阅读说明：本技术 稻瘟病抗性基因Pik-zh的功能特异性分子标记与应用 (Functional specific molecular marker of rice blast resistance gene Pik-zh and application ) 是由 杨德卫 何旎清 郑向华 黄凤凰 程朝平 叶宁 于 2021-09-07 设计创作，主要内容包括：本发明公开稻瘟病抗性基因Pik-zh的功能特异性分子标记与应用,稻瘟病抗性基因Pik-zh功能特异性分子标记为Pik-zh-InDel,通过引物对SEQ ID NO.1和SEQ ID NO.2从携带有稻瘟病抗性基因Pik-zh的水稻品种的基因组DNA中扩增出来与稻瘟病抗性基因Pik-zh呈特异性带型的分子标记；其中,SEQ ID NO.1为5’-AGCAGTGGGACGAGTGGGA-3’；SEQ ID NO.2为5’-GGTAGGACACCTGCCATCA-3’；本发明获得的稻瘟病抗病基因Pik-zh的功能特异性分子能够应用于鉴别水稻品种中稻瘟病抗性基因。(The invention discloses a functional specificity molecular marker of a rice blast resistance gene Pik-zh and application thereof, wherein the functional specificity molecular marker of the rice blast resistance gene Pik-zh is Pik-zh-InDel, and a molecular marker which is in a specific banding pattern with the rice blast resistance gene Pik-zh is amplified from a genome DNA of a rice variety carrying the rice blast resistance gene Pik-zh through a primer pair SEQ ID NO.1 and SEQ ID NO. 2; wherein SEQ ID NO.1 is 5'-AGCAGTGGGACGAGTGGGA-3'; 5'-GGTAGGACACCTGCCATCA-3' for SEQ ID NO. 2; the functional specificity molecule of the rice blast disease-resistant gene Pik-zh obtained by the invention can be applied to identifying the rice blast disease-resistant gene in rice varieties.)

1. A functional specific molecular marker of a rice blast resistance gene Pik-zh is characterized in that: the rice blast resistance gene Pik-zh functional specific molecular marker is Pik-zh-InDel, and a molecular marker which is in a specific banding pattern with the rice blast resistance gene Pik-zh is amplified from the genome DNA of a rice variety carrying the rice blast resistance gene Pik-zh through a primer pair SEQ ID NO.1 and SEQ ID NO. 2; wherein, the nucleotide sequences of the primer pair SEQ ID NO.1 and SEQ ID NO.2 are as follows:

SEQ ID NO.1：5’-ACGAGTGGGATCGATACTG-3’；

SEQ ID NO.2：5’-GGTAGGACACCTGCCATCA-3’；

the rice variety carrying the rice blast resistance gene Pik-zh is a rice disease-resistant variety Zhonghua 11.

2. The functionally specific molecular marker of the rice blast resistance gene Pik-zh according to claim 1, characterized in that: the rice blast resistance gene Pik-zh comprises a gene segment SEQ ID NO.3 of a part of coded NBS-LRR type protein.

3. The method for producing a function-specific molecular marker for the rice blast resistance gene Pik-zh according to any one of claims 1 to 2, comprising the steps of:

(1) sequence comparison is carried out aiming at the Pik-zh locus of the rice blast resistance gene, and the specific insertion/deletion InDel locus of the Pik-zh and other rice blast allelic resistance genes which can be distinguished from the locus is screened;

(2) designing a gene specific primer at the InDel site by utilizing the InDel site information obtained in the step (1);

(3) carrying out PCR amplification by taking the total DNA of a rice blast resistant variety carrying the rice blast resistant gene Pik-zh as a template, wherein the obtained PCR product is the specific molecular marker Pik-zh-InDel of the rice blast resistant gene Pik-zh.

4. The use of the rice blast resistance gene Pik-zh of any one of claims 1 to 2 as a functional specific molecular marker for identifying rice blast resistance genes of rice varieties.

5. A kit for identifying rice blast resistance of a rice variety, characterized in that: comprises a primer pair with nucleic acid sequences shown as SEQ ID NO.1 and SEQ ID NO. 2.

Technical Field

The invention belongs to the technical field of biomolecular markers, and particularly relates to a functional specificity molecule of a rice blast resistance gene Pik-zh and application thereof.

Background

Rice is one of the major food crops in the world, and rice blast caused by rice blast germs is a worldwide disease and has a tendency to increase in recent years. The rice blast not only causes great yield reduction (10% -35%) of rice, but also influences the quality of rice when the rice is not harvested even if the rice is severe. From the viewpoint of grain safety and rice disease-resistant breeding, breeding of disease-resistant varieties is one of the most economical and effective methods for preventing and treating rice blast. But the traditional disease-resistant breeding strategy has the characteristics of large workload, long period and the like. Therefore, there is an urgent need to change the traditional strategy of breeding for disease resistance.

The DNA molecular marker is a genetic marker developed on the basis of polymorphism of biomacromolecules, and is currently applied to aspects such as genome mapping, gene localization, genetic diversity evaluation, functional genomics research, transgenic plant identification, molecular marker-assisted selective breeding and the like. The gene function specific molecular marker is a molecular marker developed by utilizing the specificity and the difference of DNA sequences in a target gene. The gene characteristic molecular marker is a functional molecular marker developed from functional SNP loci or InDels in a target functional gene sequence, can accurately track and locate functional alleles, has higher accuracy in the identification of crop phenotypic traits, and can efficiently screen beneficial genes required by breeding.

In the 60 s of the 20 th century, the research work of rice blast resistance gene analysis of rice varieties is first developed in Japan, 14 genes on the first 8 resistance sites are identified, and a set of identification system for rice blast resistance gene analysis is established. However, some disease-resistant genes are located at the same site, and the multiple genes and the functional sequence and the non-functional sequence are highly homologous, so that the traditional method is difficult to distinguish. Therefore, by analyzing the specific sequence difference between the target gene and the allele and developing the function specific molecular marker to select the target gene, the selection reliability is high, and the breeding pace is greatly accelerated.

As the Pik locus located at the long arm end of the 11 th chromosome contains more than 5 alleles Pik-m, Pik-p, Pik-h, Pik-s and the like with different resistance profiles and race specificities, the locus has been the hot door for the research on rice blast resistance, wherein Pik-m, Pik-p, Pik-h and Pik-s have been successfully identified and cloned, and related function specific molecular markers of the alleles have been gradually developed and applied.

The rice blast resistance gene Pik-ZH is a new main disease-resistant gene which is identified from a rice variety ZH11 (Zhonghua 11) by the applicant and has the same allele with Pik, the total length of CDs is 3438bp, the detailed nucleic acid sequence is shown in SEQ ID NO.4 by using a map-based cloning method, and further analysis on the rice blast resistance gene Pik-ZH discovers that the Pik-ZH sequence has specific difference with partial sequences of Pik, Pik-s, Pik-h and Pik-p which are cloned at the site at present, and the Pik-ZH gene can show stronger disease-resistant reaction on strains D527 and KJ201 in China. Therefore, in order to more accurately and effectively apply the broad-spectrum resistance gene Pik-zh of rice blast to rice resistance breeding work, the development of a Pik-zh specific molecular marker which truly reflects a target gene and is convenient and easy to use is urgently needed.

Disclosure of Invention

The invention provides a functional specific molecular marker and application of a rice blast resistance gene Pik-zh, creatively discovers the Pik-zh specific molecular marker which can truly reflect the broad-spectrum resistance gene Pik-zh of rice blast, and simultaneously, the Pik-zh specific molecular marker which is convenient and easy to use and has higher accuracy and stability is prepared, so that the broad-spectrum resistance gene Pik-zh of rice blast is better applied to rice resistance breeding.

The purpose of the invention is realized by the following technical scheme:

one of the purposes of the invention is to provide a functional specific molecular marker of a rice blast resistance gene Pik-zh, the functional specific molecular marker of the rice blast resistance gene Pik-zh is Pik-zh-InDel, and a molecular marker which is in a specific banding pattern with the rice blast resistance gene Pik-zh is amplified from the genome DNA of a rice variety carrying the rice blast resistance gene Pik-zh through a primer pair SEQ ID NO.1 and SEQ ID NO. 2; wherein, the nucleotide sequences of the primer pair SEQ ID NO.1 and SEQ ID NO.2 are as follows:

SEQ ID NO.1：5’-ACGAGTGGGATCGATACTG-3’；

SEQ ID NO.2：5’-GGTAGGACACCTGCCATCA-3’；

the rice variety carrying the rice blast resistance gene Pik-zh is a rice disease-resistant variety Zhonghua 11.

Further, the rice blast resistance gene Pik-zh comprises a gene segment SEQ ID NO.3 partially coding NBS-LRR type protein.

One of the purposes of the invention is also to provide a preparation method of the functional specific molecular marker of the rice blast resistance gene Pik-zh, which comprises the following steps:

(1) sequence comparison is carried out aiming at the Pik-zh locus of the rice blast resistance gene, and the specific insertion/deletion InDel locus of the Pik-zh and other rice blast allelic resistance genes which can be distinguished from the locus is screened;

(2) designing a gene specific primer at the InDel site by utilizing the InDel site information obtained in the step (1);

(3) carrying out PCR amplification by taking the total DNA of a rice blast resistant variety carrying the rice blast resistant gene Pik-zh as a template, wherein the obtained PCR product is the specific molecular marker Pik-zh-InDel of the rice blast resistant gene Pik-zh.

One of the purposes of the invention is to apply the functional specific molecular marker of the rice blast resistance gene Pik-zh to the identification of rice blast resistance genes of rice varieties.

The invention also provides a kit for identifying rice blast resistance of rice varieties, which comprises a primer pair with nucleic acid sequences shown as SEQ ID NO.1 and SEQ ID NO. 2.

The invention has the beneficial effects that:

1. the invention is a functional specific molecular marker developed aiming at a new major disease-resistant gene Pik-zh which is allelic with Pik, and the functional specific molecular marker of Pik-zh has the advantages of low cost, high efficiency, high accuracy and the like in practical application; the Pik-zh functional specific molecular marker is Pik-zh-InDel, and Pik-zh can be successfully distinguished from other non-Pik-zh genes on the site by using an electrophoresis detection method, so that the detection efficiency is high; meanwhile, the Pik-zh specific molecular marker Pik-zh-InDel provided by the invention is a dominant marker, exists in the Pik-zh gene, and has a theoretical value of 100 percent of screening capacity on Pik-zh

2. The Pik-zh functional specificity molecular marker provided by the invention is Pik-zh-InDel, only PCR is combined with agarose gel electrophoresis or non-denatured polyacrylamide gel electrophoresis, the cost is low, the accuracy rate is high, the flux is high, in addition, the specificity is high, and the Pik-zh functional specificity molecular marker is particularly suitable for agricultural production practice and germplasm resource screening, transgenic breeding, gene polymerization and MAS technology-based resistance breeding under any genetic background, and greatly improves the breeding efficiency and the accuracy.

Drawings

FIG. 1 shows the amplification of 6 selected representative varieties of total DNA as templates using primers F1 and R1 shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings, wherein the embodiments are only for explaining the present invention and do not limit the present invention in any way.

Example 1

The preparation method of the functional specific molecular marker of the rice blast resistance gene Pik-zh specifically comprises the following steps:

(1) analysis of Pik-zh insertion/deletion (InDel) sites:

downloading genome sequences of different alleles of Pik loci and corresponding loci of different rice varieties from a public database: pik-m (GenBank: GU811860.1), Pi1 (GenBank: HQ606329.1), Pik (GenBank: AB616658.1), Pik-h (GenBank: HQ662330.1), Pik-s (GenBank: HQ662329.1), Pik-p (GenBank: HM035360.1), Shuhui 498 (GenBank: CP018167.1), Nippon (Locus: LOC _ Os11g42010), then carrying out sequence alignment on the Pik-zh Locus of the rice blast resistance gene in a rice database, carrying out design of specific markers according to the functional region difference Locus, sequencing the genome sequence of the rice donor varieties Nippon, Shuhui 498 and Pik allele which have no complete corresponding region, wherein the missing and different base parts in the alignment result are identified InDel loci, and the alignment result is as follows: pik-zh:

Shuhui498：

Pik-m：

Pi1：

Pik：

Pik-h：

Pik-s：

Pik-p：

deletion of the corresponding sequence in Nipponbare

Wherein, the boxed part is the single base part of the Pik-zh gene with difference detected after the comparison, and the blank is the single base part of the missing Pik-zh gene detected after the comparison, further analysis shows that a section of sequence in the Pik-zh gene is extremely specific, and has 9bp difference compared with the corresponding sequences of Pik-m, Pi1, Pik-h, Pik-s, Pik-p and Shuhui 498, while compared with the corresponding sequence of Nipponbare, the partial sequence of Nipponbare is completely missing.

(2) Designing a gene specific primer at the InDel site by using the InDel site information obtained in the step (1):

according to the design principle of InDel markers, primer design is carried out at the InDel differential site, and the base sequences of primer pairs are shown as follows: SEQ ID NO.1 (F1): 5'-ACGAGTGGGATCGATACTG-3', respectively;

SEQ ID NO.2(R1)：5’-GGTAGGACACCTGCCATCA-3’；

(3) carrying out PCR amplification by taking the total DNA of a rice blast resistant variety carrying a rice blast resistant gene Pik-zh as a template:

in this example, 6 representative rice varieties were selected, in order: 11 of Zhonghua, 498 of Shuhui, CO39, Nipponbare, Lijiang Xinjiang black valley and Tetepu;

performing conventional PCR amplification by using the primers SEQ ID NO.1 and SEQ ID NO.2 and the total DNA of the 6 rice varieties as templates to obtain a PCR product, namely a rice blast resistance gene Pik-zh gene specific molecular marker Pik-zh-InDel;

wherein, the PCR amplification reaction system comprises the following steps:

2x Reaction Mix:16μL；

primer SEQ ID NO.1(F1) (10 μm): 1 mu L of the solution;

primer SEQ ID NO.1(F1) (10 μm): 1 mu L of the solution;

2 μ L of DNA template (20-50 ng/. mu.L);

ddH₂o: make up to 25 μ L;

the PCR temperature cycling conditions were as follows: 5 minutes at 94 ℃; 30 seconds at 94 ℃; 30 seconds at 55 ℃; 30 seconds at 72 ℃; 35 cycles; 7 minutes at 72 ℃; storing at 10 deg.C;

after the PCR reaction is finished, 10-20ul of PCR sample products are subjected to electrophoresis detection on 3% agarose for 40 minutes under the electrophoresis condition of 110V, and after the electrophoresis is finished, a gel imaging system is used for photographing to observe the electrophoresis result, wherein fragments containing the expected target size are positive, and fragments containing or not containing the target size are negative.

Example 2

The application of the functional specific molecular marker of the rice blast disease-resistant gene Pik-zh in identifying the rice blast resistant genes of rice varieties is that firstly, a primer F1 shown in SEQ ID NO.1 and a primer R1 shown in SEQ ID NO.2 are utilized to perform PCR amplification on genomes of 6 rice varieties to be detected; secondly, detecting by using agarose electrophoresis, wherein if a nucleotide fragment with the molecular weight of 108bp in a PCR product is detected, the rice variety to be detected carries the rice blast resistance gene Pik-zh; if the nucleotide fragment with the molecular weight of 108bp is not detected, the rice variety to be detected does not carry the rice blast resistance gene Pik-zh.

Referring to FIG. 1, the amplification of the total DNA of 6 selected representative varieties using the primer F1 shown in SEQ ID NO.1 and the primer R1 shown in SEQ ID NO.2 provided in example 1 as templates; referring to FIG. 1, lane 1 is DNA ladder, lane 2 is the fragment obtained by PCR using the genome of flower 11 in the rice variety of Pik-zh donor variety as a template, and is the specific banding pattern of the rice blast resistance gene Pik-zh, i.e., lane 2 is the specific molecular marker Pik-zh-InDel of Pik-zh gene, and the fragment size is 108 bp; the DNA templates in lanes 3-7 are Shuhui 498, CO39, Nipponbare, Lijiang New Yougu, and Tetepl in that order; as shown in attached figure 1, in the rice variety carrying the Pik-zh gene, an electrophoresis detection strip of a PCR amplification product is shown by 108bp, the Zhonghua 11 and the Tepu both contain the Pik-zh gene but do not contain a functional gene of the site, the 108bp strip cannot be detected by electrophoresis, and the Shuhui 498, the CO39, the Nipponbare and the Lijiang Xinjiang black valley do not contain the Pik-zh gene.

As shown in the attached figure 1, the test result is identical with the design analysis, and the functional specificity molecular marker of the resistance gene Pik-zh can identify the rice blast resistance gene Pik-zh, so that the method is suitable for agricultural production practice and germplasm resource screening, transgenic breeding, gene polymerization and MAS technology-based resistance breeding of Pik-zh under any genetic background, and greatly improves the breeding efficiency and accuracy.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

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gcgagcactg gaggagtgga ctcggttgcg ctcgcaggtg atctaagaga caagatagag 840

gtggtcggtt atggcattga ccccatcaag ctgatctccg cgctccggaa gaaggtgggc 900

catgcggagt tgctgcaggt cagccaagca aaaaaagatg tgaaggagac gacgccgatg 960

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taatcatgac tactcgtctt aattcaatag ctgagaagtg ccacactgat gacaatgatg 4200

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ttggagagat agaagaatta ggtggtgctg aagtgaaaaa atgtagggat ttgagacaca 4440

tagaggatgg tattttggac atcccatcct tacaaccatt ggcggagagt ttatgccttg 4500

gttataacca tcttcctctt tatctgagga ctttgttgtt gtactgtagt gcataccatt 4560

ggtctaacag aatcgaaagg ggtcgtctgg tcaggaggtg gattgcggaa ggatttgtgt 4620

cggaagagaa agaagcagaa ggttactttg gcgagcttat tgacagagga tggattacgc 4680

agcacggaga caacaacagt tataattact atgagatcca ccccgtgatg ctggccttcc 4740

tgagatgcaa gtccaaggag tacaattttt taacatgctt gggtctggga tctgatacta 4800

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tggactgctt gtcaagcatg agtatggatg tgtcacacac ttgcagcctt gtcgtccttg 4920

gtgacgtggc gcgatccaag ggaatcccct tctatatgtt taagcgcttg cgagtgttgg 4980

accttgaaga taataaggat atacaggatt ctcatctgca gggcatatgt gaacagttaa 5040

gcctcagagt gaggtacctt ggtctcaagg gaacgcggat ccgaaagctc cctcaggaga 5100

tgaggaagct gaagcatttg gagattttgt atgtggggag cactcggatc agtgaacttc 5160

cgcaagagat tggagagctg aagcatctgc ggattctgga cgtgagaaac acggacatca 5220

ctgagctccc actgcagata cgggagctgc agcatctgca cactctggac gtgaggaaca 5280

cgccaatcag tgagctcccg ccgcaggttg gcaagctgca gaatctcaag attatgtgcg 5340

tgaggagcac tggggttagg gagctcccaa aggagattgg ggagctgaat catctacaga 5400

ctctggacgt gagaaacacg agggtgagag agctgccatg gcaagctggc cagatctccc 5460

aatcgttgcg cgtgcttgcc ggtgacagtg gcgatggcgt gcggttgccc gaaggcgtct 5520

gcgaagctct gatcaacggt attccagggg ctacgcgtgc aaaatgcagg gaggttctgt 5580

ccatcgcgat catcgatcgt ttcggacctc cccttgttgg gatattcaaa gttcccggca 5640

gtcatatgcg tatcccgaag atgatcaaag accacttccg cgttctttct tgcctagaca 5700

tcaggctctg ccacaagctt gaggatgatg accaaaagtt cctcgccgag atgcccaacc 5760

tgcagacgct cgtgctgagg ttcgaggccc taccaagaca acccataacc atcaacggca 5820

caggcttcca gatgctggag agcttccgtg tcgacagccg ggtgccaagg atagccttcc 5880

atgaagacgc catgcccaac ctcaagcttc tcgagttcaa gttctacgcc ggcccagcaa 5940

gcaacgatcc catcggcatc accaacctga agagcctcca aaaggtggtc tttcggtgct 6000

cgccatggta caagagcgac gcccctggca tcagcgccac cattgacgtc gtgaagaaag 6060

aagccgagga gcatcccaac cggccgatca ccctcctcat caatgctggg tataaggaga 6120

tatcaattga gtcacatggg agcagtgaaa acattgcggg cagcagtggg acgagtggga 6180

tcgatactga gcctgcacag gcacagcatg ataatctccc tgctgttcga gatgactaca 6240

agggaaaagg gattcttctt gatggcaggt gtcctacctg cggccgagcg actaaaattg 6300

aagaggaaac ccaagatcga gtagcagata ttgaaattca aacagaaact actagctag 6359