Sesame seed shattering trait regulatory gene Sihec3 and its application

Abstract

This application belongs to the field of sesame molecular genetic breeding technology, specifically to a sesame seed shattering trait regulating gene Sihec3 The gene is located on the third chromosome of sesame and is a recessive control gene. The explanation rate of the phenotypic variation of the resistance to seed shattering trait is 100%. The inventors used the offspring materials of the resistant seed shattering mutant M7 as the basis and constructed the F2 population and F 2:3 The study showed that in the tested population, the sesame anti-seedling mutation trait was a recessive trait controlled by a single gene. Furthermore, using the self-constructed sesame genome fine map and sesame efficient gene localization technology, the inventors identified the key gene regulating the sesame seed shattering trait. Sihec3 and its alleles SiHEC3 Cloning and comparative studies were conducted, and corresponding sesame grain shattering resistance gene markers were developed.

Description

Technical Field

[0001] The present application belongs to the technical field of sesame molecular genetic breeding, and specifically relates to a sesame anti-seedling trait regulating gene. Sihec3 and its applications. Background Art

[0002] Sesame is one of the oldest oil crops in the world and an important high-quality specialty agricultural product in my country. Sesame seeds are rich in unsaturated fatty acids such as oleic acid and linoleic acid, and are rich in antioxidants such as sesamol and sesamolinol, which are very beneficial to human health. Therefore, it is known as the "Queen of Oils" (Zhang et al., 2019). Currently, 75 countries in the world grow sesame, mainly distributed in tropical and subtropical regions of Asia, Africa and Latin America. my country is one of the main sesame producing countries, with an annual planting area of ​​520,000 hectares of sesame and a total output of about 650,000 tons, accounting for 10-25% of the world's total output (FAO statistics from 2000-2018). Due to its high yield and excellent product quality, my country has an outstanding advantage in the world's sesame production, processing and trade.

[0003] However, due to factors such as planting scale, variety characteristics, and specialized machinery, the current sesame cultivation and harvesting are still mainly carried out by artificial planting. The low degree of mechanization has greatly restricted the sustainable development of the world's sesame industry. From the perspective of sesame variety characteristics, the main factor restricting mechanized cultivation is that most of the existing sesame varieties are capsule cracking and seed falling varieties, that is, the sesame capsules are prone to bursting after maturity, causing the seeds to scatter. If the harvest is not timely, the yield loss can reach more than 50%, and this characteristic is difficult to meet the needs of mechanized harvesting conditions. Therefore, quality improvement based on the characteristics of sesame seed falling is of great technical significance for the mechanized production of sesame. Summary of the invention

[0004] Based on the previously obtained mutant material with phenotypic resistance to grain shattering, combined with the fine map of the sesame genome and gene mapping technology, this application aims to provide a control gene for the sesame resistance to grain shattering. Sihec3 , which can lay a certain technical foundation for the cultivation of new sesame varieties with resistance to grain shattering.

[0005] The technical solution provided in this application is described in detail as follows.

[0006] Genes regulating seed shattering in sesame Sihec3 , this gene is located on the third chromosome of sesame, is a recessive control gene, and corresponds to the trait of grain shatter resistance; the explanation rate of this gene for the phenotypic variation of grain shatter resistance is 100%;

[0007] Its allele SiHEC3 compared to, Sihec3A 1049 bp deletion occurred in the -1bp~-1012bp region of the promoter and the 1-37bp region of the 5'UTR region, resulting in functional changes (i.e., when the gene promoter region and part of the 5'UTR region were deleted, the phenotype was resistant to grain shattering);

[0008] Said Sihec3 The gDNA length of the gene is 3406bp, including 2 exons. The gDNA sequence is shown in SEQ ID No.1 (UTR region includes: 1~84bp, 3217~3406bp), specifically:

[0009]

[0010] The sesame seed shattering resistance trait regulating gene Sihec3 The encoded protein includes 244 amino acids, and the specific amino acid sequence is shown in SEQ ID No. 2;

[0011] MDLSHHRFTNTTWEPYNIPMDQGHLLQDQTPFDHHPHAQWPYFPPIHSQNEPSDSSPTQLLPSPNFDQMGSAAFKASEAGDHQDDEPEEELGAMKEMMFKIAAMQPVDIDPATIRKPRRRNV RISDDPQSVAARHRRERISEKIRILQRLVPGGTKMDTASMLDEAIRYVKFLKRQIRILQANHHQPPCIGIATPTGAATAEEWVAVTTKATATATAAAGTSSSYLFGGNNNNDGAGVEAFNME;

[0012] Correspondingly, the regulatory gene for sesame seed shattering resistance Sihec3 The corresponding anti-grain shattering phenotype cDNA has a cDNA sequence length of 735 bp, as follows (compared to its allele SiHEC3 cDNA sequence, the base at position 111 mutated from A to C, resulting in the 37th amino acid of the encoded protein mutated from glutamine Q to histidine H):

[0013] .

[0014] The sesame seed shattering trait regulatory gene Sihec3 Alleles SiHEC3 , which is a completely dominant gene. The phenotype of sesame varieties containing this gene is normal seed shattering.

[0015] The allele SiHEC3 The gDNA sequence length is 3441bp, including 2 exons; the base sequence is shown in SEQID No.3; specifically as follows (UTR region includes: 1~121bp, 3264~3441bp):

[0016]

[0017] The allele SiHEC3 The amino acids encoded by it encode 244 amino acids. The specific amino acid sequence is as follows:

[0018] MDLSHHRFTNTTWEPYNIPMDQGHLLQDQTPFDHHPQAQWPYFPPIHSQNEPSDSSPTQLLPSPNFDQMGSAAFKASEAGDHQDDEPEEELGAMKEMMFKIAAMQPVDIDPATIRKPRRRNV RISDDPQSVAARHRRERISEKIRILQRLVPGGTKMDTASMLDEAIRYVKFLKRQIRILQANHHQPPCIGIATPTGAATAEEWVAVTTKATATATAAAGTSSSYLFGGNNNNDGAGVEAFNME;

[0019] Correspondingly, the allele SiHEC3 The corresponding cDNA sequence length is 735bp, and the specific base sequence is as follows:

[0020] .

[0021] To amplify the anti-grain shattering gene Sihec3 Allele SiHEC3 Promoter and gene sequences, showing the difference between the two. The present invention designed two pairs of PCR primers for amplification based on the reference genome sequence of Sesame Yuzhi No. 11, and the specific design is as follows:

[0022] Primer1F forward primer: 5'- TGACGGTCCGATTTGTAAGGTG-3',

[0023] Primer1R reverse primer: 5′ - GAAATTCGGAGACGGGAGG-3′;

[0024] Primer2F forward primer: 5'-AGCTAAATCCTCCTCTTTCACCA-3',

[0025] Primer2R reverse primer: 5′- ACAGCGTACAGGTTTCATT-3′;

[0026] When PCR amplification is performed based on two primer pairs, the genomic DNA of the shatter-resistant or shatter-resistant germplasm is used as the template, respectively:

[0027] The lengths of the amplified bands of the DNA templates of the materials with anti-seedling traits and seed-shedding materials amplified by the Primer1F / R primer pair were 1392 bp and 2432 bp respectively.

[0028] The lengths of the amplified bands of the DNA templates of the anti-seedling material and seedling material amplified by Primer 2F / R were 3410 bp and 3408 bp respectively.

[0029] The amplified products were sequenced, the sequences were spliced ​​and compared with the reference genome, and finally the sesame seed shattering resistance gene was obtained. Sihec3 The upstream 1111bp sequence and 3406bp gDNA sequence were used to obtain the alleles SiHEC3 2114 bp upstream sequence and 3441 bp gDNA sequence;

[0030] Sequence comparison results show that, in addition to the change of some bases in the promoter region, the most important changes are: relative to the allele SiHEC3 , sesame seed shattering resistance gene Sihec3 The 1012 bp and 37 bp sequences in the promoter region were deleted.

[0031] It should be explained that when the primer pairs were designed, the amplification product of the first pair of primers Primer1F / R included the promoter and part of the gene, and the amplification product of the second pair of primers Primer2F / R included part of the gene plus the bases downstream of the gene; the specific amplification sequences are as follows.

[0032] Amplification results of two pairs of PCR primers against falling grain type materials (4543 bp, including: gene upstream sequence, UTR sequence, CDS sequence and intron sequence):

[0033]

[0034] Amplification results of the falling-seed material using two pairs of PCR primers (5581 bp, including: gene upstream sequence, UTR sequence, CDS sequence and intron sequence):

[0035]

[0036] PCR detection to identify sesame seed shattering resistance gene Sihec3 and its alleles SiHEC3 A primer pair was used and named SSR1. When the primer pair was designed, considering that the gene marker site involved belonged to SV (Structure variation) mutation, the specific design was: SSR1-F sequence: 5'-TACATGGGCACAAAGAAAC-3', SSR1-R sequence: 5'-CTGGTGAAAGAGGAGGATT-3'; when the homozygous sesame anti-grain shattering or homozygous grain shattering genomic DNA was used as template, the length of the PCR amplification product was 1724bp or 684bp, respectively.

[0037] The application of the primer pair SSR1 in sesame molecular breeding is used to detect and determine whether sesame has a phenotype of resistance to grain shattering.

[0038] Sesame seed shattering resistance gene SSR1 was identified by using the primers Sihec3 and its alleles SiHEC3 The detection and differentiation method comprises the following steps:

[0039] (1) Extracting genomic DNA from the sesame sample to be tested;

[0040] (2) using the genomic DNA extracted in step (1) as a template, performing PCR amplification using the SSR1 primer pair, and detecting the amplified product by agarose gel electrophoresis;

[0041] (3) Determine whether the sesame sample to be tested is a shattering type or a shatter-resistant type based on the electrophoretic band pattern. The specific criteria for determination are:

[0042] If the length of the sample amplified fragment is 1724bp, it means that the sample contains SiHEC3 If the gene promoter and 5'UTR region are intact, the sample can be determined to be homozygous for the granuloma type; if the amplified fragment length is 684 bp, it indicates that the sample contains Sihec3 Gene ， If the gene promoter and 5'UTR region sequences are missing, the sample can be judged as a homozygous anti-grain-shedding type; if the amplified fragment results show both 1724bp and 684bp bands, the sample is judged as a heterozygous grain-shedding type.

[0043] In order to speed up the breeding of new sesame varieties suitable for mechanized production, since 2008, the Sesame Research Center of the applicant Henan Academy of Agricultural Sciences has systematically carried out research on sesame germplasm creation technologies such as physical and chemical mutagenesis, distant hybridization, and T-DNA transformation. By constructing a sesame mutant library, a distant hybridization intermediate material library, and screening related grain-shattering-resistant materials, a preliminary grain-shattering-resistant mutant M7 strain was obtained and this mutant strain was purified.

[0044] To further clarify the key genes regulating the seed shattering trait of sesame, the inventors used the seed shattering-resistant mutant M7 as a basis and used the F2 population and F2 population of the hybrid combinations of "M7 (anti-seed shattering) × Yuzhi Dw609 (seed shattering)" and "M7 (anti-seed shattering) × Xiangcheng Da Zi Bai (seed shattering)" to construct a new gene regulating the seed shattering trait of sesame. 2:3 The study showed that in the tested population, the sesame anti-seedling mutation trait was a recessive trait controlled by a single gene. Furthermore, using the self-constructed sesame genome fine map and sesame efficient gene localization technology, the inventors identified the key gene regulating the sesame seed shattering trait. Sihec3 and its alleles SiHEC3 Cloning and comparative studies were conducted, and corresponding sesame grain shattering resistance gene markers were developed.

[0045] In general, the main technical work and advantages of this application are reflected in the following aspects: (1) A gene related to sesame seed shattering resistance was cloned for the first time. Sihec3 and its alleles. The study showed that a 1049bp SV mutation occurred in the promoter and 5'UTR region of the gene, which led to the variation of the anti-grain shattering trait. The cloning of this gene provides important information for the next step to explore the regulatory mechanism of sesame seed development; (2) By developing the anti-grain shattering gene marker SSR1, it can lay the foundation for the identification of the anti-grain shattering trait of sesame germplasm resources, and then provide important technical support for quickly and accurately determining the grain shattering trait type and sample purity of the measured samples, as well as for the breeding of new sesame varieties with resistance to grain shattering; (3) The anti-grain shattering gene and its gene marker provided in this application have important technical guidance for accelerating the process of sesame molecular assisted breeding, and are of great significance for accelerating the selection and breeding of new sesame varieties with resistance to grain shattering and comprehensively improving the level of mechanized production of sesame.

[0046] In summary, this application can resist the seed shattering gene Sihec3 and its alleles SiHEC3 The preliminary research and the design of corresponding detection methods can provide an important theoretical basis for the regulation of grain shattering traits and the study of grain development mechanisms of crops such as sesame. It also provides important genetic resources for the construction of sesame molecular assisted breeding technology system and the cultivation of new varieties resistant to grain shattering, which has important scientific research value and economic application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Picture 1 Comparison of capsules and seeds between the shatter-resistant mutant and the shatter-resistant germplasm; a: capsules and seeds of the shatter-resistant sesame germplasm M7; b: capsules and seeds of the shatter-resistant sesame germplasm (Yuzhi No. 11);

[0048] Picture 2 The whole genome association analysis results of the seed shattering trait of the hybrid population of "M7×Xiangcheng Da Zi Bai" involved in the present invention;

[0049] Picture 3 The results of SV association analysis of seed shattering traits in the hybrid population of "M7×Xiangcheng Da Zi Bai";

[0050] Picture 4 The anti-grain shattering gene of the present invention Sihec3 and alleles SiHEC3 Gene and promoter sequence alignment;

[0051] Picture 5 The results of SSR1 marker amplification of some individual plants of the F2 hybridization verification population of "Yuzhi NS610×Anhui Qingyangbai" involved in the present invention; M: DL2000 (the bands from top to bottom are 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp); Lane 1: anti-grain-shattering parent; Lane 2: grain-shattering parent; Lanes 3-96: some individual plants of the F2 verification population; 43 of them are heterozygous grain-shattering type (containing 1724bp and 684bp bands); 28 are homozygous grain-shattering type (containing 1724bp band); 22 are homozygous anti-grain-shattering type (containing 684bp band);

[0052] Picture 6 The results of SSR1 marker amplification of 96 germplasm resources involved in the present invention; M: DL2000 (the bands from top to bottom are 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp); Lane 1: parent with resistance to seed shedding; Lane 2: parent with seed shedding; Lane 3-96: 96 sesame seed shedding type germplasm resources (containing a 1724bp band); DETAILED DESCRIPTION

[0053] The present application is further explained below in conjunction with the embodiments. Before introducing the specific embodiments, some experimental backgrounds in the following embodiments are briefly introduced as follows.

[0054] Sesame Ingredients:

[0055] Sesame varieties such as Yuzhi No. 11, Xiangcheng Da Zibai and Anhui Qingyangbai are all excellent sesame varieties promoted in the existing technology and are grain-shedding materials; M7 is a grain-shedding-resistant mutant created by the Sesame Research Center of Henan Academy of Agricultural Sciences, which has been successfully used to breed a new grain-shedding-resistant variety Yuzhi NS610; the relevant germplasm materials are all existing publicly reported materials and can be publicly obtained from relevant germplasm resource banks; the applicant, as a professional research institution, has long preserved the relevant germplasm materials.

[0056] Example 1

[0057] In order to accurately determine the regulatory genes that regulate the sesame grain shattering resistance trait and lay the foundation for the breeding of new varieties, the inventors conducted further detailed genetic analysis research on this trait. The specific research process is briefly introduced as follows.

[0058] 1. Analysis of the genetic background of sesame seed shattering

[0059] During the research, the inventors selected: the shatter-resistant germplasm M7 (resistant to shattering) and Xiangcheng Da Zi Bai (grain shattering) to configure a hybrid combination (the configuration is shown in Table 1 below, and the relevant phenotypic differences can be seen in Picture 1 ). The phenotypes of F1 and F2 offspring were identified and counted.

[0060] Table 1. Analysis of the offspring traits of the hybrid population of seed-shattering-resistant × seed-shattering germplasm

[0061]

[0062] Note: 2 (0.05, 1) = 3.84.

[0063] From the chi-square test results in Table 1, it can be seen that the ratios of the number of seed-shattering plants to the number of seed-shattering-resistant plants in the F2 population of the reciprocal cross between seed-shattering-resistant and seed-shattering germplasm were 3.67:1 and 3.41:1, respectively, both of which were in line with the expected value of 3:1 ( χ 2 <3.84). This result indicates that the trait of grain shattering resistance in the hybrid population of the resistant mutant M7×Xiangcheng Da Zi Bai conforms to the Mendelian inheritance model and is a recessive trait controlled by a single gene.

[0064] 2. Gene sequencing

[0065] During the construction of the aforementioned hybrid population, the inventors simultaneously collected young leaf samples of individual plants in the aforementioned F2 population (for subsequent gene sequencing and analysis), and further conducted F 2:3 The phenotypic survey and statistics of the offspring of the strains (based on the F 2:3 The seed-falling trait type of each individual plant in the offspring of the line is used to determine whether the genotype is homozygous or heterozygous).

[0066] Subsequently, based on the results of the phenotypic investigation, 29 homozygous seed-shattering plants and 35 homozygous seed-shatter-resistant plants were randomly selected from the F2. The DNA of the leaves of each sample was extracted using the modified CTAB method described by Wei Libin et al. (Simultaneous extraction of sesame DNA and RNA, Molecular Plant Breeding, 2008); and the DNA of the two parents was extracted at the same time.

[0067] Finally, the Illumina sequencing method was used to perform genome resequencing on the above 64 materials, with a sequencing coverage of ≥30×.

[0068] 3. Genome-wide association analysis of seed shattering traits in hybrid populations

[0069] First, referring to the genome data of Yuzhi No. 11 (Zhang et al. Ultra-dense SNP genetic map construction and identification of SiDt gene controlling the determinate growth habit in Sesamum indicum L, 2016, Science Reports; Zhao et al. Identification of sesame ( Sesamum indicum L.) chromosomes using the BAC-FISH system, 2018, Plant Biology, and the BWA (Burrows- Wheeler Aligner) software was used to align and splice the genome resequencing data of each individual plant.

[0070] Subsequently, GATK (The Genome Analysis Toolkit) was used to detect and filter reliable SNPs and InDels.

[0071] A total of 1,620,769 variants (SNPs, InDels) were discovered through variant mining of the two parents. Combined with the sequencing analysis results of the above parents and 64 F2 individual plant phenotypes, GLM (Generalized linear model) association analysis was performed using Tassele 5.0 software.

[0072] The final results show that (such as Picture 2 As shown in the figure, variants closely linked to the shattering resistance trait (P value less than E-250) are distributed on chromosome 3, ranging from 19659488 to 20465244, containing 3999 SNP and Indel sites.

[0073] Furthermore, based on the inventor's existing work, the SNP and Indel sites in the interval were filtered and screened. After filtering, 83 variant sites remained in the interval, and analysis showed that these 83 variant sites were sites that could not cause functional variation.

[0074] (IV) Genome-wide association analysis of SV for seed shattering traits in hybrid populations

[0075] To further locate the target gene, the inventors used existing software such as LUMPY, Manta, and GRIDSS2 to perform SV (Structure variation) analysis on two parents and 64 F2 plants. At the same time, to ensure the accuracy of SV identification, the inventors further filtered and statistically analyzed the screening results of different software.

[0076] The results showed that a total of 31,884 SVs were identified in the two parents, including 2,786 DELs (deletions), 21 INSs (insertions), 5,729 INVs (inversions), 550 DUPs (duplications) and 22,798 TRAs (translocations). The length of SVs ranged from 30 bp to 23 Mb.

[0077] Furthermore, based on the population phenotype and SV matrix information, the inventors further performed GLM (Generalized linear model) association analysis.

[0078] Based on the SNP / indel tight linkage interval and SV matrix information of the aforementioned hybrid F2 population, the inventors performed interval filtering on the above SV variation. It was preliminarily determined that there were 21 specific SVs in the anti-shattering samples within the linkage interval on chromosome 3 (the results are shown in Figure 2). Picture 3 ).

[0079] Furthermore, the 21 SVs were filtered and screened in combination with the SV matrix library constructed by the inventors in the early stage. The results showed that the remaining 11 SVs were only present in the anti-seedling F2 samples (the results are shown in Table 2 below).

[0080] Table 2. Information on 11 candidate SVs in F2 seed-shattering resistant plants

[0081]

[0082] Note: “ / ” indicates the intergenic region.

[0083] From the analysis of the results in Table 2 above, it can be seen that among the 11 SVs, SV_20120488 and SV_20099203 explained more than 85% of the variation in resistance to seed shattering, and both were DEL (deletion) types. SV_20120488 was located in the downstream region of the gene Sindi_0765000, and SV_20099203 was located in the upstream promoter region of the gene Sindi_0765000. The genome functional annotation showed that the gene Sindi_0765000 was a homologous gene of HEC3.

[0084] In summary, the inventors preliminarily speculated that SV_20099203 (1050bp deletion) affected the upstream promoter sequence of the Sindi_0765000 gene, which may have caused changes in the gene promoter activity, thereby affecting the gene function, causing the sesame seed shattering phenotype to mutate into a seed shattering resistance phenotype. Based on this, the inventors identified the Sindi_0765000 gene as the sesame seed shattering resistance target gene and named it Sihec3 .

[0085] Example 2

[0086] Based on the target gene identified in Example 1, the inventors further investigated the located sesame seed shattering gene. Sihec3 Sequence and upstream promoter sequence and allele SiHEC3 The specific process is briefly described as follows.

[0087] First, based on the work of Example 1, combined with the existing Yuzhi 11 genome fine map data, considering the sesame anti-seedling gene Sihec3 and alleles SiHEC3 As the upstream promoter sequences of each are long, the inventors designed different primer pairs to clone the target genes respectively. Sihec3 and its alleles SiHEC3 and respective promoter sequences, and finally obtain the full-length gene sequence by splicing method. The specific sequences of the designed primer pairs are:

[0088] Primer1F forward primer: 5'- TGACGGTCCGATTTGTAAGGTG -3',

[0089] Primer1R reverse primer: 5′-GAAATTCGGAGACGGGAGG-3′;

[0090] According to the previous analysis, the length of the amplified sequence in the shatter-resistant and shatter-resistant germplasms using this primer pair was 1392 bp and 2432 bp, respectively;

[0091] Primer2F forward primer: 5'-AGCTAAATCCTCCTCTTTCACCA-3',

[0092] Primer2R reverse primer: 5′-ACAGCGTACAGGTTTCATT-3′;

[0093] According to the previous analysis, the lengths of the sequences amplified by this primer pair in the shatter-resistant and shatter-resistant germplasms were 3410 bp and 3408 bp, respectively.

[0094] Subsequently, genomic DNA extracted from the shatter-resistant germplasm M7 and the shatter-type germplasm Yuzhi 11 was used as templates, and high-fidelity enzyme PrimeSTAR Max DNA Polymerase (product of Takara) was used to perform PCR amplification with the above primer pairs. During amplification, the reference setting of the 20µL PCR amplification reaction system was as follows:

[0095] DNA template, 1µL; dNTP Mix (10mM each), 0.4µL; 2×Phanta MaxBuffer, 10µL; Phanta Max Super-Fidelity DNA Polymerase, 0.4µL;

[0096] EvaGreen, 1µL; Primer F, 0.4µL; PrimerR, 0.4µL; ddH2O, 6.4µL;

[0097] The reference design of the PCR amplification reaction program is: denaturation at 98°C for 10s, annealing at 55°C for 5s, extension at 72°C for 3min and 50s, and 30 cycles; finally, extension at 72°C for 5min; the amplified product was directly subjected to subsequent electrophoresis analysis or stored at 4°C for future use.

[0098] Finally, the amplified products were detected by electrophoresis and recovered for sequencing analysis (completed by Shanghai Sangon Biotechnology Co., Ltd.). After sequencing, the sequencing results of the PCR products were spliced.

[0099] The results of the analysis showed that the anti-grain shattering gene Sihec3 The full-length genome sequence of is 3406bp, including 2 exons and 1 intron. The gene sequence is shown in SEQ ID No.1; Allele (grain falling phenotype) SiHEC3 The full-length genome sequence is 3441bp, including 2 exons and 1 intron.

[0100] The comparative analysis results show that ( Picture 4 shown): and the corresponding allele SiHEC3 The difference is that the anti-grain shattering gene Sihec3The upstream promoter region and 5'UTR region lacked 1049 bp, and accordingly, its phenotype changed from normal grain-shattering type to grain-shattering-resistant type; Sihec3 Genes and Alleles SiHEC3 There is an A / C base mutation at position 111 in the CDS region, which causes the 37th amino acid sequence of the encoded protein to mutate from glutamine to histidine. Sequence comparison with other varieties shows that this mutation site also exists in other seed-shattering germplasms and does not affect the seed-shattering phenotype.

[0101] Example 3

[0102] Based on the results of Example 2, to further confirm Sihec3 / SiHEC3 That is, the gene regulating the seed falling trait. Based on the principle of molecular breeding technology and PCR amplification technology, the inventors Sihec3 and alleles SiHEC3 Based on the differences in promoter and gene sequences, the inventors developed and designed a primer pair for PCR detection and named it SSR1. The specific sequence design is:

[0103] SSR1-F sequence: 5′ –TACATGGGCACAAAGAAAC -3′;

[0104] SSR1-R sequence: 5′ –CTGGTGAAAGAGGAGGATT -3′.

[0105] When using primer pair SSR1 to detect and determine the phenotype of sesame seed shattering, the specific steps are as follows:

[0106] (1) Extracting genomic DNA from the sesame sample to be tested;

[0107] (2) Using the genomic DNA extracted in step (1) as a template, use primers to perform PCR amplification on SSR1. During PCR amplification, the reference setting of the 15 µL PCR amplification reaction system is as follows:

[0108] Template DNA (50ng / µL), 1.0µL; 2×Phanta Max Buffer, 7.5µL; Phanta Max Super-Fidelity DNA Polymerase, 0.3µL;

[0109] dNTP Mix, 0.3µL; Forward Primer (10µM), 0.3µL; ReversePrimer (10µM), 0.3µL; EvaGreen, 0.75µL;

[0110] Ultrapure water was added to 15 µL; the PCR amplification reaction program reference design was: 98°C denaturation for 10 s, 55°C annealing for 5 s, 72°C extension for 120 s, 30 cycles; and finally 72°C extension for 5 min.

[0111] (3) Based on the comparison of the electrophoresis results of the PCR products with the control, determine whether the sample is a shed grain type or a shed grain-resistant type.

[0112] It should be noted that since the gene marker site belongs to SV mutation, the specific judgment criteria are:

[0113] If a 1724bp band appears after sample amplification, the sample contains SiHEC3 The gene sequence and the complete promoter sequence are used to determine that the sample is a homozygous granuloma type; if a 684bp band appears after sample amplification, the sample contains Sihec3 If the gene sequence is missing and the promoter sequence is deleted, the sample is judged to be homozygous for anti-grain-shattering type; if the 1724bp and 684bp bands appear at the same time after sample amplification, the sample is judged to be heterozygous for grain-shattering type.

[0114] Based on the above method, 96 natural germplasm resources and 96 individual plants of the hybrid F2 population of "Yuzhi NS610 (resistant to grain shattering) × Anhui Qingyangbai (normal grain shattering germplasm)" were randomly selected, and the inventors detected and determined their phenotypes.

[0115] The results show that (part of the results are as follows Picture 5 As shown in Figure 2), the typing results based on primer pair PCR are 100% consistent with the actual phenotype. This indicates that the target gene involved in this marker Sihec3 It is the gene regulating the sesame seed shattering trait.

[0116] In summary, it can be considered Sihec3 The gene is related to the regulation of sesame seed shattering resistance and can be used to study the growth and development regulation mechanism of sesame and other crops. The gene marker SSR1 can be used to detect the seed shattering trait type of sesame germplasm and can be directly used in the breeding of new sesame varieties with resistance to seed shattering.

Claims

1. Genes regulating sesame seed shattering resistance Sihec3 , characterized in that, This gene is located on the third chromosome of sesame and is a recessive control gene. It explains 100% of the phenotypic variation of the resistance to seed shattering trait. Sihec3 The gene is 3406 bp in length and contains 2 exons. Its gDNA is shown in SEQ ID No.

1.

2. The sesame grain falling resistance gene according to claim 1 Sihec3 The encoded protein is characterized in that The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. The sesame anti-seedling trait regulating gene according to claim 1 Sihec3 The application in sesame breeding is characterized by: Gene Sihec3 Used to regulate the phenotype of sesame grain shattering resistance.

4. PCR detection to determine the sesame anti-seedling gene of claim 1 Sihec3 and its alleles SiHEC3 Using a primer pair, characterized in that The primer pair was named SSR1, and the specific design was as follows: SSR1-F sequence: 5'-TACATGGGCACAAAGAAAC-3', SSR1-R sequence: 5'-CTGGTGAAAGAGGAGGATT-3'.

5. Use of the primer pair according to claim 4 in sesame molecular breeding, characterized in that: Used to detect and determine whether sesame has a phenotype of resistance to grain shattering.

6. Sesame seed shattering resistance gene using the primer pair of claim 4 Sihec3 and its alleles SiHEC3 The detection and differentiation method is characterized in that: The steps include: (1) Extracting genomic DNA from the sesame sample to be tested; (2) using the genomic DNA extracted in step (1) as a template, performing PCR amplification using a primer pair, and detecting the amplified product by agarose gel electrophoresis; (3) Determine whether the sesame sample to be tested is a shattering type or a shatter-resistant type based on the electrophoretic band pattern. The specific criteria for determination are: If the length of the sample amplified fragment is 1724 bp, the sample is determined to be a homozygous granuloma type; If the length of the amplified fragment is 684 bp, the sample is judged to be homozygous for shatter-resistant type; If the amplified fragment results show both 1724bp and 684bp bands, the sample is judged to be a heterozygous granuloma type.

Images