WO2019210696A1 - 梅花垂枝性状snp分子标记及其应用 - Google Patents

梅花垂枝性状snp分子标记及其应用 Download PDF

Info

Publication number
WO2019210696A1
WO2019210696A1 PCT/CN2018/121985 CN2018121985W WO2019210696A1 WO 2019210696 A1 WO2019210696 A1 WO 2019210696A1 CN 2018121985 W CN2018121985 W CN 2018121985W WO 2019210696 A1 WO2019210696 A1 WO 2019210696A1
Authority
WO
WIPO (PCT)
Prior art keywords
primer
marker
plum
pcr
benefit
Prior art date
Application number
PCT/CN2018/121985
Other languages
English (en)
French (fr)
Inventor
张启翔
卓孝康
郑唐春
程堂仁
王佳
孙丽丹
Original Assignee
北京林业大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京林业大学 filed Critical 北京林业大学
Priority to EP18917319.8A priority Critical patent/EP3789506B1/en
Publication of WO2019210696A1 publication Critical patent/WO2019210696A1/zh

Links

Images

Classifications

    • 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
    • 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
    • C12Q2600/13—Plant traits
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to the fields of molecular biology and plant molecular breeding, in particular to a SNP molecular marker of plum blossom traits and application thereof.
  • Prunus mume Sieb.et Zucc. belongs to the genus Prunus of the Rosaceae family. It is one of the top ten traditional flowers in China. It has ornamental and use value and originated in southeastern China. Years of introduction and cultivation history (Chen Junyu. Chinese plum varieties] [M]. China Forestry Publishing House, 2010). There are many kinds of plum blossoms, and the flower-flower type is rich. Among them, weeping plum is one of the nine varieties of plum blossom with unique plant type. It is deeply loved by people because of its unique tree pose and other ornamental traits such as flowers and flowers. It has an important position in garden landscaping.
  • the use of high-throughput sequencing technology to develop SNP markers in large quantities the development of trait-linked molecular markers for initial screening of plants, to achieve the purpose of molecular-assisted breeding, can greatly shorten the breeding cycle and improve breeding efficiency.
  • the invention utilizes SLAF-seq technology to develop a large number of SNP markers of plum blossom branches, and screens closely linked molecular markers, which provides an important basis for molecular marker-assisted selection breeding.
  • the object of the present invention is to provide a SNP molecular marker of plum plume traits and its application.
  • the present invention is based on the 'six-petal' plum as the female parent, and the F 1 segregating group, the straight branch and the weeping plum plum variety obtained by the father's cross-breeding as the test material, and the plum blossom weeping branches are carried out. Development of trait molecular markers and establishment of marker-assisted selection breeding systems.
  • the SNP molecular marker of plum plume traits provided by the present invention includes molecular markers Marker301243 and/or Marker311414, and their nucleotide sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
  • the invention also provides specific PCR primers for amplifying the molecular markers.
  • the marker Marker301243 primer sequence is:
  • Reverse primer 5'-CTCTGCTGGACACCCCTAAT-3'
  • the marker Marker311414 primer sequence is:
  • Reverse primer 5'-AGAGCAGGCACCCAAGTAAGT-3'
  • the invention also provides the use of the molecular marker in identifying the vertical trait of plum, comprising the following steps:
  • the PCR amplification system in step 2) was 20 ⁇ L, including: 50 ⁇ g/ ⁇ L of template DNA 2 ⁇ L, 2 ⁇ Taq PCR Master Mix 10 ⁇ L, 10 ⁇ mol/L of forward and reverse primers each of 1 ⁇ L, and ddH 2 O 6 ⁇ L.
  • the following PCR reaction procedure is used as an example: pre-denaturation at 95 °C for 2 min, denaturation at 95 °C for 20 s, annealing at 56 °C for 30 s, extension at 72 °C for 20 s, 30 cycles, and final extension at 72 °C for 5 min.
  • PCR amplification systems and reaction program selections include, but are not limited to, the amplification systems and reaction procedures described above.
  • the molecularly labeled primer in step 2) can be a pair of forward and reverse primers labeled Marker 301243 and/or a pair of forward and reverse primers labeled Marker 311414.
  • the invention also provides AS-PCR primers for amplifying the molecular markers by AS-PCR (allele-specific PCR).
  • the AS-PCR primers were designed according to the SNP positions of Marker301243 (A/T) and Marker311414 (C/T), respectively, in order to increase the primer specificity, and at the same time, the third position of the 3' end of the specific primer was introduced.
  • One mismatched base and the other primer were designed in the usual way.
  • the marker Marker301243 primer sequence is:
  • the marker Marker311414 primer sequence is:
  • Reverse primer 5'-TCATAGGTATTCTTGTCTTTCTC-3'
  • Reverse specific primer 5'-TCATAGGTATTCTTGTCTTTCTT-3'
  • the invention also provides a method for screening or identifying a plum blossom trait, the method comprising the steps of:
  • the dominant allele genotype of the plum blossom branch is TT corresponding to the SNP locus of the marker Marker301243
  • the dominant allele of the straight branch is AT; corresponding to the SNP locus of Marker311414, the dominant allele of the plum branch is GG, and the dominant allele of the straight branch is GA.
  • the AS-PCR amplification system in step 2) was 10 ⁇ L, including: 1 ⁇ L of 50 ng/ ⁇ L of template DNA, 5 ⁇ L of 2 ⁇ Taq PCR Master Mix, 0.5 ⁇ L of 10 ⁇ mol/L of forward and reverse primers, and 3 ⁇ L of ddH 2 O.
  • AS-PCR reaction procedure is used as an example: pre-denaturation at 95 ° C for 2 min, denaturation at 95 ° C for 20 s, annealing at 46-52 ° C for 30 s, extension at 72 ° C for 20 s, 30 cycles; final extension at 72 ° C 5min.
  • PCR amplification systems and reaction program selections include, but are not limited to, the amplification systems and reaction procedures described above.
  • the molecularly labeled primer in step 2) may be a pair of forward and reverse primers labeled Marker 301243 and/or a pair of forward and reverse primers labeled Marker 311414.
  • AS-PCR primers are paired with a common primer during AS-PCR operation, and the same DNA template is subjected to PCR amplification in two PCR tubes. If the 3' end of the AS-PCR primer matches the allele in the sample, efficient amplification can be performed, and conversely, efficient amplification cannot be performed.
  • the genotype of the amplified individual is identified, and according to the relationship between the genotype and the phenotype, the genotype frequency is determined to determine the SNP position. Whether the point is closely related to the plume or straight branch of the plum.
  • the present invention also provides a detection kit for screening or identifying plum plume traits containing the specific PCR and/or the AS-PCR primer.
  • the kit also includes one or more of dNTPs, DNA polymerase, Mg 2+ , PCR reaction buffer, and standard positive template.
  • the invention further provides the use of the molecular marker and/or the primer for molecular marker-assisted breeding of plum down traits.
  • the present invention utilizes SLAF-seq technology to develop molecular markers linked to the plume traits of plum blossoms, and lays a foundation for molecular marker-assisted selection breeding of plum vertical traits.
  • the SNP molecular marker obtained by the invention has good reproducibility and stable amplification in the trait identification of the plum population and the variety, and is not affected by environmental conditions.
  • the AS-PCR primer provided by the invention is used for the AS-PCR amplification reaction, and the plum blossom hanging branch and the straight branch trait can be identified by electrophoresis detection, and the operation is simple.
  • the two molecular markers selected by the invention are used for assisting selective breeding, which can realize early selection of seedlings, reduce workload, greatly shorten plum breeding cycle, and improve breeding efficiency.
  • Example 3 FIG sequencing peaks labeled 1 Marker301243 embodiment of the present invention, wherein A is the parent 'six' plum, B male parent 'station powder weeping', C straight branches of the F 1 plum, D is the F 1 Weeping plums.
  • Example 2 is a sequencing peak diagram of Marker 311414 labeled in Example 3 of the present invention, wherein A is a female 'six-petal' plum, and B is a male parent's 'powder hanging branch'.
  • FIG. 3 is an amplification result of AS-PCR in the F 1 population of Marker 301243 (Marker left 1-12) and Marker 311414 (Marker right 1-12) in Example 4 of the present invention, wherein 1-6 are vertical branches Plum, 7-12 are straight plum blossoms, M is Marker DL2000.
  • Figure 4 is a result of AS-PCR amplification of the marker Marker301243 in the parental and plum-pig varieties in Example 4 of the present invention, wherein 1-2 is the female 'six-petal' plum, and 3-4 is the male parent' powder table.
  • Weeping branch ', 5-19 is a plum blossoming variety, and M is Marker DL2000.
  • Fig. 5 is a result of AS-PCR amplification of Marker 301243 in the plum cultivar of the plum cultivar in Example 4 of the present invention, wherein 1-27 is a plum blossom cultivar and M is a Marker DL2000.
  • Figure 6 is a result of AS-PCR amplification of the marker Marker311414 in the parental and plum-pruned varieties in Example 4 of the present invention, wherein 1-2 is the female 'six-petal' plum, and 3-4 is the male parent' powder table.
  • Weeping branch ', 5-19 is a plum blossoming variety, and M is Marker DL2000.
  • Fig. 7 is a result of AS-PCR amplification of Marker 311414 labeled in the plum cultivar of the cultivar in the fourth embodiment of the present invention, wherein 1-27 is a plum blossoming variety and M is a Marker DL2000.
  • Figure 8 is a comparison diagram of the results of AS-PCR amplification in the parental and plum-pruned varieties of Marker 301243 (top) and Marker 311414 (bottom) in Example 4 of the present invention, wherein 1-2 is a female parent's six-petal 'Mei, 3-4 is the father's 'Pink Taige', 5-19 is the plum blossom variety, M is Marker DL2000.
  • Figure 9 is a comparison diagram of the results of AS-PCR amplification of Marker 301243 (top panel) and Marker 311414 (bottom panel) in the plum cultivar of the plum cultivar in Example 4 of the present invention, wherein 1-27 is a plum-branched variety and M is a Marker DL2000.
  • Figure 10 is a diagram showing the accuracy of assisted selection breeding in the F1 population by Marker 301243 and Marker 311414 in Example 4 of the present invention.
  • Figure 11 is a diagram showing the accuracy of assisted selection breeding of Marker 301243 and Marker 311414 in plum varieties in Example 4 of the present invention.
  • FIG. 12 labeled Marker301243 + Marker311414 combination groups F 1 and Plum Cultivars assisted selection accuracy of the present invention.
  • Example 1 Development of SNP molecular markers for plum buds based on SLAF-seq technology
  • test materials included 'six-petal' plum as the female parent, 'Pantai weeping' plum as the male parent and the F 1 segregating group, straight branch and weeping plum plum variety obtained by the parental cross.
  • the parents had significant differences in phenotypic traits such as weeping branches and flowers. All materials were planted in He Village, Moganshan Town, Huzhou City, Zhejiang City (30.566389°N, 119.879582°E).
  • DNA extraction was performed according to the high-efficiency plant genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.). A 1.0% agarose gel was prepared, and 3 ⁇ L of DNA was mixed and mixed with about 1 ⁇ L of loading-buffer, voltage of 150 V, and electrophoresis for 15 min to detect DNA integrity. DNA concentration and purity were determined using NANO DROP 2000 to ensure a DNA concentration of >50 ng/ ⁇ L.
  • the genomic information of plum blossom the number of molecular markers produced by different endonucleases after digestion of the plum genome and its distribution on the genome were predicted.
  • Hae III and Hpy 166II New England Biolabs, NEB were selected. , USA) two enzymes.
  • the genomic DNA of the plum was digested with endonuclease Hae III and Hpy 166II, then Klenow fragment (3'-5'exo) (NEB) and dATP were added, and the DNA end of the digested DNA was incubated with A base at 37 °C.
  • a double-barcode tag linker (PAGE-purified, Life Technologies, USA) was ligated to the end of the above A-base-bearing product by T4 ligase. Dilute DNA sample after completion of the connection, dZTP, The high-fidelity enzyme and PCR primers are mixed for subsequent PCR reactions.
  • the PCR product was purified by a nucleic acid purification kit (Beckman Coulter, High Wycombe, UK) and then mixed, and then the mixed product of different size PCR fragments was separated by a 2% agarose gel, and 6 ⁇ L of EB was added to 50 ⁇ L of the mixed DNA solution.
  • the prepared SLAF library was subjected to double-end 100 bp sequencing using IlHia's HiSeq 2500 sequencing platform (Illumina, Inc; San Diego; CA, USA) according to the manufacturer's instructions.
  • Table 1 2 pairs of primer information for Sanger sequencing
  • PCR amplification system containing 2 ⁇ L of 100 ng/ ⁇ L template DNA, 10 ⁇ L of 2 ⁇ Taq PCR Master Mix (BIOMIGA), 10 ⁇ L/L forward and reverse primers (Biosynthetic Engineering Beijing Synthesis Department) 1 ⁇ L each, ddH 2 O 10.3 ⁇ L.
  • the PCR procedure was: pre-denaturation at 95 °C for 2 min; denaturation at 94 °C for 20 s, annealing at 56 °C for 30 s, extension at 72 °C for 20 s, 30 cycles; final extension at 72 °C for 5 min.
  • the PCR product was then subjected to Sanger sequencing (Bioscience Engineering Co., Ltd. Beijing Sequencing Department), and the sequencing results of the progeny and the parent were compared to determine that the marker contained a real SNP site and was closely linked to the vertical branch trait.
  • Example 3 Establishment of an AS-PCR method based on SNP markers for genotyping
  • markers Marker301243, Marker 311414 Two markers were obtained by Sanger sequencing (ie, markers Marker301243, Marker 311414, whose nucleotide sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively), and AS-PCR primers were designed for the corresponding SNP sites. Allele-specific design was designed according to the SNP loci of Marker301243 (A/T) and Marker311414 (C/T), respectively, in order to increase primer specificity, and introduce at the 3rd position of the 3' end of the specific primer. One mismatched base and the other primer (common primer) were designed in a conventional manner, and the primers were determined to be specific by NCBI (Table 2).
  • the AS-PCR reaction system is 10 ⁇ L, 50 ⁇ g/ ⁇ L template DNA 1 ⁇ L, 2 ⁇ Taq PCR Master Mix 5 ⁇ L, 10 ⁇ mol/L forward and reverse primers each 0.5 ⁇ L, ddH 2 O 3 ⁇ L, and the PCR reaction procedure is: pre-denaturation at 95 ° C for 2 min. Denaturation at 95 ° C for 20 s, annealing at 46-52 ° C for 30 s, extension at 72 ° C for 20 s, 30 cycles; final extension at 72 ° C for 5 min.
  • the PCR products were detected by 1% agarose gel electrophoresis, and the genotypes of the F 1 population were counted to determine the dominant alleles of plum and vertical branches.
  • the SNP position corresponding to the marker Marker301243 is located at the 426th base of the sequence shown in SEQ ID NO. 1, where the base is T or A (Fig. 1), and the dominant allele of the plum blossom branch is TT, the dominant allele of the straight branch is AT;
  • the SNP corresponding to the marker Marker311414 is located at base 607 of the sequence shown in SEQ ID NO. 2, where the base is G or A, plum blossoms
  • the dominant allele genotype is GG
  • the dominant allele genotype of the straight branch is GA (Fig. 2).
  • Ref is the reference genome sequence
  • F is the forward primer sequence
  • R is the reverse primer sequence
  • S is the specific primer sequence
  • the bold base is the AS-PCR specific primer differential base.
  • the F 1 population was subjected to AS-PCR amplification verification using the obtained two markers, Marker301243 and Marker311414.
  • the identification results of the markers are shown in Figure 3 and Figure 10.
  • the accuracy of Marker301243 in the F 1 population was 91.67%, and in the straight individuals. Accuracy is as high as 100%.
  • the success rate in the F 1 population was as high as 100% (Fig. 12).
  • the obtained specific molecular marker is stable or not is also very important for its application.
  • the same primers are used to amplify the F 1 population and the plum cultivar. If a stable band can be obtained, the developed marker is stable and can be used for different plum blossoms. Identification of plant type. The stability and accuracy of the SNP markers developed were verified by using the plum varieties as materials. Taking Marker301243 as an example, it expands two bands in the straight branch, which is the AT allele type, and expands a band in the individual of the weeping branch, which is the TT allele, indicating that the two markers are better in the plum varieties. Different phenotypes were identified and have good stability (Fig. 3).
  • Marker301243 and Marker311414 molecular markers were verified in plum and shoot varieties, respectively.
  • three genotypes may occur (Marker301243 is AA, TT and AT; Marker311414 is AA (TT), GG (CC) and AG (CT)), and a pair is used.
  • the primer one specific primer and one common primer
  • Fig. 4-9 only the AA or AT genotype paired with the specific primer used can be amplified, and the TT cannot be amplified.
  • Striping take Marker301243 as an example).
  • Figure 4 shows the results of Marker301243 in the shoots of the weeping shoots.
  • the stripless species are the TT genotypes of the weeping shoots;
  • Figure 6 is the test results of the Marker311414 in the weeping shoots, and the unattached is GG(CC) Genotypes of weeping varieties.
  • Figure 8 shows the combination of Marker301243/Marker311414.
  • the combination of the corresponding genotypes can be expressed as TT/CC (bandless/bandless), A_/CC (with/without tape), TT/_T (without tape/band) .
  • TT/CC bandless/bandless
  • A_/CC with/without tape
  • TT/_T without tape/band
  • the dominant genotype of the SNP locus in the straight shoot variety is AT, and the specific primer can amplify the band.
  • Figure 5 shows the results of Marker30124 in the straight shoots. It can be seen from the figure that most of the straight branches have bands, which are expressed as AT genotypes.
  • Figure 7 shows the results of Marker311414 in straight shoots, which are mostly strips.
  • CT genotype shows the combination of Marker301243/Marker311414.
  • the combination of the corresponding genotypes can be expressed as A_/_T (with band/band), A_/CC (with/without band), TT/_T (without band/band) , TT / CC (without / no belt).
  • FIG. 3 is the result of amplification of AS-PCR in the F 1 population by Marker 301243 and Marker 311414.
  • Figures 4-9 show the amplification stability of AS-PCR in the plum varieties of Marker 301243 and Marker 311414.
  • the molecular marker-assisted selection breeding established by the invention can realize early selection of seedlings, reduce workload, improve the selection efficiency of plum blossom traits, and greatly shorten the breeding cycle.
  • the invention provides a SNP molecular marker of plum plume trait and application thereof.
  • the SNP molecular marker of plum plump traits provided by the present invention specifically includes the molecular markers Marker301243 and/or Marker311414, and the nucleotide sequences thereof are shown in SEQ ID NO. 1 and SEQ ID NO.
  • the invention utilizes SLAF-seq technology to develop two SNP molecular markers Marker301243 and Marker311414, and provides sequence amplification primers and allele-specific PCR primers.
  • the molecular markers Marker301243 and/or Marker311414 and their primers have high accuracy, stability and repeatability when identifying plum varieties. It can be used for molecular-assisted selection breeding to achieve early selection of seedlings, rapid identification of plum blossom traits, reduction of workload, and greatly shortening plum breeding time, with good economic value and application prospects.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Botany (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

梅花垂枝性状SNP分子标记及其应用，包括分子标记Marker301243和/或Marker311414，其核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。利用SLAF-seq技术开发获得2个梅花垂枝性状SNP分子标记Marker301243和Marker311414，并提供了其序列扩增引物和等位特异性PCR引物。所述分子标记Marker301243和/或Marker311414及其引物可用于鉴定梅花品种以及分子辅助选择育种。

Description

梅花垂枝性状SNP分子标记及其应用
交叉引用
本申请要求2018年5月3日提交的专利名称为“梅花垂枝性状SNP分子标记及其应用”的第201810415993.7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领域，具体地说，涉及梅花垂枝性状SNP分子标记及其应用。
背景技术
梅花(Prunus mume Sieb.et Zucc.)隶属于蔷薇科(Rosaceae)李属(Prunus)，是我国十大传统名花之一，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。
张杰(张杰.梅花高密度遗传图谱构建及部分观赏性状QTL分析[D].北京林业大学,2016.)利用特异性位点扩增片段测序(SLAF-seq,Specific-Locus Amplified Fragment Sequencing)技术对梅花进行全基因组范围内的分子标记开发，构建了梅花标记密度最大的一张遗传连锁图谱，并将垂枝性状定位到梅花7号染色体10.54Mb-11.68Mb区间，同时得到10个可能与梅花垂枝性状紧密连锁的SLAF分子标记。
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，达到分子辅助育种的目的，可大大缩短育种的周期，提高育种效率。本发明利用SLAF-seq技术开发大量的梅花垂枝的SNP标记，筛选紧密连锁的分子标记，为其分子标记辅助选择育种提供重要基础。
发明内容
为解决现有技术中存在的梅花垂枝性状筛选周期长、效率低等问题，本发明的目的是提供梅花垂枝性状SNP分子标记及其应用。
为了实现本发明目的，本发明是以‘六瓣’梅为母本，‘粉台垂枝’为父本杂交获得的F 1分离群体、直枝和垂枝梅花品种为试材，开展梅花垂枝性状分子标记的开发及标记辅助选择育种体系的建立。
本发明提供的梅花垂枝性状SNP分子标记，包括分子标记Marker301243和/或Marker311414，它们的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
本发明还提供用于扩增所述分子标记的特异性PCR引物。
标记Marker301243引物序列为：
正向引物：5′-TGAGAATGGACAATGAGCGT-3′
反向引物：5′-CTCTGCTGGACACCCCTAAT-3′
标记Marker311414引物序列为：
正向引物：5′-CTTAGGGAATGGTGTCGCTT-3′
反向引物：5′-AGAGCAGGCACCCAAGTAAGT-3′
本发明还提供所述分子标记在鉴定梅花垂直性状中的应用，包括以下步骤：
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，利用扩增所述分子标记的引物，进行PCR扩增反应；
3)对应于标记Marker301243的SNP位点，位于SEQ ID NO.1所示序列的第426位碱基，该处碱基为T或A，梅花垂枝的优势等位基因型为TT，直枝的优势等位基因型为AT；对应于标记Marker311414的SNP位点，位于SEQ ID NO.2所示序列的第607位碱基，该处碱基为G或A，梅花垂枝的优势等位基因型为GG，直枝的优势等位基因型为GA。
其中，步骤2)中PCR扩增体系为20μL，包括：50ng/μL模板DNA 2 μL，2×Taq PCR Master Mix 10μL，10μmol/L正反向引物各1μL，ddH 2O6μL。
在本发明的具体实施方式中，使用了以下PCR反应程序作为示例：95℃预变性2min，95℃变性20s，56℃退火30s，72℃延伸20s，30个循环；72℃终延伸5min。
本领域技术人员应该知晓，PCR的扩增体系和反应程序可以根据所用DNA聚合酶不同或其它需要调整其中各组分的体积和/或用量以及各反应的温度和时间，因此，本发明所述的PCR扩增体系和反应程序选择包括但不限于上述扩增体系和反应程序。
需要指出的是，在利用本发明提供的2个SNP分子标记Marker301243和Marker311414进行梅花垂枝性状鉴定或者梅花垂枝性状分子标记辅助育种时，本领域技术人员根据需要可以选择Marker301243和Marker311414中任意一种分子标记或Marker301243和Marker311414两种分子标记的组合。两标记结合鉴定，显著提高鉴定的准确率。
因此，步骤2)中所述分子标记的引物可以为标记Marker301243的正反向引物对和/或标记Marker311414的正反向引物对。
本发明还提供用于AS-PCR(等位基因特异性PCR)扩增所述分子标记的AS-PCR引物。
所述AS-PCR引物，分别根据Marker301243(A/T)和Marker311414(C/T)的SNP位点进行设计，为了提高引物特异性，同时在特异性引物3’末端的倒数第3位置引入1个错配碱基，另一条引物(公共引物)按常规方法进行设计。
标记Marker301243引物序列为：
正向引物：5′-TGGAAACTGAATAGATGCGAT-3′
反向公共引物：5′-GGTGAAAGAGACATCAGAAAAT-3′
正向特异性引物：5′-TGGAAACTGAATAGATGCGAA-3′
标记Marker311414引物序列为：
正向公共引物：5′-CATCTAAAATAAAATCTCAAAGG-3′
反向引物：5′-TCATAGGTATTCTTGTCTTTCTC-3′
反向特异性引物：5′-TCATAGGTATTCTTGTCTTTCTT-3′
本发明还提供一种筛选或鉴定梅花垂枝性状的方法，所述方法包括以下步骤：
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，利用扩增所述分子标记的AS-PCR引物，进行AS-PCR扩增反应；
3)检测PCR扩增产物，根据AS-PCR扩增产物条带的有无，判断对应的SNP位点，其中，对应于标记Marker301243的SNP位点，梅花垂枝的优势等位基因型为TT，直枝的优势等位基因型为AT；对应于标记Marker311414的SNP位点，梅花垂枝的优势等位基因型为GG，直枝的优势等位基因型为GA。
其中，步骤2)中AS-PCR扩增体系为10μL，包括：50ng/μL模板DNA 1μL，2×Taq PCR Master Mix 5μL，10μmol/L正反向引物各0.5μL，ddH 2O 3μL。
在本发明的具体实施方式中，使用以下AS-PCR反应程序作为示例：95℃预变性2min，95℃变性20s，46-52℃退火30s，72℃延伸20s，30个循环；72℃终延伸5min。
本领域技术人员应该知晓，PCR的扩增体系和反应程序可以根据所用DNA聚合酶不同或其它需要调整其中各组分的体积和/或用量以及各反应的温度和时间，因此，本发明所述的PCR扩增体系和反应程序选择包括但不限于上述扩增体系和反应程序。
步骤2)中所述分子标记的引物可以为标记Marker301243的正反向引物对和/或标记Marker311414的正反向引物对。
本领域技术人员公知，AS-PCR操作过程中需将两条AS-PCR引物与公共引物分别配对使用，在两个PCR管中对同一DNA模板进行PCR扩 增。若AS-PCR引物的3’末端与样品中的等位位点相匹配，便能进行有效扩增，反之则不能进行有效扩增。本发明根据AS-PCR的两个特异性引物分别与公共引物配对扩增时，鉴定扩增个体的基因型，根据基因型与表型的关联情况，通过统计基因型频率，以确定该SNP位点是否与梅花的垂枝或直枝性状紧密相关。
本发明还提供含有所述特异性PCR和/或所述AS-PCR引物的用于筛选或鉴定梅花垂枝性状的检测试剂盒。所述试剂盒还包括dNTPs、DNA聚合酶、Mg 2+、PCR反应缓冲液和标准阳性模板中的一种或多种。
本发明进一步提供所述分子标记和/或所述引物在梅花垂枝性状分子标记辅助育种中的应用。
本发明具有以下优点：
(一)本发明利用SLAF-seq技术开发梅花垂枝性状连锁的分子标记，奠定了梅花垂直性状分子标记辅助选择育种的基础。
(二)本发明获得的SNP分子标记在梅花群体和品种的性状鉴定中重复性好、能够稳定扩增，且不受环境条件的影响。
(三)本发明提供的AS-PCR引物用于AS-PCR扩增反应，可通过电泳检测鉴定梅花垂枝和直枝性状，操作简单。
(四)本发明提供的鉴定梅花垂枝性状的方法，通过对基因型频率的统计，发现两标记在梅花F 1群体和品种均具有较高的鉴定准确率：利用Marker301243在F 1群体中准确率最高为91.67％，在直枝个体中准确率高达100％；同时利用标记组合Marker301243+Marker311414，在F 1群体中准确率率高达100％。利用标记组合Marker301243+Marker311414在梅花品种中鉴定的准确率为89.13％，且在梅花垂枝品种中准确率高达100％。
(五)将本发明筛选出的2个分子标记用于辅助选择育种，可以实现苗期提早选择，减少工作量，大大缩短梅花育种周期，提高育种效率。
附图说明
图1本发明实施例3中标记Marker301243的测序峰图，其中A为母 本‘六瓣’梅，B为父本‘粉台垂枝’，C为F 1代直枝梅花，D为F 1代垂枝梅花。
图2本发明实施例3中标记Marker311414的测序峰图，其中A为母本‘六瓣’梅，B为父本‘粉台垂枝’。
图3为本发明实施例4中标记Marker301243(Marker左侧1-12)和Marker311414(Marker右侧1-12)在F 1群体中AS-PCR的扩增结果，其中1-6均为垂枝梅花，7-12均为直枝梅花，M为Marker DL2000。
图4为本发明实施例4中标记Marker301243在父母本及梅花垂枝品种中AS-PCR的扩增结果，其中1-2为母本‘六瓣’梅，3-4为父本‘粉台垂枝’，5-19为梅花垂枝品种，M为Marker DL2000。
图5为本发明实施例4中标记Marker301243在梅花直枝品种中AS-PCR的扩增结果，其中1-27为梅花直枝品种，M为Marker DL2000。
图6为本发明实施例4中标记Marker311414在父母本及梅花垂枝品种中AS-PCR的扩增结果，其中1-2为母本‘六瓣’梅，3-4为父本‘粉台垂枝’，5-19为梅花垂枝品种，M为Marker DL2000。
图7为本发明实施例4中标记Marker311414在梅花直枝品种中AS-PCR的扩增结果，其中1-27为梅花直枝品种，M为Marker DL2000。
图8为本发明实施例4中标记Marker301243(上图)和Marker311414(下图)在父母本及梅花垂枝品种中AS-PCR的扩增结果对比图，其中1-2为母本‘六瓣’梅，3-4为父本‘粉台垂枝’，5-19为梅花垂枝品种，M为Marker DL2000。
图9为本发明实施例4中标记Marker301243(上图)和Marker311414(下图)在梅花直枝品种中AS-PCR的扩增结果对比图，其中1-27为梅花直枝品种，M为Marker DL2000。
图10为本发明实施例4中标记Marker301243和Marker311414在F1群体中辅助选择育种准确性。
图11为本发明实施例4中标记Marker301243和Marker311414在梅 花品种中辅助选择育种准确性。
图12为本发明实施例4中标记Marker301243+Marker311414组合在F 1群体中和梅花品种中辅助选择育种准确性。
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：基于SLAF-seq技术开发梅花垂枝性状SNP分子标记
1、实验材料
供试材料包括‘六瓣’梅为母本，‘粉台垂枝’梅为父本及以其为亲本杂交获得的F 1分离群体、直枝和垂枝梅花品种。亲本在垂枝、花色等表型性状上差异显著，所有材料均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
选取F 1群体中直枝和垂枝株型的单株各20株。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)说明进行DNA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。
3、梅花性状分子标记的开发
根据梅花基因组信息进行酶切预测，预测不同的内切酶对梅花基因组酶切后所产生的分子标记数量及其在基因组上的分布位置，最终选定Hae Ⅲ和Hpy 166Ⅱ(New England Biolabs,NEB,USA)两种酶。接着用内切酶 Hae Ⅲ和Hpy 166Ⅱ对梅花基因组DNA进行消化处理，然后加入Klenow片段(3’-5’exo)(NEB)和dATP，37℃温育使消化后的DNA末端接上A碱基，通过T4连接酶在上述带有A碱基的产物末端连上双条形码标签接头(PAGE-purified,Life Technologies,USA)。连接完成之后将稀释的DNA样品，dZTP，
Figure PCTCN2018121985-appb-000001
高保真酶和PCR引物混匀进行后续的PCR反应。PCR产物通过核酸纯化试剂盒(Beckman Coulter,High Wycombe,UK)纯化之后再混合，然后利用2％的琼脂糖凝胶对不同大小的PCR片段混合产物进行分离，在50μL混合DNA溶液中加入6μL EB，然后依次加入点样孔中，同时加入6μL DL1000 Maker，110V电泳约50min。电泳后，将胶放于BioRad紫外成像仪(ChemiDoc TM MP)切取214-294bp的条带，放入称量好的离心管中，使用QIAquick Gel Eztraction Kit进行胶纯化回收，再将回收产物溶于50μL EB溶液。胶回收得到的产物进行稀释后，根据制造商说明，利用Illumina公司的HiSeq2500测序平台(Illumina,Inc；San Diego；CA,USA)对制备好的SLAF文库进行双末端100bp测序。
实施例2：SNP分子标记的扩增
根据梅花垂枝性状差异性标记设计引物，具体如下：
根据Sanger测序结果，选择2个Marker(Marker301243、Marker311414)，利用Primer Premier 5.0设计引物，引物序列见表1。
表1用于Sanger测序的2对引物信息
Figure PCTCN2018121985-appb-000002
随机挑选若干株直枝和垂枝个体进行PCR扩增。PCR扩增体系20μL，含100ng/μL模板DNA 2μL，2×Taq PCR Master Mix(BIOMIGA)10μL，10mol/L正向和反向引物(生工生物工程股份有限公司北京合成部) 各1μL，ddH 2O 10.3μL。PCR程序为：95℃预变性2min；94℃变性20s，56℃退火30s，72℃延伸20s，30个循环；72℃终延伸5min。后将PCR产物进行Sanger测序(生工生物工程股份有限公司北京测序部)，将子代与亲本的测序结果进行对照，确定该标记含有真实的SNP位点，且与垂枝性状紧密连锁。
以上两个实施例，基于数量性状基因(quantitative trait locus，QTL)分析和SLAF-index分析，发现Marker301243和/或Marker311414与梅花垂枝条性状显著关联。通过PCR扩增和Sanger测序，确定了Marker301243和/或Marker311414存在真实的SNP位点，并在子代中发生分离。
实施例3：基于SNP标记建立AS-PCR方法进行基因分型
利用Sanger测序获得2个标记(即标记Marker301243、Marker311414，其核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示)，对相应的SNP位点设计AS-PCR引物。等位基因特异性设计时，分别根据Marker301243(A/T)和Marker311414(C/T)的SNP位点进行设计，为了提高引物特异性，同时在特异性引物3’末端的倒数第3位置引入1个错配碱基，另一条引物(公共引物)按常规方法进行设计，所设引物利用NCBI检测特异性(表2)。AS-PCR操作过程中需将两条AS-PCR引物与公用引物分别配对使用，在两个PCR管中对同一DNA模板进行PCR扩增。若AS-PCR引物的3’末端与样品中的等位位点相匹配，便能进行有效扩增，反之则不能进行有效扩增。
AS-PCR反应体系为10μL，50ng/μL模板DNA 1μL，2×Taq PCR Master Mix 5μL，10μmol/L正反向引物各0.5μL，ddH 2O 3μL，PCR反应程序为：95℃预变性2min，95℃变性20s，46-52℃退火30s，72℃延伸20s，30个循环；72℃终延伸5min。扩增结束后用1％琼脂糖凝胶电泳检测PCR产物，统计F 1群体单株的基因型，确定梅花垂枝和直枝性状的优势等位基因型。其中，对应于标记Marker301243的SNP位点，位于SEQ ID NO.1所示序列的第426位碱基，该处碱基为T或A(图1)， 梅花垂枝的优势等位基因型为TT，直枝的优势等位基因型为AT；对应于标记Marker311414的SNP位点，位于SEQ ID NO.2所示序列的第607位碱基，该处碱基为G或A，梅花垂枝的优势等位基因型为GG，直枝的优势等位基因型为GA(图2)。
用于AS-PCR扩增的2对标记引物信息见表2。
表2用于AS-PCR扩增的2对引物信息
Figure PCTCN2018121985-appb-000003
注：Ref为参考基因组序列；F为正向引物序列；R为反向引物序列；S为特异引物序列；加粗碱基为AS-PCR特异性引物差异碱基。
实施例4：Marker301243和Marker311414分子标记的应用
对Marker301243和Marker311414分子标记的准确性、稳定性、重复性进行检测，具体如下：
用得到的2个标记Marker301243和Marker311414对F 1群体进行AS-PCR扩增验证，标记的鉴定结果见图3和图10，Marker301243在F 1群体中准确率最高为91.67％，且在直枝个体中准确率高达100％。同时利用标记组合Marker301243+Marker311414进行表型鉴定时，在F 1群体中成功率高达100％(图12)。由此可见，开发的SNP标记可以用于梅花F 1群体及品种中垂枝性状的辅助选择育种中。
获得的特异性分子标记是否稳定对其应用也是非常重要的，通过相同引物对F 1群体和梅花品种进行扩增，如果能获得稳定的条带，说明开发 的标记是稳定的，可用于梅花不同株型的鉴定。以梅花品种为材料，验证所开发的SNP标记的稳定性及准确率。以Marker301243为例，其在直枝个体扩出两条带，为AT等位基因型，在垂枝个体中扩出一条带，为TT等位基因型，说明2个标记在梅花品种均能较好地鉴定不同的表型，具有很好的稳定性(图3)。进一步地，分别在梅花垂枝和直枝品种中验证Marker301243和Marker311414分子标记的特异性和稳定性。对于两个分子标记的等位基因型，可能会出现3种基因型情况(Marker301243为AA，TT和AT；Marker311414为AA(TT)，GG(CC)和AG(CT))，而使用一对引物(一条特异性引物与一条公共引物)扩增时(图4-图9)，则只有与所用特异性引物配对的AA或AT基因型个体可以被扩增出条带，TT则无法扩增出条带(以Marker301243为例)。图4为Marker301243在垂枝品种中的检测结果，其中，无条带的为TT基因型的垂枝品种；图6为Marker311414的在垂枝品种中的检测结果，无带的为GG(CC)基因型垂枝品种。图8为Marker301243/Marker311414相结合，二者结合对应基因型可表示为TT/CC(无带/无带)，A_/CC(有带/无带)，TT/_T(无带/有带)。从图8可以看出，大部分垂枝个体为纯合型，表现为TT/CC基因型组合，呈现无条带。同理，而直枝品种中SNP位点的优势基因型为AT，特异引物均能扩增出条带。图5为Marker30124在直枝品种中的检测结果，由图可知大部分直枝个体均有条带，表现为AT基因型；图7为Marker311414在直枝品种中的检测结果，多表现为有条带，为CT基因型。图9为Marker301243/Marker311414相结合，二者结合对应基因型可表示为A_/_T(有带/有带)，A_/CC(有带/无带)，TT/_T(无带/有带)，TT/CC(无带/无带)。因此，通过统计Marker301243/Marker311414特异的带型，即可对获得相应的鉴定结果。从图9可以看出，大部分直枝个体为杂合型，表现为AT/CT基因型组合，呈现两条带，少数出现与垂枝性状相同的基因型，表现为一条带或无带(如图9个体18)。可见分子标记Marker301243和Marker311414在梅花品种中AS-PCR的扩增具有较高的 稳定性和可靠性(图4-图9)。Marker301243在F1群体中鉴定的准确率显著高于梅花品种，Marker311414则反之(图11)。利用标记组合Marker301243+Marker311414进行表型鉴定时，在梅花品种中成功率为89.13％，且在梅花垂枝品种中达100％(图12)。
其中，图3为标记Marker301243和Marker311414在F 1群体中AS-PCR的扩增结果。图4-图9为标记Marker301243和Marker311414在梅花品种中AS-PCR的扩增稳定性。
本发明建立的分子标记辅助选择育种可以实现苗期提早选择、减少工作量，提高了梅花垂枝性状的选择效率，大大缩短了育种周期。
虽然上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供梅花垂枝性状SNP分子标记及其应用。本发明提供的梅花垂枝性状SNP分子标记具体包括分子标记Marker301243和/或Marker311414，其核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。本发明利用SLAF-seq技术开发获得2个梅花垂枝性状SNP分子标记Marker301243和Marker311414，并提供了其序列扩增引物和等位特异性PCR引物。所述分子标记Marker301243和/或Marker311414及其引物在鉴定梅花品种时，具有较高的准确性、稳定性且重复性好。将其用于分子辅助选择育种，可实现苗期提早选择，快速鉴定梅花垂枝性状，减少工作量，大大缩短梅花育种时间，具有较好的经济价值和应用前景。

Claims (10)

  1. 梅花垂枝性状SNP分子标记，其特征在于，包括分子标记Marker301243和/或Marker311414，它们的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
  2. 用于扩增权利要求1所述分子标记的特异性PCR引物，其特征在于，
    标记Marker301243引物序列为：
    正向引物：5′-TGAGAATGGACAATGAGCGT-3′，
    反向引物：5′-CTCTGCTGGACACCCCTAAT-3′；
    标记Marker311414引物序列为：
    正向引物：5′-CTTAGGGAATGGTGTCGCTT-3′，
    反向引物：5′-AGAGCAGGCACCCAAGTAAGT-3′。
  3. 权利要求1所述分子标记在鉴定梅花垂枝性状中的应用，其特征在于，包括以下步骤：
    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，利用权利要求1所述分子标记的引物，进行PCR扩增反应；
    3)对应于标记Marker301243的SNP位点，位于SEQ ID NO.1所示序列的第426位碱基，该处碱基为T或A，梅花垂枝的优势等位基因型为TT，直枝的优势等位基因型为AT；对应于标记Marker311414的SNP位点，位于SEQ ID NO.2所示序列的第607位碱基，该处碱基为G或A，梅花垂枝的优势等位基因型为GG，直枝的优势等位基因型为GA。
  4. 根据权利要求3所述的应用，其特征在于，步骤2)中的PCR扩增体系为20μL，包括：50ng/μL模板DNA 2μL，2×PCR Master Mix 10μL，10μmol/L正反向引物各1μL，ddH 2O 6μL。
  5. 用于AS-PCR扩增权利要求1分子标记的AS-PCR引物，其特征在于，
    标记Marker301243引物序列为：
    正向引物：5′-TGGAAACTGAATAGATGCGAT-3′，
    反向公共引物：5′-GGTGAAAGAGACATCAGAAAAT-3′，
    正向特异性引物：5′-TGGAAACTGAATAGATGCGAA-3′；
    标记Marker311414引物序列为：
    正向公共引物：5′-CATCTAAAATAAAATCTCAAAGG-3′，
    反向引物：5′-TCATAGGTATTCTTGTCTTTCTC-3′，
    反向特异性引物：5′-TCATAGGTATTCTTGTCTTTCTT-3′。
  6. 一种筛选或鉴定梅花垂枝性状的方法，其特征在于，包括以下步骤：
    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，利用权利要求1所述分子标记的AS-PCR引物，进行AS-PCR扩增；
    3)检测PCR扩增产物，根据AS-PCR扩增产物条带的有无，判断对应的SNP位点，其中，对应于标记Marker301243的SNP位点，梅花垂枝的优势等位基因型为TT，直枝的优势等位基因型为AT；对应于标记Marker311414的SNP位点，梅花垂枝的优势等位基因型为GG，直枝的优势等位基因型为GA。
  7. 根据权利要求6所述的方法，其特征在于，步骤2)中AS-PCR扩增体系为10μL，包括：50ng/μL模板DNA 1μL，2×PCR Master Mix 5μL，10μmol/L正反向引物各0.5μL，ddH 2O 3μL。
  8. 含有权利要求2和/或权利要求5所述引物的用于筛选或鉴定梅花垂枝性状的检测试剂盒。
  9. 根据权利要求8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反应缓冲液和标准阳性模板中的一种或多种。
  10. 权利要求1所述分子标记和/或权利要求2或权利要求5所述引物在梅花分子标记辅助育种中的应用。
PCT/CN2018/121985 2018-05-03 2018-12-19 梅花垂枝性状snp分子标记及其应用 WO2019210696A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP18917319.8A EP3789506B1 (en) 2018-05-03 2018-12-19 Prunus mume pendulous trait snp molecular markers and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810415993.7 2018-05-03
CN201810415993.7A CN108715902B (zh) 2018-05-03 2018-05-03 梅花垂枝性状snp分子标记及其应用

Publications (1)

Publication Number Publication Date
WO2019210696A1 true WO2019210696A1 (zh) 2019-11-07

Family

ID=63899646

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/121985 WO2019210696A1 (zh) 2018-05-03 2018-12-19 梅花垂枝性状snp分子标记及其应用

Country Status (3)

Country Link
EP (1) EP3789506B1 (zh)
CN (1) CN108715902B (zh)
WO (1) WO2019210696A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112575104A (zh) * 2020-12-11 2021-03-30 黑龙江省科学院大庆分院 一种工业***性状相关基因快速定位方法
CN112725515A (zh) * 2021-02-23 2021-04-30 广东省农业科学院环境园艺研究所 一种蝴蝶兰花底色snp分子标记引物组合物及其应用
CN113930535A (zh) * 2021-10-11 2022-01-14 广东工业大学 一种梅片树ssr分子标记、引物、试剂盒及开发方法与应用
CN114457178A (zh) * 2021-12-07 2022-05-10 浙江省农业科学院 用于预测杨梅果实可溶性固形物的kasp分子标记及其应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108715902B (zh) * 2018-05-03 2020-09-25 北京林业大学 梅花垂枝性状snp分子标记及其应用
CN109554493B (zh) * 2018-12-10 2021-09-17 北京林业大学 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
CN109680090A (zh) * 2018-12-21 2019-04-26 中国计量大学 一种鉴定茭白表型特征的分子标记及其应用、获取方法
CN110343749A (zh) * 2019-08-14 2019-10-18 苏州泓迅生物科技股份有限公司 一种检测pon1基因snp位点的引物组、引物组的设计方法及其应用
CN110819732B (zh) * 2019-10-29 2022-09-02 北京林业大学 梅花垂枝性状紧密连锁的纯合snp分子标记及其检测方法与应用
CN110698550B (zh) * 2019-11-11 2021-07-06 北京林业大学 一种快速鉴定真梅/杏梅品系的分子检测方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103184277A (zh) * 2011-12-30 2013-07-03 北京林业大学 梅花遗传图谱构建方法
CN108715902A (zh) * 2018-05-03 2018-10-30 北京林业大学 梅花垂枝性状snp分子标记及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103184277A (zh) * 2011-12-30 2013-07-03 北京林业大学 梅花遗传图谱构建方法
CN108715902A (zh) * 2018-05-03 2018-10-30 北京林业大学 梅花垂枝性状snp分子标记及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN JUNYU: "China Mei Flower Cultivars", 2010, CHINA FORESTRY PUBLISHING HOUSE
ZHANG JIE: "Prunus mume", 2016, BEIJING FORESTRY UNIVERSITY, article "Construction of High-Density Genetic Map and QTL Analysis for Some Ornamental Traits"
ZHANG, . JIE: "Construction of . . High-Density Genetic Map and QTL Analysis of Ornametal Traits in Mei", CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE (AGRICULTURE SCIENCE AND TECHNOLOGY), 15 August 2016 (2016-08-15), pages 1 - 146, XP009524795 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112575104A (zh) * 2020-12-11 2021-03-30 黑龙江省科学院大庆分院 一种工业***性状相关基因快速定位方法
CN112725515A (zh) * 2021-02-23 2021-04-30 广东省农业科学院环境园艺研究所 一种蝴蝶兰花底色snp分子标记引物组合物及其应用
CN112725515B (zh) * 2021-02-23 2021-09-28 广东省农业科学院环境园艺研究所 一种蝴蝶兰花底色snp分子标记引物组合物及其应用
CN113930535A (zh) * 2021-10-11 2022-01-14 广东工业大学 一种梅片树ssr分子标记、引物、试剂盒及开发方法与应用
CN113930535B (zh) * 2021-10-11 2023-06-23 广东工业大学 一种梅片树ssr分子标记、引物、试剂盒及开发方法与应用
CN114457178A (zh) * 2021-12-07 2022-05-10 浙江省农业科学院 用于预测杨梅果实可溶性固形物的kasp分子标记及其应用
CN114457178B (zh) * 2021-12-07 2023-08-08 浙江省农业科学院 用于预测杨梅果实可溶性固形物的kasp分子标记及其应用

Also Published As

Publication number Publication date
CN108715902B (zh) 2020-09-25
EP3789506A1 (en) 2021-03-10
EP3789506C0 (en) 2023-10-18
EP3789506A4 (en) 2022-03-02
EP3789506B1 (en) 2023-10-18
CN108715902A (zh) 2018-10-30

Similar Documents

Publication Publication Date Title
WO2019210696A1 (zh) 梅花垂枝性状snp分子标记及其应用
Balsalobre et al. GBS-based single dosage markers for linkage and QTL mapping allow gene mining for yield-related traits in sugarcane
CN109554493B (zh) 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
CN108165653B (zh) 用于鉴定辣椒熟性的InDel分子标记及其应用
Katula-Debreceni et al. Marker-assisted selection for two dominant powdery mildew resistance genes introgressed into a hybrid grape population
CN105274094B (zh) Snp标记及其应用
Ueno et al. Scanning RNA-Seq and RAD-Seq approach to develop SNP markers closely linked to MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don
CN110499389B (zh) 与烟草抗斑萎病位点rtsw紧密连锁的共显性标记引物、鉴别方法及其应用
CN106498068B (zh) 一种与烟草tmv抗性基因n紧密连锁的共显性ssr标记及其应用
CN108642201B (zh) 与谷子株高性状相关的snp标记及其检测引物和应用
WO2017088144A1 (zh) 用于白菜种质资源多样性分析及分子育种的snp组合及其应用
CN112011640B (zh) 用于鉴定西瓜果实pH的KASP分子标记、引物及应用
CN110551843B (zh) 可区分烟草抗斑萎病位点rtsw纯合杂合基因型的共显性标记引物、区分方法及其应用
Akai et al. De novo genome assembly of the partial homozygous dihaploid potato identified PVY resistance gene (Rychc) derived from Solanum chacoense
CN110499390B (zh) 用于烟草抗斑萎病rtsw基因辅助选择的分子标记引物、辅助选择的方法及其应用
CN109554494B (zh) 水稻抗褐飞虱bph9复等位基因的通用共显性分子标记及其检测方法和应用
CN105543366B (zh) 水稻抗稻瘟病基因Pi25基因内特异SNP共显性分子标记开发及应用
CN107619875B (zh) 一种用于鉴定西瓜果实形状的***缺失标记位点、引物及应用
CN113249509A (zh) 用于美洲黑杨和小叶杨种间杂交子代的鉴别引物和鉴定方法
CN113832251B (zh) 用于检测番茄花叶病毒病抗性的snp位点组合及其应用
CN112921112B (zh) 鉴定万寿菊瓣型的caps分子标记、检测引物及检测试剂盒
CN110819732B (zh) 梅花垂枝性状紧密连锁的纯合snp分子标记及其检测方法与应用
CN110499388B (zh) 鉴别烟草抗斑萎病位点rtsw等位基因类型的共显性标记引物组、鉴别方法及其应用
CN109652579B (zh) 水稻抗稻瘟病基因Pi2的共显性分子标记及其检测方法与应用
CN108715901B (zh) 一种与谷子株高性状相关的snp标记及其检测引物和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18917319

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2018917319

Country of ref document: EP

Effective date: 20201203