Molecular markers related to leaf margin notch traits in Brassica rapa and their applications

Abstract

The present invention discloses a molecular marker related to the leaf margin puncture trait of a Brassica plant and its application. The present invention provides a method for identifying the leaf margin puncture trait of a plant: detecting the genotype of the plant based on Indel LMR, the leaf margin phenotype of the A genotype plant is leaf margin puncture, the leaf margin phenotype of the B genotype plant is leaf margin entire, and the leaf margin phenotype of the H genotype plant is leaf margin puncture; genotype judgment method: if both homologous chromosomes do not contain Indel LMR, it is A genotype; if both homologous chromosomes contain Indel LMR, it is B genotype; if one homologous chromosome contains Indel LMR and the other homologous chromosome does not contain Indel LMR, it is H genotype; Indel LMR is shown in SEQ ID NO: 1. The present invention develops a molecular marker closely linked to the leaf margin puncture trait, and the use of molecular marker-assisted selection can accurately and stably select seedlings in the early stage, thereby reducing the workload and accelerating the breeding process.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a molecular marker related to leaf margin notch traits of Brassica plants and an application thereof. Background Art

[0002] Leaves are important organs of plants, playing an important role in the accumulation of photosynthetic products, gas exchange, nutrient distribution and water transport. In order to better adapt to the environment during the evolution process, plant leaves have produced leaf structures of various shapes, which are divided into entire, shallowly lobed, deeply lobed and even individual leaflet units according to the leaf margin fissures from shallow to deep. In-depth exploration and identification of new genes and molecular regulatory mechanisms that control leaf morphology, and the cultivation of new varieties of Brassica plants with diverse morphologies are effective ways to meet the consumption needs of different regions and markets and improve environmental adaptability.

[0003] Crops of Brassica campestris in the genus Brassica of the family Cruciferae include Chinese cabbage subspecies (ssp. pekinensis), common cabbage subspecies (ssp. chinensis) and turnip subspecies (ssp. rapa) (Reference: Discussion on the Classification of Chinese Cabbage, Acta Horticulturae Sinica, May 1980, Lin Weishen). There are a lot of morphological variations in the leaves of crops, and the leaf morphology of different crops is very different. As the nutritional organ of plant photosynthesis, moderate leaf fissures are beneficial to improve the plant type, improve the ventilation and light transmission of the group, enhance the photosynthesis efficiency, and reduce the occurrence of diseases and insect pests. In addition, leaf shape is closely related to plant stress resistance. Studies have shown that the production of leaf margin fissures in plants improves the stress resistance (drought resistance, high temperature resistance, frost resistance) of plants and their adaptability to strong light and weak light environments. Therefore, precise positioning of leaf margin notch QTL and gene function analysis can not only accelerate the analysis of the molecular mechanism of leaf margin notch formation, but also provide excellent genetic resources and available molecular markers for breeding work, which has important theoretical and application value.

[0004] Leaf notch is a complex quantitative trait that is affected by multiple signal pathways. The leaf notch phenotype can only be displayed after the true leaves are formed and unfolded. Traditional breeding methods are limited by the development period and are time-consuming and laborious. Developing molecular markers that are closely linked to the leaf notch trait and using molecular marker-assisted selection can accurately and stably select seedlings (or even seeds) at an early stage, thereby reducing the workload and accelerating the breeding process. Summary of the invention

[0005] The purpose of the present invention is to provide a molecular marker related to the leaf margin notch trait of Brassica rapa plants and its application.

[0006] The present invention provides a method for identifying or assisting in identifying the leaf margin trait of a plant, comprising the following steps: detecting the genotype of the plant based on Indel LMR, the leaf margin phenotype of the A genotype plant is or is a candidate for leaf margin rift, the leaf margin phenotype of the B genotype plant is or is a candidate for leaf margin entire, and the leaf margin phenotype of the H genotype plant is or is a candidate for leaf margin rift;

[0007] The determination method of the genotype based on Indel LMR is as follows: if both homologous chromosomes of the plant do not contain the Indel LMR, its genotype is genotype A; if both homologous chromosomes of the plant contain the Indel LMR, its genotype is genotype B; if one homologous chromosome of the plant contains the Indel LMR and the other homologous chromosome does not contain the Indel LMR, its genotype is genotype H;

[0008] The Indel LMR is a DNA molecule, as shown in SEQ ID NO:1.

[0009] The present invention also provides a method for identifying or assisting in identifying the leaf margin trait of a plant, comprising the following steps: detecting the genotype of the plant based on Indel LMR, the leaf margin phenotype of the A genotype plant is or is a candidate for leaf margin rift, the leaf margin phenotype of the B genotype plant is or is a candidate for leaf margin entire, and the leaf margin phenotype of the H genotype plant is or is a candidate for leaf margin rift;

[0010] The method for determining the genotype based on Indel LMR is as follows:

[0011] Using the plant's genomic DNA as a template, a specific primer pair was used for PCR amplification; if the PCR amplification showed only one amplification product of 384 bp, the genotype was A; if the PCR amplification showed only one amplification product of 1701 bp, the genotype was B; if the PCR amplification showed two amplification products of 384 bp and 1701 bp, respectively, the genotype was H;

[0012] The specific primer pair consists of IndelLMR-F and IndelLMR-R; IndelLMR-F is a single-stranded DNA molecule shown in SEQ ID NO: 2; IndelLMR-R is a single-stranded DNA molecule shown in SEQ ID NO: 3.

[0013] The present invention also provides a DNA molecule (Indel marker), named Indel LMR, as shown in SEQ ID NO: 1.

[0014] The present invention also protects the use of the Indel LMR as a detection target in identifying or assisting in identifying the leaf margin notch trait of a plant.

[0015] The present invention also provides a specific primer pair consisting of IndelLMR-F and IndelLMR-R; IndelLMR-F is a single-stranded DNA molecule shown in SEQ ID NO: 2; IndelLMR-R is a single-stranded DNA molecule shown in SEQ ID NO: 3.

[0016] The invention also provides the use of the specific primer pair in identifying or assisting in identifying the leaf margin notch trait of a plant.

[0017] The invention also provides a kit for identifying or assisting in identifying the leaf margin notch trait of a plant, comprising the specific primer pair.

[0018] The present invention also protects the use of any of the above methods in plant breeding.

[0019] The present invention also protects the use of the Indel LMR in plant breeding.

[0020] The present invention also protects the use of the specific primer pair in plant breeding.

[0021] The goal of plant breeding is to select plants with a leaf margin phenotype of notched leaf margins.

[0022] The goal of plant breeding is to select plants with a leaf margin phenotype of entire leaf margin.

[0023] Any of the above plants is a Brassica plant.

[0024] Any of the above plants is a plant of the subspecies Brassica rapa or a plant of the subspecies Brassica pekinensis.

[0025] Any of the above plants is a progeny of a plant of the subspecies Brassica rapa and a plant of the subspecies Brassica pekinensis as parents.

[0026] The offspring having the rapa subspecies plant and the rapa subspecies plant as parents may be a hybrid offspring of the rapa subspecies plant and the rapa subspecies plant.

[0027] The offspring having the rapa subspecies plant and the rapa subspecies plant as parents may be self-fertilized offspring of a hybrid of the rapa subspecies plant and the rapa subspecies plant.

[0028] The plant of the subspecies Brassica rapa may specifically be the Brassica rapa inbred line "MM".

[0029] The Chinese cabbage subspecies plant may specifically be the Chinese cabbage inbred line "Bai Yang".

[0030] The leaf margin notch phenotype can only be displayed after the true leaves are formed and unfolded. The traditional breeding method is limited by the development period and is time-consuming and labor-intensive. The present invention develops a molecular marker closely linked to the leaf margin notch. The molecular marker-assisted selection can achieve accurate and stable selection of early seedlings (even seeds), thereby reducing the workload and accelerating the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the electrophoresis diagram of the PCR amplification products of some of the tested plants in Example 2. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0033] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0034] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0035] The turnip inbred line "MM" (i.e., "European turnip line MM" in the literature) is a turnip inbred line with deep leaf margin cleavage from Western Europe. The Chinese cabbage inbred line "Bai Yang" (i.e., "Chinese cabbage line BY" in the literature) is a Chinese cabbage leaf margin full inbred line from Taiwan, China. Both inbred lines are recorded in the following documents: Yu Shuancang, Zhang Fenglan, Zhao Xiang, Yu Yangjun, Zhang Deshuang, Zhao Xiuyun, Wang Weihong. Animproved Brassica rapa genetic linkage map and locus-specific variations in a doubled haploid population. Plant Mol Biol Rep, 2013, 31: 558-568. The public can obtain it from Beijing Academy of Agriculture and Forestry Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0036] Example 1. Acquisition of Indel markers related to leaf margin notching in Brassica rapa and establishment of a detection method

[0037] 1. Acquisition of genetic populations

[0038] The turnip inbred line "MM" was used as the female parent and the cabbage inbred line "Bai Yang" was used as the male parent to obtain F 1 Generation of seeds, cultivation of F 1 The seed generation is F 1 Generation plants. 1 The generation plants were treated with free microspore culture to double the chromosomes, and a haploid doubled population, namely the DH population, was obtained. The DH population contained 80 individual plants.

[0039] The turnip inbred line "MM" was used as the female parent and the cabbage inbred line "Bai Yang" was used as the male parent to obtain F 1 Generation of seeds, cultivation of F 1 The seed generation is F 1 Generation plants. 1 The plants of the first generation were self-pollinated to obtain F 2 Generation of seeds, cultivation of F 2 The seed generation is F 2 Generation plants. 2 Generation F 2 Generation Separating Population, referred to as F 2 Group. 2 The population contained 893 individuals.

[0040] 2. QTL analysis of leaf margin notch

[0041] The following plants were cultured in a greenhouse: plants of the inbred line "MM" of turnip, plants of the inbred line "Bai Yang" of Chinese cabbage, and plants of the F obtained in step 1. 1 The first generation of plants, each individual plant of the DH population obtained in step 1, and the F 2 Individual plants in a population.

[0042] Young leaves of the plants were taken to extract genomic DNA. The SNP and Indel markers that were screened in the parents and were polymorphic, well-typed, and evenly distributed on 10 chromosomes were used to identify the genotypes of individual plants in the population. 2 The leaf margin notch phenotype of individual plants in the group was investigated.

[0043] Combining the genotype and phenotype data of individual plants in the DH population, a genetic linkage map was constructed, and the major QTL controlling leaf margin notch was located on chromosome A10, which could explain 58.3% of the phenotypic variation. The major QTL was named QTL-LobeA10.

[0044] In order to further verify and narrow the target range, F 2 The QTL-LobeA10 was finely mapped by exchanging individuals with extreme phenotypes in the population, and finally QTL-LobeA10 was located in an interval of about 164 kb between markers A10-15686749 and A10-15850960.

[0045] 3. Acquisition of molecular markers and design of primers

[0046] The resequencing information of the parental materials with leaf margin lobed and leaf margin entire in the localized interval was used to align the interval with the reference gene sequence of Brassicaceae Database (http: / / brassicadb.cn). The sequence variations found in the sequence alignment were associated with the leaf margin lobed trait and an Indel was found that was closely linked to the leaf margin lobed trait. The Indel is shown in SEQ ID NO: 1.

[0047] A specific primer pair consisting of IndelLMR-F and IndelLMR-R was designed for the Indel site.

[0048] IndelLMR-F (SEQ ID NO: 2): 5'-ACGGGAAGATCAAGGATG-3';

[0049] IndelLMR-R (SEQ ID NO: 3): 5'-AGGGGAAGCTACGATAACAC-3'.

[0050] 4. Establishment of detection method

[0051] 1. Take young leaves of the plant and extract genomic DNA.

[0052] 2. Using genomic DNA as a template, perform PCR amplification using the specific primer pair designed in step 3.

[0053] 3. Result judgment

[0054] If PCR amplification shows only one amplification product of 384 bp, the genotype of the plant is recorded as genotype A (neither of the two homologous chromosomes contains the Indel), and the leaf margin notch trait of the plant is predicted to be leaf margin notch.

[0055] If PCR amplification shows only one amplification product of 1701 bp, the genotype of the plant is recorded as genotype B (both homologous chromosomes contain the Indel), and the leaf margin puncture trait of the plant is predicted to be entire leaf margin.

[0056] If PCR amplification shows two amplification products of 384 bp and 1701 bp respectively, the genotype of the plant is recorded as H genotype (one homologous chromosome contains the Indel, and the other homologous chromosome does not contain the Indel), and the leaf margin notch trait of the plant is predicted to be leaf margin notch.

[0057] The turnip inbred line "MM" was subjected to the above steps, and PCR amplification showed only one amplification product, as shown in SEQ ID NO: 4.

[0058] The above steps were performed on the Chinese cabbage inbred line "Bai Yang", and PCR amplification showed only one amplification product, as shown in SEQ ID NO:5.

[0059] Example 2: Application of Indel in Identifying Leaf Margin Notch Traits of Brassica Plants

[0060] The turnip inbred line "MM" was used as the female parent and the cabbage inbred line "Bai Yang" was used as the male parent to obtain F 1 Generation of seeds, cultivation of F 1 The seed generation is F 1 The F 1 After self-pollination, the F 2 Generation of seeds, cultivation of F 2 The seed generation is F 2 Generation of plants.

[0061] From F 2 180 plants were randomly selected from the first generation of plants and used as test plants.

[0062] The turnip inbred line "MM" (denoted by P1) and the cabbage inbred line "Bai Yang" (denoted by P2) were used as test plants.

[0063] The genotype detection and phenotype prediction of the test plants were performed according to the method of step 4 of Example 1.

[0064] At the same time, the leaf margin fissure characteristics of the mature test plants were actually observed.

[0065] The electrophoresis of PCR amplification products of some tested plants is shown in Figure 1 .

[0066] The results are shown in Table 1. Among the 139 materials with actual leaf margin puncture phenotypes, 45 were genotype A and 94 were genotype H, with an identification accuracy of 100%. The 41 materials with actual leaf margin entire phenotypes were all genotype B, with an identification accuracy of 100%. The results show that the Indel molecular markers discovered by the present invention can be used to assist in the identification of leaf margin puncture traits.

[0067] The Indel molecular marker discovered in the present invention is named Indel LMR.

[0068] Table 1 Indel LMR in 180 copies of F 2 Genotyping and phenotyping statistics in population materials

[0069]

[0070]

[0071]

[0072] Example 3: Application of Indel markers in molecular marker-assisted selection for identifying the leaf margin notch trait of Brassica plants.

[0073] The plant materials used for the test are as follows:

[0074] Leaf margin split varieties (all of which are turnip germplasm resources disclosed in the prior art): Shenlu Wenzhou Pancai, Dongsheng Xiaoturnip, Huangchang Turnip, Ziyuan Turnip, Huangyuan Turnip, Jinlong Buluke, Lvlingke Turnip No. 1, Wenzhou Pancai, Ziding Turnip, Jinqiu Turnip, Changbai Ginseng Turnip, Yuan Turnip, Xinjiang Qiamagu, Changbai Turnip, Disease-resistant Chang Turnip, a total of 15;

[0075] Varieties with entire leaf margins (all of which are Chinese cabbage germplasm resources disclosed in the prior art): Beijing Chunhuang No. 3, Beijing Xiawang, Dongguan 19, Beijing Orange Heart, Beijing Autumn No. 5, Beijing New No. 3, Beijing Autumn 76, Beijing Jian 70, Beijing Chunhuang, Beijing Chunbai, Beijing Yanhuangye, Beijing Chunlu, Beijing No. 68, Beijing Daniuxin, and Beijing Autumn 701, a total of 15 copies.

[0076] The genotype detection and phenotype prediction of the test plants were performed according to the method of step 4 of Example 1.

[0077] At the same time, the leaf margin notch characteristics of the mature plants of the test plants were actually observed.

[0078] The results are shown in Table 2. Among the 15 materials with leaf margin rifting, 12 were genotype A and 3 were genotype H, with an identification accuracy of 100%. The 15 materials with entire leaf margin phenotype were all genotype B, with an identification accuracy of 100%. The results show that the Indel molecular marker discovered by the present invention can be used for molecular marker-assisted breeding of leaf margin rifting traits.

[0079] Table 2 Statistics of genotyping and phenotype of Indel LMR in 30 natural population materials

[0080] serial number Material Name Phenotype genotype serial number Material Name Phenotype genotype 1 Dongsheng Turnip Crack A 16 Jingchun Yellow No. 3 Full edge B 2 Yellow turnip Crack A 17 King of Jingxia Full edge B 3 Purple Turnip Crack A 18 Winter Crown 19 Full edge B 4 Yellow turnip Crack A 19 Beijing Orange Heart Full edge B 5 Golden Dragon Bruker Crack A 20 Beijing Autumn No. 5 Full edge B 6 Green Collar Kewu No.1 Crack A 21 Beijing New No. 3 Full edge B 7 Wenzhou Plate Crack A 22 Jingqiu 76 Full edge B 8 Purple Top Turnip Crack A 23 Beijing Arrow 70 Full edge B 9 Golden Turnip Crack A 24 Jing Chun Huang Full edge B 10 Shenlu Wenzhou Plate Crack A 25 Beijing Spring White Full edge B 11 Changbai Ginseng Turnip Crack A 26 Jingyan Yellow Leaf Full edge B 12 Turnip Crack A 27 Beijing Spring Green Full edge B 13 Xinjiang Qiamagu Crack H 28 Beijing No. 68 Full edge B 14 Changbai Turnip Crack H 29 Beijing beef heart Full edge B 15 Disease-resistant turnip Crack H 30 Jingqiu 701 Full edge B

[0081] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims. Sequence Listing <110> Beijing Academy of Agricultural and Forestry Sciences <120> Molecular markers related to leaf margin notch traits in Brassica rapa and their applications <130> GNCYX220416 <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1317 <212> DNA <213> Brassica campestris <400> 1 ttagttaatg aacaaaactt aggttcaccc cctagggtga acctttaaat tcacctcacc 60 tctaatagcc aatcataatg ccacatagat taatggataa aaacaattaa atattttttt 120 aaaaatcaaa atagttaaaa ataataataa taacgccaat tttaacgacg ttaaaaacgc 180 caactaaacc ctaaatccaa accgtaacct taaacccgaa actctaaaca ctaaacctaa 240 actctatacc ctaaaaccca aacctaaacc ctaaaccaaa accctatacc ctaaatccaa 300 accgtaacct taaaccctaa actctaaacc ttaaacccaa actctatacc cttaaaccca 360 aacctaaacc ctaaaacccaa atcctatacc ctaaaataa accctgtacg ttaaaaccta 420 aaccctaaac ccaaatacta taccctaaaa cccgaaccta aacccaaacc ctatactcta 480 aatccaaacc ctatacctta aaacctaaac cctaaaccct aaacccaaac tatatacccg 540 aaattcaaac tctagatcct aaattctaaa ccctaaaccc tagaccttaa atcctaaacc 600 caaaccttaa atcctaaatt taaaccataa accataaatc caaaccctat atcctaaact 660 caaatcctag accctaaatc taaattgtaa atcctatacc caaatccaaa actaaaatat 720 ttagattaat gttgatattg ttcttttaga atttaaggtt tggatttaga atctacaatt 780 taagttcatg atttagggta tagaattggg tttagggttt aaggttaaat ttagggtcta 840 atgtttgggt ttaggattta agatctaggg tttatagttt agggtttaga gtttgggttt 900 agggtataga gtttggatct agggtttagg ttttaaggta tagggtttgg atttagagta 960 tagggttcgg gtttagggta tagaatttgg gttttgggtt ttgggtttag ggtttaggtt 1020 ttagggtata tggtttggat ttagggtata ggtttgggtt tagggtttag gtttgggttt 1080 tagggtatag agttcgggtt tagattttagg atttagggtt taggtttgga tttagggttt 1140 agttaaggtt tttaatatcg taaagttgac gttattatta ttatttttta attattttag 1200 tttttaaaaa aattatttaa ttgtttttat ccattaatct atgtggcatt atgattggtt 1260 cttagaggtg aggtgaattt aaaggttcac cctagggggt gaacctaagt tttgttc 1317 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 acgggaagat caaggatg 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 agggggaagct acgataacac 20 <210> 4 <211> 384 <212> DNA <213> Brassica campestris <400> 4 acgggaagat caaggatgtt aaatctcatc aatggagaag aaaaaaaaaa aaaacaattg 60 aagaccacag ttaagattct agggcgaatc gagaaaataa tgcagagaaa atatttaaaa 120 gattttctca ttcttattag ctagtttaat attgaatatg ataattaatt ttatgtttt 180 aaagttttca tggttttttt atatatagca ttttaagctt aaatttgtat tcaattttat 240 atatttagtt aatacaaatt aagagttttg tctatattcc tggttttcag ttagaacatt 300 ggtgttttca acgaattaaa aaaagacatt aattttatat atttttagac taaataagat 360 ttttgtgtta tcgtagcttc ccct 384 <210> 5 <211> 1701 <212> DNA <213> Brassica campestris <400> 5 acgggaagat caaggatgtt aaatctcatc aatggaagag aaagaaaaaa aaaacaattg 60 aagaccacag ttaagattct agggcgaatc gagaaaataa tgcagagaaa atatttaaaa 120 gattttctca ttattattat ctagtttaat attgaatatg ataattaatt ttatgtttt 180 aaagttttca tggtttttt atatatagca ttttagactt aagtttatt tcaattttat 240 atatttagtt aatgaacaaa acttaggttc accccctagg gtgaaccttt aaattcacct 300 cacctctaat agccaatcat aatgccacat agattaatgg ataaaaacaa ttaaatattt 360 ttttaaaaaat caaaatagtt aaaaataata aataacgc caattttaac gacgttaaaa 420 acgccaacta aaccctaaat ccaaaccgta accttaaacc cgaaactcta aacactaaac 480 ctaaactcta taccctaaaa cccaaaccta aaccctaaac caaaacccta taccctaaat 540 ccaaaccgta accttaaacc ctaaactcta aaccttaaac ccaaactcta tacccttaaa 600 cccaaaccta aaccctaaac ccaaatccta taccctaaat ataaaccctg tacgttaaaa 660 cctaaaccct aaacccaaat actataccct aaaacccgaa cctaaaccca aaccctatac 720 tctaaatcca aaccctatac cttaaaacct aaaccctaaa ccctaaaccc aaactatata 780 cccgaaattc aaactctaga tcctaaattc taaaccctaa accctagacc ttaaatccta 840 aacccaaacc ttaaatccta aatttaaacc ataaaccata aatccaaacc ctatatccta 900 aactcaaatc ctagacccta aatctaaatt gtaaatccta tacccaaatc caaaactaaa 960 atatttagat taatgttgat attgttcttt tagaatttaa ggtttggatt tagaatctac 1020 aatttaagtt catgatttag ggtatagaat tgggtttagg gtttaaggtt aaatttaggg 1080 tctaatgttt gggtttagga tttaagatct agggtttata gtttagggtt tagagtttgg 1140 gtttagggta tagagtttgg atctagggtt taggttttaa ggtatagggt ttggatttag 1200 agtatagggt tcgggtttag ggtatagaat ttgggttttg ggttttgggt ttagggttta 1260 ggttttaggg tatatggttt ggatttaggg tataggttttg ggtttagggt ttaggtttgg 1320 gttttagggt atagagttcg ggtttagatt taggatttag ggtttaggtt tggatttagg 1380 gtttagttaa ggtttttaat atcgtaaagt tgacgttatt attattttt tttaattatt 1440 ttagttttta aaaaaattat ttaattgttt ttatccatta atctatgtgg cattatgatt 1500 ggttcttaga ggtgaggtga attaaaggt tcaccctagg gggtgaacct aagttttgtt 1560 cttagttaat acaaatgaag agttttgtct atattcctgg ttttcagtta gaacattggt 1620 gttttcaaag aattaaaaaa agatattaat tttatatatt tttagattaa ataagatttt 1680 tgtgttatcg tagcttcccc t 1701

Claims

1. A method for identifying or assisting in identifying the leaf margin trait of a plant, comprising the following steps: detecting the genotype of the plant based on Indel LMR, the leaf margin phenotype of the A genotype plant is or is a candidate for leaf margin rift, the leaf margin phenotype of the B genotype plant is or is a candidate for leaf margin entire, and the leaf margin phenotype of the H genotype plant is or is a candidate for leaf margin rift; The method for determining the genotype based on Indel LMR is as follows: The genomic DNA of the test plant was used as a template and a specific primer pair was used for PCR amplification; if the PCR amplification showed only one amplification product of 384 bp, the genotype was genotype A; if the PCR amplification showed only one amplification product of 1701 bp, the genotype was genotype B; if the PCR amplification showed two amplification products of 384 bp and 1701 bp respectively, the genotype was genotype H; The specific primer pair consists of IndelLMR-F and IndelLMR-R; IndelLMR-F is a single-stranded DNA molecule shown in SEQ ID NO: 2; IndelLMR-R is a single-stranded DNA molecule shown in SEQ ID NO: 3; The plant is a rapa subspecies plant or a rapa subspecies plant or a progeny of a rapa subspecies plant and a rapa subspecies plant as parents.

2. Application of specific primer pairs in identifying or assisting in identifying the leaf margin notch traits of plants; The specific primer pair consists of IndelLMR-F and IndelLMR-R; IndelLMR-F is a single-stranded DNA molecule shown in SEQ ID NO: 2; IndelLMR-R is a single-stranded DNA molecule shown in SEQ ID NO: 3; The plant is a rapa subspecies plant or a rapa subspecies plant or a progeny of a rapa subspecies plant and a rapa subspecies plant as parents.

3. Application of the method of claim 1 in plant breeding; the goal of the plant breeding is to breed plants with notched leaf margin phenotype or entire leaf margin phenotype; the plants are Brassica rapa subspecies plants or Brassica pekinensis subspecies plants or offspring with Brassica rapa subspecies plants and Brassica pekinensis subspecies plants as parents.

4. Application of specific primer pairs in plant breeding; The specific primer pair consists of IndelLMR-F and IndelLMR-R; IndelLMR-F is a single-stranded DNA molecule shown in SEQ ID NO: 2; IndelLMR-R is a single-stranded DNA molecule shown in SEQ ID NO: 3; The goal of plant breeding is to breed plants with notched leaf margins or entire leaf margins. The plant is a rapa subspecies plant or a rapa subspecies plant or a progeny of a rapa subspecies plant and a rapa subspecies plant as parents.

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