Molecular marker linked with leaf margin cleavage gene BjA10. LL of brassica juncea and application of molecular marker

Abstract

The invention relates to the field of plant breeding and plant genetics, and discloses seven SSR primer pair sequences and two IP molecular marker primer pair sequences which are linked with leaf margin cleavage gene BjA10. LL of brassica juncea, leaf margin cleavage 9B16 and leaf margin whole margin 9B18 materials of brassica juncea are used as parents to construct F2 segregation populations, and the F2 segregation populations are used as parents. Carrying out fine positioning and map-based cloning on the cleavage gene BjA10. LL of the mustard type rape by combining technologies such as SSR (Simple Sequence Repeat) and IP (Internet Protocol) molecular markers; the result lays a theoretical foundation for clarification of a molecular mechanism of leaf margin cleavage regulation of the mustard type rape and application of leaf shape characters to genetic improvement of the rape.

Description

Technical Field

[0001] The invention relates to the field of plant breeding and plant genetics, and in particular to a molecular marker linked to a mustard-type rapeseed leaf margin notch gene BjA10.LL and its application. Background Art

[0002] Rapeseed is an important oil crop in my country and even in the world. It is an important source of vegetable oil for human consumption and an important source of protein in animal feed. There are currently three main rapeseed cultivars in my country, namely Brassica napus L., Brassica rapa L. and Brassica juncea L. Among them, Brassica juncea L. has strong resistance to a variety of abiotic and biotic stresses, and is more resistant to drought, heat, barrenness, pests and diseases, lodging, pod cracking, and has a high oil content (>45%). Leaves are the main organs for photosynthesis, respiration and transpiration in plants, and play a very important role in the growth and development of plants. The leaf margin morphology is diverse, and the leaf margin rifting trait has the characteristics of early expression, easy identification, long expression period and stability. It is an ideal morphological marker in breeding and can be used in plant shape improvement, hybrid identification and purity identification.

[0003] Molecular markers are genetic markers based on DNA molecule polymorphism. In a broad sense, they refer to detectable proteins or DNA sequences that need to have genetic characteristics. In a narrow sense, they are specific DNA fragments that reflect the special differences in the genome between individuals or groups of organisms. SSR (Simple Sequence Repeats), also known as microsatellite DNA, is a molecular marker technology based on PCR using specific primers. It is usually a simple repetitive sequence that is mainly distributed in the genome of eukaryotic organisms and is repeated multiple times in tandem as a main sequence consisting of 2-6 bases. Since the flanking sequence of a specific microsatellite site in the genome is usually a single, highly conserved sequence, the flanking sequence can be used to synthesize primers for PCR amplification to amplify a single microsatellite site. Changes in the number of repeats at the microsatellite site will lead to changes in the length of the amplified product between individuals, thus being polymorphic.

[0004] Compared with other molecular markers, SSR markers have the following characteristics: (1) large number, able to cover the entire genome, and highly polymorphic; (2) multiple alleles, large amount of information; (3) inheritance according to Mendel's principle, showing codominance; (4) stable, easy to operate and use, and consume little DNA; (5) each locus is defined by a designed primer sequence, which facilitates communication and collaboration between different laboratories in design. SSR has been widely used in gene positioning, genetic linkage map construction, genetic diversity analysis and hybrid vigor prediction research in crops such as rice, wheat, corn and rapeseed.

[0005] IP marker, i.e. intron polymorphism marker, is a new type of molecular marker based on PCR technology. Its basic principle is the sequence variation between allelic introns. Introns are usually non-coding sequences with large variations and high polymorphism, while exons are coding sequences with high conservation, so primers with strong universality can be constructed. When designing IP markers, the focus is on amplifying introns, so primers should be designed on the exons on both sides of the introns. Among them, the template required for designing primers comes from the exon sequence, and the location of primer design is determined by the intron position. IP markers have the advantages of abundant quantity, high polymorphism, simple operation, and stable polymorphism, which are similar to SSR markers. In addition, IP markers are directly marked in genes, and genes need to exist in the genome of this region. The genetic linkage map constructed using IP markers can directly reflect the linkage relationship of genes; according to the homology relationship of genes, IP markers developed between different species can establish a corresponding relationship, which is conducive to the study of comparative genomics. IP markers have been successfully applied to the study of rapeseed gene positioning.

[0006] At present, the genetic mechanism of leaf margin fissure formation in mustard rapeseed is still unclear, and there are relatively few studies on its gene location. Therefore, developing more molecular markers with accurate position information will help to achieve the initial and fine positioning of the target gene, and then complete the isolation and cloning of the target gene, providing clues for the evolutionary study of leaf shape and even the evolutionary pattern of species. Summary of the invention

[0007] In view of this, the present invention provides a molecular marker linked to the Brassica juncea leaf margin notch gene BjA10.LL and its application.

[0008] The specific technical solution of the invention is as follows:

[0009] A molecular marker linked to the leaf margin notch gene BjA10.LL of Brassica juncea, characterized in that the 9 specific primer pairs corresponding to the molecular marker include at least one of primer pair SSR41, primer pair SSR59, primer pair SSR80, primer pair SSR85, primer pair SSR91, primer pair SSR124, primer pair SSR143, IP primer pair IP9, and primer pair IP12; wherein,

[0010] Primer pair SSR41:

[0011] Upstream primer (F): 5'-CGTACATAATAACTGTGGTGCG-3';

[0012] Downstream primer (R): 5'-ATATGAAGTCAGTGTATACGATAC-3';

[0013] Primer pair SSR59:

[0014] Upstream primer (F): 5'-CCATATCTAGCACTTAAAGCTG-3';

[0015] Downstream primer (R): 5'-ACAACGCATTCGATAGTAAGGGA-3';

[0016] Primer pair SSR85:

[0017] Upstream primer (F): 5'-TGGTCTGATCGAATTGACATGG-3';

[0018] Downstream primer (R): 5'-TGAGAATAGAACCGAGTCTATGC-3';

[0019] Primer pair SSR91:

[0020] Upstream primer (F): 5'-GAAGCACCCATTATCAACTC-3';

[0021] Downstream primer (R): 5'-ATCAACTTCTGCGTAGAGTG-3';

[0022] Primer pair SSR124:

[0023] Upstream primer (F): 5'-CTTGAACCCTTCTATCCC-3';

[0024] Downstream primer (R): 5'-ACCCCATCTTCGCAACAG-3';

[0025] Primer pair SSR143:

[0026] Upstream primer (F): 5'-ACCTGGTGGATCCGTTTG-3';

[0027] Downstream primer (R): 5'-GCTGTCATTACAGAGACC-3';

[0028] Primer pair IP9:

[0029] Upstream primer (F): 5'-TCCACAATCTTCGTCACC-3';

[0030] Downstream primer (R): 5'-TTAGCTCCTCGTTCGCCC-3';

[0031] Primer pair IP12:

[0032] Upstream primer (F): 5'-TCCAAACCCGCGAGTTGC-3';

[0033] Downstream primer (R): 5'-AGTAGAGGAGGAAGAGG-3'.

[0034] Furthermore, it is used to identify the leaf shape traits of Brassica juncea.

[0035] The object of the present invention also includes protecting a method for distinguishing leaf shape traits of Brassica juncea.

[0036] A method for distinguishing leaf shape traits of Brassica juncea, comprising the following steps:

[0037] Step 1, Brassica juncea genomic DNA;

[0038] Step 2, amplifying the DNA sequence of step 1 using the above primer pairs;

[0039] Step 3: Sequencing the amplified product obtained in step 2 to distinguish whether the leaf shape of Brassica juncea is an entire leaf or a lobed leaf.

[0040] A kit is used in identifying leaf shape traits of Brassica juncea, the kit comprising specific primers, wherein the specific primers are at least one of primer pair SSR41, primer pair SSR59, primer pair SSR80, primer pair SSR85, primer pair SSR91, primer pair SSR124, primer pair SSR143, IP primer pair IP9, and primer pair IP12.

[0041] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0042] The present invention relates to the field of plant breeding and plant genetics, and discloses 7 SSR primer pair sequences and 2 IP molecular marker primer pair sequences linked to a mustard rapeseed leaf margin nicking gene BjA10.LL. The mustard rapeseed leaf margin nicking 9B16 and leaf margin entire 9B18 materials are used as parents to construct an F2 segregation population, and the mustard rapeseed nicking gene BjA10.LL is precisely located and positionally cloned by combining SSR, IP molecular marker and other technologies. The results will clarify the molecular mechanism of the regulation of mustard rapeseed leaf margin nicking and lay a theoretical foundation for using leaf shape traits for rapeseed genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The results of molecular marker validation in parents and F2 plants with entire leaves and cleaved leaves. PE represents DNA from 5 plants with entire leaves; PL represents DNA from 5 plants with cleaved leaves; F1 represents DNA from 6 plants with F1 generation; F2E represents DNA from 6 plants with entire leaves in F2 generation; and F2L represents DNA from cleaved leaves in F2 generation. Arrows represent target bands.

[0044] Figure 2 The genetic and physical maps of the leaf margin notch gene BjA10.LL in Brassica juncea. DETAILED DESCRIPTION

[0045] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0046] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0047] The embodiments of the present application are described in detail below. Examples of the embodiments are shown in the drawings, but unnecessary detailed descriptions may be omitted. For example, detailed descriptions of well-known matters and repeated descriptions of actually identical structures may be omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate understanding by those skilled in the art. In addition, the drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter described in the claims.

[0048] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as those commonly understood by technicians in the technical field to which this application belongs; the terms used in this application are only for the purpose of describing specific embodiments and are not intended to limit this application; unless otherwise specified, the numerical values ​​of the parameters mentioned in this application can be measured using various measurement methods commonly used in the art (for example, they can be tested according to the methods given in the embodiments of this application).

[0049] The terms "including" and "having" in the specification and claims of the present application and any modifications thereof are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0050] In the description of the present application, "A and / or B" may include the case of A alone, the case of B alone, or any of the cases of A and B, where A and B are only used for examples and may be any technical feature connected by "and / or" in the present application.

[0051] If not otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0052] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0053] Example 1

[0054] The SSR and IP molecular marker primers linked to the leaf margin notch gene were obtained by the following steps:

[0055] In the early stage of this study, the Brassica 50K SNP and colinearity analysis were used to preliminarily locate the gene in the 18.16-19.32Mb segment of chromosome A10 and the 0.51-0.53Mb segment of chromosome B02 of Brassica juncea.

[0056] like Figure 1 and Figure 2 As shown in the figure, based on the above research results and combined with the information of the mustard genome, we first used SSR Hunter software to find SSR sites in the target interval and used Primer 5.0 to design SSR primers. A total of 179 pairs of SSR primers were designed, of which 155 pairs were located on chromosome A10 and 24 pairs were located on chromosome B02. Through mixed pool screening and small population verification, we finally obtained 7 stable polymorphic SSR molecular markers (SSR41, SSR59, SSR80, SSR85, SSR91, SSR124, SSR143), all located on chromosome A10 of mustard rapeseed, and used the F2 population to block the target gene between SSR124 and SSR143.

[0057] Furthermore, comparison with the reference genome information of Brassica juncea revealed that a total of 49 genes were included between the molecular markers SSR124 and SSR143. Therefore, IP markers were developed every 2-3 genes in this target interval, and primers were designed using Primer 5.0 software based on the exons of the selected genes. A total of 17 pairs of primers were designed, and through mixed pool screening and small group verification, two IP markers, IP9 and IP12, were still polymorphic in individual plants, and the polymorphism was stable.

[0058] Then, the above 9 stable molecular markers were selected to detect 839 individuals in the F2 population, and the genetic distance between the marker and the target gene was calculated using the number of exchange individuals detected. SSR91 screened 89 exchange individuals, SSR59 screened 34 exchange individuals, SSR124 screened 25 exchange individuals, IP9 screened 11 exchange individuals, IP12 screened 3 exchange individuals, SSR143 screened 13 exchange individuals, SSR80 screened 38 exchange individuals, SSR85 screened 79 exchange individuals, and SSR41 screened 82 exchange individuals. The whole genome DNA of all exchange individuals was re-extracted and the test was repeated to ensure the accuracy of the exchange individuals.

[0059] At the same time, the genetic map and physical map of the gene were constructed using the exchanged plant information. From the genetic map, it can be seen that markers SSR91, SSR59, and SSR124 are located on one side of the target gene, with genetic distances of 19.6cM, 9.5cM, and 3.1cM, respectively; markers IP12, IP9, SS143, SSR80, SSR85, and SSR41 are located on the other side of the target gene, with genetic distances of 0.7cM, 2.4cM, 4.1cM, 8.6cM, 15.8cM, and 24.7cM, respectively. Markers SSR124 and IP12 are closest to the target gene, with genetic distances of 3.1cM and 0.7cM, respectively, limiting the target gene to within 3.8cM.

[0060] Finally, the above 9 marker sequences linked to the target gene were aligned to the reference genome of Brassica juncea, the physical positions of each marker on the chromosome were obtained, and the physical map of the target gene was constructed. By comparing the genetic map and the physical map, it can be seen that the order of the markers on the two maps is basically the same, indicating that the constructed genetic map is relatively accurate. The physical positions of the nearest markers IP12 and SSR124 on both sides of the target gene on the chromosome are 19,010,522bp and 18,911,075bp respectively, and the physical distance between the markers is 0.1Mb.

[0061] The specific operations are as follows:

[0062] 1. Screening of molecular marker gene pool construction

[0063] Five individuals from the entire leaf parent and five individuals from the cleaved leaf parent were selected. Then, based on the results of the seedling phenotypic investigation of the F2 segregation population, six individuals from the entire leaf population and six individuals from the cleaved leaf population were selected from the F2 segregation population, and their DNAs were mixed in equal amounts to construct the parental and offspring entire leaf DNA pool and cleaved leaf DNA pool, which were named the entire leaf parent pool (PE), cleaved leaf parent pool (PL), entire leaf generation pool (F2E) and cleaved leaf generation pool (F2L), respectively. At the same time, the individuals that constitute the entire leaves and cleaved leaves can be used as small populations for subsequent verification of polymorphic markers.

[0064] 2. Extraction of Genomic DNA from Single Strains of Brassica juncea

[0065] At the five-leaf stage of rapeseed, leaf type identification was performed on the parental materials of entire leaf 9B18 and lobed leaf 9B16 and the positioning population. Leaves were sampled from individual plants, stored in liquid nitrogen and transported back to the laboratory, and stored at -80°C for later use. The CTAB method was used to extract the whole genome DNA of the leaves. The extracted DNA samples were tested by 1% agarose gel electrophoresis to ensure that the extracted genomic DNA was not degraded; the contamination level of the extracted DNA protein and organic matter was tested by a spectrophotometer, and the concentration of the qualified genomic DNA was adjusted to 50ng / μl.

[0066] 3. Design of primers for SSR and IP molecular markers

[0067] SSR primer design: The results of the Brassica napus chip positioning were mapped to the Brassica juncea genome. The region where the target gene was located, between 17.07–17.38 Mb of chromosome A10, was compared to the reference genome information of Brassica juncea. The genome sequence between the locked segments was retrieved, and the SSR loci were found using SSR Hunter software and SSR primers were designed using Primer 5.0. The primer design principles were: amplification length of 150–350 bp, Tm value set to 55–60°C, and primer sequence length of 20–22 bp. SSR primers were synthesized by Xi'an Qingke Biological Company. 179 pairs of SSR primers were designed and synthesized.

[0068] IP primer design: Referring to the alignment of the mustard genome sequence, it was found that the locked segment ChromosomeA1018.9–19.1Mb contained a total of 49 genes. Gene annotation was performed on these genes, and IP primers were developed for genes that may be related to traits using the annotation information. IP markers were developed for every 2 genes in this region, and primers were designed using Primer 5.0 software based on the exons of the selected genes, referring to the primer design principles.

[0069] 4. PCR amplification and electrophoresis analysis:

[0070] SSR markers: The PCR amplification system and SSR reaction procedures were based on the method of Lu Guangyuan et al. (2003). A 10 μL PCR system was used, including 2 μL DNA, 5 μL 2×Es Taq Master Mix (Novozyme, China), 0.6 μL of 10 μM forward and reverse primers, and 1.8 μL ddH2O. PCR reaction cycle parameters: 95°C, 1 min; 94°C, 1 min, 35°C, 1 min, 72°C, 1 min, 5 cycles in total; 94°C, 1 min; 55–60°C, 1 min; 72°C, 1 min; 34 cycles in total; 72°C, 7 min; 4°C storage. The products obtained by the PCR reaction were separated by 8% polyacrylamide gel electrophoresis (PAGE) and visualized by silver nitrate staining.

[0071] IP labeling: A 10 μL PCR system was used, including 2 μL DNA, 5 μL 2×Es Taq Master Mix (Novozyme, China), 1 μL each of 10 μM forward and reverse primers, and 1 μL ddH2O. PCR reaction cycle parameters: 95°C, 5 min; 95°C, 30 s; 55°C, 30 s; 72°C, 60 s; 35 cycles; 72°C, 10 min; 4°C storage. The products obtained by the PCR reaction were separated by 8% polyacrylamide gel electrophoresis and developed by silver nitrate staining.

[0072] 5. Polymorphic primer screening and SSR and IP molecular marker analysis

[0073] The IP primers and SSR primers designed above were used to screen polymorphic markers among the mixed pools of PE, PL, F2E, and F2L. The obtained polymorphic markers were verified by pool opening, and the confirmed polymorphic markers were verified in 839 individual plants (385 plants with entire leaves and 454 plants with slit leaves) of the F2 population to obtain exchange plants. Then, Joinmap 4.0 software was used to analyze the linkage relationship between the polymorphic markers and the target gene, and molecular genetic linkage maps and physical maps were drawn, and 7 pairs of SSR and 2 pairs of IP molecular marker primers linked to the leaf margin slit gene were obtained.

[0074] The deoxyribonucleotide primer sequences of 7 pairs of SSR primers SSR41, SSR59, SSR80, SSR85, SSR91, SSR124, SSR143 and 2 pairs of IP primers IP9, IP12 are as follows:

[0075] Primer pair SSR41:

[0076] Upstream primer (F): 5'-CGTACATAATAACTGTGGTGCG-3'.

[0077] Downstream primer (R): 5'-ATATGAAGTCAGTGTATACGATAC-3'.

[0078] Primer pair SSR59:

[0079] Upstream primer (F): 5'-CCATATCTAGCACTTAAAGCTG-3'.

[0080] Downstream primer (R): 5'-ACAACGCATTCGATAGTAAGGGA-3'.

[0081] Primer pair SSR85:

[0082] Upstream primer (F): 5'-TGGTCTGATCGAATTGACATGG-3'.

[0083] Downstream primer (R): 5'-TGAGAATAGAACCGAGTCTATGC-3'.

[0084] Primer pair SSR91:

[0085] Upstream primer (F): 5'-GAAGCACCCATTATCAACTC-3'.

[0086] Downstream primer (R): 5'-ATCAACTTCTGCGTAGAGTG-3'.

[0087] Primer pair SSR124:

[0088] Upstream primer (F): 5'-CTTGAACCCTTCTATCCC-3'.

[0089] Downstream primer (R): 5'-ACCCCATCTTCGCAACAG-3'.

[0090] Primer pair SSR143:

[0091] Upstream primer (F): 5'-ACCTGGTGGATCCGTTTG-3'.

[0092] Downstream primer (R): 5'-GCTGTCATTACAGAGACC-3'.

[0093] Primer pair IP9:

[0094] Upstream primer (F): 5'-TCCACAATCTTCGTCACC-3'.

[0095] Downstream primer (R): 5'-TTAGCTCCTCGTTCGCCC-3'.

[0096] Primer pair IP12:

[0097] Upstream primer (F): 5'-TCCAAACCCGCGAGTTGC-3'.

[0098] Downstream primer (R): 5'-AGTAGAGGAGGAAGAGG-3'.

[0099] Example 2

[0100] Application of SSR41 Primers

[0101] 1. Extraction of genomic DNA from Brassica juncea

[0102] 2-3 young leaves were selected from each plant, placed in a ziplock bag and marked with a marker, placed in liquid nitrogen and transported back to the laboratory, and stored at -80°C. The CTAB method was used to extract the whole genome DNA of the leaves. The extracted DNA samples were tested by 1% agarose gel electrophoresis to ensure that the extracted genomic DNA was not degraded; the contamination level of the extracted DNA protein and organic matter was tested by a spectrophotometer, and the concentration of the qualified genomic DNA was adjusted to 50ng / μl.

[0103] 2. PCR amplification

[0104] The entire leaf parent and the lobed leaf parent were used as templates and analyzed according to the following PCR amplification conditions and procedures.

[0105] PCR amplification conditions:

[0106] The 10 μL PCR system was as follows: 2 μL DNA, 5 μL 2×Es Taq Master Mix (Novozymes, China), 0.6 μL each of 10 μM forward and reverse primers, and 1.8 μL ddH2O.

[0107] PCR reaction program: 95°C, 1 min; 94°C, 1 min, 35°C, 1 min, 72°C, 1 min, for a total of 5 cycles; 94°C, 1 min; 55–60°C, 1 min; 72°C, 1 min; for a total of 34 cycles; 72°C, 7 min; store at 4°C.

[0108] 3. Polyacrylamide gel electrophoresis

[0109] Gel configuration: (1) Clean the glass back plate and concave plate, dry them, place them on a horizontal table, and wipe them clean with alcohol. Apply affinity silane (80 μl affinity silane + 4 ml anhydrous ethanol) evenly on the back plate, and apply 1 ml stripping silane on the concave plate, ensuring uniformity (perform in a fume hood). Then, place a seal on both sides of the back plate, stack the concave plate on the back plate, fix it with a clamp, and raise the front end of the glass plate to make it tilted.

[0110] (2) Preparation of gel: Add 460 μl APS and 46 μl TEMED (PAGE gel coagulant) to 80 ml of gel stock solution, stir rapidly, and immediately pour the gel into the gap between the glass back plate and the concave plate tilted at a suitable angle. Be careful to pour the gel at a uniform speed and avoid creating bubbles. When the gel fills the entire glass plate, lay it flat immediately, insert a comb (about 0.5 cm), and leave it for 2 hours to solidify.

[0111] Gel electrophoresis: (1) When the gel solidifies, slowly pull out the comb horizontally, remove the fixing clip, and wash the broken gel. Fix it vertically on the electrophoresis instrument with the clamp at a moderate tightness. Pour 1TBE electrophoresis solution, adjust the voltage to 1000V, the current to 100mA, and pre-electrophoresis for 10 minutes.

[0112] (2) After the pre-electrophoresis is completed, use a syringe to draw up the electrophoresis solution to rinse the gel surface to remove bubbles. Use one end of a comb to carefully scrape off excess gel to ensure that the gel surface is clean. Insert the comb until the teeth penetrate the gel surface by about 2 mm.

[0113] (3) Take 4 μl of the PCR product (with loading buffer added) and quickly spot it into the spotting space between the combs, taking care not to cross-sample. After spotting, turn on the electrophoresis instrument and run the electrophoresis for 2 h (U = 1000 V, I = 100 mA).

[0114] Silver staining: (1) Remove the comb, separate the back plate from the concave plate, and place the back plate with the glue side facing up in the silver stain solution (4g silver nitrate + 2L distilled water) for 10-15 minutes. During this time, shake the shaker continuously to ensure uniform staining.

[0115] (2) Remove the backing plate and gently rinse it with distilled water for 10 seconds to remove the silver staining solution remaining on the surface of the gel. Then place the backing plate in the color developing solution prepared 5 minutes in advance and color it for 5-10 minutes until clear bands appear. Color developing solution: 30g NaOH + 2L distilled water + 8mL formaldehyde.

[0116] (3) After development, rinse the surface of the gel gently with distilled water to remove excess color developing solution. After drying, place it on a light box to read the strips and capture images with a camera.

[0117] 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 and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A molecular marker linked to the leaf margin notch gene BjA10.LL of Brassica juncea, characterized in that: The nine specific primer pairs corresponding to the molecular markers include at least one of primer pair SSR41, primer pair SSR59, primer pair SSR80, primer pair SSR85, primer pair SSR91, primer pair SSR124, primer pair SSR143, IP primer pair IP9, and primer pair IP12; wherein, Primer pair SSR41: Upstream primer (F): 5'-CGTACATAATAACTGTGGTGCG-3'; Downstream primer (R): 5'-ATATGAAGTCAGTGTATACGATAC-3'; Primer pair SSR59: Upstream primer (F): 5'-CCATATCTAGCACTTAAAGCTG-3'; Downstream primer (R): 5'-ACAACGCATTCGATAGTAAGGGA-3'; Primer pair SSR85: Upstream primer (F): 5'-TGGTCTGATCGAATTGACATGG-3'; Downstream primer (R): 5'-TGAGAATAGAACCGAGTCTATGC-3'; Primer pair SSR91: Upstream primer (F): 5'-GAAGCACCCATTATCAACTC-3'; Downstream primer (R): 5'-ATCAACTTCTGCGTAGAGTG-3'; Primer pair SSR124: Upstream primer (F): 5'-CTTGAACCCTTCTATCCC-3'; Downstream primer (R): 5'-ACCCCATCTTCGCAACAG-3'; Primer pair SSR143: Upstream primer (F): 5'-ACCTGGTGGATCCGTTTG-3'; Downstream primer (R): 5'-GCTGTCATTACAGAGACC-3'; Primer pair IP9: Upstream primer (F): 5'-TCCACAATCTTCGTCACC-3'; Downstream primer (R): 5'-TTAGCTCCTCGTTCGCCC-3'; Primer pair IP12: Upstream primer (F): 5'-TCCAAACCCGCGAGTTGC-3'; Downstream primer (R): 5'-AGTAGAGGAGGAAGAGG-3'.

2. The use of a molecular marker linked to the leaf margin notch gene BjA10.LL of Brassica juncea as claimed in claim 1, characterized in that: Used to identify leaf shape traits of Brassica juncea.

3. A method for distinguishing leaf shape traits of Brassica juncea, characterized in that: The following steps are involved: Step 1, Brassica juncea genomic DNA; Step 2, amplifying the DNA sequence of step 1 using the primer pair as claimed in claim 1; Step 3: Sequencing the amplified product obtained in step 2 to distinguish whether the leaf shape of Brassica juncea is an entire leaf or a lobed leaf.

4. Use of a kit for identifying leaf shape traits of Brassica juncea, characterized in that: The kit comprises specific primers, which are at least one of the primer pair SSR41, primer pair SSR59, primer pair SSR80, primer pair SSR85, primer pair SSR91, primer pair SSR124, primer pair SSR143, IP primer pair IP9, and primer pair IP12 as shown in claim 1.

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