNA shown as a sequence 1 in a sequence table; (b4) is single-stranded DNA shown in a sequence 2 in a sequence table.

4. A method for detecting wheat genotypes, wherein the genotypes comprise a TT genotype, a TC genotype and a CC genotype, the method comprises the following steps: detecting the 51 st nucleotide corresponding to the sequence 4 in the sequence table in the chromosome of the wheat to be detected, wherein if the two chromosomes of the wheat to be detected are g1) chromosomes, the wheat to be detected is TT genotype wheat; if the two chromosomes of the wheat to be detected are g2) chromosomes, the wheat to be detected is CC genotype wheat; if one of the two chromosomes of the wheat to be detected is the chromosome of g1) below and the other chromosome of g2) below, the wheat to be detected is TC genotype wheat;

5. The method of claim 4, wherein: detecting the nucleotide corresponding to the 51 st position of the sequence 4 in the sequence table in the wheat chromosome to be detected by using the PARMS _ IWB12253 primer set described in claim 2 or 3.

6. A method of detecting resistance to wheat stripe rust comprising: detecting the genotype of a wheat to be tested according to the method of claim 4 or 5, wherein the wheat to be tested for the TT genotype has a higher stripe rust resistance or is more candidate than the wheat with the CC genotype.

7. A method of wheat breeding comprising: detecting the genotype of wheat according to the method of claim 4 or 5, selecting wheat with TT genotype or TC genotype as parent for breeding.

8. The wheat disease-resistant molecular marker as claimed in claim 1.

9. Substance with any of the following Y1) -Y4), comprising the PARMS _ IWB12253 primer set described in claim 2 or 3:

y1) detecting the disease-resistant molecular marker of wheat;

y3) or assisting in detecting the wheat stripe rust resistance;

10. Any of the following applications:

H1) the use of a wheat disease-resistant molecular marker as defined in claim 1 in wheat breeding;

H2) the use of a substance for detecting a wheat disease-resistant molecular marker as defined in claim 1 in wheat breeding;

H3) the application of a substance for detecting the wheat disease-resistant molecular marker in claim 1 in preparing products for detecting or assisting in detecting wheat stripe rust resistance;

H4) use of the method of claim 4 or 5 for detecting or aiding in the detection of wheat stripe rust resistance.