Molecular markers for early flowering traits of Brassica napus and methods for obtaining and using the markers

Abstract

The present invention discloses a molecular marker for early flowering traits of Brassica napus, a method for obtaining the marker and an application thereof. The mutant ebm induced by EMS in early flowering materials of rapeseed is identified through field flowering period survey, and an F2 segregation population of the mutant and double 9 in late flowering source materials is constructed. The early flowering gene is initially located by using the mixed pool resequencing (BSA‑seq) technology, and SNP molecular markers that can be used for early flowering trait screening and gene positioning are screened in this interval. The molecular markers include SNP molecular markers 1‑11 that are homozygous for both parents. The present invention provides new molecular markers for the excavation of flowering mechanism and molecular breeding of rapeseed, and has important application prospects for enhancing seed purity and the selection of flowering period materials in the hybrid breeding process of rapeseed.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a molecular marker of early flowering traits of Brassica napus and an acquisition method and application thereof. Background Art

[0002] Single nucleotide polymorphism (SNP) is widely present in the genomes of animals and plants. It is a DNA sequence polymorphism caused by the variation of a single nucleotide, including single base conversion, transversion, insertion and deletion. It is a new generation of molecular markers after microsatellite markers. Compared with microsatellite markers, SNPs have a large distribution density, a large number, and rich polymorphism; a low mutation rate, high stability and accuracy; there is no need to detect the fragment length, only the "presence or absence" detection to determine the polymorphism, which makes it easy to achieve automated screening. SNP markers have the following applications: (1) They can be used for gene-disease association studies and to locate disease target genes; (2) They can be used to screen key genes for animal and plant breeding, and by comparing the genetic differences of different phenotypes, key SNPs such as stress resistance and high yield can be screened; (3) They can be used to infer evolutionary relationships between species and identify kinship within species. They have great application potential in medicine, agronomy, and forestry.

[0003] Optimal flowering time plays a vital role in crop production and plant life cycle. Early maturing varieties inhibit vegetative growth and have a greater impact on vegetable yield. In contrast, late flowering or inhibited flowering not only reduces seed yield but also increases the residual harmful biomass for crop rotation. Therefore, understanding the regulatory mechanism of flowering time is crucial for the optimization of crop breeding. In the model plant Arabidopsis thaliana, the molecular basis of flowering regulation has been widely studied. Flowering time is mainly mediated by genetic factors in four major pathways, namely autonomous, photoperiodic, vernalization-induced and signal transduction. So far, a large number of key genes involved in flowering regulation have been cloned, such as FLOWERING LOCUS T (FT), FLOWERING LOCUS C (FLC) and CONSTANS (CO).

[0004] Rapeseed (Brassica napus L., 2n=38, AACC) is an important oil crop grown for many years in the world. Flowering time is an important determinant of potential yield formation, so appropriate flowering time is a major goal of rapeseed breeding. At present, QTL mapping studies based on biparental mapping populations have shown that chromosomes A2, A3, C1, C2, C3, C5 and C9 carry candidate homologous genes such as FT, FLC, VIN3, CO, Phytochrome A, FY, LFY, AP1, TFL1 and TFL2. Most of these genes are related to vernalization, photoperiod, autonomous pathways and floral meristem differentiation. These studies show that flowering traits are extremely complex and are controlled by multiple loci located on different chromosomes. However, the understanding of the characteristics of candidate genes for flowering in rapeseed and the development of molecular markers associated with flowering genes are still very limited. The density of molecular marker distribution determines the size of the gene positioning region. Therefore, it is urgent to develop molecular markers associated with flowering for the gene positioning of flowering genes. Summary of the invention

[0005] In view of the defects in the prior art, the present invention proposes an application of identification of early-flowering materials of rapeseed, preliminary positioning of early-flowering genes and development of molecular markers. The present invention identifies the EMS-induced mutant ebm of early-flowering materials of rapeseed through field flowering period survey, constructs an F2 segregation population of the early-flowering material and double 9 in the late-flowering source material, uses the pooled resequencing (BSA-seq) technology to preliminarily position the early-flowering gene, and screens out SNP molecular markers that can be used for early-flowering trait screening and gene positioning in this interval.

[0006] The present invention provides a molecular marker for early flowering of Brassica napus, wherein the molecular marker comprises at least one of SNP marker 1, SNP marker 2, SNP marker 3, SNP marker 4, SNP marker 5, SNP marker 6, SNP marker 7, SNP marker 8, SNP marker 9, SNP marker 10, and SNP marker 11;

[0007] The SNP markers 1 to 11 are all located on chromosome A2 of rapeseed;

[0008] The SNP marker 1 is base A or G, the SNP marker 1 is located at position 364 of the nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO.1;

[0009] The SNP marker 2 is a base T or C, the SNP marker 2 is located at the 200th position of the nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO.2;

[0010] The SNP marker 3 is a base T or A, the SNP marker 3 is located at position 152 of the nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO.3;

[0011] The SNP marker 4 is a base G or T, the SNP marker 4 is located at position 261 of the nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO.4;

[0012] The SNP marker 5 is base A or G, the SNP marker 5 is located at position 262 of the nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO.5;

[0013] The SNP marker 6 is a base G or A, the SNP marker 6 is located at position 493 of the nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO.6;

[0014] The SNP marker 7 is a base G or C, the SNP marker 7 is located at position 256 of the nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO.7;

[0015] The SNP marker 8 is base A or C, the SNP marker 8 is located at position 153 of the nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO.8;

[0016] The SNP marker 9 is base A or G, the SNP marker 9 is located at position 362 of the nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO.9;

[0017] The SNP marker 10 is a base G or A, the SNP marker 10 is located at position 413 of the nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO.10;

[0018] The SNP marker 11 is base A or G, and the SNP marker 11 is located at the 284th position of the nucleic acid, and the nucleic acid has a nucleotide sequence shown in SEQ ID NO.11.

[0019] The present invention also provides a primer pair for detecting the SNP molecular markers 1 to 11.

[0020] Furthermore, the sequence of the primer pair is:

[0021]

[0022]

[0023] The primer pair ES-1 is used to amplify the nucleotide sequence shown in SEQ ID NO.1;

[0024] The primer pair ES-2 is used to amplify the nucleotide sequence shown in SEQ ID NO.2;

[0025] The primer pair ES-3 is used to amplify the nucleotide sequence shown in SEQ ID NO.3;

[0026] The primer pair ES-4 is used to amplify the nucleotide sequence shown in SEQ ID NO.4;

[0027] The primer pair ES-5 is used to amplify the nucleotide sequence shown in SEQ ID NO.5;

[0028] The primer pair ES-7 is used to amplify the nucleotide sequence shown in SEQ ID NO.6;

[0029] The primer pair ES-10 is used to amplify the nucleotide sequence shown in SEQ ID NO.7;

[0030] The primer pair ES-12 is used to amplify the nucleotide sequence shown in SEQ ID NO.8;

[0031] The primer pair ES-20 is used to amplify the nucleotide sequence shown in SEQ ID NO.9;

[0032] The primer pair ES-40 is used to amplify the nucleotide sequence shown in SEQ ID NO.10;

[0033] The primer pair ES-47 is used to amplify the nucleotide sequence shown in SEQ ID NO.11.

[0034] The present invention also provides a method for obtaining the molecular marker of early flowering of rapeseed, comprising the following steps:

[0035] (1) Screening early-flowering rapeseed varieties;

[0036] (2) hybridizing the early-flowering rapeseed variety screened in step (1) with the late-flowering variety from which it was originally derived to obtain an F2 population;

[0037] (3) Screening out several early-flowering and late-flowering rapeseed samples from the F2 population in step (2), extracting genomic DNA from the early-flowering samples and the late-flowering samples, optimizing them to similar concentrations, and then mixing them in equal amounts to generate two extreme pools, together with the screened early-flowering variety and the two parental DNAs of the late-flowering variety of the original source, a total of four samples were used for library construction according to a standard 150 bp double-end sequencing protocol, followed by high-throughput sequencing;

[0038] (4) Check and filter the sequencing data, annotate reliable polymorphic variants, and select high-quality SNPs / InDels with a read coverage depth of at least 4 for BSA-seq analysis;

[0039] (5) The Euclidean distance and SNP index methods were used to detect potential SNP candidate loci associated with early flowering traits, and the overlapping regions based on these two methods were defined as candidate regions;

[0040] (6) Design primer pairs in the candidate region and perform PCR using early-flowering rapeseed varieties and late-flowering varieties of the original source as templates to verify whether there are SNP sites.

[0041] Furthermore, in step (6), it is verified whether there is a SNP site. If there is a SNP site, sequencing verification can obtain a difference in the single base corresponding to the SNP site between the parental species.

[0042] Furthermore, the early-flowering rapeseed variety is a material derived from the original variety through ethyl methanesulfonate mutagenesis, named ebm, and the late-flowering variety of the original source is Zhongshuang 9 (ZS9).

[0043] The present invention also provides application of the molecular marker or the primer pair in early flowering screening of rapeseed.

[0044] The present invention also provides the use of the molecular marker or the primer pair in the positioning of rapeseed early flowering gene.

[0045] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0046] 1. Although there have been some reports on the research of flowering traits of rapeseed, so far, compared with other traits, the research on the molecular mechanism of flowering is slightly less. The work carried out in rapeseed in the present invention has enriched the research on flowering.

[0047] 2. The present invention develops molecular markers for the initial positioning interval produced by BSA, and the obtained polymorphic molecular markers are helpful for the precise positioning of early flowering genes.

[0048] 3. The SNP molecular marker of the present invention can be applied to the location of genes between 5.39-10.84M on rapeseed A2 chromosome.

[0049] 4. The present invention uses BSA-seq technology to initially locate the early flowering gene in the constructed F2 population, and the obtained candidate gene interval provides a reference for the study of the molecular mechanism of rapeseed flowering, which is of great significance.

[0050] 5. The early-flowering material ebm identified in the present invention provides a new genetic resource for the breeding of early-flowering materials and serves as a guide and reference for the breeding of early-flowering varieties.

[0051] 6. The discovery of ebm, an early-flowering rapeseed material, has important theoretical guiding significance for conducting research on the flowering mechanism of crops and exploring the molecular mechanism of flowering.

[0052] 7. The selection of early-flowering rapeseed materials has always been valued by rapeseed breeders. The identification of early-flowering materials ebm provides breeders with an excellent source of early-flowering materials. Since the long growth cycle of late-flowering rapeseed has a huge impact on rapeseed yield and crop rotation, the selection of early-flowering varieties is particularly important. This has laid a solid foundation for the cloning of early-flowering genes in the next step; it provides new breeding materials for further breeding of rapeseed varieties with early-flowering characteristics; and it has important practical guiding value in the breeding practice, variety improvement and variety promotion of early-flowering crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0054] Figure 1 It is a schematic diagram of the phenotype of the early-flowering mutant ebm of Brassica napus of the present invention;

[0055] A: Top, comparison of ZS9 and ebm mutant plants at the seedling stage, Scale bar = 25 cm; Bottom, comparison of flowering traits of the main inflorescence of ZS9 and ebm mutant plants at the flowering stage, Scale bar = 25 cm;

[0056] B: Comparison of agronomic traits such as plant height, first branch height, number of branches, length of main inflorescence, number of siliques in main inflorescence and 1000-grain weight between ZS9 and ebm mutant plants at maturity, as well as comparison of the time of bud appearance, initial flowering and full flowering;

[0057] C: Frequency distribution of flowering time of the F2 population obtained by hybridization of ZS9 and ebm.

[0058] Figure 2 This is the initial location of the early flowering-related gene of Brassica napus of the present invention;

[0059] A: The upper, middle and lower Manhattan plots represent the SNP variation frequencies of pool L and pool E at the whole genome level, as well as the difference between the two pool frequencies. L is late flowering, and E is early flowering;

[0060] B: The upper, middle and lower Manhattan plots represent the SNP variation frequencies of the L pool and E pool on chromosome A02, as well as the difference between the two pool frequencies. L is late flowering and E is early flowering. The candidate region is between 5.39-10.84Mb in A02.

[0061] Figure 3It is a SNP polymorphism molecular marker developed in the initial positioning interval and flanks in the present invention.

[0062] Figure 4 This is the fine positioning process of the early flowering gene in the present invention; based on the BSA-seq results, the developed molecular markers were used to screen 60 and 460 F2 generation early flowering phenotype plants for exchange, and the number of exchange plants under the molecular marker was represented below the vertical line, and the candidate gene was finally positioned between ES-20 and ES-7. DETAILED DESCRIPTION

[0063] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0064] The method for obtaining the molecular marker of early flowering of rapeseed of the present invention comprises the following steps:

[0065] (1) Screening early-flowering rapeseed varieties;

[0066] (2) hybridizing the early-flowering rapeseed variety screened in step (1) with the late-flowering variety from which it was originally derived to obtain an F2 population;

[0067] (3) Screening out a number of early-flowering and late-flowering samples from the F2 population in step (2), extracting genomic DNA from the early-flowering samples and the late-flowering samples, optimizing them to similar concentrations, and then mixing them in equal amounts to generate two extreme pools, together with the screened early-flowering rapeseed variety and the two parental DNAs of the late-flowering variety of the original source, a total of four samples were used for library construction according to a standard 150 bp double-end sequencing protocol, followed by high-throughput sequencing;

[0068] (4) Check and filter the sequencing data, annotate reliable polymorphic variants, and select high-quality SNPs / InDels with a read coverage depth of at least 4 for BSA-seq analysis;

[0069] (5) The Euclidean distance and SNP index methods were used to detect potential SNP candidate loci associated with early flowering traits, and the overlapping regions based on these two methods were defined as candidate regions;

[0070] (6) Design primer pairs in the candidate region and perform PCR using early-flowering rapeseed varieties and late-flowering varieties of the original source as templates to verify whether there are SNP sites; if there are SNP sites, the amplification product of the primer pairs used is the SNP molecular marker.

[0071] The following describes the principles and features of the present invention by taking the screening of early-flowering materials and the specific operation of BSA-seq as examples. In the examples, the early-flowering material ebm and its original late-flowering variety ZS9 are used as examples to illustrate the method for obtaining molecular markers of the present invention. However, those skilled in the art can also obtain corresponding molecular markers through other early-flowering materials and their original late-flowering varieties. The materials and reagents used in the following examples, unless otherwise specified, can be purchased from the company through commercial channels.

[0072] Example 1: Screening of early-flowering material ebm

[0073] According to normal conditions for rapeseed growth, 263 rapeseed varieties collected by our laboratory worldwide through project cooperation were sown in the Wuchang Experimental Base of Wuhan Oil Crops Research Institute for three consecutive years from 2013 to 2015. The width of the bed was 2m, the row spacing was 0.33m, 15 plants were planted in each row, 3 biological replicates were planted, and 3 rows were planted in each biological replicate for the statistics and investigation of the days of initial flowering and full flowering. The initial flowering period was defined as the time from sowing to the flowering of the main inflorescence of 25% of the plants in the plot, and the full flowering period was defined as the time from sowing to the flowering of the branches of 50% of the plants in the plot. Among these 263 Brassica napus materials, we identified an ebm mutant with earlier initial and full flowering periods, which was derived from Brassica napus Zhongshuang 9 (ZS9) treated with EMS mutagenesis.

[0074] Example 2: Investigation of agronomic traits of early-flowering materials

[0075] The ebm and ZS9 materials were planted in the Wuchang Experimental Base of Wuhan Oil Crops Research Institute, with a width of 2m and a row spacing of 0.33m. Fifteen plants were planted in each row, and three biological replicates were planted. Each biological replicate had three rows. The phenotypes of various agronomic traits at maturity and the budding and flowering periods were investigated. The results are as follows: Figure 1 All data in the figure are the mean ± standard deviation of 3 biological replicates (10 plants were measured in each replicate). Asterisks indicate significant differences between the two lineages. As can be seen from the figure, there are no significant differences between the two lineages in agronomic traits, but in terms of flowering time, ebm is significantly earlier than ZS9.

[0076] Example 3: Genetic analysis to determine the inheritance pattern of the early flowering trait

[0077] In March 2016, the early-flowering mutant ebm and ZS9 were reciprocally crossed, and the reciprocal F1 generation was planted in September 2016. The phenotype of the reciprocal F1 generation was investigated during the flowering period in March 2017, and the F1 generation was bagged and self-pollinated. In September 2017, the F2 population of self-pollinated offspring of the reciprocal F1 generation was planted, and the number of plants with different flowering types was counted during the flowering period in March 2018. The flowering time of a single plant in the F2 population was defined as the time from sowing to the flowering of the first flower. The chi-square test was used to verify whether the early flowering trait conformed to Mendelian inheritance, and the results showed that the early flowering of this mutant was controlled by a single dominant nuclear gene.

[0078] Segregation ratio of F2 generation

[0079]

[0080] Example 4: Preliminary localization of early flowering traits using BSA-seq technology

[0081] (1) Extraction of genomic DNA

[0082] During the flowering period in March 2018, the phenotypes of individual plants in the F2 generation segregating population of the ZS9×ebm hybrid F1 offspring were investigated in the field, and 60 plants each with extremely early and late flowering were selected, and high-quality genomic DNA of these samples was extracted using a kit.

[0083] (2) Library construction and sequencing (BSA-seq)

[0084] After all genomic DNA extraction is completed, the concentration is measured, and the DNA concentration of the extreme phenotype plants is quantified, and the equal amounts are mixed evenly to form two mixed pools L-pool (extreme late-flowering plants) and E-pool (extreme early-flowering plants). After the library is constructed, Qubit 2.0 is used for preliminary quantification, and the library is diluted to 1ng / μL. Then, the insert size of the library is detected using Agilent2100. After the insert size meets expectations, the effective concentration of the library is accurately quantified using the Q-PCR method (library effective concentration>2nM) to ensure the quality of the library. After the library inspection is qualified, different libraries are pooled according to the effective concentration and the target data volume requirements for Illumina HiSeq TM PE150 sequencing.

[0085] (3) Bioinformatics analysis

[0086] i. Perform quality control on the Raw data obtained from the machine to obtain Clean data

[0087] ii. Align clean reads to the reference genome

[0088] The clean reads of each sample were aligned with the reference genome using BWA (Burrows-Wheeler Aligner) (Li and Durbin, 2009).

[0089] iii. Detect and annotate SNPs and InDels

[0090] Genome Analysis Toolkit (GATK) software was used to identify SNPs, InDels, and large-fragment variations in all samples. The SnpEff tool was then used to annotate reliable polymorphic variations, and finally high-quality SNPs / InDels with a reads coverage depth of at least 4 were screened for BSA-seq analysis.

[0091] iv. Calculation of SNP-index and definition of candidate regions

[0092] The distribution of SNP-index on chromosomes is plotted. By default, 1Mb is selected as the window and 10kb as the step size. The average value of SNP-index in each window is calculated to reflect the SNP-index distribution of the offspring. According to the results of △SNP-index, the candidate regions that control the early flowering phenotype can be clearly obtained. Figure 2 As shown, the results showed that the candidate region was between 5.39-10.84Mb of A02.

[0093] Example 5: Development of initial positioning interval and flanking molecular markers

[0094] First, the candidate regions for initial positioning were determined based on the results of BSA-seq, and then based on the results of the comparison of the clean reads data of the parental resequencing with the reference genome, the homozygous and polymorphic SNP sites in the parents were extracted within and on both sides of the candidate region as candidate sites for the development of molecular markers. In order to verify the SNP markers with true polymorphism between the two parental lineages, multiple pairs of different primers were evenly designed at different positions in the candidate region to amplify a sequence of about 500-2000bp containing the target SNP variation.

[0095] The PCR reaction system is (50 μL): 2.5 μL cDNA, 5 μL 10× Buffer, 4 μL dNTP (2.5 mM), 2.5 μL each of forward / reverse primer (10 μM), 1 μL Taq enzyme (5 U / μL) and 32.5 μL ddH2O. Add the sample on ice and mix well. The PCR reaction conditions are: 94℃5min; 94℃30sec, 52-58℃30-120sec (varies according to different product lengths); 72℃30sec, 30 cycles; 72℃10min. Detect by 1% agarose gel electrophoresis.

[0096] Sanger sequencing was performed on the products that could amplify bands in both parents. The peak results of sequencing are shown in Figure 3 Finally, the SNP markers with base differences between parents at the same site verified by sequencing were retained, as shown in Table 1.

[0097] Table 1 SNP molecular markers

[0098]

[0099] (1) For ES-1, the 364th base of the SNP molecular marker sequence from the 5' end is the SNP site n; n is A or G.

[0100] (2) For ES-2, the 200th base from the 5' end of the sequence of the SNP molecular marker is the SNP site n; n is T or C.

[0101] (3) For ES-3, the 152nd base of the SNP molecular marker sequence from the 5' end is the SNP site n; n is T or A.

[0102] (4) For ES-4, the 261st base of the SNP molecular marker sequence from the 5' end is the SNP site n; n is G or T.

[0103] (5) For ES-5, the 262nd base of the SNP molecular marker sequence from the 5' end is the SNP site n; n is A or G.

[0104] (6) For ES-7, the 493rd base of the SNP molecular marker sequence from the 5' end is the SNP site n; n is G or A.

[0105] (7) For ES-10, the 256th base of the SNP molecular marker sequence from the 5' end is the SNP site n; n is G or C.

[0106] (8) For ES-12, the 153rd base from the 5' end of the sequence of the SNP molecular marker is the SNP site n; n is A or C.

[0107] (9) For ES-20, the 362nd base of the SNP molecular marker sequence from the 5' end is the SNP site n; n is A or G.

[0108] (10) For ES-40, the 413th base of the SNP molecular marker sequence from the 5' end is the SNP site n; n is G or A.

[0109] (11) For ES-47, the 284th base of the SNP molecular marker sequence from the 5' end is the SNP site n; n is A or G.

[0110] Based on the BSA-seq results, the molecular markers developed above were used to screen and exchange individual plants of 60 and 460 F2 plants with early flowering phenotypes. The results are as follows Figure 4 The vertical line below represents the number of exchanged plants under the molecular marker. Finally, the candidate gene was located between ebm-20 and ebm-7.

[0111] In summary of the above embodiments, the use of the above-mentioned SNP molecular markers can clearly distinguish early-flowering varieties from late-flowering varieties. The identification method is simple, efficient, has a clear selection target, is not affected by the environment, and can effectively find the exchange individual plants in the positioning group. It is applied to the fine positioning of the early-flowering gene between 5.39-10.84M of the A2 chromosome.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0113]

[0114]

[0115]

Claims

1. A SNP molecular marker for early flowering in Brassica napus, characterized in that: The SNP molecular marker is shown in SEQ ID NO.6, and the polymorphism at position 493 of the nucleotide sequence shown in SEQ ID NO.6 is G or A.

2. Use of the SNP molecular marker described in claim 1 in early flowering screening of Brassica napus.

3. Use of the SNP molecular marker described in claim 1 in the localization of early flowering genes in Brassica napus.

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