Gene for cultivating long PRL brassica napus, molecular marker and application

Abstract

The invention belongs to the technical field of rape breeding and molecular biology, and particularly relates to a gene for cultivating long PRL brassica napus, a molecular marker and application. The gene is BnaC03g08100D, and the nucleotide sequence of the gene is as shown in SEQ ID NO. 1. The molecular marker is located at the 3821562 basic group of a C03 chromosome in BnaC03g08100D, and the basic group is T or G. Detection primers of the molecular marker are as shown in SEQ ID NO. 2 and SEQ ID NO. 3. The invention provides a new target for genetic improvement and molecular breeding of rape root systems, and provides support for subsequent identification and cultivation of long PRL brassica napus varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of rapeseed breeding and molecular biology, and in particular relates to a gene, a molecular marker and an application thereof for cultivating long PRL Brassica napus. Background Art

[0002] Rapeseed is one of the most important oil crops in the world. Improving its yield and quality is of great significance for ensuring the supply of edible oil and improving agricultural production efficiency. The root system is an important organ for plants to absorb water and mineral nutrients, and its development directly affects the yield and adaptability of crops. The root system of Brassica napus is a taproot system with a short main root that is shallow in the soil. The root system is usually underdeveloped, which limits its absorption and utilization of soil nutrients. Therefore, cultivating rapeseed varieties with highly developed root systems is of great significance for improving the efficient use of nutrients and increasing yields of rapeseed.

[0003] In recent years, with the continuous development of sequencing technology, genetic variations significantly associated with target traits can be identified, and targeted improvements of target traits can be achieved through molecular marker-assisted selection and other means. Genome-wide association analysis (GWAS) is to detect genetic variations in individuals within a population across the entire genome to obtain genotype data, conduct phenotypic investigations on individuals within the population to obtain phenotypic data, perform statistical analysis on genotype data and phenotypic data, and screen out genetic variations that are most likely to be significantly associated with the phenotype according to the significance threshold, and identify candidate genes that are significantly associated with the trait. Compared with humans, plants have abundant germplasm resources in nature, which contain abundant genetic variations, laying the foundation for using GWAS to explore major effect genes of important agronomic traits. With the development of sequencing technology and the continuous release of reference genomes of various crops, GWAS has become a conventional means of analyzing important agronomic traits of crops such as Arabidopsis, soybeans and rapeseed.

[0004] In the prior art, there are few studies on molecular markers related to taproot length (PRL) of Brassica napus, and more studies are conducted on molecular markers related to other traits such as seed oil content and 1000-grain weight. For example, patent CN114736986A discloses a SNP molecular marker-related detection primer or probe for a major QTL locus of the oil content trait of Brassica napus seeds and its application. The SNP molecular marker is closely linked to the major QTL locus of the oil content trait of Brassica napus seeds, and the major QTL locus of the oil content trait of Brassica napus seeds is located between bases 11378925 and 11559658 on chromosome A07 of Brassica napus. For example, CN114854894A discloses a SNP molecular marker, related detection primers or probes, and applications of the major QTL site of the thousand-grain weight trait of Brassica napus. The SNP molecular marker is tightly linked to the major QTL site of the thousand-grain weight trait of Brassica napus. The major QTL site of the thousand-grain weight trait of Brassica napus is located between bases 20082973 and 20310902 on chromosome A07 of Brassica napus.

[0005] Therefore, it is necessary to explore the genes related to taproot length in Brassica napus and further develop molecular markers that can be used for long PRL-assisted breeding. Summary of the invention

[0006] One of the purposes of the present invention is to provide a gene for screening and / or cultivating long PRL Brassica napus, which gene is significantly associated with the root development of Brassica napus, providing support for the subsequent screening and cultivation of long PRL Brassica napus varieties.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A gene used for screening and / or breeding long PRL Brassica napus, the gene being BnaC03g08100D.

[0009] Furthermore, the nucleotide sequence of the BnaC03g08100D is shown in SEQ ID NO.1.

[0010] The second object of the present invention is to provide a molecular marker based on the aforementioned BnaC03g08100D gene, which provides support for the identification and cultivation of long PRL Brassica napus varieties.

[0011] To achieve the above object, the present invention adopts the following technical solutions:

[0012] Based on the molecular marker of the aforementioned gene, the molecular marker is located at the 3821562nd base of chromosome C03 in the BnaC03g08100D, and the base is T or G.

[0013] Furthermore, the long PRL B. napus varieties carry the G allele, and the short PRL B. napus varieties carry the T allele.

[0014] A third object of the present invention is to provide a primer pair for amplifying the aforementioned molecular markers.

[0015] To achieve the above object, the present invention adopts the following technical solutions:

[0016] The primer pair used to amplify the aforementioned molecular marker comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.2 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.3.

[0017] A fourth object of the present invention is to provide a method for identifying the length of PRL in Brassica napus varieties based on the aforementioned molecular markers.

[0018] To achieve the above object, the present invention adopts the following technical solutions:

[0019] The method for identifying the length of PRL of Brassica napus varieties by using the aforementioned molecular markers comprises the following steps:

[0020] (1) Extracting DNA from Brassica napus;

[0021] (2) using the DNA obtained in step (1) as a template and the aforementioned primer pair to perform PCR amplification to obtain an amplified product;

[0022] (3) Using BsPHi to perform restriction digestion and electrophoresis analysis on the amplified product obtained in step (2), and identifying the Brassica napus variety with long PRL.

[0023] Furthermore, if the amplified product is cut into two fragments of 332bp and 148bp by BsPHi, it indicates that the Brassica napus is a short PRL variety; if the amplified product is not cut by BsPHi, it contains a 480bp fragment but does not contain 332bp and 148bp fragments, it indicates that the Brassica napus is a long PRL variety.

[0024] A fifth object of the present invention is to provide a method for cultivating Brassica napus with long PRL.

[0025] To achieve the above object, the present invention adopts the following technical solutions:

[0026] The method for cultivating long PRL Brassica napus comprises the following steps:

[0027] 1) Using the aforementioned method for identifying the length of PRL of Brassica napus varieties, a Brassica napus variety with a long PRL was identified;

[0028] 2) using the long PRL Brassica napus variety identified in step 1) for breeding and / or large-scale planting.

[0029] The sixth object of the present invention is to provide an application of the aforementioned gene, the aforementioned molecular marker and / or the aforementioned primer pair in screening and / or cultivating Brassica napus varieties with long PRL.

[0030] The seventh object of the present invention is to provide an application of the aforementioned gene, the aforementioned molecular marker and / or the aforementioned primer pair in identifying Brassica napus PRL.

[0031] The beneficial effects of the present invention are:

[0032] The present invention investigates the taproot length of 393 Brassica napus varieties, identifies 48 genetic regions stably associated with PRL through GWAS, and develops a molecular marker (Bn_C3_RPL_marker) that can be used for long PRL-assisted breeding based on the functional SNP of the BnaC03g08100D gene. The molecular marker can be used to identify and cultivate Brassica napus varieties with long PRL. The present invention helps to reveal the genetic mechanism of the development of the root system of Brassica napus, and also provides new targets for the genetic improvement and molecular breeding of the root system of rapeseed, which is expected to improve the nutrient absorption efficiency and stress resistance of rapeseed in the future, thereby further promoting the development of the rapeseed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a root system survey diagram of the association analysis population of Brassica napus under the paper culture system;

[0034] Figure 2 This is the frequency distribution diagram of the taproot length of the association analysis population of Brassica napus;

[0035] Figure 3 This is the result of genome-wide association analysis of PRL in Brassica napus;

[0036] Figure 4 This is the result of identifying the superior alleles of long PRL in the natural population of Brassica napus; Figure 4 -A is the difference analysis result of PRL between rapeseed varieties carrying A allele in chrA01_5960845 (A / T) and PRL of rapeseed varieties carrying T allele; Figure 4 -B is a diagram showing the difference analysis results between the PRL of rapeseed varieties carrying the G allele in chrA01_13740784 (G / A) and the PRL of rapeseed varieties carrying the A allele; Figure 4 -C is a graph showing the difference between the PRL of rapeseed varieties carrying the G allele in chrA01_15083269 (G / A) and the PRL of rapeseed varieties carrying the A allele; Figure 4-D is the difference analysis result of PRL between rapeseed varieties carrying T allele in chrA01_21872007 (T / C) and rapeseed varieties carrying C allele; Figure 4 -E is a graph showing the difference between the PRL of rapeseed varieties carrying the G allele in chrA02_2476122 (G / T) and the PRL of rapeseed varieties carrying the T allele; Figure 4 -F is the difference analysis result of PRL between rapeseed varieties carrying G allele in chrA02_22151710 (G / T) and rapeseed varieties carrying T allele; Figure 4 -G is a graph showing the difference between the PRL of rapeseed varieties carrying the T allele in chrA02_23742862 (T / C) and the PRL of rapeseed varieties carrying the C allele; Figure 4 -H is a graph showing the difference between the PRL of rapeseed varieties carrying the C allele in chrA03_1872078 (C / T) and the PRL of rapeseed varieties carrying the T allele; Figure 4 -I is a diagram showing the difference analysis results between the PRL of rapeseed varieties carrying the A allele in chrA03_2558982 (A / G) and the PRL of rapeseed varieties carrying the G allele; Figure 4 -J is a diagram showing the difference analysis results between the PRL of rapeseed varieties carrying the G allele in chrA03_3553290 (G / A) and the PRL of rapeseed varieties carrying the A allele; Figure 4 -K is a graph showing the difference between the PRL of rapeseed varieties carrying the T allele in chrA05_2691748 (T / C) and the PRL of rapeseed varieties carrying the C allele; Figure 4 -L is the difference analysis result of PRL between rapeseed varieties carrying C allele in chrA05_18278870 (C / T) and PRL of rapeseed varieties carrying T allele; Figure 4 -M is the difference analysis result of PRL between rapeseed varieties carrying T allele in chrA05_19830592 (T / A) and PRL of rapeseed varieties carrying A allele; Figure 4 -N is the difference analysis result of PRL between rapeseed varieties carrying G allele in chrA05_22509378 (G / A) and PRL of rapeseed varieties carrying A allele; Figure 4 -O is the difference analysis result of PRL between rapeseed varieties carrying A allele in chrA07_19487178 (A / G) and rapeseed varieties carrying G allele;

[0037] Figure 4 -P is the difference analysis result of PRL between rapeseed varieties carrying G allele in chrA09_27929092 (G / A) and rapeseed varieties carrying A allele; Figure 4 -Q is the difference analysis result of PRL between rapeseed varieties carrying G allele in chrA09_31692815 (G / C) and rapeseed varieties carrying C allele; Figure 4 -R is the difference analysis result of PRL between rapeseed varieties carrying G allele in chrA10_16639945 (G / A) and PRL of rapeseed varieties carrying A allele; Figure 4 -S is the difference analysis result of PRL between rapeseed varieties carrying T allele in chrC01_10729080 (T / G) and rapeseed varieties carrying G allele; Figure 4 -T is the difference analysis result of PRL between rapeseed varieties carrying A allele in chrC01_24517961 (A / C) and rapeseed varieties carrying C allele; Figure 4 -U is the difference analysis result of PRL between rapeseed varieties carrying G allele in chrC02_319556 (G / A) and rapeseed varieties carrying A allele; Figure 4 -V is the difference analysis result of PRL between rapeseed varieties carrying G allele in chrC02_42483651 (G / A) and PRL of rapeseed varieties carrying A allele; Figure 4 -W is a graph showing the difference between the PRL of rapeseed varieties carrying the T allele in chrC03_3821562 (T / G) and the PRL of rapeseed varieties carrying the G allele; Figure 4 -X is the difference analysis result of PRL between rapeseed varieties carrying G allele in chrC03_4097772 (G / C) and PRL of rapeseed varieties carrying C allele;

[0038] Figure 5 This is the result of identifying the superior alleles of long PRL in the natural population of Brassica napus; Figure 5 -A is a diagram showing the difference analysis results between the PRL of rapeseed varieties carrying the A allele in chrC03_4746982 (A / G) and the PRL of rapeseed varieties carrying the G allele; Figure 5 -B is a diagram showing the difference analysis results between the PRL of rapeseed varieties carrying the G allele in chrC03_5485602 (G / A) and the PRL of rapeseed varieties carrying the A allele; Figure 5 -C is the difference analysis result of PRL between rapeseed varieties carrying C allele in chrC03_21830592 (C / T) and PRL of rapeseed varieties carrying T allele; Figure 5 -D is the difference analysis result of PRL between rapeseed varieties carrying C allele in chrC03_30265320 (C / A) and PRL of rapeseed varieties carrying T allele; Figure 5-E is a graph showing the difference between the PRL of rapeseed varieties carrying the G allele in chrC03_51977184 (G / A) and the PRL of rapeseed varieties carrying the A allele; Figure 5 -F is the difference analysis result of PRL between rapeseed varieties carrying A allele in chrC04_8891786 (A / T) and PRL of rapeseed varieties carrying T allele; Figure 5 -G is the difference analysis result of PRL between rapeseed varieties carrying G allele in chrC04_38564890 (G / C) and PRL of rapeseed varieties carrying C allele; Figure 5 -H is a graph showing the difference between the PRL of rapeseed varieties carrying the G allele in chrC05_1509973 (G / A) and the PRL of rapeseed varieties carrying the A allele; Figure 5 -I is a diagram showing the difference analysis results between the PRL of rapeseed varieties carrying the G allele in chrC05_11018618 (G / A) and the PRL of rapeseed varieties carrying the A allele; Figure 5 -J is a graph showing the difference between the PRL of rapeseed varieties carrying the A allele in chrC05_39198011 (A / T) and the PRL of rapeseed varieties carrying the T allele; Figure 5 -K is a graph showing the difference between the PRL of rapeseed varieties carrying the A allele in chrC07_3424413 (A / T) and the PRL of rapeseed varieties carrying the T allele; Figure 5 -L is the difference analysis result of PRL between rapeseed varieties carrying T allele in chrC07_8382100 (T / C) and rapeseed varieties carrying C allele; Figure 5 -M is the difference analysis result of PRL between rapeseed varieties carrying G allele in chrC07_13449494 (G / A) and PRL of rapeseed varieties carrying A allele; Figure 5 -N is the difference analysis result of PRL between rapeseed varieties carrying G allele in chrC07_42348526 (G / C) and rapeseed varieties carrying C allele; Figure 5 -O is the difference analysis result of PRL between rapeseed varieties carrying T allele in chrC08_13814667 (T / C) and rapeseed varieties carrying C allele; Figure 5 -P is the difference analysis result of PRL between rapeseed varieties carrying T allele in chrC08_25362930 (T / C) and rapeseed varieties carrying C allele; Figure 5 -Q is the difference analysis result of PRL between rapeseed varieties carrying T allele in chrC08_27603182 (T / C) and rapeseed varieties carrying C allele; Figure 5-R is the difference analysis result of PRL between rapeseed varieties carrying A allele in chrC08_31132724 (A / G) and rapeseed varieties carrying G allele; Figure 5 -S is the difference analysis result of PRL between rapeseed varieties carrying G allele in chrC09_26602878 (G / A) and PRL of rapeseed varieties carrying A allele;

[0039] Figure 6 This is a graph showing the correlation analysis results between the number of superior alleles carried by rapeseed varieties in natural rapeseed populations and PRL;

[0040] Figure 7 The results of the differential analysis of PRL between rapeseed varieties carrying different alleles in chrC03_3821562 (T / G);

[0041] Figure 8 Electropherogram showing the development of a molecular marker based on chrC03_3821562(T / G) within BnaC03g08100D. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0043] In order to enhance the understanding of the present invention, some key technical and scientific terms are clearly defined below. Unless otherwise specifically defined in this article, all other technical and scientific terms follow the meanings generally accepted and understood in the field to which the present invention belongs. It should be emphasized that the scope of the present invention is not limited to the specific methods, compositions or applications described, but allows reasonable flexibility and adjustment in these aspects. At the same time, please understand that the terms used in this article are intended to illustrate specific embodiments, rather than to be interpreted restrictively.

[0044] In addition, all literature cited herein, including but not limited to patents, patent applications, academic papers, textbooks and further citations therein, are deemed to be incorporated herein by reference as a whole to the extent not directly cited. If there is any inconsistency or conflict between the contents of these cited documents or similar materials and this application, especially in terms of term definitions, term usage, technical descriptions, etc., the contents of this application shall prevail.

[0045] In the embodiments of the present invention, some key technical and scientific terms are defined as follows:

[0046] Genome-wide association study (GWAS): A high-throughput, unbiased genetic research method that aims to identify genetic loci that are significantly associated with complex traits by comparing the correlation between genetic variations (such as SNPs) and specific phenotypes (such as disease states, physiological indicators, etc.) at the genome-wide level in different individuals. GWAS is based on the principle of linkage disequilibrium (LD), that is, adjacent genetic variants tend to be inherited together, so pathogenic variants can be captured indirectly through marker SNPs.

[0047] SNP site: Single Nucleotide Polymorphism (SNP) site refers to the DNA sequence polymorphism caused by the variation of a single nucleotide within the gene sequence.

[0048] In the embodiment of the present invention, the sequence information involved is shown in Table 1.

[0049] Table 1. List of biological sequences

[0050] name Sequence number Involved rights BnaC03g08100D SEQ ID NO: 1 Right 2 BnaC03g08100D-F SEQ ID NO: 2 Right 5 BnaC03g08100D-R SEQ ID NO: 3 Right 5

[0051] Example 1. Phenotypic variation of PRL in association analysis population of Brassica napus

[0052] The paper culture method was used to investigate the taproot length phenotype of the natural population of Brassica napus. Four culture racks were placed in each light culture room, and three columns were placed in each culture rack. Each column contained 32 pieces of phosphorus-free paper, and one plant was planted on each side of each piece of phosphorus-free paper. Each experiment was able to complete the simultaneous culture of 768 plants. The indoor conditions of the light culture room were 16 hours of light / 8 hours of darkness, and the indoor temperature was 18°C. The Hogland nutrient solution formula was used, and the pH was adjusted to 5.6 every time the nutrient solution was changed. Ultrapure water was added every 3 days from the time of sowing to keep the culture paper moist, and the culture was harvested on the 14th day. When harvesting, the root system was first photographed with a camera, and two-dimensional photos of the root system were collected, and the root system pictures were analyzed using Root Reader 2D software. First, the scale of the image was obtained with the help of Image J software, and then the parameters were set (Halfkernel size was 7 and 1, Min fixed threshold was 0, Percent above local average was 0.08 and 0.2, Max fixed threshold was 255, Dust removal filter was 75, Filling filter was 3, Scale 96.47pi / cm), and the taproot length (PRL) was obtained by manually clicking on both ends of the taproot. The phenotypic data were preliminarily sorted using Microsoft Excel (2007) software, and the mean, maximum, minimum and coefficient of variation of PRL were calculated. The correlation between different phenotypes was calculated using R software.

[0053] This example statistically analyzed the taproot lengths of 393 rapeseed-related populations under phosphorus-free paper culture conditions. Figure 1-2 As shown, in this population, the maximum PRL is 22.7 cm, the minimum is 8.24 cm, and the span is 14.46 cm, indicating that the PRL of this population has a wide range of phenotypic variations, see Figure 2 The skewness (0.391) and kurtosis (0.461) of this population were both close to 0, indicating that the PRL of this population was approximately normally distributed and could be used for subsequent genome-wide association analysis.

[0054] Example 2. Genome-wide association analysis of PRL in the Brassica napus association analysis population

[0055] Previously, we performed whole-genome resequencing on 505 samples of Brassica napus, and developed more than 10 million SNP sites based on the Darmor reference genome. For details, see the article "Tang S, Zhao H, Lu S, Yu L, Zhang G, Zhang Y, Yang QY, Zhou Y, Wang X, Ma W, Xie W and Guo L. Genome-and transcriptome-wide association studies provide insights into the genetic basis of natural variation of seed oil content in Brassica napus. Mol Plant, 2021, 14: 470-487". In this study, 393 samples of rapeseed were selected for subsequent genome-wide association analysis. First, the VCFtools software was used to filter the above SNP sites according to the screening criteria of near allele frequency > 0.05 and missing rate < 0.2, and 4.6 million SNP sites were obtained for this GWAS analysis. The MLM model was used to perform GWAS analysis on the taproot length of 393 rapeseed samples, with a mean of 1×10 -5 As the threshold, SNP loci significantly associated with taproot length were screened.

[0056] In this study, two MLM models in Tassel 5.0 software were used to conduct genome-wide association analysis on PRL in 393 Brassica napus varieties under paper culture conditions. A total of 48 SNP loci significantly associated with it were identified. The phenotypic explained variance of these SNP loci ranged from 6.51% to 12.19%, as shown in Table 2. Figure 3 . The above SNP sites significantly associated with PRL are distributed on chromosomes A01, A02, A03, A05, A07, A09, A10, C01, C02, C03, C04, C05, C07, C08, and C09. Subsequently, 36 long PRL alleles were identified based on the above SNPs significantly associated with PRL. For example, the T allele in chrA01_5960845 (A / T) and the A allele in chrA01_13740784 (G / A) are both long PRL alleles, see for details. Figure 4 and Figure 5 At the same time, there is a very significant positive correlation between the number of long PRL alleles carried by the variety and PRL (p<0.0001, R:0.5902), see Figure 6 .

[0057] Table 2. SNP sites significantly associated with PRL identified by GWAS analysis

[0058]

[0059]

[0060] Example 3. Molecular marker development

[0061] SnapGene software was used to identify whether the functional SNP significantly associated with RPL was located at the restriction site. AmplifX software was used to design specific primers about 500 bp upstream and downstream of the functional SNP for developing molecular markers: BnaC03g08100D-F and BnaC03g08100D-R. The nucleotide sequences of BnaC03g08100D-F and BnaC03g08100D-R are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively. Six varieties with short root length carrying the G allele and six varieties with long root length carrying the T allele were selected for verification. After DNA was extracted, PCR amplification was performed using the above primers, and the PCR products were digested with restriction endonucleases corresponding to the target SNP to verify the accuracy of the newly developed molecular markers.

[0062] Among the SNP sites significantly associated with PRL, the SNP chrC03_3821562 (T / G) located within the gene BnaC03g08100D is located in the specific recognition sequence of the restriction endonuclease BsPHi. Among the 393 varieties in the association analysis population, 145 varieties carry the T allele in chrC03_3821562 (T / G) and 208 varieties carry the G allele in chrC03_3821562 (T / G). The PRL of the two types of Brassica napus varieties is significantly different, see Figure 7 .

[0063] As shown in Table 3, the Brassica napus varieties L900, L902, L916, L924, L794 and L740 carry the T allele in chrC03_3821562 (T / G), and the PRL range of these rapeseed varieties is 8.02cm~11.46cm; the Brassica napus varieties L16, L22, L64, L254, L450 and L454 all carry the G allele in chrC03_3821562 (T / G), and the PRL range of these rapeseed varieties is 14.41cm~17.27cm. First, primers BnaC03g08100D-F and BnaC03g08100D-R were used to amplify the above varieties, and then the amplified fragments of the above varieties were digested with BsPHi. The amplified fragments of the six short PRL varieties were cut into two fragments of 332 bp and 148 bp by BsPHi, while the amplified fragments of the six long PRL varieties could not be cut by BsPHi and were 480 bp. For details, see Figure 8.

[0064] In summary, the present invention developed a molecular marker based on chrC03_3821562 (T / G) that can be used to identify long PRL varieties and named it Bn_C3_PRL_marker.

[0065] Table 3. Information on 10 varieties used for molecular marker development

[0066] Variety name chrC03_3821562(T / G) Root length (cm) L900 T 8.02 L902 T 8.5 L916 T 9.31 L924 T 10.28 L794 T 11.8 L740 T 11.46 L16 G 14.41 L22 G 14.36 L64 G 16.61 L254 G 15.31 L450 G 17.62 L454 G 17.27

Claims

1. A gene for screening and / or cultivating long PRL Brassica napus, characterized in that: The gene is BnaC03g08100D.

2. The gene according to claim 1, characterized in that The nucleotide sequence of BnaC03g08100D is shown in SEQ ID NO.

1.

3. A molecular marker based on the gene according to any one of claims 1 to 2, characterized in that: The molecular marker is located at the 3821562nd base of chromosome C03 in BnaC03g08100D, and the base is T or G.

4. The molecular marker according to claim 3, characterized in that The long PRL B. napus varieties carry the G allele, while the short PRL B. napus varieties carry the T allele.

5. A primer pair for amplifying a molecular marker according to any one of claims 3 to 4, characterized in that: The primer pair includes an upstream primer whose nucleotide sequence is shown as SEQ ID NO.2 and a downstream primer whose nucleotide sequence is shown as SEQ ID NO.

3.

6. A method for identifying the length of PRL in Brassica napus varieties based on the molecular markers according to any one of claims 3 to 4, characterized in that: The steps include: (1) Extracting DNA from Brassica napus; (2) using the DNA obtained in step (1) as a template and the primer pair described in claim 5 to perform PCR amplification to obtain an amplified product; (3) Using BsPHi to perform restriction digestion and electrophoresis analysis on the amplified product obtained in step (2), and identify the Brassica napus variety with long PRL.

7. The method according to claim 6, characterized in that If the amplified product is cut into two fragments of 332bp and 148bp by BsPHi, it indicates that the Brassica napus is a short PRL variety; if the amplified product is not cut by BsPHi, it contains a 480bp fragment but does not contain 332bp and 148bp fragments, it indicates that the Brassica napus is a long PRL variety.

8. A method for cultivating long PRL Brassica napus, characterized in that: The steps include: 1) using the method described in any one of claims 6 to 7 to identify a long PRL Brassica napus variety; 2) using the long PRL Brassica napus variety identified in step 1) for breeding and / or large-scale planting.

9. Use of the gene according to any one of claims 1 to 2, the molecular marker according to any one of claims 3 to 4 and / or the primer pair according to claim 5 in screening and / or breeding Brassica napus varieties with long PRL.

10. Use of the gene according to any one of claims 1 to 2, the molecular marker according to any one of claims 3 to 4 and / or the primer pair according to claim 5 in identifying PRL of Brassica napus.

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