SNP (Single Nucleotide Polymorphism) molecular marker related to brassica napus branching number, primer and application

Abstract

The invention relates to the technical field of molecular biology, in particular to an SNP (Single Nucleotide Polymorphism) molecular marker related to the brassica napus branching number, a primer and application. The SNP molecular marker related to the position of the rape genome chrA09: 46507122 comprises a nucleotide sequence as shown in SEQ ID NO. 1 and a nucleotide sequence as shown in SEQ ID NO. 2; the basic group at the chrA09: 46507122 position of the rape genome is polymorphic, and the basic group is T or C. The invention further discloses a method for identifying the rape genome chrA09: 46507122 position of the rape genome. The primer is a primer for amplifying the SNP molecular marker. The application is the application of the SNP molecular marker in rape multi-branch character breeding. The method can provide possibility for genetic improvement of the branch number character of the oilseed rape, and further facilitates cultivation of high-yield oilseed rape varieties.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a SNP molecular marker, a primer and an application thereof related to the branch number of rapeseed. Background Art

[0002] At present, some rapeseed varieties have low yields and low planting benefits, and farmers are not very enthusiastic about planting. Research on branching-related traits is of great significance for breeding high-yield rapeseed varieties. Rapeseed yield is determined by the number of siliques per plant, the number of seeds per silique, and the thousand-grain weight. A large number of field test results from previous studies have shown that the number of siliques per plant contributes the most to the seed yield per plant. Branching-related traits such as the number of branches and the number of siliques in the main inflorescence are the main factors determining the number of siliques per plant, and are significantly positively correlated with the number of siliques per plant. In addition, branching-related traits are relatively stable under different environments. By increasing the number of branches and the number of siliques in the main inflorescence, the number of siliques per plant and the yield per plant can be significantly increased. Therefore, it is of great significance to deeply understand the genetic basis and molecular mechanism of rapeseed branching and develop corresponding molecular markers to improve breeding efficiency and breed high-yield rapeseed varieties.

[0003] In summary, there is a need for a SNP molecular marker, primer and application related to the branch number of rapeseed to provide a possibility for genetic improvement of the branch number trait of rapeseed, thereby contributing to the breeding of high-yield rapeseed varieties. Summary of the invention

[0004] The present invention aims to provide a SNP molecular marker, primer and application related to the number of rapeseed branches. The specific technical scheme is as follows:

[0005] In the first aspect, the present invention provides a SNP molecular marker related to the number of rapeseed branches, and the SNP molecular marker related to the chrA09:46507122 position of the rapeseed genome includes the nucleotide sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.2; the base at the chrA09:46507122 position of the rapeseed genome is polymorphic, and the base is T or C.

[0006] Optionally, the base of the nucleotide at position 102 of the nucleotide sequence shown in SEQ ID NO.1 is T; and the base of the nucleotide at position 102 of the nucleotide sequence shown in SEQ ID NO.2 is C.

[0007] In a second aspect, the present invention provides a primer for amplifying the SNP molecular marker associated with the branch number of rapeseed, characterized in that it includes the nucleotide sequence shown in SEQ ID NO.3, the nucleotide sequence shown in SEQ ID NO.4 and the nucleotide sequence shown in SEQ ID NO.5.

[0008] In a third aspect, the present invention provides an application of the SNP molecular marker associated with the number of rapeseed branches in the breeding of rapeseed with multiple branching traits.

[0009] The application of the technical solution of the present invention has at least the following beneficial effects:

[0010] The present invention provides a SNP molecular marker, primer and application related to the number of rape branches, which can provide a possibility for genetic improvement of the number of rape branches, thereby helping to cultivate high-yield rape varieties. Specifically, the present invention uses a SNP molecular marker related to the position of chrA09:46507122 in the rape genome, which can be used in rape multi-branch trait breeding, identify rape individuals with a large number of branches, and facilitate the selection of favorable genotype single plants for seed retention to increase the number of branches of the offspring, thereby helping to cultivate high-yield rape varieties. DETAILED DESCRIPTION

[0011] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0012] Example:

[0013] A method for obtaining a SNP molecular marker related to the number of rapeseed branches is as follows:

[0014] 1) Experimental Materials

[0015] "Lt1" is a Brassica napus variety bred by Hunan Crop Research Institute. This variety has multiple branches, stable traits, 100% penetrance, and an average number of branches of about 14. 20B is a conventional normal branching line.

[0016] 2) Group building

[0017] Hybridize "Lt1" with normal plant material 20B to obtain F1. Self-pollinate the F1 individual to obtain the F2 population, and backcross the F1 individual with the recessive parent 20B to obtain the BC1F1 population. Use the F2 population and BC1F1 population for preliminary positioning. According to the preliminary positioning results, use molecular marker-assisted selection to obtain the BC1F2 population through self-pollination of the individual with heterozygous genotype at the target site, and backcross the individual with heterozygous genotype with 20B to obtain the BC2F1 population. Use the BC1F2 population and BC2F1 population for fine positioning.

[0018] 3) Trait genetic analysis

[0019] SPSS 19.0 (SPSS Inc., Chicago, IL, USA) was used to analyze the mean, standard deviation, and coefficient of variation of the traits. SPSS 19.0 was used to analyze the normal distribution using the Kolmogorov-Smirnov test. 2 The phenotypic variation of each trait was displayed using frequency distribution histograms in Excel (Microsoft Corp., Redmond, WA, USA). Multiple comparisons between different genotypes of the same trait were performed using SPSS 19.0 software.

[0020] 4) Preliminary positioning based on the BSA method of rapeseed 60K SNP chip

[0021] In the BC1F1 population, based on the branch number phenotype, normal plant types with more than 3 branches and normal plant types with 1 branch were selected, and 40 plants were selected for each of the two phenotypes. Among them, a Bulk pool was constructed by mixing every 10 plants with more than 3 branches and every 10 plants with 1 branch, and a total of 4 groups of parallel extreme phenotype Bulk pools were constructed. Construct 4 groups of parallel extreme phenotype Bulk pools. The 4 groups of parallel extreme phenotype Bulk pools and two parents, a total of 10 samples, were subjected to BSA analysis using the rapeseed 60KSNP chip to obtain SNPs near the target gene. The rapeseed 60K SNP chip related experiments were completed on the rapeseed group chip platform of Huazhong Agricultural University.

[0022] After obtaining the genotype data, the IF function of Excel (Microsoft Corp., Redmond, WA, USA) was used to screen for polymorphic SNPs. The screening criteria were: (1) polymorphism between the parents; (2) consistent genotypes among the four multi-branch pools; (3) the same genotype among the four normal plant pools; (4) polymorphism between the multi-branch pool and the normal plant pool.

[0023] Analysis of 42,090 SNPs in the rapeseed 50K SNP chip revealed that 16,064 SNPs were polymorphic in both parents. Further analysis of these 16,064 SNPs revealed that 237 SNPs were polymorphic between the two phenotypic pools. Aligning these differential markers to the Darmor-bzh reference genome revealed that 201 SNPs were located on chromosome A09, accounting for 81.7% of the total polymorphic SNPs, and were mainly located in the 15-Mb to 19-Mb segment of A09, indicating that genes related to branch formation may be located on chromosome A09 of rapeseed. The potential target QTL (i.e., Quantitative trait locus, which represents the quantitative trait locus of branch-related traits) is determined based on the distribution of polymorphic SNPs on chromosomes.

[0024] 5) Preliminary localization based on the BSA method of whole genome resequencing

[0025] In the F2 population, based on the branch number phenotype, normal plant types with more than 3 branches and normal plant types with 1 branch were selected. 200 plants were selected for each of the two phenotypes, and the DNA was extracted and then mixed in equal amounts. The two mixed pools and the parents were sent to Huazhi Biotechnology Co., Ltd. and sequenced using Illumina HiSeq 4000, PE150. The QTL-Seq method was used for analysis to verify the initial positioning results and preliminarily narrow down the positioning segment (Reference: Takagi H, Abe A, Yoshida K, Kosugi S, Natsume S, Mitsuoka C, Uemura A, Utsushi H, Tamiru M, Takuno S, Innan H, Cano LM, Kamoun S, Terauchi R (2013) QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. Plant J 74(1): 174-183. doi: 10.1111 / tpj.12105)

[0026] 6) Fine mapping of genes related to multiple branches

[0027] 128-hole plug trays were used to sow the BC1F1, BC1F2 and BC2F1 segregating populations, and a total of 10,000 plants were expected to be sown. A 96-well deep-well plate was used for sampling to achieve high-throughput sampling. Based on the initial positioning results, KASP markers were developed for the positioning segment, and the plants of the segregating population were screened for exchange plants using KASP. The development and analysis of KASP markers were sent to Huazhi Biotechnology Co., Ltd. for completion. For the key exchange plants, after harvesting from the cross, two rows of each strain were planted for progeny testing to ultimately determine the segment where the multi-branching gene was located. In this segment, the KASP marker was developed using the SNP at the chrA09:46507122 position of the rapeseed genome. The base at the chrA09:46507122 position of the rapeseed genome is polymorphic, and the base is T or C. Among them, when the base at position 46507122 of the rapeseed genome chrA09: is T, it shows more branches; when the base at position 46507122 of the rapeseed genome chrA09: is C, it shows less branches. The SNP molecular marker associated with position 46507122 of the rapeseed genome chrA09: includes the nucleotide sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.2. The base of the nucleotide at position 102 of the nucleotide sequence shown in SEQ ID NO.1 is T; the base of the nucleotide at position 102 of the nucleotide sequence shown in SEQ ID NO.2 is T.

[0028] Identification of dominant alleles

[0029] Step S1: randomly selecting 200 individual plants in the BC1F2 population, taking plant leaves at the seedling stage, and extracting genomic DNA from Brassica napus using the CTAB (specifically hexadecyltrimethylammonium bromide) method;

[0030] Step S2, using the genomic DNA extracted in step S1 as a template, and using the primer set SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 to detect the SNP molecular marker; specifically, FAM and HEX fluorescent linker sequences are connected to the 5′ ends of the SEQ ID NO.3 and the SEQ ID NO.4, respectively; when using the primer set, the SEQ ID NO.3, the SEQ ID NO.4 and the SEQ ID NO.5 are used in combination; PCR amplification is performed using the primer set; the PCR amplification system is shown in Table 1; wherein, 2×KASP Master Mix is ​​a product of LGC Company, and the product catalog number is KBS-1016-002.

[0031] Table 1 PCR amplification system

[0032] Final concentration Actual usage SEQ ID NO.5 0.42μM 0.0033μL SEQ ID NO.3 0.17μM 0.0013μL SEQ ID NO.4 0.17μM 0.0013μL 2 × KASPMasterMix 1× 0.3945μL Ultrapure water 0.3995μL DNA (dry) 20ng-50ng Total volume 0.8μL

[0033] The PCR reaction program (specifically Touchdown PCR reaction program) includes: denaturation at 94°C for 15 min; denaturation at 95°C for 20 s, annealing and extension at 65°C-56°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 0.8°C in each cycle; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 30 cycles.

[0034] At the same time, a blank control without adding template DNA to the reaction system was set up, and one blank control was set up for each PCR plate.

[0035] The PCR amplification products were scanned using the bidirectional single excitation plate reader PHERAstar, with FAM excitation wavelength of 485 nm and emission wavelength of 520 nm; HEX excitation wavelength of 528 nm and emission wavelength of 560 nm; system reference fluorescence ROX excitation wavelength of 575 nm and emission wavelength of 610 nm. Three replicates were set for each PCR amplification product sample.

[0036] After the PCR amplification reaction is completed, the KASP reaction product is read by a scanner, and the results of the fluorescence scan are automatically converted into a graph. Kraken software is used to analyze the scanning data of the two-way single excitation plate reader PHERAstar.

[0037] Step S3: After maturity, the branching characteristics of the 200 selected plants were investigated, and the number of branches of each plant was counted, corresponding to its SNP genotype, and the phenotypic differences in the number of branches of different genotypes of the SNP site chrA09:46507122T>C were compared. For chrA09:46507122T>C, the branch numbers were T / T: 6.7, T / C: 10.2, C / C: 15.4, and the dominant allele was C.

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A SNP molecular marker related to the number of rapeseed branches, characterized in that: The SNP molecular marker associated with the chrA09:46507122 position of the rapeseed genome includes the nucleotide sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.2; the base at the chrA09:46507122 position of the rapeseed genome is polymorphic, and the base is T or C.

2. The SNP molecular marker associated with the number of rapeseed branches according to claim 1, characterized in that: The base of the 102nd nucleotide of the nucleotide sequence shown in SEQ ID NO.1 is T; the base of the 102nd nucleotide of the nucleotide sequence shown in SEQ ID NO.2 is C.

3. A primer for amplifying the SNP molecular marker associated with the branch number of rapeseed as claimed in claim 1, characterized in that: It includes the nucleotide sequence shown in SEQ ID NO.3, the nucleotide sequence shown in SEQ ID NO.4 and the nucleotide sequence shown in SEQ ID NO.

5.

4. Use of the SNP molecular marker associated with rapeseed branch number as claimed in claim 1 in rapeseed multi-branching trait breeding.