A molecular marker, primers and applications of SNPs related to branch number in rapeseed

Abstract

This invention relates to the field of molecular biology, specifically to a SNP molecular marker, primers, and applications related to the number of branches in rapeseed. The SNP molecular marker associated with the rapeseed genome position chrA09:46507122 includes the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2; the base at position chrA09:46507122 in the rapeseed genome exhibits polymorphism, with the base being either T or C. The primers are used to amplify this SNP molecular marker. The application is the use of this SNP molecular marker in breeding rapeseed for the high branching trait. This invention can provide possibilities for the genetic improvement of the branching trait in rapeseed, thereby contributing to the breeding of high-yielding rapeseed varieties.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a SNP molecular marker, primer, and application related to the number of branches in rapeseed. Background Technology

[0002] Currently, some rapeseed varieties suffer from low yields and low economic benefits, leading to low farmer enthusiasm for cultivation. Research on branching-related traits is crucial for breeding high-yielding rapeseed varieties. Rapeseed yield is determined by the total number of siliques per plant, the number of seeds per silique, and the thousand-seed weight. Extensive field trials have shown that the total number of siliques per plant contributes most significantly 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 total number of siliques per plant, showing a significant positive correlation. Furthermore, branching-related traits are relatively stable under different environmental conditions. Increasing the number of branches and the number of siliques in the main inflorescence can significantly increase the total number of siliques per plant and the yield per plant. Therefore, a deeper understanding of the genetic basis and molecular mechanisms of rapeseed branching, and the development of corresponding molecular markers, are of great significance for improving breeding efficiency and cultivating high-yielding rapeseed varieties.

[0003] In summary, there is a need for a molecular marker, primer, and application of SNP related to the number of branches in rapeseed to provide a possibility for the genetic improvement of the branching trait in rapeseed, thereby helping to breed high-yielding rapeseed varieties. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular marker, primer, and application of SNPs related to the number of branches in rapeseed. The specific technical solution is as follows:

[0005] In a first aspect, the present invention provides a SNP molecular marker related to the number of branches in rapeseed, wherein the SNP molecular marker related to the chrA09:46507122 position in 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 in the rapeseed genome exhibits polymorphism, and its base is T or C.

[0006] Optionally, the nucleotide at position 102 of the nucleotide sequence shown in SEQ ID NO.1 is T; and 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 primers for amplifying SNP molecular markers related to the number of branches in rapeseed, characterized in that they include the nucleotide sequences shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

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

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

[0010] This invention provides a SNP molecular marker, primers, and applications related to the number of branches in rapeseed, which can provide possibilities for the genetic improvement of the branching trait in rapeseed, thereby contributing to the breeding of high-yielding rapeseed varieties. Specifically, this invention uses an SNP molecular marker associated with the rapeseed genome location chrA09:46507122, which can be applied in the breeding of rapeseed with multiple branches. This allows for the identification of rapeseed individuals with a high number of branches, facilitating the selection of advantageous genotypes for seed production to increase the number of branches in offspring, thus contributing to the breeding of high-yielding rapeseed varieties. Detailed Implementation

[0011] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art are within the scope of protection of the present invention.

[0012] Example:

[0013] A method for obtaining SNP molecular markers related to the branch number of rapeseed is as follows:

[0014] 1) Experimental materials

[0015] "Lt1" is a Brassica napus flowering cultivar bred by the Hunan Provincial Crop Research Institute. This cultivar is characterized by multi-branching, stable traits, 100% penetrance, and an average of about 14 branches. 20B is a conventional, normally branching cultivar.

[0016] 2) Group building

[0017] The "Lt1" genotype was crossed with the normal plant type material 20B to obtain the F1 generation. F1 plants were self-crossed to obtain the F2 population. Simultaneously, F1 plants were backcrossed with the recessive parent 20B to obtain the BC1F1 population. The F2 and BC1F1 populations were used for initial mapping. Based on the initial mapping results, marker-assisted selection was used to select heterozygous plants at the target locus. These heterozygous plants were then self-crossed to obtain the BC1F2 population. Simultaneously, heterozygous plants were backcrossed with 20B to obtain the BC2F1 population. The BC1F2 and BC2F1 populations were used for fine mapping.

[0018] 3) Genetic analysis of traits

[0019] Statistical analysis of traits, including mean, standard deviation, and coefficient of variation, was performed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Normality analysis was conducted using the Kolmogorov-Smirnov test in SPSS 19.0. Chi-square tests (χ² tests) were performed on the F2 and BC1F1 populations. 2 The test was performed in SAS 8.1 software (SAS Inc., Cary, NC, USA). Frequency distribution histograms were used in Excel 1 (Microsoft Corp., Redmond, WA, USA) software to display phenotypic variation for each trait. Multiple comparisons between different genotypes of the same trait were performed using SPSS 19.0 software.

[0020] 4) Initial localization using the BSA method based on the rapeseed 60K SNP chip.

[0021] In the BC1F1 population, based on the branching number phenotype, 40 plants of normal plant type with more than 3 branches and 40 plants of normal plant type with 1 branch were selected for each phenotype. Each group of 10 plants with more than 3 branches and each group of 10 plants with 1 branch were combined to construct a Bulk pool, resulting in a total of 4 parallel extreme phenotype Bulk pools. These 10 samples (4 parallel extreme phenotype Bulk pools and two parents) were analyzed using a rapeseed 60K SNP chip (BSA) to obtain SNPs near the target genes. The rapeseed 60K SNP chip experiments were conducted on the rapeseed microarray platform at Huazhong Agricultural University.

[0022] After obtaining the genotype data, polymorphic SNPs were screened using the IF function in Excel (Microsoft Corp., Redmond, WA, USA). The screening criteria were: (1) polymorphism between the parents; (2) identical genotypes among the four multi-branched pools; (3) the same genotype among the four normal type pools; and (4) polymorphism between the multi-branched pool and the normal type pool.

[0023] Analysis of 42,090 SNPs from the 50K SNP microarray in rapeseed revealed that 16,064 SNPs exhibited polymorphism between the parents. Further analysis of these 16,064 SNPs revealed that 237 SNPs showed polymorphism between the two phenotypic pools. Aligning these differentially expressed markers to the Darmor-bzh reference genome, 201 SNPs were found to be located on chromosome A09, accounting for 81.7% of the total polymorphic SNPs, primarily in the 15-Mb to 19-Mb region of A09, indicating that branching-related genes may be located on the rapeseed A09 chromosome. The distribution of polymorphic SNPs on the chromosome was used to determine the locations of potential target QTLs (Quantitative Trait Locus, representing quantitative trait loci associated with branching).

[0024] 5) Initial localization based on BSA method using whole-genome resequencing

[0025] In the F2 population, based on the branching number phenotype, 200 normal-type plants with more than 3 branches and 200 normal-type plants with 1 branch were selected from each phenotype. DNA was extracted from each phenotype and then pooled in equal volumes. The two pools and the parents were sent to Huazhi Biotechnology Co., Ltd. for sequencing using an Illumina HiSeq 4000 PE150. QTL-Seq was used to analyze the preliminary localization results and to initially narrow down the localization region (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 lociin 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 multibranching

[0027] The BC1F1, BC1F2, and BC2F1 segregating populations were sown using 128-well seed trays, with a total of 10,000 individual plants expected to be sown. High-throughput sampling was achieved using 96-well deep-well plates. Based on the initial localization results, KASP markers for localization regions were developed. Individual plants from the segregating populations were screened for exchange plants using KASP markers. KASP marker development and analysis were performed by Huazhi Biotechnology Co., Ltd. For key exchange plants, after self-crossing, two rows of each line were planted for progeny testing to ultimately determine the region containing the multi-branching gene. Within this region, KASP markers were developed using the SNP at position chrA09:46507122 in the rapeseed genome. The base at position chrA09:46507122 in the rapeseed genome exhibits polymorphism, with either T or C bases. Specifically, when the base at position chrA09:46507122 in the rapeseed genome is T, it exhibits multi-branching; when the base at position chrA09:46507122 in the rapeseed genome is C, it exhibits few branches. The SNP molecular markers associated with position chrA09:46507122 in the rapeseed genome include the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2. The nucleotide sequence shown in SEQ ID NO.1 has a base of T at position 102; the nucleotide sequence shown in SEQ ID NO.2 also has a base of T at position 102.

[0028] Identification of dominant alleles

[0029] Step S1: In the BC1F2 population, 200 individual plants were randomly selected. During the seedling stage, plant leaves were taken and genomic DNA was extracted from rapeseed using the CTAB (specifically, hexadecyltrimethylammonium bromide) method.

[0030] Step S2: Using the genomic DNA extracted in step S1 as a template, the SNP molecular markers are detected using primer sets SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5. Specifically, FAM and HEX fluorescent adapter sequences are ligated to the 5′ ends of SEQ ID NO.3 and SEQ ID NO.4, respectively. When using the primer set, SEQ ID NO.3, SEQ ID NO.4, and 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. Among them, 2×KASP Master Mix is ​​a product of LGC Corporation, product catalog number 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 (dried) 20ng-50ng Total volume 0.8μL

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

[0034] Meanwhile, a blank control was set up in the reaction system without template DNA, with one blank control set up for each PCR plate.

[0035] PCR amplification products were scanned using a two-way single-excitation plate reader, Pherastar. The excitation wavelength for FAM was 485 nm, and the emission wavelength was 520 nm; the excitation wavelength for HEX was 528 nm, and the emission wavelength was 560 nm; the system reference fluorescence ROX was excited at 575 nm and emitted at 610 nm. Each PCR amplification product sample was replicated in triplicate.

[0036] After the PCR amplification reaction was completed, the KASP reaction products were scanned using a scanner to read the fluorescence data, and the results of the fluorescence scans were automatically converted into images. The scan data from the Pherastar two-way single-excitation plate reader were analyzed using Kraken software.

[0037] Step S3: After maturity, the branching traits of 200 selected individual plants were examined, and the number of branches of each individual plant was counted and matched with its SNP genotype. The phenotypic differences in the number of branches among different genotypes of the SNP locus chrA09:46507122T>C were compared. For chrA09:46507122T>C, the branching numbers were T / T: 6.7, T / C: 10.2, and C / C: 15.4, with C being the dominant allele.

[0038] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a SNP molecular marker related to the number of branches in rapeseed in breeding for the multi-branching trait of rapeseed, characterized in that, The SNP molecular markers associated with the chrA09: 46507122 position in the rapeseed genome include the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2; the base at the chrA09: 46507122 position in the rapeseed genome exhibits polymorphism, with the base being either T or C; The nucleotide at position 102 of the nucleotide sequence shown in SEQ ID NO.1 is T; the nucleotide at position 102 of the nucleotide sequence shown in SEQ ID NO.2 is C; Specifically, when the base at position chrA09: 46507122 in the rapeseed genome is T, it exhibits multi-branching; when the base at position chrA09: 46507122 in the rapeseed genome is C, it exhibits few branches.