Molecular marker closely related to cold tolerance and early flowering traits in brassica and application thereof

Abstract

The application discloses a molecular marker closely related to a cold-tolerant early flowering character of rapeseed and application, and takes 176 rapeseed variety resources with different genetic sources, different cold tolerance and different flowering period as research materials. A molecular marker related to seedling stage cold tolerance and early flowering is screened on an A07 chromosome through whole genome correlation analysis. The molecular marker can be used for breeding of cold-tolerant and early-maturing varieties, and is simple and easy to operate. The method is easy to implement, solves problems existing in conventional breeding methods, such as that selection and identification must be carried out in the later period, and accelerates the pace of rapeseed quality improvement.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to molecular markers closely related to the cold resistance and early flowering traits of rapeseed seedlings and their application in breeding for late-sowing and early-maturing varieties. Background Technology

[0002] Rapeseed (Brassica napus L.) is an important oilseed crop in my country, with an annual planting area of ​​over 100 million mu (approximately 6.67 million hectares), accounting for about 20% of the world's total production. Rapeseed oil production accounts for more than 50% of China's domestic oilseed crop oil production, making it the largest source of edible vegetable oil in China. In recent years, my country's vegetable oil self-sufficiency rate has been only 32%, indicating a significant supply-demand gap. Rapeseed cultivation in my country is divided into two major regions: winter rapeseed and spring rapeseed. Winter rapeseed is mainly planted in the Yangtze River basin, accounting for about 90% of the country's rapeseed area and yield.

[0003] In double-cropping rice areas, the area capable of forming a rice-rice-oilseed rape planting pattern reaches 12.7 million mu, mainly distributed in Hubei, Hunan, Jiangxi, and Anhui provinces. Currently, the continuously delayed harvest period of late rice leads to tight rice-oilseed rape rotation stubble and low sowing temperatures, causing widespread cold damage stress during the germination and seedling stages of rapeseed, resulting in slow seedling growth and severely impacting yield. Furthermore, there is a lack of new rapeseed varieties with short growth periods that are tolerant to late sowing. Therefore, developing molecular markers through technological innovation to genetically improve the early-maturing traits of rapeseed to tolerate late sowing can lay a theoretical foundation and provide technical guidance for breeding new early-maturing and highly resistant rapeseed varieties in triple-cropping areas. This is an important way to ensure rapeseed yield in rice-rice-oilseed rape rotation areas and expand the rapeseed planting area in winter fallow fields. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular marker closely related to the cold-resistant and early-flowering traits of rapeseed, and the application of this molecular marker in the breeding of late-sown, early-maturing rapeseed. This marker can assist in screening for cold-resistant and early-flowering materials, effectively improving the efficiency of rapeseed breeding.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A molecular marker closely related to the cold-resistant and early-flowering trait of rapeseed was obtained through the following method:

[0007] (1) Using 176 rapeseed varieties with different genetic origins and large variations in cold resistance and flowering time as research materials, the variety information, resequencing data and single nucleotide polymorphism (SNP) markers developed for the population have been published on the database platform BnaSNPDB (https: / / www.ncbi.nlm.nih.gov / sra);

[0008] (2) Each material in the population was planted in a seedling tray (32 holes per tray, 58mm×58mm per hole). The indoor growth conditions were 22℃, 16 hours of light / 8 hours of darkness. When the seedlings reached the 4-leaf stage, the cold resistance was assessed by lowering the temperature from -2℃ to -4℃, and then to -6℃. Each temperature was maintained for one hour. After the treatment, the seedlings were placed at 22℃ to recover for 3 days, and then the survival rate was counted.

[0009] (3) Each material in the population was planted at the Wuchang Experimental Base according to the late sowing (November 1st), and the flowering time of each material was counted;

[0010] (4) Genome-wide association analysis was conducted on the BnaSNPDB platform (https: / / bnapus-zju.com / gwas) using the seedling cold-resistance phenotype and flowering phenotype under late-sowing conditions of rapeseed germplasm resources. The EMMAX (EfficientMixed-Model Association eXpedited) model was used to obtain the -log phenotype for each SNP. 10 P-value; significance P<10 -5 To establish a standard, loci were screened on chromosome A07, and candidate gene BnaA07g11930D was obtained within these loci. The nucleotide sequence of this gene was obtained from a public database (https: / / www.genoscope.cns.fr / brassicanapus / ). Primers were designed, and PCR amplification, sequence determination, and alignment were performed using DNA from different rapeseed varieties as templates. PCR primers were redesigned at sequence variation sites to convert the gene into the functional marker BnaA07g11930D-197. The forward primer sequence is 5′-TGGATTTAGAGGTGCGATTGA-3′ (SEQ ID NO.1), and the reverse primer sequence is 5′-CGATTCTTCCAACTCCAACTT-3′ (SEQ ID NO.2).

[0011] Molecular markers closely related to the cold-resistant and early-flowering traits of rapeseed contain molecular markers with the sequence shown in SEQ ID NO.4.

[0012] A kit for identifying the cold-resistant and early-flowering traits of rapeseed contains primers for amplifying the molecular marker shown in SEQ ID NO. 4. Preferably, the primer sequences are as shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0013] Compared with the prior art, the present invention has the following advantages and effects:

[0014] This invention establishes a novel molecular marker in rapeseed that is associated with cold resistance during the seedling stage and early flowering for the first time. It effectively solves the shortcomings of conventional breeding methods, such as the susceptibility of cold resistance to environmental influences, the requirement that flowering can only be identified in the later stages, and the large workload and long cycle of population identification. It can significantly improve the breeding process of late-sown, early-maturing rapeseed varieties. Attached Figure Description

[0015] Figure 1 This is the association analysis result of cold resistance phenotype data in rapeseed seedlings. The horizontal axis represents the physical distance between chromosomes, and the vertical axis represents the p-value after -log10. The dots represent SNP markers, and the arrows indicate the locations of significantly associated molecular markers.

[0016] Figure 2 This is the result of an association analysis of flowering period phenotypic data for rapeseed after late sowing. The horizontal axis represents the physical distance between chromosomes, and the vertical axis represents the p-value after -log10. The dots represent SNP markers, and the arrows indicate the locations of significantly associated molecular markers.

[0017] Figure 3 The results show the detection of the functional marker BnaA07g11930D-197 in materials with different cold resistance and flowering times. M is the molecular weight standard 100bp DNA ladder; R4815, R4220, and R4795 are non-cold-resistant late-flowering varieties, and R5057, R5058, and R5103 are cold-resistant early-flowering varieties, with molecular weights of 209bp and 197bp, respectively. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Example 1: Molecular marker BnaA07g11930D-197 closely related to the cold resistance and early flowering traits of rapeseed.

[0019] This embodiment uses 176 rapeseed varieties with different genetic origins and significant variations in cold resistance and flowering time as examples to construct a natural population. The following details the development method of molecular markers related to cold resistance and early flowering traits:

[0020] (I) Assessment of Cold Resistance in Groups

[0021] 176 rapeseed varieties with different genetic origins, seed cold resistance, and flowering time were used as research materials (provided by Zhejiang University). The seedlings were exposed to 16 hours of light and 8 hours of darkness. Cold resistance was assessed when the seedlings reached the 4-leaf stage. The temperature was lowered from -2℃ to -4℃ and then to -6℃, with each temperature maintained for one hour. After the treatment, the seedlings were placed at 22℃ to recover for 3 days, and the survival rate was then counted.

[0022] (II) Statistics on flowering period after late sowing

[0023] Plant materials were planted in the field and sown on the delayed sowing date (November 1st). Three replicates were set up, with three rows in each replicate (30 plants in total). A randomized block design was used, and field management was carried out using conventional methods. The flowering time was recorded according to the standard of 20% flowering per plant in each replicate. The average of the three replicate plots was calculated to represent the flowering time of each variety.

[0024] (III) DNA Extraction

[0025] Young leaves of all varieties were collected during the seedling stage, and total DNA was extracted using the CTAB method. The specific steps are as follows:

[0026] (1) Place a leaf with a diameter of about 1 cm into a 2 ml centrifuge tube, crush it with a steel ball on a sample crusher, add 800 μl of CTAB extraction solution (50 mmol / L Tris-HCl pH 8.0; 20 mmol / L EDTA pH 8.0; 50 mmol / L NaCl, 1 g / L CTAB) and shake well, then incubate in a 65℃ water bath for 30 min.

[0027] (2) Take out the centrifuge tube, add 400 μl of pure chloroform solution, shake on a shaker (100 rpm) for 10 min, centrifuge at 12000 rpm for 10 min at room temperature, and aspirate the supernatant (about 400 μl) into a new centrifuge tube;

[0028] (3) Add two volumes of anhydrous ethanol (about 800 μl) to the supernatant, mix by inversion, and let stand at -20℃ for 30 min. Then centrifuge at 12000 rpm for 15 min to obtain DNA precipitate at the bottom of the tube. After the DNA precipitate has air-dried naturally, add 100 μl of double-distilled water to dissolve it and use it for subsequent PCR detection.

[0029] (iv) Correlation Analysis

[0030] Genome-wide association analysis (GWA) was conducted on the BnaSNPDB platform (https: / / bnapus-zju.com / gwas) using the seedling cold-resistance phenotype and flowering time phenotype under delayed sowing conditions of rapeseed germplasm resources. The EMMAX (EfficientMixed-Model Association eXpedited) model was used to obtain the -log phenotype for each SNP. 10 P-value. Assuming significance, P < 10. -5 As a standard, loci were obtained by screening on chromosome A07.

[0031] (V) Functional Marker Conversion and Detection

[0032] The candidate gene BnaA07g11930D was obtained within the locus. The nucleotide sequence of this gene was obtained from a public database (https: / / www.genoscope.cns.fr / brassicanapus / ). Primers were designed, and PCR amplification, sequencing, and alignment were performed using DNA from different rapeseed varieties as templates. PCR primers were redesigned at the sequence variation sites to convert the gene into the functional marker BnaA07g11930D-197. The forward primer sequence is 5′-TGGATTTAGAGGTGCGATTGA-3′, and the reverse primer sequence is 5′-CGATTCTTCCAACTCCAACTT-3′. Using DNA from three cold-hardy late-flowering rapeseed varieties (R4815, R4220, R4795) and three cold-hardy early-flowering varieties (R5057, R5058, R5103) as templates, PCR amplification and comparison were performed, with Zhongshuang 11 as the reference sequence (https: / / yanglab.hzau.edu.cn / BnIR). A 12bp sequence was found to be inserted in the cold-hardy late-flowering rapeseed varieties.

[0033] Example 2: Application of functional molecular markers related to cold resistance and early flowering traits in rapeseed in rapeseed quality breeding.

[0034] Using 176 varietal resources from Example 1 used for association analysis as research materials, the seedling survival rate from frost damage ranged from 0% to 100%, and the flowering period after late sowing ranged from 128 to 168 days. Identification was performed using the functional marker BnaA07g11930D-197, following the steps below:

[0035] (1) Using total rapeseed DNA as a template, PCR amplification was performed using primers with functional marker BnaA07g11930D-197. The forward primer was 5′-TGGATTTAGAGGTGCGATTGA-3′, and the reverse primer was 5′-CGATTCTTCCAACTCCAACTT-3′.

[0036] (2) The total PCR volume is 20 μl, and the specific components are as follows:

[0037]

[0038]

[0039] (3) PCR amplification program: 95.0℃ pre-denaturation for 5 min; 95.0℃ denaturation for 30 s, 58.0℃ annealing for 30 s, 72.0℃ extension for 20 s, for a total of 32 cycles; 72.0℃ extension for 5 min. After separation by 3% agarose gel electrophoresis, specific bands with molecular weights of 209 bp and 197 bp were obtained;

[0040] (4) Among the 176 tested materials, 67 materials showed a specific 209bp (sequence as shown in SEQ ID NO.3) band pattern, with an average survival rate of 25% after cold damage and an average flowering time of 141 days after late sowing; 106 materials showed a specific 197bp (sequence as shown in SEQ ID NO.4) band pattern, with an average survival rate of 63% after cold damage, which was significantly higher than that of the materials showing the 209bp band pattern (P<0.05), and an average flowering time of 131 days after late sowing, which was significantly earlier than that of the materials showing the 209bp band pattern (P<0.01);

[0041] The above identification results show that by identifying and screening with functional molecular markers in breeding, materials with a specific band of 197bp are retained and materials with a band of 209bp are eliminated. This allows for the selection of cold-resistant and early-flowering materials at the seedling stage. Under late-sowing conditions, the cold resistance of seedlings is increased by 38%, and flowering can be advanced by 10 days, thus accelerating the breeding process of late-sowing and early-maturing rapeseed varieties.

Claims

1. The application of molecular markers in the identification or breeding of cold-resistant and early-flowering traits in rapeseed, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

4. Rapeseed containing the molecular marker has the trait of cold resistance and early flowering.

2. The application of primers in identifying the cold-resistant and early-flowering trait of rapeseed, characterized in that, The primers are used to amplify the molecular markers of the nucleotide sequence shown in SEQ ID NO.

4.

3. The application according to claim 2, characterized in that, The primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2.

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