SSR (Simple Sequence Repeat) molecular marker closely linked with rape pod number trait locus

Abstract

The invention provides an SSR (Simple Sequence Repeat) molecular marker closely linked with a rape pod number character site, and belongs to the technical field of molecular biology and genetic breeding. The SSR molecular marker provided by the invention is located on the C06 chromosome of the rape, and the genetic position of the SSR molecular marker is 32.0-33.9 Mb and corresponds to the position between the 33916285 basic group and the 33982616 basic group of the C06 chromosome. The marker position of the SSR molecular marker closely linked with the rape pod number provided by the invention is closely linked with a new site (namely, an area between the 32146644th basic group and the 32146656th basic group of the rape C06 chromosome) capable of controlling the rape pod number character, and early breeding of the rape pod number character can be realized by combining a primer pair for detecting the SSR molecular marker; the breeding process of the rape is greatly promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and genetic breeding, and particularly relates to an SSR molecular marker closely linked to the silique number trait locus of rapeseed. Background Art

[0002] Rapeseed is the largest oil crop in China, accounting for about 20% of the world's rapeseed production. Rapeseed oil is the largest source of domestic edible vegetable oil, accounting for 57.2% of the total domestic edible vegetable oil, and plays a very important role in the national edible oil supply security strategy. In addition, rapeseed oil has a fatty acid composition similar to that of diesel and is a green renewable energy source. In the context of the continuous expansion of urbanization scale and the further reduction of cultivated land area in China, improving the oil yield per unit area of rapeseed (= yield per unit area × oil content) has become one of the most urgent tasks in rapeseed production in China at present, and is a fundamental issue related to the sustainable development of the rapeseed industry in China.

[0003] In recent years, breakthroughs have been made in high oil content breeding of rapeseed in China, but the yield per unit area is relatively low. This has seriously affected the enthusiasm of farmers to grow rapeseed, restricting the economic benefits of rapeseed and the international competitiveness of the rapeseed industry. At the same planting density, the yield per unit area of rapeseed depends on the yield per plant, and the yield per plant is composed of three components (the number of siliques per plant, seed weight, and 1000-seed weight). Studies have shown that there is a negative correlation to varying degrees among the three components of rapeseed yield per plant, but the correlation coefficient is often not large (Bai Guiping et al., 2016), which indicates that the yield can be increased by increasing a single yield component (such as the number of siliques).

[0004] The rapeseed silique number trait is a typical quantitative genetic trait, showing a continuous distribution and being easily affected by the environment. Traditional breeding methods mainly rely on phenotypic selection, with low selection efficiency and long cycles, and can no longer fully meet the current needs of rapeseed production. With the development of molecular biology and molecular genetics, breeders are gradually realizing the transition from phenotypic selection to genotypic selection for traits. Molecular marker-assisted breeding is a new breeding method that effectively combines molecular genetics with traditional phenotypic selection. Its basic principle is to directly use molecular markers closely linked to and co-segregating with the target trait gene to screen the selected individuals for the target region and the entire genome during the rapeseed breeding process, so as to achieve the purpose of improving the selection efficiency of the target trait and shortening the breeding cycle. Using molecular markers closely linked to the silique number trait can make up for the deficiencies of conventional breeding methods, and moreover, multiple excellent loci can be aggregated to greatly improve the breeding efficiency.

[0005] Due to its advantages such as wide distribution in the genome, high polymorphism, co-dominant inheritance, and good repeatability, SSR markers have become one of the most important molecular marker types in crop genetic research and breeding applications. Compared with SNP (single nucleotide polymorphism) markers, SSR marker detection is simpler and more suitable for popularization and application in conventional breeding projects.

[0006] Currently, there are some reports on QTL mapping of the silique number trait in rapeseed, but most of the loci cannot be replicated and are difficult to be directly applied to rapeseed breeding. Lu et al. detected silique number associated loci on chromosomes A01, A02, A03, A08, A09, A10, C04, and C06 using an association population, but none of them could be replicated (Lu et al., 2018). Especially in the interval of 32.0 - 33.9 Mb on chromosome C06, no SSR markers that can be used in molecular breeding practice have been found yet. Molecular markers for the analgesic effect of sufentanil are very important for achieving individualized use of sufentanil. Summary of the Invention

[0007] To solve the above problems, the present invention provides a molecular marker for detecting the silique number trait of rapeseed, and the molecular marker is a nucleic acid molecule; the nucleic acid molecule contains a nucleic acid fragment between the 32146644th base and the 32146656th base of chromosome C06 of rapeseed. The region between the 32146644th base and the 32146656th base of chromosome C06 of rapeseed is the major QTL locus for the silique number trait.

[0008] The present invention also provides an SSR molecular marker closely linked to the silique number trait locus of rapeseed, and the marker position of the SSR molecular marker is closely linked to the above-mentioned molecular marker.

[0009] In an embodiment of the present invention, the SSR molecular marker is located on chromosome C06 of rapeseed, and its genetic position is 32.0 - 33.9 Mb, corresponding to the region between the 33916285th base and the 33982616th base of chromosome C06.

[0010] The present invention also provides the application of a reagent for detecting the above-mentioned molecular marker and / or the above-mentioned SSR molecular marker in the preparation of a product for detecting the silique number trait of rapeseed.

[0011] In an embodiment of the present invention, the product includes a detection kit.

[0012] The present invention also provides a product for detecting the silique number trait of rapeseed, and the product comprises primer pairs for amplifying the above SSR molecular markers; the primer pairs include the forward primer snpC06-1F shown in the nucleotide sequence SEQ ID NO.1 and the reverse primer snpC06-1R shown in the nucleotide sequence SEQ ID NO.2.

[0013] In one embodiment of the present invention, the product comprises a detection kit.

[0014] The present invention also provides a rapeseed breeding method, and the rapeseed breeding method comprises: obtaining a genetic linkage map related to the silique number trait of rapeseed based on the above SSR molecular markers; screening rapeseed with optimized silique number trait according to the genetic linkage map.

[0015] In one embodiment of the present invention, the rapeseed breeding method comprises the following steps:

[0016] (1) Plant the selected single-plant seeds of the F3 generation of the cross between "352" and "M201" in the field, take samples before final thinning, and extract the total DNA of leaves by the CTAB method.

[0017] (2) Use SSR molecular markers to conduct assistant selection on the offspring of the two parents ("352" and "M201"), and only retain the single plants with the same banding pattern as "M201", and retain the plants with the silique number exceeding the average silique number of the F3 population offspring. Identifying the major QTL locus of the silique number trait of rapeseed by the SSR molecular markers of the present invention can effectively predict the silique number of rapeseed, thereby improving the selection efficiency of high-yield rapeseed breeding and accelerating the breeding process.

[0018] The present invention also provides the application of the above molecular marker or the above SSR molecular marker or the above product or the above rapeseed breeding method in rapeseed breeding.

[0019] The technical solution of the present invention is realized in the following manner:

[0020] The screening method for the major QTL locus of the silique number trait of rapeseed provided by the present invention comprises the following steps:

[0021] (1) Hybridize the rapeseed lines "352" and "M201", and the hybrid F1 is self-crossed for multiple generations to generate a recombinant inbred line segregation population.

[0022] (2) Use the CTAB method (Doyle et al. 1987) to extract the total DNA of the leaves of the parents "352" and "M201" and the recombinant inbred line segregation population. The reagents used in the process include extraction solution (1.4M NaCl, 100mM Tris, pH 8.0, 20mM EDTA, pH 8.0, 2% CTAB), chloroform, isopentyl alcohol, and absolute ethanol.

[0023] (3) Synthesize SNP primers publicly available and self-developed for Brassica napus, perform PCR amplification on parental DNA, electrophorese the products in a denaturing polyacrylamide gel, and determine the sizes of the bands after staining and developing, then screen for polymorphic primers;

[0024] (4) Perform molecular marker analysis on the recombinant inbred line segregating population using the polymorphic primers to obtain genotype data;

[0025] (5) Input the genotype data of the recombinant inbred line segregating population into Joinmap 4.0 software to construct a genetic linkage map;

[0026] (6) Input the genotype data of the recombinant inbred line population (limited to the markers mapped on the genetic map) and the silique number trait data into WinQTLcart 2.5 software for QTL mapping. Among them, the QTL located on linkage group C06 can be repeatedly detected, and the effect value and contribution rate are relatively large.

[0027] Using the above technical measures, the applicant finally obtained the major QTL locus for the silique number trait in Brassica napus. This major QTL locus is located on chromosome C06 of Brassica napus and is tightly linked to the SSR marker self-developed by the applicant. Its primer sequences are snpC06-1F: GCTGACGAGCTGTGGTGT, and the reverse primer snpC06-1R: CGTGAACCACCGCTACGA. The contribution rate, additive effect, and dominant effect on the silique number of Brassica napus were measured using WinQTLCart 2.5 software. The effect value and contribution rate of the said QTL locus are relatively high, which plays a key role in regulating the silique number of Brassica napus. These primers can be used for map-based cloning, high-yield breeding of Brassica napus, and molecular marker-assisted selection.

[0028] Compared with the prior art, the advantages of the present invention are as follows:

[0029] 1. The molecular marker for detecting the silique number trait in Brassica napus provided by the present invention is a nucleic acid molecule; the nucleic acid molecule contains a nucleic acid fragment between the 32,146,644th base and the 32,146,656th base on chromosome C06 of Brassica napus. In conventional breeding methods, the phenotypic identification of the silique number trait has to wait until the maturity stage for seed testing, which is time-consuming, laborious, and has low selection efficiency (the silique number phenotype is greatly affected by the environment). By detecting the major QTL locus for the silique number trait, elimination can be carried out at the seedling stage, which not only saves production costs but also greatly improves the selection efficiency. Moreover, the position of the major QTL for silique number in the present invention is clear, the detection method is convenient and fast, and it is not affected by the environment.

[0030] 2. The SSR molecular marker closely linked to the silique number trait locus provided by the present invention is located on chromosome C06 of rapeseed, and its genetic position is 32.0 - 33.9 Mb, corresponding to the base positions from the 33,916,285th to the 33,982,616th on chromosome C06. By detecting the SSR molecular marker closely linked to the silique number trait of the present invention, the number of siliques can be predicted, and then single plants with more siliques can be accurately and quickly screened. The marker position of the SSR molecular marker closely linked to the silique number of rapeseed provided by the present invention is closely linked to a new locus that can control the silique number trait of rapeseed (i.e., the region between the 32,146,644th and 32,146,656th base positions on chromosome C06 of rapeseed). Combining with the primer pair for detecting the SSR molecular marker, early breeding of the silique number trait of rapeseed can be realized, greatly promoting the breeding process of rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the frequency distribution of silique number in the recombinant inbred line population. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The following embodiments are provided to better further understand the present invention, which is not limited to the described optimal embodiments, and does not constitute a limitation to the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other existing technologies falls within the protection scope of the present invention.

[0033] For those not specified in the following embodiments regarding specific experimental steps or conditions, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not specified in terms of the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0034] Example 1: Construction of the silique number segregating population of rapeseed and trait determination

[0035] In this example, rapeseed lines "352" and "M201" were selected as parents. After obtaining F1 through artificial hybridization, self-crossing was carried out for multiple generations to construct a recombinant inbred line segregating population containing 172 single plants. Standardized field management was uniformly implemented, and the silique number of the main inflorescence and 3 primary branches of each plant was identified by seed inspection at the silique maturity stage (50 days after the end of flowering). Figure 1 The seed inspection data of the silique number showed that the silique number in the population was normally distributed, proving the quantitative genetic characteristics of the silique number trait.

[0036] Example 2: Development and synthesis of primers

[0037] Based on the phenotypic data and genotype analysis results of the recombinant inbred line segregation population constructed in Example 1, first, fine mapping was performed on the target interval of 32.0 - 33.9 Mb on chromosome C06. The sequence information of the parents "352" and "M201" in this interval was obtained through whole-genome resequencing technology (Illumina HiSeq X Ten platform, 150 bp paired-end sequencing, average depth 30×), and data quality control was performed using the SOAPnuke software.

[0038] The MISA software was used to scan for SSR loci in the target interval, with the minimum repeat number set to 10 times to identify SSR loci. Through sequence alignment analysis, SSR loci with obvious length polymorphisms between the two parents were screened out (located between the 32146644th base and the 32146656th base on chromosome C06 of rapeseed, and this SSR locus was tightly linked to the region between the 32146644th base and the 32146656th base on chromosome C06 of rapeseed). This locus showed stable codominant inheritance characteristics. The Primer Premier 5.0 software was used to design primers, and after multiple optimizations, the optimal primer pair was finally determined: forward primer snpC06-1F (5'-GCTGACGAGCTGTGGTGT-3') and reverse primer snpC06-1R (5'-CGTGAACCACCGCTACGA-3').

[0039] The primer synthesis was entrusted to Shanghai Sangon Biotech Co., Ltd. To verify the primer specificity, 20 recombinant inbred line individual plants were selected for PCR amplification. The reaction system (20 μL) included: 10 μL of 2×Taq PCR Master Mix, 0.5 μL of each forward and reverse primer (10 μM), 1 μL of template DNA a (50 ng / μL), and 8 μL of ddH2O. The amplification program was: pre-denaturation at 94°C for 5 min; 35 cycles (94°C for 30 s, 58°C for 30 s, 72°C for 30 s); final extension at 72°C for 10 min. The amplification products were detected by 2% agarose gel electrophoresis. The results showed that all samples obtained a single bright band, the size was consistent with the expectation, and different allelic genotypes could be clearly distinguished, proving that the primers had high specificity and stability.

[0040] Example 3: Screening of primer polymorphisms

[0041] 1. Randomly select 10 plants from each of the parents, mix the DNA in equal amounts, and use it as the template for screening primers.

[0042] 2. Use the dissolved primers to perform PCR amplification on the parental DNA.

[0043] (1) DNA extraction:

[0044] DNA extraction was performed using the CTAB method, which was modified based on Doyle's method to better suit DNA extraction from rapeseed. The operation process is as follows:

[0045] ① Label the centrifuge tubes, add an appropriate amount of leaves, and then add steel beads;

[0046] ② Place the centrifuge tubes on the adapter of the tissue disruptor and immerse them in liquid nitrogen;

[0047] ③ After 40 s in liquid nitrogen, place them in the tissue disruptor for disruption for 1 min;

[0048] ④ Add 500 μL of 2% CTAB solution by mass fraction to the centrifuge tubes and then incubate in a 65 °C water bath for 30 min, and shake well repeatedly during the process;

[0049] ⑤ Add an equal volume (500 μL) of a 24:1 solution mainly composed of chloroform and gently invert up and down;

[0050] ⑥ Centrifuge at 12000 rpm for 8 min in an eppendorf high-speed centrifuge, transfer the supernatant obtained by centrifugation, and add 1000 μL of -20 °C ice ethanol;

[0051] ⑦ Centrifuge at the same speed as in step 6 for 5 min, discard the supernatant, add 1000 μL of 76% ethanol by mass fraction for rinsing, and then air-dry the centrifuge tubes at room temperature;

[0052] ⑧ Add 200 - 300 μL of TE Buffer solution containing RNase and store at -20 °C for later use.

[0053] (2) Reaction system:

[0054] 2 μL of DNA template, 1 μL of 10× Buffer (Mg2+), 0.8 μL of dNTPs (2.5 mM each), 0.2 μL of Taq E (5 u / μL), 0.5 μL of Primer F / Primer R, 5 μL of ddH2O, with a total volume of 10 μL.

[0055] (3) PCR reaction program:

[0056] Denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 59 °C for 45 s, extension at 72 °C for 45 s, with the annealing temperature decreasing by 0.5 °C for each cycle, for 10 cycles; denaturation at 94 °C for 30 s, annealing at 54 °C for 45 s, extension at 72 °C for 45 s, for 20 cycles; in the first 10 cycles, the annealing temperature decreases by 0.5 °C per cycle until it reaches 54 °C, and then the following 20 cycles are carried out; extension at 72 °C for 5 min; store at 4 °C.

[0057] (4) 6% polyacrylamide gel electrophoresis:

[0058] A: Preparation of 6% Polyacrylamide Gel

[0059] Clean the long glass plate with clean water and let it dry. Place it horizontally. First, wipe it with clean absorbent paper and absolute ethanol twice. After the alcohol has completely evaporated, apply the adhesive and let it dry. Place seals on both sides and set aside. Clean the short glass plate with absolute ethanol twice as well. After the ethanol has evaporated, apply the silanizing agent and let it dry. Place the short glass plate upside down on the long glass plate and fix both sides with clips. Prepare the gel: 60 mL of 6% polyacrylamide gel + 300 μL of 10% ammonium persulfate + 30 μL of TEMED. After stirring evenly, pour it between the two glass plates, insert a comb to form the sample loading gel slot, fix it with a clip, press a heavy object on it. After 2 hours, the gel plate solidifies and electrophoresis can be carried out.

[0060] B: Gel Electrophoresis

[0061] Remove the solidified gel plate, remove the clips and the comb, fix it on the electrophoresis tank, pour in the electrophoresis buffer 5×TBE solution, set the electrophoresis parameters as voltage U = 2000 v, current I = 100 mA, power P = 80 w. Preheat the electrophoresis for 20 min, insert the sample loading comb, and prepare for sample loading. Before sample loading, add 10 μL of loading buffer to the selected amplification system. The loading buffer contains formamide, xylene cyanol, and bromophenol blue. Then, denature it in a boiling water bath for 5 min. After denaturation, quickly place it in an ice box. After cooling, it can be used for sample loading. Load 100 bp DNA ladder Marker on both sides of the sample loading slot, quickly load the samples in sequence, and then start electrophoresis. After 70 min, the electrophoresis is completed.

[0062] C: Silver Staining and Development

[0063] ① Rinsing: After peeling the gel after electrophoresis from the glass plate, put it into a plate filled with distilled water, gently shake it for 5 - 6 s, pour it out to wash away the residual electrophoresis solution on the surface of the gel;

[0064] ② Staining: Transfer the gel into a 0.1% silver nitrate solution and shake it for staining for 8 - 10 min;

[0065] ③ Washing with water: Transfer the stained gel into distilled water and rinse it 2 - 3 times to wash away the residual silver nitrate on the surface of the gel;

[0066] ④ Development: Transfer the rinsed gel into the developing solution (1.5% NaOH, 0.4% formaldehyde) for development until the bands are clearly shown;

[0067] ⑤ Rinsing: Pour out the developing solution and rinse the film with tap water 2 - 3 times;

[0068] ⑥ Preservation: Place the rinsed gel on the film viewing lamp for statistical analysis and photography.

[0069] D: Band Pattern Interpretation

[0070] Place the glass plate that has been naturally dried after development on the film reading table and observe the position difference of the two parental bands with the naked eye.

[0071] Example 4: Genotype analysis of recombinant inbred line population, construction of genetic linkage map and QTL positioning

[0072] (1) DNA from 172 individual plants of the recombinant inbred line population was extracted using the CTAB method (see Example 3);

[0073] (2) Polymorphic primers were selected to perform PCR amplification on the DNA of 172 individuals in the recombinant inbred line population, and then the PCR products were subjected to polyacrylamide gel electrophoresis, development, staining and band type interpretation. Differential molecular markers can be divided into two categories: one is co-dominant markers, that is, the differential bands are manifested as variations in position (i.e., the size of the amplified product), and the band types of the isolated population are read as A, B and H, respectively, indicating that they are derived from "M201", "352" and heterozygous band types; the other is dominant markers, that is, the differential bands are manifested as the presence or absence of variation, and are read as A, C (at this site, "352" has a band, and the isolated population has no band and reads A, and the band has C) and B, D (at this site, "M201" has a band, and the isolated population has no band and reads B, and the band has D).

[0074] (3) Obtaining molecular marker genotype data by interpreting the molecular marker band patterns obtained after staining.

[0075] (4) Joinmap4.0 software was used to perform linkage analysis on the molecular marker genotype data of the recombinant inbred line population to construct a molecular marker genetic linkage map.

[0076] (5) Based on the genetic map, the genotype data of the recombinant inbred line population, and the silique number phenotypic data of the two populations, QTL detection was performed using QTLCart2.5 software. A major QTL locus was detected near the SSR molecular marker marker of the C06 linkage group. Its LOD value and contribution rate were large, and the enhancing allele originated from the parent "M201".

[0077] Table 1 Basic information of the major QTL for silique number in linkage group C06

[0078]

[0079] Example 5: Application of SSR molecular markers in assisted selection of rapeseed silique number traits

[0080] (1) F3 seeds were obtained by self-pollination of individual “352” × “M201” plants in bags during field planting.

[0081] (2) Before final thinning, label and sample individual F3 plants, and extract total leaf DNA (see Example 3). Use SSR molecular markers to determine their genotypes, and only retain the individual plants with the same band pattern as "M201". All the remaining individual plants with the same band pattern as "352" and the heterozygous ones are pulled out (because SSR molecular markers are co-dominant markers, so retain the individual plants with band pattern B and pull out the individual plants with band patterns A and H).

[0082] (3) Harvest F3 individual plants at maturity and conduct a census of the number of siliques per plant. The results show that the number of siliques per main raceme of the individual plants with the same SSR molecular marker genotype as "M201" significantly exceeds the population average of "352". It can be seen that eliminating at the seedling stage not only saves production costs but also greatly improves the selection efficiency, and thus can quickly screen out multi-silique plant lines for increasing rapeseed yield.

[0083] Table 2 Census data of the number of siliques per plant of F3 individual plants obtained by assisted selection using SSR marker snpC06-1

[0084]

[0085]

[0086] Note: A and B represent the molecular marker band patterns derived from "M201" and "352" respectively.

[0087] Obviously, the above examples are only for clear illustration and not limitations on the implementation methods. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. A molecular marker for detecting the trait of the number of rapeseed siliques, characterized in that, The molecular marker is a nucleic acid molecule; the nucleic acid molecule comprises a nucleic acid fragment between the 32,146,644th base and the 32,146,656th base of rapeseed chromosome C06.

2. An SSR molecular marker tightly linked to the rapeseed silique number trait locus, characterized in that, The marker position of the SSR molecular marker is tightly linked to the molecular marker described in claim 1.

3. The SSR molecular marker according to claim 2, wherein, The SSR molecular marker is located on rapeseed chromosome C06, and its genetic position is 32.0 - 33.9 Mb, corresponding to the region between the 33,916,285th base and the 33,982,616th base of chromosome C06.

4. Use of a reagent for detecting the molecular marker described in claim 1 and / or the SSR molecular marker described in claim 2 or 3 in the preparation of a product for detecting the rapeseed silique number trait.

5. The application according to claim 4, characterized in that The product includes a detection kit.

6. A product for detecting the trait of the number of rapeseed pods, characterized in that, The product includes a primer pair for amplifying the SSR molecular marker described in claim 2 or 3; the primer pair includes a forward primer snpC06-1F shown in the nucleotide sequence SEQ ID NO.1 and a reverse primer snpC06-1R shown in the nucleotide sequence SEQ ID NO.

2.

7. The product according to claim 6, wherein, The product includes a detection kit.

8. A rapeseed breeding method, characterized in that, The rapeseed breeding method includes: obtaining a genetic linkage map related to the rapeseed silique number trait based on the SSR molecular marker described in claim 2 or 3; screening rapeseed with optimized silique number trait according to the genetic linkage map.

9. Use of the molecular marker described in claim 1 or the SSR molecular marker described in claim 2 or 3 or the product described in claim 6 or 7 or the rapeseed breeding method described in claim 8 in rapeseed breeding.

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