CN113046456B - Primer for identifying stripe rust resistance of wheat and application thereof - Google Patents
Primer for identifying stripe rust resistance of wheat and application thereof Download PDFInfo
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Abstract
The invention discloses a primer for identifying the stripe rust resistance of wheat and application thereof. The invention firstly discloses a primer for identifying the stripe rust resistance of wheat, which is a primer pair YrZM175-CAPS1 consisting of a single-stranded DNA molecule shown by SEQ ID No.1 and a single-stranded DNA molecule shown by SEQ ID No.2 and/or a primer pair YrZM175-CAPS5 consisting of a single-stranded DNA molecule shown by SEQ ID No.3 and a single-stranded DNA molecule shown by SEQ ID No. 4. The invention further discloses an application and a method of the primer in identification of the stripe rust resistance of wheat. The invention develops the closely linked molecular marker, provides a good tool for applying the stripe rust resistant gene YRZM175 to wheat stripe rust resistant molecular marker assisted breeding, and plays an important role in wheat stripe rust resistant breeding.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer for identifying the stripe rust resistance of wheat and application thereof.
Background
Wheat is an important worldwide food crop, and the planting area of the whole world is stabilized at 2 multiplied by 10 every year 8 km 2 The above. China is one of the countries with the largest wheat planting area and the largest consumption in the world, and the wheat yield directly influences the life of Chinese people and the national food safety. The high and stable yield of wheat has important influence on the social stability and agricultural development of China. Wheat stripe rust is caused by Puccinia striiformis f.sp.tritici, and is one of serious diseases endangered worldwide. Wheat stripe rust is an air-borne fungal disease, is attacked under low-temperature and high-humidity conditions, is popular in low-latitude areas with low-temperature humidity at night and high-latitude areas with cold weather and high humidity, and has the characteristics of wide harm area, high frequency and extremely serious loss. Wheat stripe rust damages leaves, leaf sheaths, stems and ears of wheat, and the damaged parts have green fading spots and then generate yellow summer spore piles, so that the physiological function of the wheat is seriously damaged, the grain number of the wheat per ear is reduced, the thousand seed weight is reduced, the quality is deteriorated, and when the disease is serious, the wheat cannot be spiced, thereby causing the failure of production. The wheat stripe rust disease can cause the yield reduction of wheat by 10% ~ up to ten percent in moderate epidemic years20 percent, the yield reduction of the pandemic year is about 30 percent, and the yield reduction of the pandemic year is up to more than 50 percent. Therefore, the effective prevention and control of the stripe rust have important significance for guaranteeing the grain safety. The cultivation and planting of the disease-resistant variety are the most economic, safe and effective measures for preventing and treating the wheat stripe rust.
The wheat stripe rust resistance genes named at present are distributed at 82 sites (Yr 1-Yr 82), and except Yr18, yr29, yr30, yr36, yr39, yr46, yr48, yr49 and Yr52, the genes are micro-effect genes (adult plant disease resistance genes), and most of the named stripe rust resistance genes are main effect disease resistance genes with physiological specialization. The high resistance expression of major disease resistance genes and the easy field selection characteristics are well liked by breeders, however, because they develop resistance against specific pathogenic races, there is a risk of resistance loss in the case of recurrent races. In order to avoid the large-area loss of resistance due to the singularization of the source of resistance, the development and use of new disease-resistant genes and their linked markers are required.
Disclosure of Invention
The invention aims to solve the technical problem of how to efficiently detect or assist in detecting the wheat stripe rust resistant gene YrZM175 so as to breed the wheat stripe rust resistant gene YrZM 175.
In order to solve the technical problems, the invention firstly provides a primer; the primer is used for identifying or assisting in identifying the stripe rust resistance of wheat and/or breeding or assisting in breeding stripe rust resistance wheat and/or detecting or assisting in detecting the allele of the stripe rust resistance gene YrZM175 of wheat.
The primer comprises a primer pair YrZM175-CAPS1 and/or a primer pair YrZM175-CAPS5; the primer pair YrZM175-CAPS1 comprises an upstream primer F1 and a downstream primer R1;
the upstream primer F1 is (a 1) or (a 2) as follows:
(a1) A single-stranded DNA molecule shown as SEQ ID No. 1;
(a2) A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.1 and has the same function as SEQ ID No. 1;
the downstream primer R1 is (a 3) or (a 4) as follows:
(a3) A single-stranded DNA molecule shown as SEQ ID No. 2;
(a4) A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.2 and has the same function as SEQ ID No. 2;
the primer pair YrZM175-CAPS5 comprises an upstream primer F2 and a downstream primer R2;
the upstream primer F2 is (b 1) or (b 2) as follows:
(b1) A single-stranded DNA molecule represented by SEQ ID No. 3;
(b2) A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.3 and has the same function as SEQ ID No. 3;
the downstream primer R2 is (b 3) or (b 4) as follows:
(b3) A single-stranded DNA molecule represented by SEQ ID No. 4;
(b4) A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.4 and has the same function as SEQ ID No. 4.
In the above primers, each single-stranded DNA molecule in the primer pair is packaged separately.
The invention further provides a kit; the kit is a kit for identifying or assisting in identifying the stripe rust resistance of wheat and/or breeding or assisting in breeding stripe rust resistance wheat and/or detecting or assisting in detecting the allele of the stripe rust resistance gene YrZM175 of wheat.
The kit comprises the primer.
The kit also comprises restriction enzyme EagI and/or restriction enzyme HhaI. The restriction endonuclease EagI and a primer pair YrZM175-CAPS1 are used in a set; the restriction enzyme HhaI is used in combination with the primer pair YrZM175-CAPS5.
The kit also comprises other reagents used for PCR reaction and/or enzyme digestion reaction.
The primer or the kit is applied to any one of the following 1) to 6):
1) Identifying or assisting in identifying the stripe rust resistance of the wheat to be detected;
2) Preparing a product for identifying or assisting in identifying the stripe rust resistance of the wheat to be detected;
3) Detecting or assisting in detecting the YrZM175 allele of the wheat stripe rust resistance gene to be detected;
4) Preparing a product for detecting or assisting in detecting the yellow rust resistance gene YrZM175 allele of the wheat to be detected;
5) Breeding or assisting in breeding the stripe rust resistant wheat;
6) Preparing and breeding or assisting in breeding the stripe rust resistant wheat product.
The invention also provides a method for identifying or assisting in identifying the stripe rust resistance of wheat to be detected.
The method for identifying or assisting in identifying the stripe rust resistance of the wheat to be detected comprises the following steps of A1) and/or A2):
a1 Taking genome DNA of wheat to be detected as a template, carrying out PCR amplification on the YrZM175-CAPS1 by adopting the primer to obtain an amplification product, and carrying out enzyme digestion on the amplification product by EagI to obtain an enzyme digestion product; judging the stripe rust resistance of the wheat to be detected according to the enzyme digestion product:
if the amplification product of the wheat to be detected can be cut into two fragments of 620bp-680bp and 720bp-780bp by EagI enzyme, the wheat to be detected has or is candidate to have the stripe rust resistance property;
if the amplification product of the wheat to be detected cannot be digested by EagI, the wheat to be detected has or is candidate to have the stripe rust-sensitive character;
a2 Taking genome DNA of wheat to be detected as a template, carrying out PCR amplification on the YrZM175-CAPS5 by adopting the primer to obtain an amplification product, and carrying out enzyme digestion on the amplification product by using HhaI to obtain an enzyme digestion product; judging the stripe rust resistance of the wheat to be detected according to the enzyme digestion product:
if the amplification product of the wheat to be detected can be cut into two fragments of 50bp-100bp and 520bp-570bp by HhaI enzyme, the wheat to be detected has or is candidate to have the stripe rust resistance property;
if the amplification product of the wheat to be detected cannot be digested by the HhaI enzyme, the wheat to be detected has or is candidate to have the stripe rust-sensitive character.
In the method, the two fragments of 620bp-680bp and 720bp-780bp can be obtained if the amplification product of the wheat to be detected can be cut by EagI enzyme, and specifically, the two fragments of 648bp and 755bp can be obtained if the amplification product of the wheat to be detected can be cut by EagI enzyme;
if the amplification product of the wheat to be detected can be cut into two fragments of 50bp-100bp and 520bp-570bp by the HhaI enzyme, the two fragments are specifically cut into two fragments of 87bp and 549bp by the HhaI enzyme.
In a specific embodiment of the invention, the method for identifying or assisting in identifying the stripe rust resistance of the wheat to be detected is the following A1) and/or A2):
a1 Taking genome DNA of wheat to be detected as a template, carrying out PCR amplification on the YrZM175-CAPS1 by adopting the primer to obtain an amplification product, and carrying out enzyme digestion on the amplification product by EagI to obtain an enzyme digestion product; judging the stripe rust resistance of the wheat to be detected according to the enzyme digestion product:
if the amplification product of the wheat to be detected can be cut into two fragments of 648bp and 755bp by EagI enzyme, the wheat to be detected has or is candidate to have the stripe rust resistance property;
if the amplification product of the wheat to be detected cannot be digested by EagI, the wheat to be detected has or is candidate to have the stripe rust-sensitive character;
a2 Taking genome DNA of wheat to be detected as a template, carrying out PCR amplification on the YrZM175-CAPS5 by adopting the primer to obtain an amplification product, and carrying out enzyme digestion on the amplification product by using HhaI to obtain an enzyme digestion product; judging the stripe rust resistance of the wheat to be detected according to the enzyme digestion product:
if the amplification product of the wheat to be detected can be cut into two fragments of 87bp and 549bp by HhaI enzyme, the wheat to be detected has or is candidate to have the stripe rust resistance property;
if the amplification product of the wheat to be detected cannot be digested by the HhaI enzyme, the wheat to be detected has or is candidate to have the stripe rust-sensitive character.
The invention further discloses a method for detecting or assisting in detecting the alleles of the stripe rust resistant gene YRZM175 to be detected.
The method for detecting or assisting in detecting the YrZM175 allele of the stripe rust resistant gene to be detected comprises the following steps B1) and/or B2):
b1 Taking genome DNA of wheat to be detected as a template, carrying out PCR amplification on YrZM175-CAPS1 by adopting the primer to obtain an amplification product, and carrying out enzyme digestion on the PCR amplification product by EagI to obtain an enzyme digestion product; judging the allele of the wheat stripe rust resistance gene YrZM175 to be detected according to the enzyme digestion product:
if the amplification product of the wheat to be detected can be cut into two fragments of 620bp-680bp and 720bp-780bp by EagI enzyme, the wheat to be detected contains or is candidate to contain the YrZM175 disease-resistant allele;
if the amplification product of the wheat to be detected can not be cut by EagI enzyme, the wheat to be detected contains or candidate contains YrZM175 susceptible allele;
b2 Taking genome DNA of wheat to be detected as a template, carrying out PCR amplification on the YrZM175-CAPS5 by adopting the primer to obtain an amplification product, and carrying out enzyme digestion on the PCR amplification product by using HhaI to obtain an enzyme digestion product; judging the allele of the wheat stripe rust resistance gene YrZM175 to be detected according to the enzyme digestion product:
if the amplification product of the wheat to be detected can be cut into two fragments of 50bp-100bp and 520bp-570bp by HhaI enzyme, the wheat to be detected contains or is candidate to contain the YRZM175 disease-resistant allele;
if the amplification product of the wheat to be tested can not be digested by HhaI, the wheat to be tested contains or is candidate to contain the YRZM175 susceptible allele.
In the method, the two fragments of 620bp-680bp and 720bp-780bp can be obtained if the amplification product of the wheat to be detected can be cut by EagI enzyme, and specifically, the two fragments of 648bp and 755bp can be obtained if the amplification product of the wheat to be detected can be cut by EagI enzyme;
if the amplification product of the wheat to be detected can be cut into two fragments of 50bp-100bp and 520bp-570bp by the HhaI enzyme, the two fragments are specifically cut into two fragments of 87bp and 549bp by the HhaI enzyme.
In a specific embodiment of the invention, the method for detecting or assisting in detecting the YrZM175 allele of the stripe rust resistant gene to be detected is B1) and/or B2) as follows:
b1 Taking genome DNA of wheat to be detected as a template, carrying out PCR amplification on the YrZM175-CAPS1 by adopting the primer to obtain an amplification product, and carrying out enzyme digestion on the PCR amplification product by EagI to obtain an enzyme digestion product; judging the wheat stripe rust resistant gene YrZM175 allele to be detected according to the enzyme digestion product:
if the amplification product of the wheat to be detected can be cut into two fragments of 648bp and 755bp by EagI enzyme, the wheat to be detected contains or candidate contains YrZM175 disease-resistant allele;
if the amplification product of the wheat to be detected can not be digested by EagI, the wheat to be detected contains or candidate contains YrZM175 susceptible allele;
b2 Taking genome DNA of wheat to be detected as a template, carrying out PCR amplification on the YrZM175-CAPS5 by adopting the primer to obtain an amplification product, and carrying out enzyme digestion on the PCR amplification product by using HhaI to obtain an enzyme digestion product; judging the allele of the wheat stripe rust resistance gene YrZM175 to be detected according to the enzyme digestion product:
if the amplification product of the wheat to be detected can be cut into two fragments of 87bp and 549bp by HhaI enzyme, the wheat to be detected contains or candidate contains the YrZM175 disease-resistant allele;
if the augmentation production of the wheat to be detected can not be digested by HhaI, the wheat to be detected contains or candidate contains the YRZM175 susceptible allele.
The invention also provides a method for breeding the stripe rust resistant wheat.
The method for breeding the stripe rust resistant wheat comprises the following steps:
according to the method for identifying or assisting in identifying the stripe rust resistance of the wheat to be detected, the wheat with the stripe rust resistance is bred;
or the like, or, alternatively,
and (3) breeding the wheat containing the disease-resistant allele of the stripe rust resistant gene YrZM175 according to the method for detecting or assisting in detecting the stripe rust resistant gene YrZM175 allele of the wheat to be detected.
In practice, the yellow rust resistant parent containing the YrZM175 disease resistance allele can be crossed with the susceptible parent without the allele, and the single plant containing the YrZM175 disease resistance allele is selected in a segregating generation by using the primer combination, the incision enzyme and the method until a stable strain homozygous for the YrZM175 disease resistance allele is bred.
The primer, the kit and the application of the method in wheat breeding also belong to the protection scope of the invention.
Above, stripe rust resistant wheat is wheat with an invasive IT = 0-2; the wheat susceptible to stripe rust is the wheat with the infection type IT = 3-4.
In the above, the wheat to be tested is the wheat containing the stripe rust resistant gene YrZM175 or the filial generation thereof with other wheat, such as the filial generation of the wheat variety "midge 175", the wheat variety "midge 175" and the wheat variety "Avocet S" (e.g. F1 generation, F2 generation, F3 generation.. Times), the derivative of the wheat variety "midge 175", or the filial generation of the wheat variety "midge 175" with other wheat varieties (e.g. F1 generation, F2 generation, F3 generation.. Times.).
In the above, the stripe rust may be stripe rust caused by 32 (CYR 32) in the stripe rust epidemic race.
The method takes wheat 175 in the stripe rust resistant variety and the stripe rust susceptible variety Avocet S as parents to construct a population, and utilizes the stripe rust strain CYR32 to cross offspring F 1 And F 2 The single plant is subjected to seedling-stage disease resistance identification, differential SNP among anti-influenza pools is searched by using a cluster segregation analysis method in combination with RNA sequencing (BSR-Seq), closely linked molecular markers YrZM175-CAPS1 and YrZM175-CAPS5 are developed, a good tool is provided for the application of the anti-stripe rust gene YrZM175 in wheat stripe rust resistant molecular marker assisted breeding, and an important role is played in wheat disease resistance breeding.
Drawings
FIG. 1 shows the primer pair YrZM175-CAPS1 as the challenge parent and F 2 And (5) amplification and enzyme digestion results of the population strains.
FIG. 2 shows the primer pair YrZM175-CAPS5 as the challenge parent and F 2 And (5) amplification and enzyme digestion results of the population strains.
FIG. 3 is a linkage diagram of 2 SSR markers, 1 InDel marker and 5 CAPS markers with the disease-resistant gene.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The source of each test material in the following examples of the invention:
miao 175 (Triticum aestivum) is described in the following documents: wheat 175, chen Xinmin, china breed, 7 th 2009, is a new examined wheat variety. Zhongmai 175 is bred by the national wheat improvement center of the institute of crop science of Chinese academy of agricultural sciences, and the combination is BPM-27/Jing 411, which passes the national variety approval in 2008. The medium wheat 175 is a main cultivar popularized in large area in northern winter wheat areas, and has excellent properties of high yield, disease resistance, lodging resistance, prematurity and the like.
Avocet S (Triticum aestivum) is described in: the observation of disease resistance variation of different wheat varieties, long Ling, guizhou agricultural science, 10 th 2009.
The wheat variety Mingxian 169 can be obtained from the national crop germplasm resource bank.
The rust stripe subspecies CYR32 is described in the following documents: the name and characteristics of No. 32 in the Chinese wheat stripe rust fungus stripe, mo Anmin, journal of plant protection, no.4 of 2003.
The method for detecting disease resistance by inoculating puccinia striiformis in the following examples of the invention is as follows: planting the wheat to be tested in a plastic pot of 9cm multiplied by 9cm, when the wheat seedlings grow to one heart and one leaf, uniformly inoculating the spores of the puccinia striiformis microspecies CYR32 to the leaves of the wheat by adopting a sweeping method (the inoculating method is that the mingxian 169 attached with fresh spores is carefully moved to the upper part of the wheat to be tested, then the spores on the leaves are uniformly swept to the leaves of each piece of the wheat to be tested), and recording the infection type of the wheat plant to be tested 14 days after inoculation (the mingxian 169 plant serving as a susceptible control is fully attacked, and the infection type IT = 4), wherein the infection type grading standard adopts a 6-grade standard (namely IT =0, 0; 1, 2, 3 or 4) (Bariana and McIntosh 1993), and is detailed in a table 1.
TABLE 1 wheat stripe rust seedling stage infection type grading Standard
| Infection Type (IT) | Symptoms and signs |
| 0 | The leaf does not produce any symptoms |
| 0； | Small necrotic spots are generated on the leaves, the distribution is sporadic, and summer spore heap is not generated |
| 1 | Necrotic spots are produced on the leaves, sporadic small-scattered summer sporangium masses on the necrotic spots |
| 2 | Necrotic spots are generated on the leaves, and very small summer sporangia masses which are more born on the necrotic spots |
| 3 | The leaf with the tablet is faded, the sporangium is large and the number is large |
| 4 | The leaf is not faded and a large amount of summer spore heap is grown on the leaf |
Note: wheat (R) resistant to stripe rust is rated 0-2, and wheat (S) susceptible to stripe rust is rated 3-4.
Example 1 acquisition of molecular marker YrZM175-CAPS1 primer set and YrZM175-CAPS5 primer set for anti-stripe rust Gene
Use of puccinia striiformis subspecies CYR32 to treat wheat 175 (disease-resistant variety), avocet S (susceptible variety) and F thereof 1 And F 2 The generation groups are subjected to seedling-stage resistance identification, and the results are shown in table 2. The results show that: wheat 175 in the parent is completely resistant to diseases, avocet S in the parent is completely susceptible to diseases, F 1 All against diseases, F 2 The anti-cold separation is 3: 1 (X) 2 =1.06, p > 0.05), indicating that the medium wheat 175 contains a dominant disease-resistant gene. At F 2 In the population, 50 extremely resistant single plants (0;) and 50 extremely sensitive single plants (4) are selected, and the leaves of the 50 extremely resistant single plants and the 50 extremely sensitive single plants are taken to respectively form an anti-disease pool and an infection pool.
Wheat 175, avocet S and F in Table 2 2 Seedling stage infection of offspring to CYR32
All SSR markers on 2AL were used, as Migmai 175, avocet S and Migmai 175/Avocet SF 2 And (3) performing PCR amplification by using the genome DNA of the single strain as a template, and screening the polymorphic SSR marker. And (3) carrying out 6% denaturing polyacrylamide gel electrophoresis detection on PCR amplification products marked by all SSRs, and carrying out silver staining and color development. 2 SSR markers Xgwm382 and Xwmc658 on 2AL in parent and F 2 The single plants have polymorphism, and are close to the linkage distance of the stripe rust resistant gene YrZM175, namely 5.12cM and 6.51cM respectively.
The anti-disease pool and the susceptible pool were analyzed by cluster segregation analysis combined with RNA sequencing (BSR-Seq) to obtain 13 differential SNPs closely linked to the yellow rust resistant gene YrZM 175. Using genomic DNA of medium wheat 175 and Avocet S as templates, 13 differential SNPs were developed as markers. Markers developed using 13 different SNPs, zhongmai 175/Avocet S F 2 The PCR detection is carried out by taking the genome DNA of the single strain as a template, the result shows that the linkage distance between the primer pair YrZM175-CAPS1 and the YrZM175 is the nearest 0.81cM, and the PCR detection result is shown in figure 1. In FIG. 1, M is Marker, pr is Mianmai 175, ps is Avocet S, R is disease-resistant single plant, and S is susceptible single plant. After the amplification product of the primer pair YrZM175-CAPS1 is subjected to EagI enzyme digestion and 2% agarose gel electrophoresis detection, two band types appear, namely a band type A1 (with specific fragments of 648bp and 755 bp) and a band type A2 (with specific fragment of 1403 bp), the specific DNA fragments of 648bp and 755bp can be displayed after the amplification and the enzyme digestion of Migmai 175 and a disease-resistant single strain, and the specific DNA fragments of 1403bp can be displayed after the amplification and the enzyme digestion of Avocet S and a disease-susceptible single strainSpecific DNA fragments.
By adopting a genome mining method, 1 InDel marker YrZM175-InD1 and 4 CAPS markers YrZM175-CAPS2, yrZM175-CAPS3, yrZM175-CAPS4 and YrZM175-CAPS5 are developed by using a reference genome (Triticum _ aestivum IWGSC reference v 1.0) of Chinese spring as a sequence template and genomic DNAs of Chinese wheat 175 and Avocet S as PCR templates. 175/Avocet S F Migmai 2 The genomic DNA of the individual plants was used as a template, and PCR detection was carried out, and the results showed that the linkage distance between YrZM175-InD1 and YrZM175-CAPS5 and YrZM175 was closest, and the PCR detection results of 0.6cM and 0.27cM, and YrZM175-CAPS5 are shown in FIG. 2. In FIG. 2, M is Marker, pr is Mianmai 175, ps is Avocet S, R is disease-resistant single plant, and S is susceptible single plant. After the amplification product of the primer pair YrZM175-CAPS5 is subjected to HhaI enzyme digestion, 2% agarose gel electrophoresis detection shows that two band types appear, namely a band type B1 (with specific fragments of 87bp and 549 bp) and a band type B2 (with specific fragments of 636 bp), the specific DNA fragments of 87bp and 549bp can be displayed after the amplification enzyme digestion of Migmai 175 and a disease-resistant single strain, and the specific DNA fragment of 636bp can be displayed after the amplification enzyme digestion of Avocet S and a disease-sensitive single strain.
175/Avocet S F Migmai 2 The genomic DNA of the individual strain was used as a template, and PCR amplification was carried out using the above 8 markers (2 SSR markers Xgwm382 and Xwmc658, 1 InDel marker YrZM175-InD1, and 5 CAPS markers YrZM175-CAPS1, yrZM175-CAPS2, yrZM175-CAPS3, yrZM175-CAPS4, and YrZM175-CAPS 5) to obtain PCR amplification products. The gene linkage map was drawn by treatment with MapMaker3.0, and as shown in FIG. 3, 8 markers each linked to the anti-stripe rust gene at a genetic distance of from 0.27cM to 6.51cM, and the anti-stripe rust gene YRZM175 was located between YRZM175-CAPS1 and YRZM175-CAPS5 at a distance of 0.81cM and 0.27cM from the two markers, respectively.
The primer pair YrZM175-CAPS1 is as follows:
upstream primer (SEQ ID NO. 1): 5'-CTTTGCAATGCCACAGTATGG-3';
downstream primer (SEQ ID NO. 2): 5'-GATCCCATCAATTTTGCCTTG-3'.
The YrZM175-CAPS5 primer pair is as follows:
upstream primer (SEQ ID NO. 3): 5'-GCAATATCTTTAGGCACCGC-3';
downstream primer (SEQ ID NO. 4): 5'-TTACGCATTATTTCTCACACGG-3'.
Example 2 screening of wheat for stripe rust resistance Using the YrZM175-CAPS1 primer set and the YrZM175-CAPS5 primer set
1. Obtaining the strains to be tested
1. Hybridizing by taking Migmai 175 as a female parent and Avocet S as a male parent to obtain F 1 And (5) seed generation.
2、F 1 The plant grown by the generation seed is F 1 Plant generation, F 1 Selfing the plant to obtain F 2 And (5) seed generation.
3. Planting F 2 Seed generation to obtain F 2 And (4) generating individuals, and randomly selecting 10 individuals from the generation individuals.
4. Each individual obtained in step 3 was selfed to produce lines, and 10 individuals in total produced 10 lines, which were designated as Z1 line to Z10 line in sequence.
2. Screening of stripe rust resistant wheat from test strains by using YrZM175-CAPS1 primer pair and YrZM175-CAPS5 primer pair
Taking the strain from Z1 to Z10 (F) 2 Selfing progeny of the generation individual plant), taking the Zhongmai 175 and the Avocet S as the wheat to be detected, and respectively carrying out the following detection:
1. extracting the genome DNA of the wheat leaves to be detected, and mixing and extracting 10 single plants in each strain.
2. And (2) taking the genomic DNA extracted in the step (1) as a template, and performing PCR amplification by adopting a YrZM175-CAPS1 primer pair to obtain an amplification product.
And (3) PCR reaction system: 2 XPCR Taq Mix 7.5. Mu.l, ddH 2 O 4.5μl，Forward Primer1μl，Reverse Primer 1μl，DNA 1μl。
Procedure for PCR amplification: 5min at 94 ℃; 37 cycles at 94 ℃ 30s, 56 ℃ 35s, 72 ℃ 45s; 10min at 72 ℃.
3. And (3) carrying out enzyme digestion on the amplification product obtained in the step (2) to obtain an enzyme digestion product.
An enzyme digestion reaction system: 10 × CutSmart Buffer 2 μ l, ddH 2 O7.75. Mu.l, eagI 0.25. Mu.l, PCR amplification product 10. Mu.l.
The enzyme digestion reaction conditions are as follows: 4h at 37 ℃.
4. And (3) carrying out 2% agarose gel electrophoresis on the enzyme digestion product obtained in the step (3).
Detecting the stripe rust resistance of the wheat to be detected according to the following standard:
if the enzyme digestion product has fragments of 648bp and 755bp, the wheat to be detected carries the wheat stripe rust resistant gene YrZM175 disease-resistant allele, and the wheat to be detected has the stripe rust resistant character, namely the wheat to be detected is stripe rust resistant wheat;
if the enzyme digestion product does not have fragments of 648bp and 755bp, the wheat to be detected carries the wheat stripe rust resistant gene YrZM175 susceptible allele, and the wheat to be detected does not have the stripe rust resistant character, namely the wheat to be detected is stripe rust resistant wheat.
The results show that: the strains Z1, Z2, Z4, Z7, Z9 and Z10 are all the same as the banding pattern of the Zhongmai 175 and show specific fragments with 648bp and 755bp, so that the strains Z1, Z2, Z4, Z7, Z9 and Z10 are identified as stripe rust resistant wheat; the band patterns of the Z3, Z5, Z6 and Z8 strains are identical to those of Avocet S, showing a specific fragment of 1403bp, but not of 648bp and 755bp, thus identifying the Z3, Z5, Z6 and Z8 strains as stripe rust susceptible wheat.
5. And (4) carrying out sequencing verification on the PCR amplification product obtained in the step (2), wherein the result is consistent with the result obtained in the step (4).
6. And (3) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and adopting a YrZM175-C4PS5 primer pair to obtain an amplification product.
And (3) PCR reaction system: 2 XPCR Taq Mix 7.5. Mu.l, ddH 2 O 4.5μl，Forward Primer 1μl，Reverse Primer 1μl，DNA 1μl。
Procedure for PCR amplification: 5min at 94 ℃; 30s at 94 ℃, 35s at 54 ℃ and 40s at 72 ℃ for 37 cycles; 10min at 72 ℃.
7. And (4) carrying out enzyme digestion on the PCR product obtained in the step (6) to obtain an enzyme digestion product.
An enzyme digestion reaction system: 10 × CutSmart Buffer 2 μ l, ddH 2 O7.75. Mu.l, hhaI 0.25. Mu.l, PCR amplificationThe product was increased by 10. Mu.l.
The enzyme digestion reaction conditions are as follows: 4h at 37 ℃.
8. And (4) carrying out 2% agarose gel electrophoresis on the enzyme digestion product obtained in the step (7).
Detecting the stripe rust resistance of the wheat to be detected according to the following standard:
if the enzyme digestion product has 87bp and 549bp fragments, the wheat to be detected carries a wheat stripe rust resistance gene YrZM175 disease-resistant allele, and the wheat to be detected has the character of stripe rust resistance, namely the wheat to be detected is stripe rust resistance wheat;
if the enzyme digestion product does not have the fragments of 87bp and 549bp, the wheat to be detected carries the wheat stripe rust resistant gene YrZM175 susceptible allele, and the wheat to be detected does not have the character of stripe rust resistance, namely the wheat to be detected is stripe rust resistant wheat.
The results show that: the strains Z1, Z2, Z4, Z7, Z9 and Z10 are all the same as the zone type of the Zhongmai 175 and show specific fragments with fragments of 87bp and 549bp, so that the strains Z1, Z2, Z4, Z7, Z9 and Z10 are identified as stripe rust resistant wheat; the band patterns of the Z3, Z5, Z6 and Z8 strains were identical to those of Avocet S, showing a specific fragment of 636bp, not 87bp and 549bp, thus identifying the Z3, Z5, Z6 and Z8 strains as stripe rust susceptible wheat.
9. And (4) performing sequencing verification on the PCR amplification product obtained in the step (6), wherein the sequencing verification is consistent with the result obtained in the step (8).
3. Inoculation of rust stripe for detection of disease resistance
And (3) respectively detecting the disease resistance of each wheat to be detected in the step two according to the method for detecting the disease resistance by inoculating the puccinia striiformis, and the results are shown in table 3. The strain Z1, the strain Z2, the strain Z4, the strain Z7, the strain Z9 and the strain Z10 are disease-resistant strains, and the strain Z3, the strain Z5, the strain Z6 and the strain Z8 are disease-sensitive strains.
Wheat 175, avocet S and F in Table 3 2 Seedling stage infection type of selfing progeny of generation individual plant to CYR32
| Name of plant | Infection type |
| Zhongmai 175 | 10 plants are infected with IT =0 |
| Avocet S | 10 plants are infected with IT =4 |
| Z1 | 1 plant infection type IT =0,5 plant infection type IT =0; |
| Z2 | the infection type IT =0 of 13 plants; |
| Z3 | 4 plants infected IT =3,2 plants infected IT =4 |
| Z4 | 10 plants are infected with IT =0,2 plants are infected with IT =0; |
| Z5 | 5 plants infected IT =3,3 plants infected IT =4 |
| Z6 | 3 plants infected IT =3,4 plants infected IT =4 |
| Z7 | 6 plants infected IT =0,3 plants infected IT =0； |
| Z8 | 4 plants infected IT =3,4 plants infected IT =4 |
| Z9 | The infection type IT =0 of 11 plants; |
| Z10 | 8 plants are infected with IT =0,2 plants are infected with IT =0; |
the results show that the molecular markers YrZM175-CAPS1 and YrZM175-CAPS5 can be applied to assist in identifying and screening wheat with stripe rust resistance.
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
<110> institute of crop science of Chinese academy of agricultural sciences
<120> primer for identifying stripe rust resistance of wheat and application thereof
<130> GNCFY192465
<160> 4
<170> PatentIn version 3.5
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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Claims (9)
1. A primer set, characterized in that: the primer group comprises a primer pairYrZM175-CAPS1And/or primer pairsYrZM175-CAPS5；
The primer pairYrZM175-CAPS1Comprises an upstream primer F1 and a downstream primer R1;
the upstream primer F1 is a single-stranded DNA molecule shown in SEQ ID No. 1;
the downstream primer R1 is a single-stranded DNA molecule shown in SEQ ID No. 2;
the primer pairYrZM175-CAPS5Comprises an upstream primer F2 and a downstream primer R2;
the upstream primer F2 is a single-stranded DNA molecule shown in SEQ ID No. 3;
the downstream primer R2 is a single-stranded DNA molecule shown in SEQ ID No. 4.
2. A kit, characterized in that: the kit comprises the primer set of claim 1; the kit also comprises restriction enzyme EagI and/or restriction enzyme HhaI.
3. Use of the primer set of claim 1 or the kit of claim 2 in any one of:
1) Identifying or assisting in identifying the stripe rust resistance of the wheat to be detected;
2) Preparing a product for identifying or assisting in identifying the stripe rust resistance of the wheat to be detected;
3) Detection or auxiliary detection of wheat stripe rust resistance gene to be detectedYrZM175An allele;
4) Preparation detection or auxiliary detection of wheat stripe rust resistance gene to be detectedYrZM175The product of an allele.
4. Use according to claim 3, characterized in that: the wheat to be detected contains a stripe rust resistance geneYrZM175Or progeny of a cross thereof with other wheat.
5. Use of the primer set of claim 1 or the kit of claim 2 in any one of:
1) Breeding or assisting in breeding the stripe rust resistant wheat;
2) Preparing and breeding or assisting in breeding the stripe rust resistant wheat product.
6. A method for identifying or assisting in identifying the stripe rust resistance of wheat to be detected is characterized by comprising the following steps: the method is A1) and/or A2) as follows:
a1 Using genomic DNA of wheat to be tested as a template, and the primer set according to claim 1YrZM175-CAPS1The PCR amplification is carried out, and the PCR amplification is carried out,obtaining an amplification product, and carrying out enzyme digestion on the amplification product by using EagI to obtain an enzyme digestion product; judging the stripe rust resistance of the wheat to be detected according to the enzyme digestion product:
if the amplification product of the wheat to be detected can be cut into two fragments of 648bp and 755bp by EagI enzyme, the wheat to be detected has or is candidate to have the stripe rust resistance property;
if the amplification product of the wheat to be detected cannot be digested by EagI, the wheat to be detected has or is candidate to have the stripe rust-sensitive character;
a2 Using genomic DNA of wheat to be tested as a template, and the primer set according to claim 1YrZM175-CAPS5Carrying out PCR amplification to obtain an amplification product, and carrying out enzyme digestion on the amplification product by using HhaI to obtain an enzyme digestion product; judging the stripe rust resistance of the wheat to be detected according to the enzyme digestion product:
if the amplification product of the wheat to be detected can be cut into two fragments of 87bp and 549bp by HhaI enzyme, the wheat to be detected has or is candidate to have the stripe rust resistance property;
if the amplification product of the wheat to be detected cannot be subjected to enzyme digestion by the HhaI, the wheat to be detected has or is candidate to have the stripe rust-sensitive character.
7. Detection or auxiliary detection of stripe rust resistance gene to be detectedYrZM175A method of allelic generation, characterized by: the method comprises the following steps B1) and/or B2):
b1 Using genomic DNA of wheat to be tested as a template, and the primer set according to claim 1YrZM175-CAPS1Carrying out PCR amplification to obtain an amplification product, and carrying out enzyme digestion on the PCR amplification product by using EagI to obtain an enzyme digestion product; judging the wheat stripe rust resistance gene to be detected according to the enzyme digestion productYrZM175Allele:
if the amplification product of the wheat to be detected can be cut into two fragments of 648bp and 755bp by EagI enzyme, the wheat to be detected contains or candidate containsYrZM175A disease resistance allele;
if the amplification product of the wheat to be detected can not be digested by EagI, the wheat to be detected contains or candidate containsYrZM175A disease-sensitive allele;
b2 Modulo the genomic DNA of the wheat to be testedA plate using the primer set according to claim 1YrZM175-CAPS5Carrying out PCR amplification to obtain an amplification product, and carrying out enzyme digestion on the PCR amplification product by using HhaI to obtain an enzyme digestion product; judging the wheat stripe rust resistance gene to be detected according to the enzyme digestion productYrZM175Allele:
if the amplification product of the wheat to be detected can be cut into two fragments of 87bp and 549bp by HhaI enzyme, the wheat to be detected contains or candidate containsYrZM175A disease resistance allele;
if the amplification product of the wheat to be detected can not be digested by HhaI, the wheat to be detected contains or candidate containsYrZM175A disease-sensitive allele.
8. A method for breeding yellow rust resistant wheat is characterized in that the method is C1) and/or C2):
c1 Selecting wheat having the stripe rust resistance trait according to the method of claim 6;
c2 The method according to claim 7, wherein the selection of the gene containing the stripe rust resistance geneYrZM175Wheat with disease resistance alleles.
9. The method according to claim 6 or 7, characterized in that: the wheat to be detected contains a stripe rust resistance geneYrZM175Or a progeny of a cross thereof with another wheat.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132570A (en) * | 2015-09-22 | 2015-12-09 | 中国农业科学院作物科学研究所 | Primer combination assisting in screening stripe-rust-resistant wheat and application of primer combination |
| WO2017079286A1 (en) * | 2015-11-03 | 2017-05-11 | Two Blades Foundation | Wheat stripe rust resistance genes and methods of use |
| CN108977440A (en) * | 2018-09-05 | 2018-12-11 | 中国农业科学院作物科学研究所 | A kind of molecular labeling and application method for 895 stripe rust resisting QTL of wheat in detecting |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132570A (en) * | 2015-09-22 | 2015-12-09 | 中国农业科学院作物科学研究所 | Primer combination assisting in screening stripe-rust-resistant wheat and application of primer combination |
| WO2017079286A1 (en) * | 2015-11-03 | 2017-05-11 | Two Blades Foundation | Wheat stripe rust resistance genes and methods of use |
| CN108977440A (en) * | 2018-09-05 | 2018-12-11 | 中国农业科学院作物科学研究所 | A kind of molecular labeling and application method for 895 stripe rust resisting QTL of wheat in detecting |
Non-Patent Citations (2)
| Title |
|---|
| Development of sequence tagged site and cleaved amplified polymorphic sequence markers for wheat stripe rust resistance gene Yr5;Xianming Chen等;《Crop Sci.》;20031130;第43卷;第2058-2063页 * |
| Identification of a new stripe rust resistance gene in Chinese winter wheat Zhongmai 175;Lu Jia-ling等;《Journal of Integrative Agriculture》;20161231;第15卷(第11期);第2461-2468页 * |
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