CN109797240B - Molecular marker and primer closely linked with wheat stripe rust resistant gene Yr26 and application thereof - Google Patents

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a molecular marker closely linked with a wheat stripe rust resistance gene Yr26, a primer and application thereof, wherein the marker is positioned at 326913074-326914227 of a wheat 1B chromosome, the nucleotide sequence of the marker is shown as SEQ ID NO.1 in a sequence table, the molecular marker WRS1633 is favorable for establishing a wheat molecular marker assisted breeding system, the molecular marker is separated from the Yr26 gene together, and the molecular marker can be simply, rapidly and high-flux applied to wheat molecular assisted breeding.

Description

Molecular marker and primer closely linked with wheat stripe rust resistant gene Yr26 and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a molecular marker and a primer closely linked with a wheat stripe rust resistance gene Yr26 and application thereof.

Background

Wheat stripe rust is an important disease affecting the safe production of wheat in the world, and causes millions of tons of yield loss every year. National stripe rust has been a pandemic that has occurred many times in China. Since the 21 st century, due to the continuous emergence and development of new highly pathogenic races, wheat stripe rust in the northwest and southwest regions is in continuous disasters and presents a high tendency. Because of mainly relying on chemical prevention and control, pesticide residue and environmental pollution become more and more serious, and serious threats are caused to the food production safety and ecological environment in China. The cultivation of wheat varieties with durable and stable disease resistance is the most economical and environment-friendly measure for continuously and effectively controlling stripe rust.

In the 70's of the last century, Sida junior Liu and professor Chenpedu, etc. utilized tetraploid cone wheat gamma 80-1 to hybridize with haynaldia villosa, then the offspring was backcrossed with hexaploid common wheat for many times, and then the wheat-haynaldia villosa 6VS/6AL translocation line was identified and bred from the progeny. The parent source of the hybrid is Yangmai No. 5/4/gamma 80-1/Haynaldia villosa// Ningmai No. 6/3/Yangmai No.2, and the translocation line comprises germplasms such as 92R137, 92R89, 92R90 and 92R149, which are called 92R series for short. Then the gene Pm21 of the powdery mildew resistance gene on 6VS and the gene of the stripe rust resistance gene on 1BL are positioned in sequenceYr26. Because the translocation line has good resistance and is widely applied to wheat breeding in the last 90 th century, breeders utilize the translocation line to successively breed eighteen small wheat disease-resistant new varieties such as an endowheat series (No. 8-11), a stone wheat 14, a Yuanzhong 175 and a Yangmai 18. The accumulated popularization area of the new varieties exceeds six million mu, and the yield of the wheat is increased by 15 hundred million kilograms (http:// www.cutech.edu.cn/cn/gxkj/2013/01/1354173454039838. htm).

Yr26Initially localized to the short arm of the 1B chromosome by equine involution, etc. (1 BS), and reacted withXgwm11、Xgwm18AndXgwm413linkage; the Wangchunmei and the like use the special cytogenetic materials (lack body and lack line) of the Chinese spring wheatYr26The C-1BL6-0.32 region located on the long arm of chromosome 1B was developedYr26The gene flanking markers are recentlyXwe173(1.4 cM) andXbarc181(6.7 cM); the post-tensioned xiaojuan and the like develop and design a plurality of closer linkage markers by comparing genomics and utilizing the collinear region of the wheat EST sequence information with rice and brachypodium distachyon and draw a finer genetic map. In addition, other researchers have found that the major sources of stripe rust resistance are found in the research on ' Guinong's line ' material cultivated by professor Zhang Qingqing Duty of Guizhou universityYr26(ii) a While Chuanmai 42 and its derivative line are introduced and cultivated from CIMMYT 'synthetic wheat' from Sichuan academy of agricultural sciencesThe column material is analyzed by research, and the resistance source is fromYrCH42(Li et al 2006) which was then confirmedYrCH42AndYr26（Yr24) Are the same gene or the same allele.

Due to the fact thatYr26The wide application of the traditional Chinese medicine can cause the variation of small species of the stripe rust, and the traditional Chinese medicine is found in Sichuan for the first time in 2008Yr26The frequency of small toxic species V26, V26, is continuously increased in the following years, and the small toxic species V26 exist in large quantities in northwest, southwest and other areas, and pose a threat to wheat grain production in China. But comprisesYr26The gene varieties are widely planted, can not be completely eliminated in a short period, andYr26still maintaining good resistance to CYR32 and CYR33, which are still in epidemic status, and thus, can pass throughYr26Improve and prolong the disease resistance by polymerizing with other genes in the adult plant stageYr26The molecular markers with more closely linked genes are particularly urgent.

Disclosure of Invention

The invention provides a molecular marker and a primer closely linked with a wheat stripe rust resistance gene Yr26 and application thereof, which are simply, rapidly and high-flux applied to wheat molecular assisted breeding.

The first purpose of the invention is to provide a wheat stripe rust resistance geneYr26Closely linked molecular markersWRS1633The marker is located at the 326913074-326914227 th site of wheat 1B chromosome, and the wheat stripe rust resistance geneYr26Closely linked, and the nucleotide sequence is shown as SEQ ID NO.1 in the sequence table.

The second purpose of the invention is to provide a wheat stripe rust resistance geneYr26A primer of a closely linked molecular marker, the sequence of the primer being:

the upstream primer WRS1633F is shown as SEQ ID NO.2 in the sequence table;

the downstream primer WRS1633R is shown as SEQ ID NO.3 in the sequence table.

The third purpose of the invention is to provide a molecular marker in the wheat stripe rust resistance geneYr26The application in auxiliary breeding in the aspect of detecting or resisting the stripe rust of wheat.

A fourth object of the present invention is to provideThe primer is planted in the wheat stripe rust resistant geneYr26The application in auxiliary breeding in the aspect of detecting or resisting the stripe rust of wheat.

The invention provides a method for detecting the stripe rust resistance gene of wheat by using the primerYr26The method specifically comprises the following steps:

s1, extracting genome DNA of wheat to be detected;

s2, performing PCR amplification by taking the genomic DNA of the wheat as a template and taking an upstream primer WRS1633F and a downstream primer WRS1633R as primers;

s3, agarose gel electrophoresis detection, if the size of the amplified product fragment is 1364bp, the wheat to be detected contains the stripe rust resistant geneYr26If 1364 amplified bands cannot be detected, the wheat to be detected does not contain the stripe rust resistance geneYr26。

Preferably, the primer is used for detecting the stripe rust resistance gene of the wheatYr26The method of (1) comprises:

the PCR system is as follows: 10 XBuffer 1.5. mu.l, 25. mu.M/. mu.l MgCl₂ Mu.l of 0.9. mu.l, 0.2. mu.l of 2.5 mM each of 4 dNTPs, 1.0. mu.l of 10. mu.M each of WRS1633R and WRS1633F, 0.15. mu.l of 5U/. mu.l of Taq enzyme, 1.5. mu.l of 50 ng/. mu.l template DNA, and DDH₂O to 15 μ l;

reaction procedure: 1) 5 minutes at 94 ℃; 2) 30 seconds at 94 ℃, 1 minute at 55 ℃ and 1 minute at 72 ℃, and circulating from the second step for 35 cycles; 3) 10 minutes at 72 ℃.

By adopting the technical scheme, the invention has the beneficial effects that the invention provides the wheat stripe rust resistance geneYr26The closely linked molecular markers are beneficial to the establishment of the wheat molecular marker-assisted breeding systemYr26The gene is co-separated, and the molecular marker of the invention can be simply, conveniently, rapidly and high-flux applied to wheat molecular assisted breeding.

Drawings

FIG. 1, utilizingWRS1633Primer group for susceptible parent Avocet S and disease-resistant parent 92R137 and F₇The electrophoresis chart of the amplification result of the recombinant inbred line population, wherein lanes 1-10 are respectively Avocet S, 92R137 and ARF₇-3、ARF₇-7、ARF₇-12、ARF₇-34、ARF₇-2、ARF₇-4、ARF₇-8、ARF₇-25。

Detailed Description

The present invention is described in detail below with reference to specific examples, but it should be understood that the scope of the present invention is not limited by the specific examples. The following examples are generally conducted under conventional conditions, and the materials are commercially available as the materials, and the steps thereof will not be described in detail since they do not relate to the invention.

The invention provides a wheat stripe rust resistance geneYr26The closely linked molecular marker WRS1633 is located at 326913074-326914227 of wheat 1B chromosome, is closely linked with wheat stripe rust resistant gene Yr26, and has the nucleotide sequence shown in SEQ ID NO. 1. In the genetic linkage map (map drawn by using more than 20000 individuals of F2 generations), the molecular marker is cosegregated with wheat stripe rust resistance gene Yr26, or in more than 20000 samples of wheat, the sample with stripe rust resistance contains the molecular marker. Wherein, the leaf blade does not produce rust stripe spores and has the symptom of anaphylactic necrosis reaction, and the leaf blade is rust stripe resistance.

The molecular marker WRS1633 is obtained as follows:

taking the wheat genome DNA of the tested wheat material (after the hybridization of the wheat material 92R137 and the susceptible material Avocet S F)₁F produced by selfing₂Individuals with stripe rust resistance in generations), extracting wheat genome DNA by referring to conventional molecular biology operation steps; designing a primer; PCR amplification is carried out by taking the genome DNA of the wheat to be tested as a template.

Primers were derived from genome re-sequencing performed in the previous laboratory to capture sequencing, verified by sanger sequencing.

An upstream primer WRS1633F: 5'-gagaggaaacaagcgagcga-3' shown as SEQ ID NO. 2;

a downstream primer WRS1633R: 5'-cttcgtcttgtggcattccg-3' shown as SEQ ID NO. 3;

the PCR system is as follows:10 XBuffer 1.5. mu.l, 25. mu.M/. mu.l MgCl₂ Mu.l of 0.9. mu.l, 0.2. mu.l of 2.5 mM each of 4 dNTPs, 1.0. mu.l of 10. mu.M each of WRS1633R and WRS1633F, 0.15. mu.l of 5U/. mu.l of Taq enzyme, 1.5. mu.l of 50 ng/. mu.l template DNA, and DDH₂O to 15 μ l;

reaction procedure: 1) 5 minutes at 94 ℃; 2) 30 seconds at 94 ℃, 1 minute at 55 ℃ and 1 minute at 72 ℃, and circulating from the second step for 35 cycles; 3) 10 minutes at 72 ℃.

The amplification sequence is determined to be SEQ ID NO.1, and the PCR product is a molecular marker WRS1633 closely linked with the wheat stripe rust resistant gene Yr 26.

Based on the same invention concept, the invention also provides the wheat stripe rust resistance gene of the primerYr26The application in detection specifically comprises the following steps:

using genome DNA of wheat to be detected as a template, amplifying by using primers WRS1633F (shown as SEQ ID NO. 2) and WRS1633R (shown as SEQ ID NO. 3) to obtain an amplification product, and sequencing and performing gel electrophoresis on the obtained amplification product.

The specific PCR reaction system is shown in Table 1:

TABLE 1

Components	Sample addition amount (μ l)
		10×Buffer	1.5
MgCl2 （25μM）	0.9
		dNTPs （2.5 mM）	0.8
WRS1633R（10μM）	1.0
		WRS1633F（10μM）	1.0
Taq enzyme (5U/. mu.l)	0.15
		DNA (50ng/μl)	1.5
DDH2O	9.15
		Total of	15

The PCR procedure was: 1) 5 minutes at 94 ℃; 2) 30 seconds at 94 ℃, 1 minute at 55 ℃ and 1 minute at 72 ℃, and circulating from the second step for 35 cycles; 3) 10 minutes at 72 ℃.

FIG. 1 is a drawing ofWRS1633Amplification results of the primer pairs for the susceptible parent Avocet S, the disease-resistant parent 92R137, and the F7 recombinant inbred line population, lanes 1-10 are Avocet S, 92R137, and ARF, respectively₇-3、ARF₇-7、ARF₇-12、ARF₇-34、ARF₇-2、ARF₇-4、ARF₇-8、ARF₇-25. The results show thatWRS1633Can be in disease-resistant parent 92R137 and containsYr26The progeny of the strain successfully amplifies a PCR product with the size of 1364bp, while the susceptible parent does not containYr26The progeny of (a) does not have such a PCR product, indicating the use of molecular markersWRS1633The wheat stripe rust resistance can be detected.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> northwest agriculture and forestry science and technology university

<120> molecular marker closely linked with wheat stripe rust resistance gene Yr26, obtaining method and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1364

<212> DNA

<213> Artificial sequence

<400> 1

gagaggaaac aagcgagcga tcggctgaca ctgacaaaat gagaaaggaa gaaacaaata 60

aaagatctga gaaaataatc gatggtatgt ggcacatcca agaacaggtg gtaccaaata 120

tatacataat gaaatttcta gcttttattc aacatagaat ttagtactag tttccgaatc 180

tttggtgcag catgattaca aaaacctgtt tctattgaaa ctggaatgcc tacgcatatc 240

tgtaaataaa atagaagtac agttctttgg ttgtataatg ctaaactgtc cagttaaaat 300

atttgaagga ccttgccttg cacaaagaac tggccatcac taagttccta aagtgagtag 360

gtacggtgag ctaaaggata catataagct aaaatgttgg ataacgcatg catgccattt 420

atataaatca tttgtgatgt aactgcctaa ctggtaatag gtgcatacca tttatgccca 480

ccaacttcaa atgtagggga acggacaaag tgatccaagt ccagttcaag gaaattgttc 540

atgttccaag tgtatgtccc tttgacgaac tccttcttct gaacaaagag gttctgaact 600

gtcgtaggct tcttctgaac cacaacagcc ttcttttcag gagaagagac atcaattttt 660

aatatctcca caccgaagac acagctatca tcaactagaa aagcagatga tttcagtagc 720

tcctgaagag gaatcaagca atgctccttc gagaagatat tcttgaaacc aaagttgtag 780

ctagctgcac ttaggtaaag gttagaactt ggaagattag acagcgaaga acttgtatgc 840

aagaagcaaa tattaccaaa taatatctaa ctaaccttca ccctagttaa gaaagcaagg 900

tagtgaatta catccagatg atgataatca aatttaaaga gaaacatgcg tatgctatct 960

gaaccataat agactcaagt gtgaaacttg taatgtctca agtgagatac atgtgtatgc 1020

catcagaacc atccaaattc ctgaaggtct acagagaacc ttgacagcaa agcaaataca 1080

acgcgacttg ttaatgccaa aacattgtac ttgaccatat tttgatattt acagatattg 1140

acttgtaatg atagaatcat ggtatttata tctcaaactt atgacacaca gaggagtatg 1200

gattcatagc tataatatga tggacgtgct tgaaaaaaca catccatgag actacctttg 1260

catccacagt acattcgtct tgaatggttg tatattgaca actcaaacac cggatgcacc 1320

atgtgacctg gcaccaagct tggtcggaat gccacaagac gaag 1364

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

gagaggaaac aagcgagcga 20

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<400> 3

cttcgtcttg tggcattccg 20

Claims (6)

1. Wheat stripe rust resistance geneYr26Closely linked molecular markers, wherein the molecular markers areWRS1633The marker is located at the 326913074-326914227 th site of the wheat 1B chromosome, and the nucleotide sequence is shown as SEQ ID NO. 1.

2. The wheat stripe rust resistance gene as claimed in claim 1Yr26The primer of the closely linked molecular marker is characterized in that the sequence of the primer is as follows:

the upstream primer WRS1633F is shown as SEQ ID NO.2 in the sequence table;

the downstream primer WRS1633R is shown as SEQ ID NO.3 in the sequence table.

3. The molecular marker of claim 1 in wheat stripe rust resistance geneYr26The application in auxiliary breeding in the aspect of detecting or resisting the stripe rust of wheat.

4. The primer of claim 2 in wheat stripe rust resistance geneYr26The application in auxiliary breeding in the aspect of detecting or resisting the stripe rust of wheat.

5. Use according to claim 4, characterized in that the wheat stripe rust resistance geneYr26The detection specifically comprises the following steps:

s1, extracting genome DNA of wheat to be detected;

s2, performing PCR amplification by taking the genomic DNA of the wheat as a template and taking an upstream primer WRS1633F and a downstream primer WRS1633R as primers;

s3, agarose gel electrophoresis detection, if the size of the amplified product fragment is 1364bp, the wheat to be detected contains the stripe rust resistant geneYr26If the 1364bp amplification band cannot be detected, the wheat to be detected does not contain the stripe rust resistance geneYr26。

6. The use of claim 5, wherein in S2, the PCR system is: 10 XBuffer 1.5. mu.l, 25. mu.M MgCl₂ Mu.l of 0.9. mu.l, 0.2. mu.l of 2.5 mM each of 4 dNTPs, 1.0. mu.l of 10. mu.M each of WRS1633R and WRS1633F, 0.15. mu.l of 5U/. mu.l of Taq enzyme, 1.5. mu.l of 50 ng/. mu.l template DNA, and DDH₂O to 15 μ l;

reaction procedure: 1) 5 minutes at 94 ℃; 2) 30 seconds at 94 ℃, 1 minute at 55 ℃ and 1 minute at 72 ℃, and circulating from the second step for 35 cycles; 3) 10 minutes at 72 ℃.

Images