CN102559911B - Method for assisting in identifying powdery mildew resistant plants and special primers for method - Google Patents

Abstract

The invention discloses a method for assisting in identifying powdery mildew resistant plants and special primers for the method. The special primers comprises an Xcfd50 primer pair consisting of DNA shown in a sequence 1 and a sequence 2 in a sequence table, and also can comprise an XMag633 primer pair consisting of DNA shown in sequences 3 and 4. The special primers can be used for assisting in identifying the powdery mildew resistant plants. The primer composition and the method can assist in identifying the powdery mildew resistant plants, and have the advantages of simple operation, low cost and capacity of realizing early breeding. The primer composition and the method can be used for genetic breeding of plants, and have significance for breeding new varieties of identifying powdery mildew resistant plants.

Description

Method for auxiliary identification of powdery mildew resistant plants and special primers thereof

technical field

The invention relates to a method for assisting identification of powdery mildew resistant plants and special primers thereof.

Background technique

Wheat is an important food crop in my country, which is closely related to national food security, national economic development, social stability and improvement of people's living standards. Wheat powdery mildew is a worldwide wheat disease. With the popularization of dwarf and semi-dwarf wheat varieties and the improvement of water and fertilizer conditions, the increase of planting density has resulted in closed canopy of wheat fields, making the damage caused by powdery mildew in my country more and more serious, and the area and scope of the disease have continued to expand. Key diseases have risen to become major diseases, posing a serious threat to my country's food production and safety.

Breeding and promoting disease-resistant varieties has been recognized as the most economical, effective and safe way to control wheat powdery mildew, and the key to breeding disease-resistant varieties is to continuously explore and effectively use new sources of resistance. Breeding wheat varieties containing multiple powdery mildew resistance genes is one of the effective ways to broaden the resistance spectrum, improve resistance and maintain resistance. Therefore, it is of great significance for molecular assisted breeding to discover new disease resistance genes and their closely linked molecular markers, and to carry out timely mapping of new disease resistance genes.

SSR markers, also known as microsatellite (Microsatellite) markers, are simple repeating sequences consisting of a group of 1 to 6 nucleotides in series with a repeat number between 5 and 10. They are ubiquitous in higher organisms and randomly distributed throughout the genome, forming polymorphisms in DNA sequences due to their different repeat times. Both sides of each SSR locus are generally relatively conservative single-copy sequences, so the polymorphism of this tandem repeat sequence can be easily detected by PCR amplification by designing specific primers for the conserved sequences at both ends.

SSR molecular marker technology has the following characteristics: (1) There are many loci and abundant in quantity, covering the entire chromosome set; (2) Each locus has many allelic forms, and the polymorphism is high; (3) Most of them are co-dominant Markers can distinguish homozygous and heterozygous genotypes, thereby providing relatively complete genetic information at a single locus; (4) The quality of template DNA is not high, the amount of DNA used is small, and it is easy to use PCR technology to analyze; (5) The operation is simple and the results are reproducible; (6) The chromosome positions of most SSR markers are known, which can be conveniently applied to researches such as gene mapping and identification of exogenous genetic material. Wheat SSR markers are transferable between wheat close species and within the genus, that is, most of the SSR primers designed based on the sequence information of common wheat can also play a role in close species of wheat and amplify normal DNA fragment.

Li et al. (2009) used SSR markers to analyze the powdery mildew resistance gene from wild emmer wheat material IW2, and found that the powdery mildew resistance gene was located between the SSR markers Xbarc84 and Xwmc687, with genetic distances of 3.2 cM and 2.0 cM, respectively. The gene was located on the chromosome 3BL in the material of Chinese Spring Deficiency-four system, double-end system and deletion line, and the powdery mildew resistance gene was named Pm41. Hua et al. (2009) used SSR markers to analyze the common wheat powdery mildew resistance line P63 from wild emmer wheat, and found that the powdery mildew resistance gene is located between the SSR markers Xwmc257 and Xgwm148, and the genetic distances are 16cM and 6.7cM, respectively. , and located the gene on chromosome 2BS, the powdery mildew resistance gene was named Pm42.

Contents of the invention

The purpose of the present invention is to provide a method for assisting identification of powdery mildew resistant plants and special primers thereof.

The invention provides the application of a primer pair A (Xcfd50 primer pair) in the preparation of a kit for assisting in the identification of powdery mildew resistant plants; the primer pair A is a single-stranded DNA shown in sequence 1 of the sequence listing and the sequence of the sequence listing Primer pairs consisting of single-stranded DNA shown in 2. The plant is wheat. The wheat can specifically be wheat material 87-1, wheat material IW132, wheat material Nongda 212, wheat material Nongda 211, offspring (including hybrid offspring, backcross offspring) obtained with wheat material 87-1 and wheat material IW132 as initial parents ), the offspring of the hybrid with the wheat material Nongda 212 (or the wheat material Nongda 211) and its selfed progeny.

The present invention also protects the application of the primer pair A and primer pair B (XMag633 primer pair) in the preparation of a kit for assisting in the identification of powdery mildew resistant plants; DNA and a primer pair composed of single-stranded DNA shown in sequence 4 of the sequence listing. The plant is wheat. The wheat can specifically be wheat material 87-1, wheat material IW132, wheat material Nongda 212, wheat material Nongda 211, offspring (including hybrid offspring, backcross offspring) obtained with wheat material 87-1 and wheat material IW132 as initial parents ), the offspring of the hybrid with the wheat material Nongda 212 (or the wheat material Nongda 211) and its selfed progeny.

The invention also protects a kit for assisting identification of powdery mildew resistant plants, including the pair of primers. The kit may also include the primer pair B.

The invention also protects the application of the primer pair A in assisting identification of powdery mildew resistant plants.

The invention also protects the application of the primer pair A and the primer pair B in assisting identification of powdery mildew resistant plants.

The present invention also protects a method for assisting identification of powdery mildew-resistant plants, including step A; said step A includes the following steps: using the genomic DNA of the plant to be tested as a template, and using said primers to perform PCR amplification on A; if There are two specific fragments in the PCR amplification product, and the plant to be tested is a candidate powdery mildew resistant plant; if the PCR amplification product does not have the two specific fragments, the plant to be tested is a candidate powdery mildew susceptible plant Plant; one of the two specific fragments is 505-515bp, and the other is 485-495bp.

The method can also include step B; said step B includes the following steps: using the genomic DNA of the plant to be tested as a template, using the primers to perform PCR amplification on B; if there is a specific fragment in the PCR amplification product, The plant to be tested is a candidate powdery mildew resistant plant; if the PCR amplification product does not have the specific fragment, the plant to be tested is a candidate powdery mildew susceptible plant; the specific fragment is 565-575bp DNA fragments.

The plant is wheat. The wheat can specifically be wheat material 87-1, wheat material IW132, wheat material Nongda 212, wheat material Nongda 211, offspring (including hybrid offspring, backcross offspring) obtained with wheat material 87-1 and wheat material IW132 as initial parents ), the offspring of the hybrid with the wheat material Nongda 212 (or the wheat material Nongda 211) and its selfed progeny.

The identification results of the step A and the step B can be mutually verified. If the identification results of the same test plant in the step A and the step B are consistent, the test plant is a further determined candidate powdery mildew resistant plant or a further determined candidate powdery mildew susceptible plant. If the identification results of the same test plant in the step A and the step B are inconsistent, other existing methods can be combined to determine the resistance of the test plant to powdery mildew.

Wild emmer wheat (Triticum dicoccoides, 2n=4×=28, AABB) is the secondary gene source of common wheat, which has good resistance to wheat leaf rust, stem rust and powdery mildew, and is used in the genetic improvement of wheat disease resistance It has great application potential. Wild emmer is the tetraploid ancestor of common wheat, and it is easy to successfully hybridize with common wheat. The hybrid F1 is partially or completely fertile, and its disease resistance can be easily transferred to common wheat through hybridization and backcrossing. Therefore, it is of great practical significance to fully explore the powdery mildew resistance source of wild emmer wheat and introduce it into common wheat to increase the diversity and gene accumulation of wheat disease resistance genes. The discovery of the powdery mildew resistance gene Ml7K65 further confirmed the role of wild emmer in the resistance of common wheat to powdery mildew, and also provided a basis for the breeding of new wheat varieties. The present invention has screened two molecular markers closely linked with powdery mildew resistance gene M17K65: Xcfd50 and Xmag633. These two markers can be effectively used for marker-assisted selection of M17K65 gene. Using this marker as a guidepost, the fine mapping of the M17K65 gene can be carried out or the M17K65 gene can be approached by chromosome walking method, thus laying the foundation for the cloning of the M17K65 gene.

The primer composition and method provided by the invention can assist in the identification of powdery mildew resistant plants, and have the advantages of simple operation, low cost and early breeding. The primer composition and method of the invention can be used for plant genetic breeding, and has great value for breeding new varieties of powdery mildew-resistant plants.

Description of drawings

Figure 1 shows the results of PCR identification of 7K65/Nongda 211-F2 generation plants using Xcfd50 primer pair.

Fig. 2 is the result of PCR identification of 7K65/Nongda 212-F2 generation plants using Xcfd50 primer pair.

Fig. 3 is the result of PCR identification of 7K65/Nongda 211-F2 generation plants using XMag633 primer pair.

Fig. 4 is the result of PCR identification of 7K65/Nongda 212-F2 generation plants using XMag633 primer pair.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Wheat material 87-1 (common wheat susceptible to wheat powdery mildew): Handan Academy of Agricultural Sciences, Hebei Province; the public can obtain it from China Agricultural University; References: Zhang Liansong, Huawei, Guan Haiying, Li Genqiao, Zhang Hongtao, Xie Chaojie, Yang Zuomin, Sun Qixin, Liu Zhiyong Molecular marker mapping of powdery mildew resistance gene MlWE29 introduced from wild emmer into common wheat, Acta Crops Sinica 2009, 35(6): 998-1005.

Wheat material IW132 (wild emmer wheat with high resistance to powdery mildew): University of Haifa, Israel; the public can obtain it from China Agricultural University; References: Xie Chaojie, Sun Qixin, Yang Zuomin Identification of wild emmer wheat seedling stage disease resistance in Israel. Acta Milo Crops 2003, 23(2): 39-42.

Wheat material Morocco (common wheat susceptible to wheat powdery mildew): The public can obtain it from China Agricultural University; references: Liu Huiyuan, K Suenaga, He Zhonghu, Wang Zhulin, Liang Shanshan, Ma Jun, M Bernard, PSourdille, Xia Xianchun common wheat white powder QTL analysis of disease adult plant resistance. Acta Crops 2006, 32(2): 197-202.

Wheat material Nongda 212 (common wheat susceptible to wheat powdery mildew): a new wheat variety cultivated by the Wheat Group of the School of Agronomy and Biotechnology, China Agricultural University. The public can purchase it from Beijing Tongzhou District Seed Company.

Wheat material Nongda 211 (common wheat susceptible to wheat powdery mildew): a new wheat variety cultivated by the Wheat Group of the School of Agronomy and Biotechnology, China Agricultural University. The public can purchase it from Beijing Tongzhou District Seed Company.

Wheat material Xue Zao (common wheat susceptible to wheat powdery mildew): Institute of Crop Research, Beijing Academy of Agricultural Sciences; the public can obtain it from China Agricultural University; References: Zhang Liansong, Huawei, Guan Haiying, Li Genqiao, Zhang Hongtao, Xie Chaojie, Yang Zuomin, Sun Qixin, Liu Zhiyong Molecular marker mapping of powdery mildew resistance gene MlWE29 introduced from wild emmer into common wheat. Acta Crops 2009, 35(6): 998-1005.

Physiological race E09: Institute of Plant Protection, Chinese Academy of Agricultural Sciences; the public can obtain it from China Agricultural University; references: Zhang Liansong, Huawei, Guan Haiying, Li Genqiao, Zhang Hongtao, Xie Chaojie, Yang Zuomin, Sun Qixin, Liu Zhiyong Molecular marker mapping of powdery mildew gene MlWE29. Acta Crops 2009, 35(6): 998-1005.

The division of resistance and susceptibility of plants to the pathogen of wheat powdery mildew is based on the response type of plants at seedling stage after inoculation with the pathogen of wheat powdery mildew. The grading standards are shown in Table 1.

Table 1 Grading standard of seedling stage response type of plants inoculated with wheat powdery mildew pathogenic bacteria (Liu et al.1999)

Embodiment 1, the acquisition of plant material

1. Acquisition of disease-resistant parent 7K65

1. A wheat material 87-1 (as the male parent) was crossed with a wheat material IW132 (as the female parent) to obtain 10 seeds, namely the F1 generation.

2. Cross 10 F1 generation wheat plants (as the female parent) with 1 wheat material Morocco to obtain 50 seeds, namely the BC1 generation.

3. Sow 50 BC1 generation seeds, inoculate wheat powdery mildew pathogenic bacteria (No. E09 physiological race) at the seedling stage, select the seedlings (total 2 strains) that show disease resistance after 15 days of inoculation of pathogenic bacteria, treat after heading It was crossed with 1 wheat material 87-1 (the resistant seedlings were used as the female parent, and the wheat material 87-1 was used as the male parent), and 23 seeds were obtained, which was the BC2 generation.

4. Sow 23 BC2 generation seeds, and harvest self-bred seeds respectively.

5. Plant 23 groups of self-bred seeds obtained in step 4 in the experimental field of China Agricultural University Science Park, and each group of seeds has 1 row, and inoculate wheat powdery mildew pathogenic bacteria (No. E09 physiological race) at the seedling stage respectively, wherein the row number All the plants in the "7K65 row" showed homozygous resistance to wheat powdery mildew, and the plants in this row (homozygous powdery mildew resistant materials) were reserved for follow-up research, which was the disease-resistant parent 7K65. The pedigree of the resistant parent 7K65 is: IW132/87-1//Morocco/3/87-1.

2. Acquisition of 7K65/Nongda 212-F2 generation disease-resistant segregation population

1. A disease-resistant parent 7K65 (as the female parent) was crossed with a wheat material Nongda 212 (as the male parent) to obtain 18 seeds, which were the F1 generation.

2. Self-cross 18 F1 generation wheat plants to obtain 247 seeds, namely 7K65/Nongda 212-F2 generation.

3. Acquisition of 7K65/Nongda 211-F2 generation disease-resistant segregation population

1. A disease-resistant parent 7K65 (as the female parent) was crossed with a wheat material Nongda 211 (as the male parent), and 20 seeds were harvested, which was the F1 generation.

2. Self-cross 20 F1 generation wheat plants to obtain 268 grains, namely 7K65/Nongda 211-F2 generation.

Embodiment 2, the powdery mildew resistance identification of plant material

The disease resistance of 7K65/Nongda 212-F2 generation plants (247 plants) and 7K65/Nongda 211-F2 generation plants (268 plants) were identified, and the wheat material Xuezao was used as a susceptible control (3 plants). The specific methods are as follows (performed in a greenhouse):

1. Inoculate the wheat material Xue Zao with the physiological race E09 of powdery mildew pathogenic bacteria using natural propagation and artificial brushing (1 time a day during the growth period of the seedlings), and repeat the inoculation several times until smaller spots appear on the wheat leaves. Thick mycelium layer, a large number of spores and lesions are connected into sheets, which can be used as a breeding pot.

2. Plant the grains (seeds) to be tested in plastic seedling trays, and plant about 15 seeds in each hole. When the seeds germinate and grow to the stage of one leaf and one heart, place the breeding pot around the seedling tray, and the powdery mildew pathogenic spores Seedlings of the test plants were inoculated by natural propagation.

3. 15 days after the inoculation (placement of the multiplication pot), when the susceptible control has fully developed the disease, record the disease resistance of the plant to be tested according to observation, and recheck it after another 3 days to accurately record its disease resistance.

The results showed that the results of the two observations recording the disease resistance of the tested plants were consistent. Among the 247 7K65/Nongda 212-F2 generation plants, 174 individual plants were resistant to the disease, and 73 individual plants were susceptible to the disease. The chi-square test results showed that χ ² _3∶1 = 2.73, (χ ² _0.05 , 1 = 3.840 ) (P<0.05), in line with the segregation ratio of a single gene of 3:1, indicating that the powdery mildew resistance traits of this combination are controlled by a dominant single gene. Among the 268 7K65/Nongda 211-F2 generation plants, 196 individual plants were resistant to the disease, and 72 individual plants were susceptible to the disease. The chi-square test results showed that χ ² _3∶1 = 0.497, (χ ² _0.05, 1 = 3.84 ) (P<0.05), in line with the 3:1 monogene segregation ratio, which also indicated that the powdery mildew resistance traits of this combination were controlled by a dominant single gene. Temporarily named the disease resistance gene as Ml7K65.

Embodiment 3, the PCR identification of plant material

1. Association analysis of SSR markers and plant powdery mildew resistance

The resistance and susceptibility pools were constructed using the Bulked Segregant Analysis (BSA) method: the DNAs of 10 homozygous disease-resistant and 10 homozygous disease-resistant individuals were selected from the F2 individual plants and mixed in equal amounts to form DNA resistance pool and DNA susceptibility pool are used to screen molecular markers linked to disease resistance genes. Finally, it was found that the Xcfd50 primer pair and the XMag633 primer pair were closely linked to the M17K65 gene, so that the two pairs of primers could be used to help identify the powdery mildew resistance of plants.

2. Preparation of primer composition

The primer composition consisted of the Xcfd50 primer pair and the XMag633 primer pair.

The sequences of the upstream and downstream primers of the Xcfd50 primer pair (the annealing temperature applied to PCR is 60.0° C.) are as follows:

Upstream primer (sequence 1 of the sequence listing): 5'-TTCTGCAACATTTTTTGTCCCA-3';

Downstream primer (sequence 2 of the sequence listing): 5'-CGTATGATCCTAACGAGGGC-3'.

The sequences of the upstream and downstream primers of the XMag633 primer pair (the annealing temperature applied to PCR is 52.0° C.) are as follows:

Upstream primer (sequence 3 of the sequence listing): 5'-CGCCAAAACTCTGGGACG-3';

Downstream primer (sequence 4 of the sequence listing): 5'-GAAAGAAGGACCGTGCTACAAG-3'.

The above four primers were synthesized separately.

3. PCR identification of plant material (application of Xcfd50 primer pair)

7K65/Nongda 212-F2 generation plants (247 plants) and 7K65/Nongda 211-F2 generation plants (268 plants) were identified by PCR, and wheat material 87-1, wheat material Nongda 212 and wheat material Nongda 211 were used as negative controls , wheat material IW132 and disease-resistant parent 7K65 were used as positive controls, and the specific methods were as follows:

1. Cut the leaves of each seedling in Example 2, and extract genomic DNA.

2. Using the genomic DNA in step 1 as a template, perform PCR amplification with the Xcfd50 primer pair to obtain a PCR amplification product.

3. Perform 8% polyacrylamide gel electrophoresis on the PCR amplified product in step 2, then stain with silver staining, and finally count the experimental results. Part of the results are shown in Figure 1 (R represents a resistant individual plant, and S represents a susceptible individual plant).

Among the 174 resistant individual plants, 163 showed two specific bands of 510bp and 490bp by electrophoresis. Among 73 susceptible individuals, only 13 showed two specific bands of 510bp and 490bp in electrophoresis. The results showed that using Xcfd50 primers to amplify the genomic DNA of the tested plants by PCR, according to whether the amplified products had DNA fragments of 510bp and 490bp, could assist in identifying whether the tested plants were powdery mildew resistant or powdery mildew susceptible plants. Each PCR amplification product was sequenced and verified, which was consistent with the electrophoresis result.

4. PCR identification of plant material (application of XMag633 primer pair)

7K65/Nongda 212-F2 generation plants (247 plants) and 7K65/Nongda 211-F2 generation plants (268 plants) were identified by PCR, and wheat material 87-1, wheat material Nongda 212 and wheat material Nongda 211 were used as negative controls , wheat material IW132 and disease-resistant parent 7K65 were used as positive controls, and the specific methods were as follows:

1. Cut the leaves of each seedling in Example 2, and extract genomic DNA.

2. Using the genomic DNA in step 1 as a template, perform PCR amplification with the XMag633 primer pair to obtain a PCR amplification product.

3. Perform 8% polyacrylamide gel electrophoresis on the PCR amplified product in step 2, then stain with silver staining, and finally count the experimental results. Part of the results are shown in Figure 2 (R represents a resistant individual plant, and S represents a susceptible individual plant). Each PCR amplification product was sequenced and verified, which was consistent with the electrophoresis result.

Among the 174 resistant individual plants, 171 showed a specific band of 570bp by electrophoresis. Of the 73 susceptible individuals, only 9 showed a specific band of 570bp in electrophoresis. The results showed that using XMag633 primers to amplify the genomic DNA of the tested plants by PCR, according to whether the amplified product had a 570bp DNA fragment, could assist in identifying whether the tested plants were powdery mildew resistant or powdery mildew susceptible plants.

Claims (9)

1. the application of primer pair first in the test kit of preparation assistant identification powdery mildew disease-resistant wheat; The primer pair that described primer pair first is comprised of single stranded DNA shown in the sequence 2 of single stranded DNA shown in the sequence 1 of sequence table and sequence table.

2. primer pair first and the primer pair B application in the test kit of preparation assistant identification powdery mildew disease-resistant wheat; The primer pair that described primer pair first is comprised of single stranded DNA shown in the sequence 2 of single stranded DNA shown in the sequence 1 of sequence table and sequence table; The primer pair that described primer pair B is comprised of single stranded DNA shown in the sequence 4 of single stranded DNA shown in the sequence 3 of sequence table and sequence table.

3. the test kit of an assistant identification powdery mildew disease-resistant wheat, comprise the primer pair first; The primer pair that described primer pair first is comprised of single stranded DNA shown in the sequence 2 of single stranded DNA shown in the sequence 1 of sequence table and sequence table.

4. test kit as claimed in claim 3, it is characterized in that: described test kit also comprises primer pair B; The primer pair that described primer pair B is comprised of single stranded DNA shown in the sequence 4 of single stranded DNA shown in the sequence 3 of sequence table and sequence table.

5. the application of primer pair first in assistant identification powdery mildew disease-resistant wheat; The primer pair that described primer pair first is comprised of single stranded DNA shown in the sequence 2 of single stranded DNA shown in the sequence 1 of sequence table and sequence table.

6. primer pair first and the primer pair B application in assistant identification powdery mildew disease-resistant wheat; The primer pair that described primer pair first is comprised of single stranded DNA shown in the sequence 2 of single stranded DNA shown in the sequence 1 of sequence table and sequence table; The primer pair that described primer pair B is comprised of single stranded DNA shown in the sequence 4 of single stranded DNA shown in the sequence 3 of sequence table and sequence table.

7. the method for an assistant identification powdery mildew disease-resistant wheat, comprise the step first;

Described step first comprises the steps: that genomic dna take wheat to be measured as template, carries out pcr amplification with the primer pair first; If have two specific fragments in pcr amplification product, wheat to be measured is candidate's powdery mildew disease-resistant wheat; If do not have described two specific fragments in pcr amplification product, wheat to be measured is the susceptible wheat of candidate's Powdery Mildew; One in described two specific fragments is 505-515bp, and another is 485-495bp; The primer pair that described primer pair first is comprised of single stranded DNA shown in the sequence 2 of single stranded DNA shown in the sequence 1 of sequence table and sequence table.

8. method as claimed in claim 7, it is characterized in that: described method also comprises step second;

Described step second comprises the steps: that genomic dna take wheat to be measured as template, carries out pcr amplification with primer pair B; If have a specific fragment in pcr amplification product, wheat to be measured is candidate's powdery mildew disease-resistant wheat; If do not have a described specific fragment in pcr amplification product, wheat to be measured is the susceptible wheat of candidate's Powdery Mildew; A described DNA fragmentation that specific fragment is 565-575bp; The primer pair that described primer pair B is comprised of single stranded DNA shown in the sequence 4 of single stranded DNA shown in the sequence 3 of sequence table and sequence table.

9. method as claimed in claim 8, it is characterized in that: if same described wheat to be measured is consistent with the qualification result in described step second in described step first, this wheat to be measured is the further candidate's who determines powdery mildew disease-resistant wheat or the further candidate's who determines the susceptible wheat of Powdery Mildew.

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