阅读说明：本技术 一种文冠果抗旱基因、snp及其应用 (Xanthoceras sorbifolia drought-resistant gene, SNP and application thereof ) 是由 刘肖娟 王利兵 毕泉鑫 于海燕 李迎超 于 2021-09-27 设计创作，主要内容包括：本发明公开了一种文冠果抗旱基因、SNP及其应用。属于分子标记技术领域。本发明利用全基因组的重测序和关联分析,找到与文冠果重要表型抗旱节水相关联的遗传位点SNP1以及候选基因XsGIR1。XsGIR1的核苷酸序列如SEQ.ID.NO.1所示,变异位点SNP1为XsGIR1的205bp C→G。文冠果XsGIR1SNP标记的应用可以实现抗旱节水文冠果种质资源的快速识别,极大加快传统育种进程,是有效的遗传研究手段。(The invention discloses a xanthoceras sorbifolia drought-resistant gene, SNP and application thereof. Belongs to the technical field of molecular marking. The invention utilizes the re-sequencing and association analysis of the whole genome to find the genetic locus SNP1 and the candidate gene XsGIR1 associated with the important phenotype drought resistance and water conservation of the shinyleaf yellowhorn. The nucleotide sequence of the XsGIR1 is shown in SEQ ID No.1, and the variation site SNP1 is 205bp C → G of the XsGIR 1. The application of the xanthoceras sorbifolia XsGIR1SNP marker can realize the rapid identification of drought-resistant water-saving xanthoceras sorbifolia germplasm resources, greatly accelerate the traditional breeding process, and is an effective genetic research means.)

1. A xanthoceras sorbifolia drought-resistant gene is named as XsGIR1, and the nucleotide sequence of the gene is as follows:

ATGAGTCGAAGAAGTAATGGTCCCAAGCTTGAACTGAAGCTGAACCTATCACCTCCTAGGAATAGAGACCAGCAAGTTCAATCGCCCAACGGATCGGTTTCGTCATGGGAAATTTCAACGGAGAGTTCATGCGTGTCATCGGAGCCGGAAGACACGACCATGCATTATACAAGCAGCCCAGACACGACAAATTCAATGTTGTTGNTGGGGTGTCCTCGATGCTTTATGTATGTCATGTTGTCTGATGTCAATCCAAAATGTCCCAAGTGCAAGAGCACCGTTTTGCTTGATTTTCTCAACGAAGACAACCCCAAGAAGACTAGCAACTGA；SEQ.ID.NO.1；

wherein N is C or G;

the amino acid sequence is as follows:

MSRRSNGPKLELKLNLSPPRNRDQQVQSPNGSVSSWEISTESSCVSSEPEDTTMHYTSSPDTTNSMLLXGCPRCFMYVMLSDVNPKCPKCKSTVLLDFLNEDNPKKTSN.；SEQ.ID.NO.2；

wherein X is L or V.

2. An SNP marker related to the drought resistance trait of xanthoceras sorbifolia is named as SNP1, the SNP1 is located at the 205 th position of an XsGIR1 gene, and the nucleotide at the position is C or G.

3. A group of primer pairs for detecting the drought resistance of shinyleaf yellowhorn is characterized in that the nucleotide sequences of the primer pairs are as follows:

5’-ATGAGTCGAAGAAGTAATGG-3’；SEQ.ID.NO.3；

3’-TCAGTTGCTAGTCTTCTTGG-5’；SEQ.ID.NO.4。

4. a kit for detecting the drought resistance of xanthoceras sorbifolia bunge, which is characterized by comprising the primer pair of claim 3.

5. Use of the SNP marker according to claim 2, the primer pair according to claim 3 or the kit according to claim 4 for breeding xanthoceras sorbifolia.

6. A method for detecting the drought resistance of shinyleaf yellowhorn is characterized in that the drought resistance of the shinyleaf yellowhorn is determined by detecting SNP1 on an XsGIR1 gene, when the nucleotide of the SNP1 is C, the shinyleaf yellowhorn is not drought-tolerant, and when the nucleotide of the SNP1 is G, the shinyleaf yellowhorn is drought-tolerant.

7. The method for detecting the drought resistance trait of xanthoceras sorbifolia as claimed in claim 6, comprising the following steps:

(1) extracting the genome DNA of the shiny-leaved yellowhorn to be detected;

(2) carrying out PCR amplification on the xanthoceras sorbifolia genomic DNA by using the primer of claim 3 to obtain a PCR amplification product;

(3) and detecting and analyzing the PCR amplification product.

8. The method for detecting the drought resistance trait of xanthoceras sorbifolia as claimed in claim 7, wherein the PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 15s, extension at 72 ℃ for 30s, and 35 cycles; extending for 5min at 72 ℃; storing at 4 ℃.

9. The method for detecting the drought resistance trait of xanthoceras sorbifolia as claimed in claim 7, wherein the PCR amplification system comprises: 10 × TransTaq HiFi buffer II 2 μ L; 1.5. mu.L of 2.5mM dNTPs; 10 μ M of each of the upstream and downstream primers was 1 μ L; TransTaq HiFi DNA Polymerase 0.2 μ L; 2 mu L of DNA; ddH₂O is added to the volume of 20 mu L.

Technical Field

The invention relates to the technical field of molecular markers, in particular to a xanthoceras sorbifolia drought-resistant gene, SNP and application thereof.

Background

Xanthoceras sorbifolia (Xanthoceras sorbifolia) is a plant of Xanthoceras of Sapindaceae, deciduous sub-tree or shrub, has strong adaptability and stress resistance, is rich in germplasm resources, and is an excellent local economic tree species for fixing sand, preserving soil, conserving water source and improving ecological environment. However, the xanthoceras sorbifolia is mainly distributed in areas with water resources shortage, the normal growth and development of the xanthoceras sorbifolia are severely inhibited by drought, and the drought becomes an important limiting factor for the development of the suitable growing area, the survival rate of forestation and the high yield and high yield of the xanthoceras sorbifolia. Therefore, the method has very important practical significance for vigorously mining woody oil resources and carrying out the breeding and improvement work of the drought resistance characters of the shinyleaf yellowhorn.

In recent years, with the rapid development of sequencing technologies, genome-wide re-sequencing and association analysis (GWAS) has been widely applied to research such as detection of mutation sites, mapping of high-quality traits, and population evolution. GWAS is different from traditional molecular breeding such as Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment Length Polymorphism (AFLP) and SSR, utilizes millions of Single Nucleotide Polymorphism (SNP) markers in a genome, is an effective means for positioning complex quantitative traits, has the advantages of high resolution and high flux, can simultaneously correlate multiple complex traits and detect multiple alleles. However, there are some disadvantages in the detailed studies, such as insufficient accuracy of candidate gene determination, false positive, etc. In order to obtain a more accurate research result, on the basis of completing the sequencing of the xanthoceras sorbifolia genome, the genetic locus and the candidate gene associated with the important phenotype of the xanthoceras sorbifolia are found by utilizing the re-sequencing and association analysis of the whole genome of a larger population, so that the traditional breeding process can be greatly accelerated, and the method is an effective genetic research means.

In conclusion, providing a genetic locus and a candidate gene associated with xanthoceras sorbifolia drought resistance is a problem to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides a xanthoceras sorbifolia drought-resistant gene, SNP and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a xanthoceras sorbifolia drought-resistant gene is named as XsGIR1, and the nucleotide sequence of the gene is as follows:

ATGAGTCGAAGAAGTAATGGTCCCAAGCTTGAACTGAAGCTGAACCTATCACCTCCTAGGAATAGAGACCAGCAAGTTCAATCGCCCAACGGATCGGTTTCGTCATGGGAAATTTCAACGGAGAGTTCATGCGTGTCATCGGAGCCGGAAGACACGACCATGCATTATACAAGCAGCCCAGACACGACAAATTCAATGTTGTTGNTGGGGTGTCCTCGATGCTTTATGTATGTCATGTTGTCTGATGTCAATCCAAAATGTCCCAAGTGCAAGAGCACCGTTTTGCTTGATTTTCTCAACGAAGACAACCCCAAGAAGACTAGCAACTGA；SEQ.ID.NO.1；

wherein N is C or G;

the amino acid sequence is as follows:

MSRRSNGPKLELKLNLSPPRNRDQQVQSPNGSVSSWEISTESSCVSSEPEDTTMHYTSSPDTTNSMLLXGCPRCFMYVMLSDVNPKCPKCKSTVLLDFLNEDNPKKTSN.；SEQ.ID.NO.2；

wherein X is L or V.

An SNP marker related to the drought resistance trait of xanthoceras sorbifolia is named as SNP1, the SNP1 is located at the 205 th position of an XsGIR1 gene, and the nucleotide at the position is C or G.

Discovery of xanthoceras sorbifolia drought-resistant gene XsGIR1 and SNP marker: the invention utilizes a GWAS method to perform resequencing on 320 shinyleaf yellowhorn germplasm resources to obtain 1 significant SNP locus in 1 gene which is related to drought resistance and water conservation of shinyleaf yellowhorn. According to the comparison result of NCBI, the gene is named as XsGIR1, the cDNA total length is 330bp, 110 amino acids are coded, the nucleotide sequence is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No. 2. The SNP1 is 205bp C → G of XsGIR1, and the amino acid sequence is Leu → Val.

A group of primer pairs for detecting the drought resistance of shinyleaf yellowhorn, wherein the nucleotide sequences of the primer pairs are as follows:

5’-ATGAGTCGAAGAAGTAATGG-3’；SEQ.ID.NO.3；

3’-TCAGTTGCTAGTCTTCTTGG-5’；SEQ.ID.NO.4。

a kit for detecting the drought resistance of xanthoceras sorbifolia bunge comprises the primer pair.

The SNP marker, the primer pair or the kit is applied to the breeding of the shiny-leaved yellowhorn.

A method for detecting the drought resistance of shinyleaf yellowhorn is characterized in that the drought resistance of the shinyleaf yellowhorn is determined by detecting SNP1 on an XsGIR1 gene, when the nucleotide of the SNP1 is C, the shinyleaf yellowhorn is not drought-resistant, and when the nucleotide of the SNP1 is G, the shinyleaf yellowhorn is drought-resistant.

The 205bp of the XsGIR1 is converted from C to G, and can be recognized and cut by BseYI when the 205bp is a C site. After the PCR amplification product is cut by BseYI enzyme, the target fragment with the 205bp position as the C site is cut into two fragments of 204bp and 126bp by enzyme, and the target fragment with the mutation position G site can not be cut by enzyme due to the lack of BseYI recognition site. The large fragments which are not cut by enzyme are related to the drought tolerance of the shinyleaf yellowhorn, and the small fragments which are cut by enzyme are related to the drought tolerance of the shinyleaf yellowhorn.

Further, the method comprises the following steps:

(1) extracting the genome DNA of the shiny-leaved yellowhorn to be detected;

(2) carrying out PCR amplification on the genome DNA of the shinyleaf yellowhorn by using the primer to obtain a PCR amplification product;

(3) and detecting and analyzing the PCR amplification product.

Extracting genome DNA of the xanthoceras sorbifolia bunge resource to be detected, carrying out PCR amplification by using the primer pair, carrying out BseYI enzyme digestion on an amplification product, and observing the band condition through agarose gel electrophoresis. And identifying the drought-resistant water-saving xanthoceras sorbifolia germplasm resources according to the number of the strips, and realizing selective breeding of the drought-resistant xanthoceras sorbifolia germplasm resources.

Further, the PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 15s, extension at 72 ℃ for 30s, and 35 cycles; extending for 5min at 72 ℃; storing at 4 ℃.

Further, the PCR amplification system is shown in table 1:

TABLE 1 PCR amplification System

According to the technical scheme, compared with the prior art, the invention has the following beneficial effects: the invention utilizes the re-sequencing and association analysis of the whole genome to find the genetic locus SNP1 associated with the important phenotype drought-resistant water-saving of the shinyleaf yellowhorn and the candidate gene XsGIR 1. The application of the xanthoceras sorbifolia XsGIR1SNP marker can realize the rapid identification of drought-resistant water-saving xanthoceras sorbifolia germplasm resources, can greatly accelerate the traditional breeding process, and is an effective genetic research means.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the results of PCR in example 1 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The medicament required by the embodiment of the invention is a conventional experimental medicament purchased from a commercially available channel; the experimental methods not mentioned in the examples are conventional experimental methods, and are not described in detail herein.

Example 1

(1) Xanthoceras sorbifolia drought resistance detection method

The Water Use Efficiency (WUE) is an important index for evaluating the drought resistance of plants, and the delta 13C (carbon isotope ratio) is one of the most reliable methods for detecting the WUE at present. Based on the mean value of the delta 13C of the plants investigated in the global range (-28.74), water-saving drought-tolerant plants and water-consuming drought-intolerant plants are distinguished. In 6-month middle ten days from 2016 to 2017, the location of a test site (a shinyleaf yellowhorn germplasm resource garden in Daizhou county, Fuxin City, Liaoning province) has no effective rainfall for more than 200 continuous days, the rainfall is reduced by 80 percent compared with the mean value in the same period, meanwhile, the test site has no artificial irrigation, and severe drought is achieved according to the water industry standard (SL 424-2008). The DELTA 13C values of the leaves of the 24 adult xanthoceras sorbifolia trees were measured by PDB-based DELTA V Advantage isotope ratio mass spectrometer (Pee De Belemmnite) at 6 months 2017. The results are shown in Table 2.

TABLE 224 Delta 13C values of adult xanthoceras sorbifolia leaves

The result shows that 11 parts of xanthoceras sorbifolia bunge leaves have the delta 13C value of-26.34 to-27.39 and are drought-enduring xanthoceras sorbifolia bunge; 16 parts of xanthoceras sorbifolia bunge leaves have delta 13C values of-29.12 to 30.58, and are non-drought-enduring xanthoceras sorbifolia bunge.

(2) Extraction of xanthoceras sorbifolia genome DNA

And extracting the genome DNA of the 27 adult tree leaves with different drought resistances by using a polysaccharide polyphenol genome DNA extraction kit.

(3) PCR amplification and digestion

Taking shinyleaf yellowhorn genome DNA as a template, and taking 5'-ATGAGTCGAAGAAGTAATGG-3', SEQ.ID.NO.3 as a template; 3 '-TCAGTTGCTAGTCTTCTTGG-5' and SEQ ID NO.4 as primers, and the XsGIR1 target fragment is amplified. The amplified product is cut by BseYI enzyme, the cut product is separated by electrophoresis with 12% agarose gel, and the composition of gene bands is counted by ultraviolet development photography. The results are shown in FIG. 1.

As a result, two bands exist in 16 parts of enzyme cutting products in 27 parts of adult xanthoceras sorbifolia trees, and only one specific band exists in 11 parts of enzyme cutting products, so that the allelic variation of the XsGIR1-G is found. And this result corresponds to the above δ 13C value determination result.

Comprehensively finds that the site of the drought-enduring shinyleaf yellowhorn XsGIR 1205 bp is C, and the site of the drought-intolerant shinyleaf yellowhorn XsGIR 1205 bp is mutated into G. The XsGIR1 CAPS molecular marker is closely related to the drought resistance of the shinyleaf yellowhorn, and the developed molecular marker can be used for screening and identifying the germplasm resources of the drought-resistant water-saving shinyleaf yellowhorn.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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atgagtcgaa gaagtaatgg tcccaagctt gaactgaagc tgaacctatc acctcctagg 60

aatagagacc agcaagttca atcgcccaac ggatcggttt cgtcatggga aatttcaacg 120

gagagttcat gcgtgtcatc ggagccggaa gacacgacca tgcattatac aagcagccca 180

gacacgacaa attcaatgtt gttgntgggg tgtcctcgat gctttatgta tgtcatgttg 240

tctgatgtca atccaaaatg tcccaagtgc aagagcaccg ttttgcttga ttttctcaac 300

gaagacaacc ccaagaagac tagcaactga 330

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Met Ser Arg Arg Ser Asn Gly Pro Lys Leu Glu Leu Lys Leu Asn Leu

1 5 10 15

Ser Pro Pro Arg Asn Arg Asp Gln Gln Val Gln Ser Pro Asn Gly Ser

20 25 30

Val Ser Ser Trp Glu Ile Ser Thr Glu Ser Ser Cys Val Ser Ser Glu

35 40 45

Pro Glu Asp Thr Thr Met His Tyr Thr Ser Ser Pro Asp Thr Thr Asn

50 55 60

Ser Met Leu Leu Xaa Gly Cys Pro Arg Cys Phe Met Tyr Val Met Leu

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Ser Asp Val Asn Pro Lys Cys Pro Lys Cys Lys Ser Thr Val Leu Leu

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Asp Phe Leu Asn Glu Asp Asn Pro Lys Lys Thr Ser Asn

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