WO2017092110A1 - 芝麻花序有限基因Sidt1及其SNP标记 - Google Patents

芝麻花序有限基因Sidt1及其SNP标记 Download PDF

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WO2017092110A1
WO2017092110A1 PCT/CN2015/099095 CN2015099095W WO2017092110A1 WO 2017092110 A1 WO2017092110 A1 WO 2017092110A1 CN 2015099095 W CN2015099095 W CN 2015099095W WO 2017092110 A1 WO2017092110 A1 WO 2017092110A1
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inflorescence
sesame
gene
limited
sidt1
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French (fr)
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张海洋
苗红梅
李春
魏利斌
段迎辉
徐芳芳
王慧丽
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河南省农业科学院芝麻研究中心
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Priority to US15/499,863 priority Critical patent/US10301687B2/en
Publication of WO2017092110A1 publication Critical patent/WO2017092110A1/zh

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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/66—Pedaliaceae, e.g. sesame
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention belongs to the technical field of molecular genetic breeding, and particularly relates to a sesame inflorescence limited gene Sidt1 and a SNP marker SiDt27-1 thereof.
  • Sesame is a crop with infinite flowering and short growth period.
  • the growth period of Chinese sesame varieties is 85-95 days.
  • the performance of the plant growth and development process mainly includes: the inflorescence of the plant is infinite, and the flowering period is long, about 25-35 days.
  • the flowering period is long, about 25-35 days.
  • the mutants mainly include the following aspects in the field: (1) Single-rod, one-leaf and three-flowered, four-sided, four-sided, lower-winged leaves, dark green, hypertrophic, high chlorophyll content, light and efficient; (2) Inflorescence is limited, the plant has a typical limited growth habit, the plant height is 130-150 cm; in the Huanghuai District, after the growth and development of the variety forms 15-20 nodes, the growth point of the top of the plant stops growing and caps automatically; the top of the cap is formed after capping 4-6 capsules; this property is controlled by a recessive single gene, which can be stably inherited, and the limited habits of the breeding progeny reach over 98%; (3) the growth and development speed of the line is fast, the bud is early, and the flowering period is 20-25 days.
  • the variety has high resistance to stem blight and blight; suitable for close planting and mechanized management and harvest.
  • the sesame germplasm resource bank In addition to Yuzhi DS899, in the current world sesame germplasm resource bank, another type of inflorescence-limited sesame germplasm is also preserved (the sesame germplasm resource stock number of Henan Academy of Agricultural Sciences Sesame Research Center is 08TP092).
  • the field traits of this germplasm are mainly The performance is as follows: the plant is branched, the inflorescence is limited, the number of nodes in the capsule is 2-3 knots, the number of capsules per plant is 30-40, the knotty is low, and it is not suitable for direct production.
  • the limited traits of this germplasm are subject to single hidden Sexual gene control.
  • the main object of the present invention is to provide a gene sequence of a sesame inflorescence limited gene Sidt1, and to provide a cDNA sequence of the limited gene Sidt1 gene sequence; another object of the present invention is to provide a SNP marker SiDt27-1 for a limited gene Sidt1 gene sequence. In order to facilitate the screening and cultivation of new varieties.
  • the sesame inflorescence finite gene Sidt1 which has 1809 bp, contains 4 exons and 3 introns, is located on the 4th chromosome of sesame, and is located in the 8th linkage group of the sesame SNP genetic map, 18.0-19.2cM
  • the interpretation rate of the inflorescence limited trait is 100% (Vg/Vp); the DNA sequence thereof is shown in SEQ ID NO.
  • the cDNA sequence of the sesame inflorescence finite gene Sidt1 is 531 bp in length and encodes 176 amino acids, as shown in SEQ ID.
  • the SNP molecular marker SiDt27-1 of the sesame inflorescence limited gene Sidt1 has 92 bp, which is the base sequence of the 378th to 469th in the inflorescence limited gene Sidt1, and the specific sequence is:
  • the method for detecting the sesame inflorescence limited gene Sidt1 by using the SNP molecular marker SiDt27-1 comprises the following steps:
  • Reverse primer HSDt01-R sequence 5'TAAATCAATCAGGTCTTAAGCAGT 3';
  • the PCR amplification product was subjected to gel electrophoresis to determine whether the genomic DNA of the sesame germplasm resource to be tested contained the SNP molecular marker SiDt27-1, and the judgment rule was as follows:
  • the SNP molecular marker SiDt27-1 marker site is not included, when the primer HSDt01-2F is paired with HSDt01-R for amplification, a product with a band size of 97 bp can be amplified, and the test germplasm belongs to the dt0 type. Inflorescence is infinite, can not be used to cultivate new varieties of limited sesame inflorescence;
  • the tested germplasm is heterozygous and the inflorescence is infinite, but the progeny can be separated in shape, and the inflorescence finite material can be further obtained from the progeny.
  • the progeny can be separated in shape, and the inflorescence finite material can be further obtained from the progeny.
  • the genomic DNA extracted in the step (1) can be used as a template to PCR-amplify a specific sequence, and when PCR is amplified, the following primer sequences are used:
  • the forward primer Dt1Primer F sequence is: 5'-ATGGCAAAAATGTCATCGGACC-3';
  • the reverse primer Dt1Primer R sequence is: 5'-CTAGCGCCTTCTAGCAGCAGTC-3';
  • the PCR amplification product is further sequenced and compared with the gene sequence of the inflorescence limited gene Sidt1 (ie, the sequence in SEQ ID NO. 1). If they are the same, the tested germplasm belongs to a limited inflorescence and can be used for breeding new ones. Sesame varieties.
  • the inventors configured a hybrid combination of Yuzhi DS899 (single shot, 15-20 nodes) and 08TP092 (branch, 2-3 nodes) and investigated the characteristics of F1 and F2 generations.
  • the results showed that the F1 progeny showed limited inflorescence and 3-5 nodes; F2 progeny showed limited inflorescence and 3-15 nodes.
  • the inventors believe that in the two inflorescence-limited sesame materials of Yuzhi DS899 and 08TP092, the inflorescence limited traits are controlled by the same gene locus, but the mutant traits have some differences. Therefore, it is speculated that the two inflorescence-limited germplasm may have some differences in the sequence of the inflorescence limited gene.
  • the inflorescence-limited genes in the two germplasm were named Sidt1 (derived from Yuzhi DS899) and Sidt2 (from 08TP092).
  • the mutants of Yuzhi DS899 and 08TP092 are dt1 type and dt2 type, respectively, and the inflorescence infinite type normal strain is dt0 type.
  • the inventors identified a mutated gene locus with limited sesame inflorescence, which laid a solid foundation for the next step to explore the regulation mechanism of sesame inflorescence development and the selection of new sesame varieties with dt1 inflorescence, and promoted the molecular breeding of sesame seeds.
  • the development of research The development of research.
  • the present invention aims to develop and apply molecular markers for the obtained sesame inflorescence limited gene Sidt1, and provides technical support for accelerating the genetic basis of sesame importance and breeding of new varieties.
  • the present invention carried out genome re-sequencing and genetic map construction of sesame germplasm resources for the inflorescence-limited sesame mutant Yuzhi DS899, and cloned a sesame inflorescence limited gene Sidt1, and developed its SNP marker SiDt27-1.
  • a PCR identification method for identifying whether the gene and the marker site are contained in the germplasm resource is provided, and the method can preliminarily determine the inflorescence type of the sesame variety to be tested, and provide a reference for the cultivation of the new variety;
  • the detection method of the sesame inflorescence finite gene Sidt1 and the SNP marker SiDt27-1 provided by the invention has mature technology and good stability of the detection result, and is important for improving the efficiency of sesame breeding and improving the research level of sesame genetic breeding in China. significance.
  • the present invention first discovered the sesame inflorescence limited gene Sidt1;
  • the present invention developed the SNP marker SiDt27-1 of the sesame inflorescence limited gene Sidt1, and provided genetic information for analyzing the flower development mechanism of sesame seeds and the like;
  • the SNP molecular marker SiDt27-1 provided by the invention provides an important molecular marker for the establishment of sesame molecular assisted breeding technology, and provides a rapid detection method for screening new sesame varieties with limited inflorescence.
  • Figure 1 shows the field control of Yuzhi DS899 (dt1 type, inflorescence limited, right side) and parent Yuzhi 11 (dt0 type, inflorescence infinite, left side);
  • Figure 4 is a result of PCR amplification of primers pair HSDt01-1F, HSDt01-2F and HSDt01-R of an inflorescence limited SNP marker SiDt27-1 in a part of sesame germplasm of the present invention, wherein: lane M is DL 2000 marker, the displayed strip The bands are 750 bp, 500 bp, 250 bp and 100 bp from top to bottom; lane 1 is Yuzhi 11 material (infinite type) containing Sidt1 allele 2 (ie dt0 type); lane 2 is containing Sidt1 allele 1 (ie dt1 type) material (limited type); Lane 3 is F 1 intermediate hybrid type material of Yuzhi 11 ⁇ Yuzhi DS899 combination; Lane 4 is a PCR amplification control using water as template;
  • Figure 5 is a diagram showing the gene structure of the sesame inflorescence limited gene Sidt1 of the present invention.
  • Figure 6 is a sequence alignment of the sesame inflorescence finite gene Sidt1 of the present invention and the normal strain allele SiDt (the sesame genome is annotated as Sitfl); wherein: the arrow indicates the nucleotide sequence difference between the Sidt1 gene and the normal allele SiDt. Point; the upper row is the nucleotide sequence of the normal allele SiDt, and the lower row is the nucleotide sequence of the Sidt1 gene;
  • FIG. 7 is a result of alignment of a SiDt acid coding sequence of a sesame inflorescence finite gene Sidt1 and a normal strain allele according to the present invention; wherein: an arrow indicates an amino acid sequence difference position between the Sidt1 protein and the normal gene SiDt protein;
  • Figure 8 is a PCR verification of primer pairs HSDt01-1F, HSDt01-2F and HSDt01-R of SNP-tagged SiDt27-1 in the F 2 population of the present invention; wherein the lane M is DL 2000 marker, the bands are displayed from top to bottom respectively 200 bp and 100 bp; lanes 1-11 are Sidt1 homozygous (inflorescence limited) F 2 plants; lanes 12, 13, 19, 20 and 22 are SiDt homozygous (infinite inflorescence) F 2 plants; lanes 14-18 and 21 is a Sidt1/SiDt heterozygous (infinite inflorescence) F 2 plant;
  • Figure 9 is a result of Southern blot hybridization of the sesame inflorescence limited gene Sidt1 of the present invention and the normal strain allele SiDt; wherein: lane M is Marker; lane 1: positive plasmid hybrid blot containing the normal allele SiDt probe sequence;
  • Lanes 2-4 from left to right are HindIII-cut Yuzhi 11 (wild type infinity, dt0), Yuzhi DS899 (dt1 type limited) and S.raditum wild seed sesame (dt0 type, infinity) genomic DNA Hybridization results; lanes 5-7 from left to right were respectively HindIII and EcoRI double-cut Yuzhi No. 11 (wild type infinity, dt0), Yuzhi Ds899 (dt1 type limited) and S.raditum wild seed sesame (dt0 type) , infinite) hybridization results of genomic DNA;
  • Figure 10 is a diagram showing the application of the SNP-tagged SiDt27-1 of the present invention in the selection of breeding materials; wherein: lane M is DL2000Marker, lanes 1-20 are Sidt1 homozygous (inflorescence limited) F 2 plants; lanes 21, 29, 30, 33, 35, 37, 38 and 40 are SiDt homozygous (infinite inflorescence) F 2 plants; lanes 22-28, 31, 32, 34, 36 and 39 are Sidt1/SiDt heterozygous (infinite inflorescence) F 2 plants .
  • lane M is DL2000Marker
  • lanes 1-20 are Sidt1 homozygous (inflorescence limited) F 2 plants
  • lanes 21, 29, 30, 33, 35, 37, 38 and 40 are SiDt homozygous (infinite inflorescence) F 2 plants
  • lanes 22-28, 31, 32, 34, 36 and 39 are Sidt1/SiDt heterozygous (infinite in
  • the sesame variety Yuzhi No. 11 is an important parent material for sesame breeding in China.
  • the limited new sesame inflorescence line Yuzhi DS899 is a new variety selected from Yuzhi No. 11 by the Sesame Research Center of Henan Academy of Agricultural Sciences using EMS mutagenesis technology.
  • the company applied for the protection of the national new variety right (application number: 20150395.2), and participated in the 2015 provincial sesame new variety regional test identification. Its main features are: inflorescence is limited, the average node is 15-20, each flower is 3 flowers, single pole, capsules, and white grains.
  • the sesame resource JS012 used in this application comes from the germplasm resource bank of the Sesame Research Center of Henan Academy of Agricultural Sciences.
  • the main features are: inflorescences infinite, single flowers per branch, branches, capsules, and black grains.
  • the other sesame germplasm resources such as 08TP092, Wuning Black, Zhengzhi 98N09, and wild species involved in the examples are all from the germplasm resources of the Sesame Research Center of Henan Academy of Agricultural Sciences; these germplasm materials can be obtained from public sources. (Or directly obtained from the Germ Resource Library of the Sesame Research Center of Henan Academy of Agricultural Sciences) and planted and applied.
  • PCR reaction in the following examples was carried out using a PTC-100 (MJ research product) thermal cycler; the related enzymes, buffers and other reagent products in the PCR reaction were purchased from Shanghai Shenggong Reagent Co., Ltd.; Primers were synthesized by Huada Gene Company.
  • the DNA extraction of the plants in the PCR reaction was carried out according to the modified CTAB method of Wei Libin et al. (Synthesis of Sesame DNA and RNA, 2008, Molecular Plant Breeding).
  • the related gene sequencing in the following examples was performed by Tianjin GeneChip Biotechnology.
  • This embodiment mainly introduces the screening process of the sesame inflorescence limited gene Sidt1 and its SNP molecular marker SiDt27-1.
  • the specific process is as follows.
  • Yuhuai DS899 and Yuhuan No.11, JS102 and Wuning Black for three inflorescences of infinite germplasm for combination of positive and negative crosses (the specific combination configuration is shown in the following table) And conduct an investigation of the inflorescence type of F1 offspring.
  • the combination configuration of Yuzhi DS899 and Yuzhi No.11, JS102 and Wuning Black is:
  • the F 1 survey showed that F 1 is a normal infinite inflorescence, indicating that the mutant trait (limited inflorescence) is controlled by recessive genes. Subsequently, the three orthogonal F 2 progeny populations of Yuzhi DS899 and Yuzhi No.11, JS102 and Wuning Black were additionally planted, each group was greater than 200 plants. A field trait survey was conducted during the flowering period, and the results are shown in the table below.
  • the genetic linkage map constructed is shown in Figure 2.
  • the map has 13 linkage groups, including 3101 bin segments, 124,000 markers, and the total length of the map is 1872.2 cM, with an average of 144.0 cM/linkage group and 0.015 cM/marker interval.
  • the PCR sequence amplification method can be used to amplify the relevant sequence and further sequenced, and the related process is introduced as follows.
  • the gene of the SiDt27-1 locus was identified using Yuji No. 11 genomic data, and the gene sequence was named Sidt1. Analysis of the sequence of Sidt1 revealed that the gene was annotated as the TFL gene (SiDt) in the sesame genome (Yuzhi 11).
  • Primer premier 5.0 software was used to design primer pairs for amplifying the sesame inflorescence limited gene Sidt1.
  • the specific design is as follows:
  • the forward primer Dt1Primer F sequence is: 5'-ATGGCAAAAATGTCATCGGACC-3';
  • the reverse primer Dt1Primer R sequence is: 5'-CTAGCGCCTTCTAGCAGCAGTC-3'.
  • the PCR reaction procedure was: predenaturation at 94 ° C for 3 minutes; then denaturation at 94 ° C for 30 seconds, renaturation at 55 ° C for 30 seconds, extension at 72 ° C for 1 minute, 30 cycles; and finally extension at 72 ° C for 5 minutes.
  • the PCR amplification product was stored at 4 ° C for use, or the amplified band was directly recovered and sequenced.
  • the genomic DNA of Yuzhi 11 was amplified by the above primer pair, and the amplified product was sequenced.
  • the inflorescence-limited Sidt1 gene was sequence-aligned with the allele SiDt of the normal strain (ie, Yuzhi No. 11), and the results are shown in Fig. 6. As can be seen from Figure 6, the differences in the Sidt1 sequence are only in the inflorescence-infinite and inflorescence-limited germplasm genomes. Whether a G/A mutation has occurred in the 236th base of the coding region. When the inflorescence type is mutated from infinite to dt1 finite, the base G at position 236 of the coding region is mutated to base A, resulting in the mutation of the 79th amino acid sequence of the encoded protein from serine (S) to asparagine (N). ) ( Figure 7).
  • RNA of the young plant of Yuzhi DS899 was extracted, and the total cDNA was obtained by reverse transcription using TaKaRa RNA reverse transcription kit (TaKaRa); the relevant operation reference reverse transcription kit instruction manual Just do it;
  • the primer pair (designed by Primer Premier 5.0) used in the PCR reaction is designed as follows:
  • TFL1CDs-F (5'-ATGGCAAAAAATGTCATCGGACC-3'),
  • TFL1CDs-R (5'-CTAGCGCCTTCTAGCAGCAGTC-3');
  • the cDNA coding region of Sidt1 was amplified by PCR, and the cDNA sequence of Sidt1 gene was obtained by sequencing the PCR product; the cDNA sequence of the gene was 531 bp, encoding a total of 176 amino acids, and the sequence is shown in SEQ ID 2.
  • the primer design method is carried out in accordance with Wei et al. (Development of SNP and InDel markers via de novo transcriptome assembly in Sesamum indicum L., 2014, Molecular Breeding). In addition, it is necessary to distinguish the genomic DNA to be detected. In the different SNP alleles, researchers usually need to pay attention to the following two aspects:
  • the SNP primer pair was designed into three, and a mismatch was introduced at the 3rd position of the 3' end of the specific primer to increase the specificity of the amplification product; the principle of introducing the mismatch was: the 3' end of the primer
  • the mismatched base of the 3rd mismatch base and the SNP of the 3' end can form a stable complementary mismatch structure, ie, a strong mismatch (C/T or G/A) and a weak mismatch (C/A). Or G/T), medium mismatch (A/A, C/C, G/G or T/T) with medium mismatch type;
  • the 5' end of one of the primer sequences is randomly increased by 5 bases, the main purpose of which is to further condense the PCR products at different sites.
  • Gel electrophoresis maps can be distinguished more easily.
  • Reverse primer HSDt01-R sequence 5'TAAATCAATCAGGTCTTAAGCAGT 3';
  • the amplification product band is large.
  • the small size is 92 bp (dt1 type); when the forward primer HSDt01-2F is paired with the reverse primer HSDt01-R, the amplified product band size is 97 bp (dt0 type).
  • the Sidt1 gene is a gene that regulates the type of sesame inflorescence.
  • the F 2 population was specifically selected: 50 strains were randomly selected from the F 2 population constructed by Yuzhi DS899 and Yuzhi No. 11, JS102 and Wuning Black, respectively, and the inflorescence types of each strain were investigated in the field.
  • the PCR reaction uses a 10 ⁇ L reaction system and is set as follows:
  • Taqase enzyme (5 U/ ⁇ L), 0.2 ⁇ L;
  • the PCR reaction procedure was: pre-denaturation at 94 ° C for 3 minutes, followed by denaturation at 94 ° C for 30 seconds, renaturation at 55 ° C for 30 seconds, extension at 72 ° C for 30 seconds, 30 cycles, and finally extension at 72 ° C for 5 minutes.
  • the amplified product was stored at 4 ° C. .
  • the PCR product was analyzed by non-denaturing polyacrylamide gel electrophoresis, the gel concentration was 8-10%, the gel size was 180 mm ⁇ 120 mm ⁇ 2 mm, the electrophoresis buffer was 0.5 ⁇ TBE, and the 150 V constant voltage AC electrophoresis was 1.5 to 2 hours.
  • a silver nitrate aqueous solution with a concentration of 0.1% was added to the gel, and silver staining was performed on a horizontal shaker for 10 min; then a 2% sodium hydroxide and 0.4% formaldehyde mixed solution was added, and the color was moderately developed in a horizontal shaker. Finally, rinse the gel with water and record the reading data.
  • Sidt1 gene is a gene that causes the selfactory mutation of sesame inflorescence, which can be used to study the mechanism of flower inflorescence development of sesame and other crops.
  • Primer Premier 5.0 software was used to design primer pairs based on the sesame variety "Yuzhi 11" genomic DNA and SiDt sequence, as follows:
  • Dt-Gs forward primer 5'-GAGCCCTCTTTCAAAAACACC-3'
  • Dt-Gs reverse primer 5'-AGCAGCAACAGGGAGACCTA-3'
  • the genomic DNA of the sesame variety "Yuzhi 11" was used as a template, and the above primers were used for PCR amplification of Dt-Gs.
  • the PCR product was 459 bp in size.
  • Taqase enzyme (5 U/ ⁇ L), 0.5 ⁇ L;
  • the PCR reaction procedure was: pre-denaturation at 94 ° C for 3 minutes; denaturation at 94 ° C for 30 seconds, renaturation at 55 ° C for 30 seconds, extension at 72 ° C for 30 seconds, 30 cycles; final extension at 72 ° C for 5 minutes; storage at 4 ° C.
  • the PCR product was confirmed by 1% agarose gel electrophoresis and sequenced.
  • the PCR product was used as a Southern hybridization probe, and the probe was labeled and detected by DIG-High Prime DNA Labeling & Detection Starter Kit I (Roche Reagent); the specific procedure was carried out according to the instructions of DIG-High Prime DNA Labeling & Detection Starter Kit I. .
  • the probe fragment described in the step (1) was ligated into the pGEM-T Easy vector, and the pGEM-T Easy plasmid carrying the probe sequence was constructed for the Southern hybridization positive control;
  • the steps of ligation, recovery, purification, etc. are carried out by using a linking reagent and a recovery kit (the related products are all Promega products); the quality of the plasmid is detected by 0.8% agarose gel electrophoresis;
  • the pGEM-T Easy plasmid with the probe sequence was extracted with a DNA extraction kit (TaKaRa) and diluted to 1-5 ⁇ g/L for the next Southern hybridization; the specific steps were referenced to the DNA extraction kit. Bright.
  • the digested DNA product was subjected to 0.7% agarose gel electrophoresis, and subjected to constant voltage at 25 V and low temperature at 4 ° C overnight. Then, the membrane was transferred and hybridized, and then washed, and the results of Southern hybridization were recorded (the hybridization results are shown in Fig. 9). Show);
  • the selection process of limited-type high-yield and high-quality breeding materials was introduced, and the application process of SNP molecular marker SiDt27-1 of sessile inflorescence limited gene Sidt1 was introduced.
  • the specific process is as follows.
  • the PCR reaction uses a 10 ⁇ L reaction system and is set as follows:
  • Taqase enzyme (5 U/ ⁇ L), 0.2 ⁇ L;
  • the PCR reaction procedure was: predenaturation at 94 ° C for 3 minutes, followed by denaturation at 94 ° C for 30 seconds, renaturation at 55 ° C for 30 seconds, extension at 72 ° C for 30 seconds, 30 cycles, and finally 72 ° C for 5 minutes.
  • the reaction product was stored at 4 ° C until use.
  • the PCR product in step (2) was analyzed by non-denaturing polyacrylamide gel electrophoresis, the gel concentration was 8-10%, the gel size was 180 mm ⁇ 120 mm ⁇ 2 mm, the electrophoresis buffer was 0.5 ⁇ TBE, and the 150V constant voltage AC electrophoresis. 1.5 ⁇ 2 hours; after the electrophoresis is finished, a silver nitrate aqueous solution with a concentration of 0.1% is added to the gel, and it is placed on a horizontal shaker for silver staining for 10 min; then a 2% sodium hydroxide and 0.4% formaldehyde mixed solution is added and placed in a horizontal shake. Appropriate color development in the bed; finally rinse the gel with water and record the data.
  • the SNP marker is a SNP locus marker of sesame inflorescence, which can be used to predict the inflorescence type of sesame varieties, and is used for the breeding of sesame molecular marker-assisted breeding and new inflorescence-limited sesame varieties.

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Abstract

提供一种芝麻花序有限基因 SidtI及其SNP标记 SiDt27-1。芝麻花序有限基因 SidtI有1809bp，包含4个外显子和3个内含子，位于芝麻第4条染色体上，在芝麻SNP遗传图谱中的位置为第8连锁群，18.0－19.2cM，对花序有限性状的解释率为100％（Vg/Vp）；其DNA序列如SEQ ID NO.1所示。SNP分子标记 SiDt27-1有92bp，为花序有限基因 SidtI中第378至469处碱基序列。

Description

芝麻花序有限基因Sidt1及其SNP标记 技术领域
本发明属于分子遗传育种技术领域，具体涉及一种芝麻花序有限基因Sidt1及其SNP标记SiDt27-1。
背景技术
芝麻(Sesame indicum L.，2n＝26)是世界上最古老的优质油料作物之一，也是我国的特色农产品，在食品加工中具有重要地位。自解放以来，我国共育成芝麻品种约140个，在很大程度上推动了我国芝麻生产的快速发展。但是与其他油料作物相比，芝麻仍属于低产作物。
芝麻是花序无限型且生育期较短的农作物，通常我国芝麻品种的生育期在85-95天。植株生长发育过程的表现主要包括：植株花序无限，花期较长，约为25-35天。下部蒴果成熟开裂时，多数植株的顶端仍在进行花芽分化，植株蒴果成熟度不一致，从而影响到芝麻的产量和产品质量。
目前，在世界芝麻生产中，收获指数低、籽粒成熟度不一致、抗病抗逆性差等是限制芝麻高产稳产的主要因素，开展高产高抗病抗逆、适于机械化生产的芝麻新材料创制和新品种选育是当前我国芝麻育种研究工作的重点。
在此背景下，自2007年起，河南省农业科学院芝麻研究中心积极开展了芝麻EMS诱变育种和种质创制工作。2009年采用EMS诱变方法，从我国主导芝麻品种豫芝11号中诱变创制出了花序有限型芝麻优异新品系，命名为豫芝DS899。该突变体在田间表现主要包括以下方面：(1)植株单杆，一叶三花，蒴果四棱，下部叶片较大，叶片深绿、肥厚，叶绿素含量高，光和效率强；(2)花序有限，植株具有典型的有限生长习性，株高130-150厘米；在黄淮区，该品种生长发育形成15-20个节位后，植株顶端生长点停止生长并自动封顶；封顶后顶端形成4-6个蒴果群；该特性受隐性单基因控制，能稳定遗传，繁殖后代中有限习性植株达到98％以上；(3)该品系生长发育速度快，现蕾早，花期20-25天，结蒴集中，无空稍尖，生育期80天左右；平均单株结蒴60-80个；平均蒴粒数50-55粒，千粒重4.0-4.5g，籽粒含油量51.43％，蛋白质含量26.75％(2014年郑州收获种子)，属于高蛋白型优质育种新材料。(4)品种对茎点枯病和枯萎病抗性高；适于密植和机械化管理与收获。
除豫芝DS899外，在当前世界芝麻种质资源库中，还保存另一类花序有限型芝麻种质(河南省农科院芝麻研究中心芝麻种质资源库存编号为08TP092)。该种质的田间性状主要 表现为：植株分支型，花序有限，蒴果节位数为2-3节，单株蒴果数30-40个，结蒴性低，不适宜直接用于生产，该种质花序有限性状受单隐性基因控制。
基于现有的有限花序的芝麻种质资源进行分析，对芝麻有限花序基因进行分析和标记，对于培育新的芝麻种子资源具有十分重要研究和应用价值。
发明内容
本发明主要目的是提供一种芝麻花序有限基因Sidt1的基因序列，同时提供了该有限基因Sidt1基因序列的cDNA序列；本发明另一目的是提供了对有限基因Sidt1基因序列的SNP标记SiDt27-1，从而便于新品种的筛选和培育。
下面结合实施例对本发明做进一步的解释说明。
芝麻花序有限基因Sidt1，该基因有1809bp，包含4个外显子和3个内含子，位于芝麻第4条染色体上，在芝麻SNP遗传图谱中的位置为第8连锁群，18.0-19.2cM，对花序有限性状的解释率为100％(Vg/Vp)；其DNA序列如SEQ ID NO.1所示。
芝麻花序有限基因Sidt1的cDNA序列，长度为531bp，编码176个氨基酸，具体如SEQ ID.2所示。
需要说明的是，与正常株(dt0)基因序列的唯一不同点就在于，花序有限基因Sidt1的cDNA序列中，第236位核苷酸由G突变成了A；当G变为A时，导致编码蛋白的第79个氨基酸序列由丝氨酸(S)突变为了天冬酰胺(N)，植株花序就由无限(dt0型)变成了dt1有限型。
所述芝麻花序有限基因Sidt1的SNP分子标记SiDt27-1，有92bp，为花序有限基因Sidt1中第378至469处碱基序列，具体序列为：
CCTGATGTTCCTGGTCCTAATGATCCATATCTGAGGGAGCACCTGCACTGGTATGCTTTCATTTTTAACTGCTTAAGACCTGATTGATTTAA。
利用所述SNP分子标记SiDt27-1检测芝麻花序有限基因Sidt1的检测方法，具体包括以下步骤：
(1)提取待测芝麻种质资源的基因组DNA；
(2)以步骤(1)中所提取DNA为模板，进行PCR扩增，PCR扩增时，采用如下引物序列：
正向引物HSDt01-1F序列：5'CCTGATGTTCCTGGTCCGAA 3'；
正向引物HSDt01-2F序列；5'CTATTCCTGATGTTCCTGGTCCGAG 3'；
反向引物HSDt01-R序列：5'TAAATCAATCAGGTCTTAAGCAGT 3'；
对PCR扩增产物进行凝胶电泳，判断待测芝麻种质资源的基因组DNA中是否含有SNP分子标记SiDt27-1，判断规则如下：
如果仅含有SNP分子标记SiDt27-1标记位点，当采用引物HSDt01-1F与HSDt01-R配对进行PCR扩增时，能扩增出条带大小为92bp的产物，则该待测种质属于dt1型，花序有限，可用于培育花序有限型芝麻新品种；
如果不含有SNP分子标记SiDt27-1标记位点，当采用引物HSDt01-2F与HSDt01-R配对进行扩增时，能扩增出条带大小为97bp的产物，则该待测种质属于dt0型，花序无限，不能用于培育花序有限型芝麻新品种；
如果所扩增条带大小既有92bp的产物，也有97bp的产物，则该待测种质属于杂合型，花序无限，但其后代可以出现形状分离，从后代中可进一步获得花序有限型材料以便用于新品种的培育；
(3)为进一步准确判断待测种质资源是否含有花序有限基因Sidt1，可以步骤(1)中所提取的基因组DNA为模板，PCR扩增特定序列，PCR扩增时，采用如下引物序列：
正向引物Dt1Primer F序列为：5'-ATGGCAAAAATGTCATCGGACC-3'；
反向引物Dt1Primer R序列为：5'-CTAGCGCCTTCTAGCAGCAGTC-3'；
对PCR扩增产物进一步进行测序，并与花序有限基因Sidt1的基因序列(即SEQ ID NO.1中的序列)进行比对，如果相同，则待测种质属于花序有限，可用于培育新的芝麻品种。
2012年，发明人将豫芝DS899(单杆，节位15-20个)和08TP092(分支，节位2-3个)配置了杂交组合并调查了F1、F2代特征特性。结果显示，F1后代均表现为花序有限，节位3-5个；F2后代均表现为花序有限，节位3-15个不等。同其他研究结果相结合，发明人认为，在豫芝DS899和08TP092两个花序有限型芝麻材料中，花序有限性状受同一基因位点控制，但突变性状表现有一定差异。因此，推测2种花序有限型种质在花序有限基因的序列上可能有一定差异。随之，将2个种质中的花序有限基因分别命名为Sidt1(来源于豫芝DS899)和Sidt2(来源于08TP092)。相应地，豫芝DS899和08TP092突变体分别为dt1型和dt2型，花序无限型正常株为dt0型。
后期研究结果显示，dt1型和dt2型有限种质之间的差异在于，Sidt基因在序列的不同碱基片段发生了变异。2013年以来，发明人利用已构建的豫芝DS899(dt1型)×JS012(dt0型)的F2群体，参考芝麻群体作图、芝麻基因组精细图和重测序技术(Zhang et al.,2013；Miao and Zhang,2014；Li et al.,2013)，成功克隆了豫芝DS899的花序有限基因Sidt1，在芝 麻基因组注释中，该基因命名为Sitfl。最终，发明人确定了芝麻花序有限的突变基因位点，为下一步探明芝麻花序发育调控机理、选育dt1型花序有限芝麻新品种奠定了坚实基础，并较好地推动了芝麻分子辅助育种研究的发展。
在此基础上，本发明针对获得的芝麻花序有限基因Sidt1进行分子标记开发和应用，为加快芝麻重要性状遗传基础和新品种选育提供技术支撑。
本发明创新点主要体现在以下几个方面：
(1)本发明对花序有限型芝麻突变体豫芝DS899开展了基因组重测序和芝麻种质资源遗传图谱构建，克隆提供了一种芝麻花序有限基因Sidt1，开发了其SNP标记SiDt27-1，同时提供了一种鉴定种质资源中是否含有该基因及标记位点的PCR鉴定方法，该方法可以较为便捷和快速地初步确定待测芝麻品种的花序类型，为新品种培育提供参考；
(2)本发明所获得的芝麻花序有限基因Sidt1及SNP标记SiDt27-1序列明确，检测结果稳定可靠；本发明所涉及的花序有限基因对芝麻产量、品质以及芝麻生产等具有重要影响，并对开展芝麻等农作物花发育机理研究以及培育适于机械化生产的芝麻新品种具有重要意义；
(3)本发明所提供的芝麻花序有限基因Sidt1及其SNP标记SiDt27-1的检测方法，技术成熟，检测结果稳定性好，对提高芝麻育种工作效率、提升我国芝麻遗传育种研究技术水平具有重要意义。
与现有芝麻育种方法相比，本发明优点可以概括为：
(1)本发明首次发现了芝麻花序有限基因Sidt1；
(2)本发明开发了芝麻花序有限基因Sidt1的SNP标记SiDt27-1，为分析芝麻等农作物花发育机理提供了遗传信息；
(3)本发明所提供的SNP分子标记SiDt27-1，为建立芝麻分子辅助育种技术提供了重要分子标记，为筛选花序有限的芝麻新品种提供了快速检测方法。
附图说明
图1为豫芝DS899(dt1型，花序有限，右侧)和亲本豫芝11号(dt0型，花序无限，左侧)植株田间对照；
图2为本发明利用F2群体建立的芝麻SNP高密度遗传图谱；
图3为本发明的芝麻花序有限基因Sidt1在芝麻SNP遗传图谱中的定位结果；
图4为本发明的花序有限SNP标记SiDt27-1的引物对HSDt01-1F、HSDt01-2F和HSDt01-R在部分芝麻种质中的PCR扩增结果，其中：泳道M为DL 2000marker，显示的 条带从上到下分别为750bp、500bp、250bp和100bp；泳道1为含Sidt1等位位点2(即dt0型)的豫芝11号材料(无限型)；泳道2为含Sidt1等位位点1(即dt1型)的材料(有限型)；泳道3为豫芝11号×豫芝DS899组合的F1中间杂合型材料；泳道4为以水为模板的PCR扩增对照；
图5为本发明的芝麻花序有限基因Sidt1的基因结构；
图6为本发明的芝麻花序有限基因Sidt1与正常株等位基因SiDt(芝麻基因组注释命名为Sitfl)序列比对结果；其中：箭头指示Sidt1基因与正常等位基因SiDt的核苷酸序列差异位点；上排为正常等位基因SiDt的核苷酸序列，下排为Sidt1基因的核苷酸序列；
图7为本发明的芝麻花序有限基因Sidt1与正常株等位基因SiDt基酸编码序列比对结果；其中：箭头指示Sidt1蛋白与正常基因SiDt蛋白的氨基酸序列差异位点；
图8为本发明的SNP标记SiDt27-1的引物对HSDt01-1F、HSDt01-2F和HSDt01-R在F2群体中的PCR验证；其中泳道M为DL 2000marker，显示的条带从上到下分别为200bp和100bp；泳道1-11为Sidt1纯合(花序有限型)F2植株；泳道12、13、19、20和22为SiDt纯合(无限花序型)F2植株；泳道14-18和21为Sidt1/SiDt杂合(无限花序型)F2植株；
图9为本发明的芝麻花序有限基因Sidt1与正常株等位基因SiDt的Southern印迹杂交结果；其中：泳道M为Marker；泳道1：包含正常等位基因SiDt探针序列的阳性质粒杂交印迹；
泳道2-4从左到右分别为HindⅢ酶切的豫芝11号(野生型无限，dt0)、豫芝DS899(dt1型有限)和S.raditum野生种芝麻(dt0型，无限)基因组DNA的杂交结果；泳道5-7从左到右分别为HindⅢ和EcoRⅠ双酶切的豫芝11号(野生型无限，dt0)、豫芝Ds899(dt1型有限)和S.raditum野生种芝麻(dt0型，无限)基因组DNA的杂交结果；
图10为本发明的SNP标记SiDt27-1在育种材料选择中的应用；其中：泳道M为DL2000Marker，泳道1-20为Sidt1纯合(花序有限型)F2植株；泳道21、29、30、33、35、37、38和40为SiDt纯合(无限花序型)F2植株；泳道22～28、31、32、34、36和39为Sidt1/SiDt杂合(无限花序型)F2植株。
具体实施方式
下面结合实施例对本发明做进一步的解释说明。介绍具体实施例前，首先对本发明中所用主要芝麻种质资源及品种简要介绍如下。
现有技术中，芝麻品种豫芝11号是我国重要的芝麻育种优异亲本材料。本申请中所采 用的芝麻花序有限型新品系豫芝DS899，是由河南省农业科学院芝麻研究中心采用EMS诱变技术从豫芝11号选出的新品种。2015年3月对该品系申请了国家新品种权保护(申请号：20150395.2)，并参加了2015年度省级芝麻新品种区试鉴定。其主要特征为：花序有限，平均节位15-20个，每叶腋3花，单杆，蒴果四棱，白粒。
本申请中所采用的芝麻资源JS012，来自于河南省农业科学院芝麻研究中心种质资源库，主要特征为：花序无限，每叶腋单花，分枝，蒴果四棱，黑粒。
实施例中涉及到的08TP092、武宁黑、郑芝98N09、野生种等其他芝麻种质资源，均来自于河南省农业科学院芝麻研究中心种质资源库；这些种质材料均可从公开渠道获得(或直接从河南省农业科学院芝麻研究中心种质资源库获得)，并种植应用。
另外需要说明的是，下述实施例中PCR反应采用PTC-100(MJ research公司产品)热循环仪进行；PCR反应中相关酶类、缓冲液等试剂产品均采购于上海生工试剂公司；相关引物均由华大基因公司合成提供。PCR反应中植株DNA提取均参照魏利斌等的改良CTAB法进行(芝麻DNA和RNA同步提取方法，2008，分子植物育种)。下述实施例中相关基因测序由天津基因芯片生物公司完成。
实施例1
本实施例主要介绍一下芝麻花序有限基因Sidt1及其SNP分子标记SiDt27-1的筛选过程，具体过程如下。
一、花序有限性状遗传背景介绍
为研究分析花序有限性状，2012年，发明人选用豫芝DS899与豫芝11号、JS102和武宁黑等三个花序无限型种质进行正反交组合配置(具体组合配置如下表所示)，并对F1后代进行l花序类型调查。豫芝DS899与豫芝11号、JS102和武宁黑的正反交组合配置类型为：
Figure PCTCN2015099095-appb-000001
其中豫芝DS899(花序有限)和无限型亲本豫芝11号的性状对比如图1所示。
F1调查结果显示，F1均为正常的无限花序，说明该突变性状(有限花序)受隐性基因控制。随后，对豫芝DS899与豫芝11号、JS102及武宁黑的3个正交F2后代群体进行了加代种植，每个群体均大于200株。花期开展了田间性状调查，结果如下表所示。
F2及测交后代群体中花序有限性状分离结果：
Figure PCTCN2015099095-appb-000002
上述数据显示，花序有限型突变体与野生型性状的分离比分别为64:215、78:242和71:229。适合性检验结果显示其符合1:3分离比，表明该突变性状受1对隐性基因控制。进一步进行豫芝DS899突变体与豫芝11号、JS102及武宁黑的3个测交后代群体检测。结果显示，突变性状后代分离比例分别为94:92、108:125和78:84。适合性检验结果显示，花序有限性状在测交后代中符合1:1分离比，再次表明该突变性状受1对隐性基因控制。
二、芝麻花序有限与无限型亲本的F2遗传群体构建
2013年7月，利用纯化的花序有限型系豫芝DS899和花序无限型种质JS012重新配置杂交组合，获得F1；
2013年11月，将上述组合的F1种子采用营养钵点播，种植在河南省农业科学院芝麻研究中心三亚基地，2对真叶后，适时移栽各植株，确保株系数量大于200个。
三、F2群体SNP遗传图谱构建及花序有限基因Sidt1定位
(1)F2群体亲本及122个F2单株基因组重测序
从F2群体中随机挑选120个株系，采集120个株系单株和2个亲本单株的幼嫩叶片，提取各植株DNA，采用Illumina测序方法对122份材料进行基因组重测序，测序覆盖度≥30×。
(2)参考豫芝11基因组数据(Zhang et al.，Genome sequencing of the important oilseed crop Sesamum indicum L.，2013，Genome Biology；Miao et al.，The sesame genome project and genome sequencing.XXII International Plant and Animal Genome Conference(San Diego,USA)，2014(Conference poster abstract))，选用BWA(Burrows-Wheeler Aligner)软件将各株系的测序数据进行拼接。
(3)利用Joinmap_linkage map软件构建芝麻F2群体SNP高密度遗传图谱。所构建遗 传连锁图谱如图2所示。图谱共有13个连锁群，包含3101个bin区段，12.4万个标记，图谱全长1872.2cM，平均144.0cM/连锁群，0.015cM/标记区间。
(4)在初花期-终花期，对F2群体120个株系及2个亲本进行花序类型鉴定，共鉴定3次。结合上述SNP高密度遗传图谱，采用WinQTL cart软件，确定了1个与芝麻有限花序性状紧密连锁的区间，见图3。结果显示，该突变基因位于第8连锁群，遗传距离18.0cM-19.2cM，两标记之间的物理距离约为480kb。进一步分析发现，此区间内共包含了15个SNP/InDel多态位点。
随后，将15个候选的SNP/InDel多态位点转换为分子标记(换言之，根据已知序列设计引物并扩增获得相应序列)，具体的15个候选花序有限SNP/InDel位点分子标记如下表所示：
Figure PCTCN2015099095-appb-000003
Figure PCTCN2015099095-appb-000004
随机从上述亲本和F2群体中选出50个植株，提取DNA，利用上述15组分子标记引物对，进行关联性筛查。结果显示，仅SiDt27-1位点与花序有限性状紧密连锁(图4)。
实施例2
本实施例主要进行芝麻花序有限基因Sidt1克隆及基因序列分析。
在实施例1基础上，为获知花序有限基因Sidt1的具体序列，可采用PCR扩增方法扩增获得相关序列后进一步测序获知，相关过程介绍如下。
(1)根据上述实施例1中获得的SNP位点，利用豫芝11号基因组数据，确定了SiDt27-1位点所在的基因，将该基因序列命名为Sidt1。对Sidt1序列分析发现，该基因在芝麻基因组(豫芝11号)被注释为TFL基因(SiDt)。
随后，根据基因组数据，利用Primer premier 5.0软件设计了扩增芝麻花序有限基因Sidt1的引物对，具体设计如下：
正向引物Dt1Primer F序列为：5'-ATGGCAAAAATGTCATCGGACC-3'；
反向引物Dt1Primer R序列为：5'-CTAGCGCCTTCTAGCAGCAGTC-3'。
以豫芝DS899的DNA为模板，进行PCR扩增；
PCR反应程序为：94℃预变性3分钟；之后94℃变性30秒，55℃复性30秒，72℃延伸1分钟，循环30次；最后72℃延伸5分钟。PCR扩增产物4℃保存备用，或者直接回收扩增条带并测序。
结果显示，该基因的基因组全长序列为1809bp，共包含4个外显子和3个内含子(图5)，序列如SEQ ID1所示。
同时，利用上述引物对对豫芝11号基因组DNA进行了扩增，并对扩增产物进行了测序。
将花序有限Sidt1基因与正常株(即豫芝11号)的等位基因SiDt进行序列比对，结果如图6。从图6中可以看出，在花序无限型和花序有限型种质基因组中，Sidt1序列的差异仅 在于编码区第236位碱基是否发生了G/A突变。当花序类型由无限型突变为dt1有限型时，编码区第236位的碱基G就突变为了碱基A，导致编码蛋白的第79个氨基酸序列由丝氨酸(S)突变为了天冬酰胺(N)(图7)。
(2)依照上述DNA提取方法，提取豫芝DS899幼嫩植株的RNA，并利用TaKaRa RNA反转录试剂盒(TaKaRa公司)进行反转录获得总cDNA；相关操作参考反转录试剂盒使用说明书进行操作即可；
需要说明的是，反转录过程中，PCR反应时所采用的引物对(利用Primer Premier5.0设计获得)设计如下：
TFL1CDs-F(5'-ATGGCAAAAATGTCATCGGACC-3')，
TFL1CDs-R(5'-CTAGCGCCTTCTAGCAGCAGTC-3')；
PCR反应扩增获得Sidt1的cDNA编码区序列，对该PCR产物测序即可获得Sidt1基因的cDNA序列；该基因的cDNA序列为531bp，共编码176个氨基酸，序列如SEQ ID2所示。
(3)花序有限基因Sidt1的SNP引物设计与SNP位点验证
进行SNP位点验证时，引物设计方法参照Wei等(Development of SNP and InDel markers via de novo transcriptome assembly in Sesamum indicum L.，2014，Molecular Breeding)进行，另外需要解释的是，为区分待检测基因组DNA中不同SNP等位位点，研究人员通常需要注意以下2方面的问题：
第一，将SNP引物对设计成3条，并需在特异引物的3’末端第3位的碱基引入错配以增加扩增产物的特异性；引入错配的原则为：引物3’端的第3位错配碱基与3’末端的SNP错配碱基能形成稳定性互补的错配结构，即强错配型(C/T或G/A)与弱错配型(C/A或G/T)搭配，中等错配型(A/A、C/C、G/G或T/T)与中等错配型搭配；
第二，在含有SNP位点的2个正向或反向引物中，将其中1条引物序列的5’端随机增加5个碱基，其主要目的是为了不同位点的PCR产物在后续凝胶电泳图谱区分能够较为便捷地区分开来。
利用Primer premier 5.0软件所设计的SNP位点的引物对序列如下：
正向引物HSDt01-1F序列：5'CCTGATGTTCCTGGTCCGAA 3'；
正向引物HSDt01-2F序列；5'CTATTCCTGATGTTCCTGGTCCGAG 3'；
反向引物HSDt01-R序列：5'TAAATCAATCAGGTCTTAAGCAGT 3'；
需要说明的是，当正向引物HSDt01-1F与反向引物HSDt01-R配对时，扩增产物条带大 小为92bp(dt1型)；当正向引物HSDt01-2F与反向引物HSDt01-R配对时，扩增产物条带大小为97bp(dt0型)。
为确认Sidt1基因即是调控芝麻花序类型的基因，我们配置了三个组合F2群体进行了验证。F2群体具体为：随机从豫芝DS899分别与豫芝11号、JS102和武宁黑构建的F2群体中挑选50个株系，同时田间调查各株系单株的花序类型。
从此50个株系随机选择50个单株，采集50个单株和4个亲本单株的幼嫩叶片，提取各植株DNA，并以此作为模板，进行PCR扩增。
PCR反应采用10μL反应体系，设置如下：
模板DNA(50ng/μL)，1.0μL；
10×PCR Buffer(Mg2+)，1.0μL；
Taqase酶(5U/μL)，0.2μL；
dNTP(10mmol/L)，0.2μL；
Forward Primer 1(10μM)，0.5μL；
Forward specific Primer 2(10μM)，0.5μL；
Reverse Primer(10μM)，1.0μL；
加入超纯水5.6μL。
PCR反应程序为：94℃预变性3分钟，之后94℃变性30秒，55℃复性30秒，72℃延伸30秒，循环30次，最后72℃延伸5分钟，扩增产物4℃保存备用。
对PCR产物进行非变性聚丙烯酰胺凝胶电泳分析，凝胶浓度为8～10％，凝胶大小180mm×120mm×2mm，电泳缓冲液为0.5×TBE，150V恒压交流电电泳1.5～2小时。电泳结束后，在凝胶加入浓度为0.1％的硝酸银水溶液，置于水平摇床上渗透银染10min；再加入2％氢氧化钠和0.4％甲醛混合溶液，置于水平摇床中适度显色；最后清水漂洗凝胶并记录读取数据。
部分电泳图谱如图8所示。
结果显示，表现为花序有限的植株，其扩增结果为92bp条带；花序无限的亲本扩增结果为97bp条带；杂合型植株扩增出92bp和97bp条带，性状表现为无限型。
综上，我们可以认为该Sidt1基因是引起芝麻花序有限突变发生的基因，该基因可用于芝麻等作物花序发育机理研究。
实施例3
本实施例主要进行芝麻花序有限基因Sidt1序列特征分析。
为进一步确定芝麻花序有限基因Sidt1的特征，并同时确定其在芝麻基因组中的拷贝数，我们开展了花序有限基因Sidt1的Southern杂交印迹验证工作。
(1)花序有限基因Sidt1的Southern杂交探针设计及制备
利用Primer Premier 5.0软件，依据芝麻品种“豫芝11号”基因组DNA和SiDt序列，设计引物对，具体如下：
Dt-Gs正向引物：5'-GAGCCCTCTTTCAAAAACACC-3'，
Dt-Gs反向引物：5'-AGCAGCAACAGGGAGACCTA-3')；
以芝麻品种“豫芝11号”基因组DNA为模板，利用上述引物对Dt-Gs进行PCR扩增，PCR产物大小459bp。
PCR反应中，各成分及反应体积如下：
模板DNA(50ng/μL)，2.0μL；
10×PCR Buffer(Mg2+)，5.0μL；
Taqase酶(5U/μL)，0.5μL；
dNTP(10mmol/L)，1.0μL；
Dt-Gs正向引物(10μM)，1.0μL；
Dt-Gs反向引物(10μM)，1.0μL；
加超纯水至总体积50μL。
PCR反应程序为：94℃预变性3分钟；94℃变性30秒，55℃复性30秒，72℃延伸30秒，循环30次；最后72℃延伸5分钟；4℃保存。
PCR产物经1％琼脂糖凝胶电泳检测后测序确认。
该PCR产物即用作Southern杂交探针，利用DIG-High Prime DNA Labeling&Detection Starter Kit I(Roche试剂公司)，进行探针标记并检测；具体操作步骤参照DIG-High Prime DNA Labeling&Detection Starter Kit I的说明书进行。
(2)重组标准品质粒的构建
将步骤(1)中所述探针片段连入pGEM-T Easy载体，构建成带有探针序列的pGEM-T Easy质粒，用于Southern杂交阳性对照；
连接、回收、纯化等步骤采用连接试剂、回收试剂盒(相关产品均为Promega公司产品)完成；采用0.8％琼脂糖凝胶电泳检测质粒连接质量；
采用DNA提取试剂盒(TaKaRa公司)提取带有探针序列的pGEM-T Easy质粒，并将其浓度稀释为1-5μg/L，用于下一步Southern杂交；具体步骤参考DNA提取试剂盒使用说 明。
(3)花序有限基因Sidt1的Southern杂交印迹
选取豫芝11号、豫芝Ds899和S.raditum(野生种)等3个芝麻品种的幼嫩叶片各200克，提取各植株DNA；
分别取上述3个品种的基因组DNA各10μg，用限制性内切酶HindⅢ和EcoRⅠ内切酶进行双酶切16h，采用回收试剂盒对酶切的产物进行回收纯化；(相关酶试剂及回收试剂盒均为TaKaRa公司产品，具体操作参考试剂使用说明书进行)；
对上述酶切产物进行适当浓缩后，用地高辛标记并进行Southern杂交，具体杂交过程为：
对酶切后的DNA产物进行0.7％琼脂糖凝胶电泳，25V恒压、4℃低温过夜；然后进行转膜、杂交探针，然后进行洗膜，记录Southern杂交结果(杂交结果如图9所示)；
详细Southern杂交过程及步骤参考高辛标记Southern杂交试剂盒(Roche公司)使用说明书进行。
从Southern杂交结果可以看出，在花序有限dt1型和花序无限dt0型芝麻种质中，Sidt1及其等位基因SiDt均可被杂出，且杂交条带均为1条，大小相似。这一结果表明，在2种花序类型的栽培种和野生种种质基因组中，芝麻花序有限基因Sidt1及其等位基因均以单拷贝存在；芝麻花序有限基因Sidt1及其等位基因的基因序列差异极小。
实施例4
本实施例主要进行芝麻花序有限基因Sidt1的SNP分子标记SiDt27-1开发。
本实施例以选育花序有限型高产优质育种材料为例，主要介绍芝麻花序有限基因Sidt1的SNP分子标记SiDt27-1应用过程，具体过程如下。
(1)为选育出花序有限型高产优质育种材料，2014年从芝麻种质库中挑选郑芝98N09做母本，以豫芝DS899做父本，6月份种植于河南省农业科学院芝麻研究中心原阳基地。2014年7月，配置杂交组合，获得F1。2014年11月，在三亚基地种植F1代种子，单株罩网自交，随后获得F2代种子，群体大于200株。2015年2月，随机选取50个F2单株的饱满种子，种于海南三亚基地。花期调查F2株系后代花序类型。每个株系设定3个重复，每个重复调查10棵植株。
(2)利用实施例2中的三引物序列(正向引物HSDt01-1F、正向引物HSDt01-2F、反向引物HSDt01-R)进行SNP标记的可靠性评价；
随机挑选上述F2群体的50个单株个体和2个亲本个体，提取基因组DNA，并以此为 模板，进行PCR。
PCR反应采用10μL反应体系，设置如下：
模板DNA(50ng/μL)，1.0μL；
10×PCR Buffer(Mg2+)，1.0μL；
Taqase酶(5U/μL)，0.2μL；
dNTP(10mmol/L)，0.2μL；
Forward Primer 1(10μM)，0.5μL；
Forward specific Primer 2(10μM)，0.5μL；
Reverse Primer(10μM)，1.0μL；
加入超纯水5.6μL。
PCR反应程序为：94℃预变性3分钟，之后94℃变性30秒，55℃复性30秒，72℃延伸30秒，循环30次，最后72℃延伸5分钟，反应产物4℃保存备用。
(3)非变性聚丙烯酰胺凝胶电泳对扩增产物进行分离
对步骤(2)中PCR产物进行非变性聚丙烯酰胺凝胶电泳分析，凝胶浓度为8～10％，凝胶大小180mm×120mm×2mm，电泳缓冲液为0.5×TBE，150V恒压交流电电泳1.5～2小时；电泳结束后，在凝胶加入浓度为0.1％的硝酸银水溶液，置于水平摇床上渗透银染10min；再加入2％氢氧化钠和0.4％甲醛混合溶液，置于水平摇床中适度显色；最后清水漂洗凝胶并记录读取数据。
部分电泳图谱如图8所示。
(4)SNP分子标记检测与被检测样品表型一致性分析
从SiDt27-1位点筛选过程我们可以知晓，理论上具有SiDt27-1等位位点1(条带大小92bp，即本申请SiDt27-1位点)的植株应该表现为花序有限；具有SiDt27-1等位位点2(条带大小97bp，即SiDt27-1位点中的碱基G变为A时位点)的植株应该表现为花序无限；具有SiDt27-1等位位点1、2(条带大小92bp和97bp，)的植株为杂合体，应该表现为花序无限。
检测结果显示，在有SiDt27-1等位位点1(条带大小92bp)的20个植株中均为有限花序，可靠度为100％；在有SiDt27-1等位位点2(条带大小97bp)的15个植株中，均为无限花序，可靠度为100％。在有SiDt27-1等位位点1和2(条带大小为92bp、97bp)的15个植株为杂合子，表型均为无限花序，可靠度100％。
部分植株的PCR扩增结果如图10所示。
综上，我们可以认为该SNP标记即为芝麻花序有限基因SNP位点标记，可以用于预测芝麻品种的花序类型，用于芝麻分子标记辅助育种和花序有限型芝麻新品种的选育。

Claims (5)

  1. 芝麻花序有限基因Sidt1，其特征在于，该基因有1809bp，包含4个外显子和3个内含子，位于芝麻第4条染色体上，在芝麻SNP遗传图谱中的位置为第8连锁群，18.0-19.2cM，对花序有限性状的解释率为100％(Vg/Vp)；其DNA序列如SEQ ID NO.1所示。
  2. 权利要求1所述芝麻花序有限基因Sidt1的cDNA序列，其特征在于，该cDNA序列长度为531bp，编码176个氨基酸，具体如SEQ ID.2所示。
  3. 权利要求1所述芝麻花序有限基因Sidt1的SNP分子标记SiDt27-1，其特征在于，该分子标记SiDt27-1有92bp，为花序有限基因Sidt1中第378至469处碱基序列，具体序列为：CCTGATGTTCCTGGTCCTAATGATCCATATCTGAGGGAGCACCTGCACTGGTATGCTTTCATTTTTAACTGCTTAAGACCTGATTGATTTAA。
  4. 利用权利要求3所述SNP分子标记SiDt27-1检测芝麻花序有限基因Sidt1的检测方法，其特征在于，具体包括以下步骤：
    (1)提取待测芝麻种质资源的基因组DNA；
    (2)以步骤(1)中所提取DNA为模板，进行PCR扩增，PCR扩增时，采用如下引物序列：
    正向引物HSDt01-1F序列：5'CCTGATGTTCCTGGTCCGAA 3'；
    正向引物HSDt01-2F序列；5'CTATTCCTGATGTTCCTGGTCCGAG 3'；
    反向引物HSDt01-R序列：5'TAAATCAATCAGGTCTTAAGCAGT 3'；
    对PCR扩增产物进行凝胶电泳，判断待测芝麻种质资源的基因组DNA中是否含有SNP分子标记SiDt27-1，判断规则如下：
    如果仅含有SNP分子标记SiDt27-1标记位点，当采用引物HSDt01-1F与HSDt01-R配对进行PCR扩增时，能扩增出条带大小为92bp的产物，则该待测种质属于dt1型，花序有限，可用于培育花序有限型芝麻新品种；
    如果不含有SNP分子标记SiDt27-1标记位点，当采用引物HSDt01-2F与HSDt01-R配对进行扩增时，能扩增出条带大小为97bp的产物，则该待测种质属于dt0型，花序无限，不能用于培育花序有限型芝麻新品种；
    如果所扩增条带大小既有92bp的产物，也有97bp的产物，则该待测种质属于杂合型，花序无限，其后代会表现出性状分离，可进一步获得花序有限型材料。
  5. 如权利要求4所述利用SNP分子标记SiDt27-1检测芝麻花序有限基因Sidt1的检测方法，其特征在于，为进一步准确判断待测种质资源是否含有花序有限基因Sidt1，可以步骤(1)中所提取的基因组DNA为模板，进行PCR扩增，PCR扩增时，采用如下引物序列：
    正向引物Dt1Primer F序列为：5'-ATGGCAAAAATGTCATCGGACC-3'；
    反向引物Dt1Primer R序列为：5'-CTAGCGCCTTCTAGCAGCAGTC-3'；
    对PCR扩增产物进行测序，并与花序有限基因Sidt1的基因序列进行比对，如果相同，则待测种质属于花序有限，可用于培育新的芝麻品种，
    所述花序有限基因Sidt1的基因序列为SEQ ID NO.1中的序列。
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