WO2020118883A1 - 一种梅花垂枝性状紧密连锁的snp分子标记及其检测方法与应用 - Google Patents

一种梅花垂枝性状紧密连锁的snp分子标记及其检测方法与应用 Download PDF

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WO2020118883A1
WO2020118883A1 PCT/CN2019/073921 CN2019073921W WO2020118883A1 WO 2020118883 A1 WO2020118883 A1 WO 2020118883A1 CN 2019073921 W CN2019073921 W CN 2019073921W WO 2020118883 A1 WO2020118883 A1 WO 2020118883A1
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primer
plum
molecular marker
traits
snp
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French (fr)
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张启翔
卓孝康
郑唐春
程堂仁
王佳
孙丽丹
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北京林业大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

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  • the invention relates to the technical field of molecular biology and plant molecular breeding, in particular to a closely linked SNP molecular marker of plum blossom weeping traits and a detection method and application thereof.
  • Plum Panus mume Sieb.et Zucc. belongs to Rosaceae (Prunus), is one of the ten traditional Chinese famous flowers, has ornamental and use value, originated in southeastern China, has been 3000 years of introduction and cultivation history (Chen Junyu. Chinese Plum Variety Map [M]. China Forestry Press, 2010).
  • the variety of plum flowers is diverse, with rich colors, patterns, and plant types. Among them, weeping plum is one of the nine major varieties of plum flowers with unique plant types. It is deeply popular because of its unique tree posture and color, floral fragrance and other ornamental traits. People love it and have an important position in garden landscaping.
  • the invention utilizes GWAS and group selection methods to develop trait-marker-associated SNP markers, obtains SNP markers closely related to plum weeping traits, enables early screening of target traits, greatly shortens breeding cycle, and improves breeding efficiency , Reduced the breeding cost, and realized the molecular marker assisted selection breeding of plum blossom weeping traits.
  • the object of the present invention is to provide a SNP molecular marker with closely linked plum blossom branch traits and its detection method and application.
  • the whole genome sequencing of 214 plum varieties is carried out, and the GWAS analysis method is used to detect the sites that are significantly associated with the traits of plum branches; through group selection analysis, it is observed whether there is a selection signal in the region of the significant associated sites; combining GWAS association analysis and population Select analysis to obtain 47 SNP markers that are significantly associated with plum weeping traits, and locate the weeping traits in the two intervals of 11.1-11.8Mb and 14.2-15.2Mb of the chromosome 7 of plum blossoms.
  • the F1 segregated population of'branches' hybridization was used as material, and the 47 SNP markers that were significantly associated were verified by mass spectrometry, and it was found that the SNP marker Pm7_11182911 was most closely associated with the plum blossom weeping traits.
  • the present invention provides a SNP molecular marker closely linked to plum weeping traits, which is located at 11182911 bp of chromosome 7 of plum blossom (Prunus mume Genome v1.0 Genbank accession number: PRJNA171605; publication date: February 28, 2013 ), the polymorphism of the SNP molecular marker is A/C.
  • the present invention has developed a method for genotype identification of plum blossom weeping/straight traits.
  • the plum blossom's weeping/straight traits are identified:
  • the genotype of the SNP locus corresponding to the plum branch trait is AC, and the genotype corresponding to the straight branch is CC.
  • the present invention provides a detection primer of SNP molecular marker, the detection primer includes:
  • the detection primer when the genotype of the SNP is analyzed by analyzing the sequence of the single-base extension reaction product, the detection primer further includes a single-base extension primer:
  • the present invention provides a kit for the detection of plum blossom weeping traits, the kit includes the combination of the aforementioned forward primer F and the reverse primer R or includes the aforementioned forward primer F and reverse primer R And single-base extension primer combinations.
  • the kit also includes dNTPs, DNA polymerase, Mg 2+ , PCR reaction buffer.
  • the kit of the present invention further includes a standard positive template and SAP enzyme.
  • the present invention provides the application of the SNP molecular marker or the detection primer or the kit in the identification of plum blossom branch or straight branch traits.
  • the present invention provides a method for identifying plum plant type traits, which uses the genome of the plum to be tested as a template, uses forward primer F and reverse primer R for PCR amplification, and analyzes the sequence of the PCR amplification product. Determine the genotype of plum to be tested;
  • the sequence of the forward primer F is 5'-ACGTTGGATGTGCTTGTCAAACACAGTCCG-3'; the sequence of the reverse primer R is 5'-ACGTTGGATGGGTGTGTTTCTTTCTAACGAG-3'.
  • the PCR amplification product obtained by the above PCR amplification can be subjected to sequence analysis by using a conventional SNP typing method in the art, such as direct sequencing method, mass spectrometry analysis detection method.
  • the method for analyzing the sequence of the amplified product is Sequenom
  • the technology for SNP genotyping includes the following steps:
  • step (2) Using the alkaline phosphatase-treated product obtained in step (1) as a template to perform a single-base extension reaction, the sequence of the primer for the single-base extension reaction is: 5'-ACTAACCTCATTTCATAAGTTGA-3';
  • the genotype of the product of analyzing the extension single base extension reaction is to use Sequenom Technical and mass spectrometry analysis.
  • the method for identifying plum plant type traits includes the following steps:
  • a single base extension reaction is performed using a primer of single base extension reaction: 5'-ACTAACCTCATTTCATAAGTTGA-3';
  • the reaction system (5 ⁇ L) of the PCR amplification reaction is as follows:
  • 10 ⁇ PCR Buffer (containing Mg 2+ ) 0.625 ⁇ L, 25 mM MgCl 2 0.325 ⁇ L, dNTP (2.5 mM each) 1.0 ⁇ L, forward primer F 0.5 ⁇ L, reverse primer R 0.5 ⁇ L, 5U/ ⁇ L Taq DNA polymerase 0.1 ⁇ L genomic DNA template (50ng/ ⁇ L) 1.0 ⁇ L, add water to make up 5 ⁇ L.
  • reaction procedure is as follows: 94°C 15min; 94°C 10-20sec, 56-65°C 30sec, 72°C 1min, 45 cycles; 72°C 3-5min.
  • reaction system (7 ⁇ L) treated with alkaline phosphatase is as follows: 10 ⁇ SAP Buffer 0.17 ⁇ L, 1 U/ ⁇ L SAP enzyme 0.3 ⁇ L, PCR product of step (2) 5 ⁇ L, and water to make up 7 ⁇ L.
  • the reaction procedure is as follows: 37°C, 40 min; 85°C, 5 min.
  • the reaction system (9 ⁇ L) of the single base extension reaction is as follows: 10 ⁇ iPlex Buffer 0.2 ⁇ L, iPlex Termination Mix 0.2 ⁇ L, single base extension primer 0.804 ⁇ L, iPlex enzymes 0.041 ⁇ L, step (3)
  • the obtained alkaline phosphatase treated product was 7 ⁇ L, and 9 ⁇ L was made up with water.
  • the present invention combines the GWAS analysis and population selection method to develop the closely linked SNP molecular marker Pm7_11182911 of plum blossom weeping traits.
  • the SNP molecular marker has good repeatability and accuracy in the identification of the weeping/straight branch traits of the isolated population of plum hybrid progeny
  • the advantage of high performance has achieved the detection effect of single molecule label identification accuracy rate of more than 96%.
  • the present invention provides an efficient detection method for the SNP molecular marker Pm7_11182911, which has the advantages of high throughput, low detection cost, and no influence by environmental conditions.
  • the Pm7_11182911 molecular marker and its detection method provided by the present invention can realize the selection of weeping/straight traits at the seedling stage, greatly reducing the breeding workload, significantly shortening the breeding cycle of new weeping plum varieties, and improving the breeding efficiency In order to reduce the breeding cost, it can be used in plum molecular assisted selection breeding in practice.
  • Example 1 is a schematic diagram of a method for measuring phenotypic traits in Example 2 of the present invention, where (a) is the branching angle in the growth phase; (b) is the branching angle in the mature period; (c) is the different parts of the mature branch and the back gravity Angle of direction.
  • Example 2 is a Manhattan diagram of GWAS analysis in Example 2 of the present invention, wherein (a)-(g) are the branch angle A1 in the growth phase, the branch angle A2 in the mature stage, the angle T1 between the different parts of the branches and the direction of back gravity GWAS analysis results of the angle T2 between different parts of the branches and the direction of back gravity, T3 the angle between different parts of the branches and the direction of back gravity, T4 the angle between different parts of the branches and the direction of back gravity, and the angle T5 of different parts of the branches and the direction of back gravity.
  • FIG. 3 is a population selection analysis diagram of Example 3 of the present invention, wherein (a) is the population genetic differentiation coefficient (F ST ); (b) is the nucleic acid diversity of weeping plum (Pi); (c) is the nucleic acid diversity of weeping plum Sex (Pi); (d) is an enlarged view of population genetic differentiation coefficient and nucleic acid diversity analysis results.
  • F ST population genetic differentiation coefficient
  • Pi the nucleic acid diversity of weeping plum
  • Pi the nucleic acid diversity of weeping plum Sex
  • d is an enlarged view of population genetic differentiation coefficient and nucleic acid diversity analysis results.
  • FIG. 4 is the embodiment 3 of the present invention.
  • the marker-trait association sites for joint selection with GWAS are screened, wherein (a) is the joint screening association site; (b) is the comparison of candidate association site allele frequency and nucleic acid diversity Sex.
  • Example 5 is a graph showing the results of mass spectrometry in Example 4 of the present invention, where (a) is the AC genotype peak map of the SNP marker Pm7_11182911; (b) is the CC genotype peak map of the SNP marker Pm7_11182911.
  • FIG. 6 is an analysis diagram of the genotyping results of the SNP marker Pm7_11182911 in Example 4 of the present invention, where (a) is the frequency analysis of the genotypes of Strachy plum and weeping plum; (b) is the scatter diagram of the genotype.
  • test materials were 214 plum cultivars, collected in Wuhan Moshan Plum Germplasm Resource Garden (30.546719°N, 114.413254°E), which included conventional cultivars from 11 regions including Hubei, Yunnan, Jiangsu and Anhui ( (Such as Gongfen, cinnabar, single petal, jade dish, apricot plum, Huang Xiang, weeping branch, jumping branch and Longyou variety group).
  • the genome extraction materials of 214 plum varieties are from fresh or silica gel dried leaves.
  • CTAB Cyl trimethyl ammonium bromide
  • genomic DNA is extracted to prepare a 1.0% agarose gel.
  • Qualified DNA samples are used for library construction and sequencing.
  • the qualified DNA samples are randomly interrupted, and the DNA fragments are subjected to the steps of end repair, adding PolyA tail, adding sequencing adapter, purification, PCR amplification and library detection steps.
  • the short insert of the library is 500bp, and the long insert is 2Kb. After all the libraries passed the test, the Illumina HiSeq200 sequencing platform was used for sequencing.
  • Example 2 Mining SNP markers associated with plum weeping traits based on GWAS analysis
  • the phenotypic traits of 214 plum cultivars were collected.
  • the method of collecting phenotypic traits is shown in Figure 1.
  • the branch angles of mature branches and growing branches were measured. At the same time, the mature branches were divided into 5 parts. The angle between each part and the direction of back gravity was measured. A total of 7 phenotypic traits were obtained for GWAS analysis.
  • the ADMIXTURE1.3 software was used to calculate the group structure matrix file (Q) of 214 plum varieties, and the SPMGeDiv.1.4b software was used to calculate the kinship matrix (K).
  • GWAS analysis uses TASSEL v.5 software to select a mixed linear model (MLM), which includes fixed effects, random effects, and genetic relationships, as follows:
  • y is the phenotype
  • ⁇ is the label and Q
  • ⁇ is the genetic effect
  • X and Z are the known matrix
  • e is the residual.
  • the F1 segregated population obtained from'six petals' (mother parent) ⁇ 'Fantai weeping branch' (paternal parent) was used as the material, including 127 weeping individuals and 161 straight branches.
  • the test materials were colonized in He Village, Moganshan Town, Huzhou City, Zhejiang City (30.566389°N, 119.879582°E).
  • extract genomic DNA According to the instructions of the high-efficiency plant genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.), extract genomic DNA, prepare a 1.0% agarose gel, draw 3 ⁇ L of DNA and mix about 1 ⁇ L of loading-buffer, voltage 150V, electrophoresis for 15min, detect DNA Completeness. Use NANO DROP 2000 to determine the DNA concentration and purity to ensure that the DNA concentration is >50ng/ ⁇ L. Qualified DNA samples are used for mass spectrometry experiments.
  • PCR amplification primer 1 ACGTTGGATGTGCTTGTCAAACACAGTCCG;
  • PCR amplification primer 2 ACGTTGGATGGGTGTGTTTCTTTCTAACGAG;
  • the PCR reaction procedure is as follows:
  • the SAP reaction solution is as follows:
  • the total volume of the reaction system is 7 ⁇ L, of which 5 ⁇ L of PCR product and 2 ⁇ L of SAP mixed solution.
  • the reaction procedure is: 37°C, 40 min; 85°C, 5 min; 4°C, forever. After setting the program, start the PCR instrument for alkaline phosphatase treatment.
  • reaction liquid system is as follows:
  • the single-base extension reaction system is 7 ⁇ L of the PCR product after SAP treatment and 2 ⁇ L of the extension reaction solution, and the total volume of the reaction system is 9 ⁇ L.
  • the reaction program is as follows:
  • step (7) The product detection steps obtained in step (7) are as follows:
  • Nanodispenser SpectroCHIP chip spotting transfer the test sample from the 384-well reaction plate to the MassARRAY SpectroCHIP chip with the surface covered matrix;
  • MassARRAY Analyzer ComPmc mass spectrometry detection after transferring the sample to the SpectroCHIP chip, it can be put into the mass spectrometer for detection. Each sample on the chip is tested for 3-5sec, and the detection process is fully automated;
  • the mass genotyping results were used to calculate the genotype frequency of the F 1 segregated population obtained by cross-breeding the'six petals' (mother) ⁇ 'Pantai weeping branch' (paternal parent).
  • the genotype frequency of the individual shoots is the largest, which can accurately separate the individual shoots from the plant individuals.
  • the 161 straight-branch individuals and 127 weep-branch individuals in the F 1 population were typed, with accuracy rates of 97.5% (157/161) and 96.7% (122/127), respectively. It is expressed as AC genotype (Figure 6).
  • the invention establishes a molecular marker assisted selection breeding system for weeping plum blossoms, which can realize the early selection of weeping traits using a single marker, greatly improving breeding efficiency, reducing breeding workload, field planting area and labor, and greatly reducing breeding costs And the cycle has a pioneering value for molecular assisted selection breeding of weeping plum.
  • the invention provides a SNP molecular marker with close linkage of plum blossom weeping traits, which is located at 11182911 bp of chromosome 7 of plum blossom and the polymorphism is A/C.
  • the invention combines the GWAS analysis and the group selection method to develop the SNP molecular markers closely linked to the plum weeping traits. The accuracy of single molecular markers to identify the weeping/straight branch traits is above 96%.
  • the SNP molecular markers and detection methods of the closely linked plum weeping traits provided by the present invention can realize the selection of weeping/straight traits at the seedling stage, greatly shorten the breeding cycle, improve the breeding efficiency, reduce the breeding cost, and can be practiced It is used in plum blossom molecular assisted selection breeding, which has good economic value and application prospects.

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Abstract

本发明提供一种梅花垂枝性状紧密连锁的SNP分子标记，其位于梅花第7号染色体第11182911bp处，多态性为A/C。本发明还提供所述SNP分子标记的检测方法和其在梅花垂枝/直枝性状鉴定中的应用。

Description

一种梅花垂枝性状紧密连锁的SNP分子标记及其检测方法与应用
交叉引用
本申请要求2018年12月10日提交的专利名称为“一种梅花垂枝性状紧密连锁的SNP分子标记及其检测方法与应用”的第201811504968.2号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及分子生物学及植物分子育种技术领域，具体涉及一种梅花垂枝性状紧密连锁的SNP分子标记及其检测方法与应用。
背景技术
梅花(Prunus mume Sieb.et Zucc.)隶属于蔷薇科(Rosaceae)李属(Prunus)，是中国的十大传统名花之一，具有观赏和使用价值，起源于中国西南部，距今已有3000多年的引种栽培历史(陈俊愉.中国梅花品种图志[M].中国林业出版社,2010)。梅花的种类多样，花色、花型、株型丰富，其中垂枝梅是具有独特株型的梅花的九大品种之一，因其别致的树姿兼具花色、花香等其它观赏性状而深受人们喜爱，在园林造景中具有重要的地位。
利用高通量测序技术大量开发SNP标记，开发性状连锁的分子标记进行植株的初期筛选，达到分子标记辅助育种的目的，可大大缩短梅花的育种周期，提高育种效率。
本发明利用GWAS和群体选择方法开发性状-标记关联的SNP标记，获得了与梅花垂枝性状紧密关联的SNP标记，能够对目标性状进行早期筛选，极大地缩短了育种的周期，提高了育种效率，降低了育种成本，实现了梅花垂枝性状的分子标记辅助选择育种。
发明内容
为了解决现有技术中存在的技术问题，本发明的目的是提供一种梅花垂枝性状紧密连锁的SNP分子标记及其检测方法与应用。
本发明通过对214个梅花品种进行全基因组测序，利用GWAS分析方法检测与梅花枝条性状显著关联的位点；通过群体选择分析，观察显著关联位点区域是否存在选择信号；联合GWAS关联分析和群体选择分析，获得与梅花垂枝性状显著关联的47个SNP标记，将垂枝性状定位到梅花7号染色体11.1-11.8Mb和14.2-15.2Mb两个区间，以‘六瓣’和‘粉台垂枝’杂交的F1分离群体作为材料，利用质谱法验证显著关联的47个SNP标记，发现SNP标记Pm7_11182911与梅花垂枝性状的关联最为紧密。
首先，本发明提供一种梅花垂枝性状紧密连锁的SNP分子标记，其位于梅花第7号染色体第11182911bp处(Prunus mume Genome v1.0 Genbank登陆号：PRJNA171605；公开日期：2013年2月28日)，所述SNP分子标记的多态性为A/C。
上述位于梅花第7号染色体第11182911bp处的A/C突变，与梅花的垂枝/直枝性状紧密连锁，因此，所有包括位于梅花第7号染色体第11182911bp处的SNP位点的DNA分子标记(例如序列如SEQ ID NO.1所示DNA分子，上述SNP位点位于SEQ ID NO.1的第300位)均在本发明的保护范围中。
基于上述SNP分子标记的开发，本发明开发了梅花垂枝/直枝性状的基因型鉴定方法，通过对梅花第7号染色体第11182911bp处的碱基序列进行分析，鉴定梅花的垂枝/直枝性状：梅花垂枝性状对应的上述SNP位点的基因型为AC，直枝对应的基因型为CC。
为进行所述SNP基因型的检测，本发明提供SNP分子标记的检测引物，所述检测引物包括：
正向引物F：
5’-ACGTTGGATGTGCTTGTCAAACACAGTCCG-3’；
反向引物R：
5’-ACGTTGGATGGGTGTGTTTCTTTCTAACGAG-3’。
本发明中，当通过分析单碱基延伸反应产物的序列分析所述SNP的 基因型时，所述检测引物还包括单碱基延伸引物：
单碱基延伸引物：5’-ACTAACCTCATTTCATAAGTTGA-3’。
在此基础上，本发明提供一种用于梅花垂枝性状检测的试剂盒，所述试剂盒包括上述正向引物F和反向引物R的组合或包括上述正向引物F、反向引物R和单碱基延伸引物的组合。
此外，所述试剂盒还包括dNTPs、DNA聚合酶、Mg 2+、PCR反应缓冲液。
优选地，本发明所述的试剂盒还包括标准阳性模板和SAP酶。
进一步地，本发明提供所述的SNP分子标记或所述的检测引物或所述的试剂盒在梅花垂枝或直枝性状鉴定中的应用。
以及所述的SNP分子标记或所述的检测引物或所述的试剂盒在梅花分子辅助育种中的应用。
进一步地，本发明提供一种鉴定梅花株型性状的方法，其为以待测梅花的基因组为模板，采用正向引物F和反向引物R进行PCR扩增，分析PCR扩增产物的序列，判断待测梅花的基因型；
所述正向引物F的序列为5’-ACGTTGGATGTGCTTGTCAAACACAGTCCG-3’；所述反向引物R的序列为5’-ACGTTGGATGGGTGTGTTTCTTTCTAACGAG-3’。
本发明中，经上述PCR扩增得到的PCR扩增产物可以采用直接测序法、质谱分析检测方法等本领域常规的SNP分型方法进行序列分析。
为更好地满足高通量、高精度、高效性、低成本和广谱性分析的SNP分型的要求，优选地，所述分析扩增产物的序列的方法为采用Sequenom
Figure PCTCN2019073921-appb-000001
技术进行SNP的基因型分析，具体包括如下步骤：
(1)将所述PCR扩增产物用碱性磷酸酶处理；
(2)以步骤(1)得到的碱性磷酸酶处理的产物为模板进行单碱基延伸反应，所述单碱基延伸反应的引物的序列为：5’-ACTAACCTCATTTCATAAGTTGA-3’；
(3)分析延伸单碱基延伸反应的产物的基因型。
所述分析延伸单碱基延伸反应的产物的基因型为采用Sequenom
Figure PCTCN2019073921-appb-000002
技术和质谱检测进行分析。
为更好地满足高通量、自动化分析的要求，作为本发明的一种优选实施方式，所述鉴定梅花株型性状的方法包括如下步骤：
(1)提取待测梅花的基因组DNA；
(2)以待测植株的基因组DNA为模板，利用所述正向引物F和反向引物R进行PCR扩增反应；
(3)将PCR产物用碱性磷酸酶处理，以去除体系中游离的dNTPs；
(4)以碱性磷酸酶处理后的产物为模板，采用单碱基延伸反应的引物：5’-ACTAACCTCATTTCATAAGTTGA-3’进行单碱基延伸反应；
(5)延伸反应结束后，利用树脂纯化延伸产物；
(6)将延伸产物转移到MassARRAY SpectroCHIP芯片上，利用MassARRAY Analyzer ComPmc质谱仪检测，利用软件分析质谱检测结果，获得所述SNP位点的分型数据。
上述步骤(2)中，PCR扩增反应的反应体系(5μL)如下：
10×PCR Buffer(含Mg 2+)0.625μL、25mM MgCl 2 0.325μL、dNTP(2.5mM each)1.0μL、正向引物F 0.5μL、反向引物R 0.5μL、5U/μL Taq DNA聚合酶0.1μL基因组DNA模板(50ng/μL)1.0μL、加水补足5μL。
反应程序如下：94℃15min；94℃10-20sec，56-65℃30sec，72℃1min，45个循环；72℃3-5min。
上述步骤(3)中，碱性磷酸酶处理的反应体系(7μL)如下：10×SAP Buffer 0.17μL、1U/μL SAP酶0.3μL、步骤(2)的PCR产物5μL、加水补足7μL。
反应程序如下：37℃，40min；85℃，5min。
上述步骤(4)中，单碱基延伸反应的反应体系(9μL)如下：10×iPlex Buffer 0.2μL、iPlex Termination mix 0.2μL、单碱基延伸引物0.804μL、 iPlex enzyme 0.041μL、步骤(3)得到的碱性磷酸酶处理产物7μL、加水补足9μL。
反应程序如下：
Figure PCTCN2019073921-appb-000003
本发明的有益效果在于：
(1)本发明联合GWAS分析和群体选择方法开发梅花垂枝性状紧密连锁的SNP分子标记Pm7_11182911，该SNP分子标记在梅花杂交后代分离群体的垂枝/直枝的性状鉴定中具有重复性好、准确性高的优势，实现了单分子标记鉴定准确率达到96％以上的检测效果。
(2)本发明提供了所述SNP分子标记Pm7_11182911的高效检测方法，具有通量高、检测成本低、不受环境条件的影响等优势。
(3)本发明提供的Pm7_11182911分子标记及其检测方法可以实现垂枝/直枝性状的幼苗期选择，极大地减少了育种工作量，显著缩短了垂枝梅花新品种的培育周期，提高了育种效率，降低育种成本，可以在实践中用于梅花分子辅助选择育种。
附图说明
图1为本发明实施例2中表型性状测量方法的示意图，其中，(a)为生长期分枝角；(b)为成熟期分枝角；(c)为成熟枝条不同部位与背重力方向夹角。
图2为本发明实施例2中GWAS分析的Manhattan图，其中(a)-(g)分别为生长期分枝角A1、成熟期分枝角A2、枝条不同部位与背重力方向夹角T1、枝条不同部位与背重力方向夹角T2、枝条不同部位与背重力方 向夹角T3、枝条不同部位与背重力方向夹角T4、枝条不同部位与背重力方向夹角T5的GWAS分析结果图。
图3为本发明实施例3的群体选择分析图，其中(a)为群体遗传分化系数(F ST)；(b)为垂枝梅核酸多样性(Pi)；(c)为直枝梅核酸多样性(Pi)；(d)为群体遗传分化系数与核酸多样性分析结果的放大图。
图4为本发明实施例3群体选择与GWAS联合筛选的标记-性状关联位点，其中，(a)为联合筛选关联位点；(b)为比较候选关联位点等位基因频率及核酸多样性。
图5为本发明实施例4中质谱分析的结果图，其中，(a)为SNP标记Pm7_11182911的AC基因型峰图；(b)为SNP标记Pm7_11182911的CC基因型峰图。
图6为本发明实施例4中SNP标记Pm7_11182911的分型结果分析图，其中，(a)为直枝梅与垂枝梅基因型频率分析；(b)为基因型散点图。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：梅花全基因组SNP标记开发
1、实验材料
供试材料为214个梅花栽培品种，采集于武汉磨山梅花种质资源圃(30.546719°N，114.413254°E)，包含了来源于湖北、云南、江苏和安徽等11个地区的常规栽培品种(如宫粉、朱砂、单瓣、玉碟、杏梅、黄香、垂枝、跳枝和龙游品种群)。
2、基因组DNA的提取
214个梅花品种基因组提取材料来源于新鲜或者硅胶干燥的叶片，按照CTAB(Cetyl trimethyl ammonium bromide)法提取基因组DNA，制备1.0％琼脂糖凝胶，吸取3μL基因组DNA混合约1μL loading-buffer至上样孔，电压150V，电泳15min，检测基因组DNA的完整性；用NANO DROP 2000测定基因组DNA的浓度和纯度，保证基因组DNA的浓度>50ng/μL；用Qubit对DNA浓度进行精确定量。经检测合格的DNA样品用于建库测序。
3、建库测序
根据Illumina测序平台的要求，对检验合格的DNA样品进行随机打断，DNA片段经末端修复、加PolyA尾、加测序接头、纯化、PCR扩增和文库检测步骤。文库短插入片段为500bp，长插入片段为2Kb。所有文库检测合格后，利用Illumina HiSeq200测序平台进行测序。
4、质控检测
根据以下5个标准对rawdata进行过滤：
(1)reads错配碱基大于10％；(2)reads为低质量的序列(Phred quality scores Q≤7，短插入片段碱基超过65％，长插入片段碱基超过80％)；(3)10bp的接头序列；(4)短插入片段双端超过10bp的重叠；(5)双端序列相同的reads。经过对测序数据的严格过滤，去除符合以上5个标准的数据，得到高质量的clean data用于后续分析。
5、SNPs检测及质量控制
将高质量的测序数据通过BWA软件比对到参考基因组，利用GATK软件进行SNP calling和质量控制(质控参数：QD<2.0||FS>60.0||MQ<40.0||HaplotypeScore>13.0)。同时，对于基因型丢失超过10％的SNP进行过滤，去除最小等位基因频率(MAF)小于0.01的SNP位点，去除Hardy-Weinberg检测P<10 -6的SNP位点，去除三重或多重等位基因，只保留二等位基因。最终，获得了3,014,409个高质量SNP位点用于后续 GWAS分析。
实施例2：基于GWAS分析挖掘梅花垂枝性状关联SNP标记
1、梅花株型性状表型测定
对214个梅花栽培品种的表型性状进行采集，表型性状的采集方法如图1所示。测量成熟枝条和生长期枝条的分枝角，同时将成熟枝条分为5个部分，测量每个部分与背重力方向的夹角，共获得7个表型性状用于GWAS分析。
2、GWAS挖掘性状-标记关联位点
利用ADMIXTURE1.3软件计算214个梅花品种的群体结构矩阵文件(Q)，利用SPMGeDi v.1.4b软件计算亲缘关系矩阵(K)。GWAS分析使用TASSEL v.5软件，选择混合线性模型(MLM)，该模型包含了固定效应、随机效应和遗传关系，具体如下：
y＝Xβ+Zμ+e
其中，y为表型，β为标记和Q，μ为遗传效应，X和Z为已知矩阵，e为残差。
利用GWAS关联分析，7个表型性状均在Pm7染色体上检测到与垂枝性状显著关联的位点(图2)，关联区域位于11-12Mb和14-16Mb区域。
实施例3基于群体选择分析挖掘梅花垂枝性状受选择位点
1、群体选择分析
利用VCFtools v.0.1.15软件分析了垂枝梅与直枝梅群体遗传分化系数(F ST)和群体内核酸多样性(Pi)，发现Pm7的10-12Mb和14-15.5Mb区域受过强烈选择(图3)，该结果与GWAS结果相同，进一步确定了位于该区域内SNP标记与垂枝性状紧密关联。
2、联合群体选择与GWAS筛选候选标记
联合群体选择分析与GWAS分析结果，筛选了49个与梅花垂枝性状紧密关联的SNP标记，并对这49个SNP位点的等位基因频率和核酸多 样性进行比较，发现垂枝梅与植枝梅存在显著差异(图4)。联合群体选择与GWAS选择大大减少的了候选标记的数量，并且增加了SNP标记的可靠性。
实施例4候选SNP位点的验证
1、实验材料
以‘六瓣’(母本)ב粉台垂枝’(父本)得到的F1分离群体作为材料，其中垂枝个体127株，直枝个体161株。供试材料定植于浙江省湖州市莫干山镇何村(30.566389°N，119.879582°E)。
2、基因组DNA的提取
按照高效植物基因组DNA提取试剂盒(天根生化科技有限公司)的说明书进行基因组DNA的提取，制备1.0％琼脂糖凝胶，吸取3μL DNA混合约1μL loading-buffer，电压150V，电泳15min，检测DNA完整性。用NANO DROP 2000测定DNA浓度和纯度，保证DNA浓度>50ng/μL。质检合格的DNA样品用于质谱检测实验。
3、引物设计
根据SNP位置信息，提取47个SNP位点前后300bp序列，设计PCR反应和单碱基延伸反应引物。其中，Pm7_11182911位点的核苷酸序列如SEQ ID NO.1所示。根据序列信息，利用Genotyping Tools及MassARRAY Assay Design软件设计待测SNP位点的PCR扩增引物及单碱基延伸引物。Pm7_11182911位点的扩增引物序列如下：
PCR扩增引物1：ACGTTGGATGTGCTTGTCAAACACAGTCCG；
PCR扩增引物2：ACGTTGGATGGGTGTGTTTCTTTCTAACGAG；
单碱基延伸引物：ACTAACCTCATTTCATAAGTTGA。
4、PCR扩增
采用多重PCR扩增技术，在384孔板中进行，每个孔的反应体系为5μL，反应体系如表1所示。
表1PCR反应体系
试剂名称 每个反应(μL)
H 2O 0.95
PCR Buffer(10×,含15mM MgCl 2) 0.625
MgCl 2(25mM) 0.325
dNTP(2.5mM each) 1.0
引物使用液 1.0
HotstarTaq(5U/μL) 0.1
DNA模板 1.0
终体积 5.0
PCR反应程序如下：
Figure PCTCN2019073921-appb-000004
5、PCR产物碱性磷酸酶处理(SAP处理)
PCR反应结束后，PCR产物用SAP处理，去除游离的dNTPs。SAP反应液如下：
表2 SAP反应液的配方
试剂名称 每个反应(μL)
H 2O 1.53
SAP Buffer(10×) 0.17
SAP酶(1U/μL) 0.3
终体积 2.0
对于每个碱性磷酸酶处理反应孔，反应体系总体积为7μL，其中PCR产物5μL，SAP混合液2μL。反应程序为：37℃，40min；85℃，5min；4℃，forever。设定程序后，启动PCR仪进行碱性磷酸酶处理。
6、单碱基延伸
在碱性磷酸酶处理结束后，进行单碱基延伸反应，反应液体系如下：
表3碱性磷酸酶反应液的配方
试剂名称 每个反应(μL)
H 2O 0.755
iPlex Buffer(10×) 0.2
iPlex Termination mix 0.2
引物使用液 0.804
iPlex enzyme 0.041
终体积 2.0
对于每个反应孔，单碱基延伸反应体系为SAP处理后PCR产物7μL及延伸反应液2μL，反应体系总体积为9μL。将已加入延伸反应体系的384孔板放入PCR仪中，运行名为“extension”的反应程序，反应程序如下：
Figure PCTCN2019073921-appb-000005
7、产物纯化
取6mg树脂在384孔树脂刮板上，均匀覆盖，刮去多余的树脂，放置20min。将反应结束的384孔板1000rpm离心1min，每孔加入25μL去离子水，倒置在树脂板上面(注意固定，不能移位)，然后反置树脂板扣在384孔板上，敲击使树脂落入384孔板，封膜。以384孔板的长轴为轴心，翻转384孔板20分钟，3500rpm离心5min后备用。
8、检测
步骤(7)得到的产物检测步骤如下：
(1)Nanodispenser SpectroCHIP芯片点样，将检测样品从384孔反应板转移到表面覆盖基质的MassARRAY SpectroCHIP芯片；
(2)MassARRAY Analyzer ComPmc质谱检测，将样品转移到SpectroCHIP芯片后，即可放入质谱仪进行检测，芯片上每个样品检测3-5sec，检测过程全自动化；
(3)TYPER软件分析实验结果，获得分型数据。质谱分型结果如图5所示。
9、结果分析
利用质谱分型结果对‘六瓣’(母本)ב粉台垂枝’(父本)杂交得到的F 1分离群体的基因型频率进行计算，其中，SNP位点Pm7_11182911的直枝个体与垂枝个体的基因型频率差异最大，能够准确的将垂枝个体与植株个体分离。对F 1群体的161个直枝个体和127个垂枝条个体进行分型，准确率分别为97.5％(157/161)和96.7％(122/127)，直枝个体表现为CC基因型，垂枝个体表现为AC基因型(图6)。
本发明建立了垂枝梅花的分子标记辅助选择育种体系，利用单标记即可以实现垂枝性状的早期选择，大大提高了育种效率，减少育种工作量、田间种植面积以及劳动力，大大降低了育种成本和周期，对垂枝梅花的分子辅助选择育种具有开创性的价值。
上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
工业实用性
本发明提供一种梅花垂枝性状紧密连锁的SNP分子标记，其位于梅花第7号染色体第11182911bp处，多态性为A/C。本发明联合GWAS分析和群体选择方法开发了梅花垂枝性状紧密连锁的SNP分子标记，该SNP分子标记在梅花杂交后代分离群体的垂枝/直枝性状鉴定中重复性好、准确性高，实现了单分子标记鉴定垂枝/直枝性状的准确率达到96％ 以上。本发明提供的梅花垂枝性状紧密连锁的SNP分子标记及其检测方法可实现垂枝/直枝性状的幼苗期选择，极大缩短了育种周期，提高了育种效率，降低了育种成本，可以在实践中用于梅花分子辅助选择育种，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种梅花垂枝性状紧密连锁的SNP分子标记，其特征在于，其位于梅花第7号染色体第11182911 bp处，所述SNP分子标记的多态性为A/C。
  2. 权利要求1所述的SNP分子标记的检测引物，其特征在于，所述检测引物包括：
    正向引物F：
    5’-ACGTTGGATGTGCTTGTCAAACACAGTCCG-3’；
    反向引物R：
    5’-ACGTTGGATGGGTGTGTTTCTTTCTAACGAG-3’。
  3. 根据权利要求2所述的检测引物，其特征在于，所述检测引物还包括单碱基延伸引物：
    所述单碱基延伸引物的序列如下：
    5’-ACTAACCTCATTTCATAAGTTGA-3’。
  4. 一种用于梅花垂枝性状检测的试剂盒，其特征在于，所述试剂盒包括权利要求2或权利要求3所述的检测引物。
  5. 根据权利要求4所述的试剂盒，其特征在于，所述试剂盒还包括dNTPs、DNA聚合酶、Mg 2+、PCR反应缓冲液；
    优选地，所述试剂盒还包括标准阳性模板和SAP酶。
  6. 权利要求1所述的SNP分子标记或权利要求2或3所述的检测引物或权利要求4或5所述的试剂盒在梅花垂枝或直枝性状鉴定中的应用。
  7. 权利要求1所述的SNP分子标记或权利要求2或3所述的检测引物或权利要求4或5所述的试剂盒在梅花分子辅助育种中的应用。
  8. 一种鉴定梅花株型性状的方法，其特征在于，以待测梅花的基因组为模板，采用正向引物F和反向引物R进行PCR扩增，分析PCR扩增产物的序列，判断待测梅花的基因型；
    所述正向引物F的序列为5’-ACGTTGGATGTGCTTGTCAA ACACAGTCCG-3’；所述反向引物R的序列为5’-ACGTTGGATGGGTGTGTTTCTTTCTAACGAG-3’。
  9. 根据权利要求8所述的方法，其特征在于，所述分析扩增产物的序列的方法包括如下步骤：
    (1)将所述PCR扩增产物用碱性磷酸酶处理；
    (2)以步骤(1)得到的碱性磷酸酶处理的产物为模板进行单碱基延伸反应，所述单碱基延伸反应的引物的序列为：5’-ACTAACCTCATTTCATAAGTTGA-3’；
    (3)分析延伸单碱基延伸反应的产物的基因型。
  10. 根据权利要求9所述的方法，其特征在于，所述分析单碱基延伸反应的产物的基因型为采用Sequenom
    Figure PCTCN2019073921-appb-100001
    技术和质谱检测进行分析。
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