Molecular marker related to root expansion character of brassica plant and application of molecular marker

Abstract

The invention discloses a molecular marker related to a root expansion character of a brassica plant and application of the molecular marker. The invention provides a DNA (Deoxyribose Nucleic Acid) molecule (Inde-BrrRSN1), which is shown as SEQ ID NO: 1. The invention also provides a primer pair which is composed of a primer F as shown in SEQ ID NO: 2 and a primer R as shown in SEQ ID NO: 3. According to the molecular marker and the primer pair provided by the invention, early identification and screening can be carried out on the fleshy root and non-fleshy root characters of the brassica plant through molecular identification before character phenotype observation, so that rapid breeding of the brassica plant is realized, and the molecular marker and the primer pair have application and popularization values.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a molecular marker related to the root swelling trait of Brassica plants and an application thereof. Background Art

[0002] The formation of enlarged organs is a common phenomenon in plant evolution. Among vegetable crops, enlarged fleshy roots are the most common edible organ. Fleshy roots are modified organs, such as roots or hypocotyls, that evolved during the course of plant evolution to adapt to terrestrial environments. These modified fleshy roots provide plants with nutrients and energy, enabling them to survive in harsh environmental conditions. In agricultural production, fleshy roots, as the primary edible organ, directly impact crop yield and quality. Improving the yield and quality of fleshy roots is key to the current development of the cruciferous root vegetable industry.

[0003] Brassica campestris, a Brassica rapa subspecies, includes Chinese cabbage (Ssp. pekinensis), common cabbage (Ssp. chinensis), and turnip (Ssp. rapa). For classification information, see the following document: "A Study on the Classification of Chinese Cabbage," Acta Horticulturae Sinica, May 1980, by Lin Weishen. Turnip, also known as kohlrabi, radish, and turnip, belongs to the same family of cruciferous root vegetables as mustard, radish, and rutabaga, all with fleshy roots as edible organs. Turnip, like Chinese cabbage, a native of my country, belongs to the same Brassica rapa species and is one of the ancestors of Chinese cabbage, possessing enlarged, fleshy roots. Turnip's fleshy roots are rich in nutrients, high in dry matter content, and tender, dense root tissue, making them highly valuable for both food and medicine.

[0004] The formation and development of fleshy rhizomes is a complex process influenced by a variety of factors, including genetics, environmental factors, plant hormones, and carbon metabolites. Currently, research on the molecular mechanisms of plant root development is primarily focused on model plants (Arabidopsis, tobacco, maize, and rice), with related studies also reported in potatoes and sweet potatoes. However, the molecular mechanisms controlling root enlargement in turnip remain unclear. Studies have shown that enlargement traits such as the longitudinal diameter, transverse diameter, and root weight of turnip fleshy roots are all quantitative traits controlled by multiple genes. Therefore, precise mapping of QTLs associated with turnip root enlargement and candidate gene discovery, as well as the development of molecular markers associated with root enlargement, will provide more genetic resources for high-yield breeding of root-enlarged crops and provide a theoretical basis for the targeted improvement of storage organs in vegetable crops through molecular breeding. Summary of the Invention

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

[0006] The present invention provides a DNA molecule (Indel-BrrRSN1), as shown in SEQ ID NO: 1.

[0007] The invention also provides application of the DNA molecule as a molecular marker for the root swelling trait of Brassica rapa plants.

[0008] In the application, the Brassica plant having the DNA molecule is a Brassica plant having a root enlargement trait.

[0009] In the application, the Brassica plant not having the DNA molecule is a Brassica plant not having the root enlargement trait.

[0010] The present invention also provides the use of the DNA molecule as a molecular marker for root traits of Brassica plants; the root traits are fleshy roots or non-fleshy roots.

[0011] In the application, the Brassica plant having the DNA molecule is a plant having fleshy roots.

[0012] In the application, the Brassica plant having the DNA molecule is a plant without fleshy roots.

[0013] The present invention also provides the use of the DNA molecule in the breeding of Brassica plants; the breeding goal is to select plant germplasm with fleshy root traits from Brassica plants.

[0014] In the application, the Brassica plant having the DNA molecule is a plant having fleshy roots.

[0015] In the application, the Brassica plant having the DNA molecule is a plant without fleshy roots.

[0016] The present invention also provides a primer pair consisting of a primer F and a primer R; the primer F is shown in SEQ ID NO: 2, and the primer R is shown in SEQ ID NO: 3. The primer F and the primer R are both single-stranded DNA molecules.

[0017] The present invention also provides the use of the primer pair, which is as follows (a1) or (a2):

[0018] (a1) Use in identifying or assisting in identifying root traits of test plants;

[0019] (a2) Use in breeding or assisting in breeding plants with different root traits from test plants;

[0020] The root trait is fleshy root or non-fleshy root;

[0021] The test plants are Brassica plants.

[0022] The present invention also provides the use of the primer pair in preparing a kit; the function of the kit is as follows (b1) or (b2):

[0023] (b1) Identify or assist in identifying the root characteristics of test plants;

[0024] (b2) breeding or assisting in the breeding of plants with different root traits from the test plants;

[0025] The root trait is fleshy root or non-fleshy root;

[0026] The test plants are Brassica plants.

[0027] The present invention also provides a kit comprising the primer pair;

[0028] The functions of the kit are as follows (b1) or (b2):

[0029] (b1) Identify or assist in identifying the root characteristics of test plants;

[0030] (b2) breeding or assisting in the breeding of plants with different root traits from the test plants;

[0031] The root trait is fleshy root or non-fleshy root;

[0032] The test plants are Brassica plants.

[0033] The present invention also provides a method for screening or assisting in screening plants with different root traits, comprising the following steps:

[0034] Using the genomic DNA of the test plant as a template, PCR amplification was performed using the primer pair;

[0035] If the PCR amplification yields only one amplification product with a size between 1500-2000 bp (specifically, 1800-2000 bp, more specifically, 1909 bp), the genotype of the test plant is genotype A; if the PCR amplification yields only one amplification product with a size between 1000-1500 bp (specifically, 1100-1300 bp, more specifically, 1206 bp), the genotype of the test plant is genotype B; if the PCR amplification yields two amplification products with sizes between 1500-2000 bp (specifically, 1800-2000 bp, more specifically, 1909 bp) and 1000-1500 bp (specifically, 1100-1300 bp, more specifically, 1206 bp), respectively, the genotype of the test plant is genotype H;

[0036] Plants of genotype A are plants with a fleshy root phenotype;

[0037] Plants of genotype H are plants with a fleshy root phenotype;

[0038] Plants of genotype B are plants that do not have a fleshy root phenotype;

[0039] The test plants are Brassica plants.

[0040] The present invention also provides a method for identifying or assisting in identifying plant root traits, comprising the following steps:

[0041] Using the genomic DNA of the test plant as a template, PCR amplification was performed using the primer pair;

[0042] If the PCR amplification yields only one amplification product with a size between 1500-2000 bp (specifically, 1800-2000 bp, more specifically, 1909 bp), the genotype of the test plant is genotype A; if the PCR amplification yields only one amplification product with a size between 1000-1500 bp (specifically, 1100-1300 bp, more specifically, 1206 bp), the genotype of the test plant is genotype B; if the PCR amplification yields two amplification products with sizes between 1500-2000 bp (specifically, 1800-2000 bp, more specifically, 1909 bp) and 1000-1500 bp (specifically, 1100-1300 bp, more specifically, 1206 bp), respectively, the genotype of the test plant is genotype H;

[0043] The root characteristics of plants with genotype A are fleshy roots;

[0044] The root traits of plants with genotype H are fleshy roots;

[0045] The root characteristics of plants with genotype B are non-fleshy roots;

[0046] The test plants are Brassica plants.

[0047] The test plants are offspring plants with fleshy root plants and non-fleshy root plants as parents.

[0048] The test plant is a fleshy root plant.

[0049] The test plants are non-fleshy root plants.

[0050] As an example, the fleshy root plant is the turnip inbred line "MM".

[0051] As an example, the non-fleshy root plant is the cabbage inbred line "Bai Yang".

[0052] As an example, the fleshy root plant is Shenlu Wenzhou pancai, Dongsheng small turnip, yellow long turnip, purple round turnip, yellow round turnip, Jinlong Buluke, Green Collar Kewu No. 1, Wenzhou pancai, purple top turnip, golden ball turnip, Changbai ginseng turnip, round turnip, Xinjiang Chamagu, Changbai turnip or disease-resistant long turnip.

[0053] As an example, the non-succulent root plant is Beijing Chunhuang No. 3, Beijing Xiawang, Dongguan 19, Beijing Orange Heart, Beijing Autumn No. 5, Beijing New No. 3, Beijing Autumn 76, Beijing Arrow 70, Beijing Chunhuang, Beijing Chunbai, Beijing Yanhuangye, Beijing Chunlv, Beijing No. 68, Beijing Daniuxin or Beijing Autumn 701.

[0054] The molecular markers and primer pairs provided by the present invention can be used for early identification and screening of the fleshy and non-fleshy root traits of Brassica plants through molecular identification before the trait phenotype can be observed, thereby realizing the rapid breeding of Brassica plants and having application and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Shown are the root phenotypes of the parents and representative F1 plants (photographs of plants 80 days after sowing).

[0056] Figure 2 This is the positioning result diagram based on BSA-seq sequencing.

[0057] Figure 3 The electrophoresis diagram of PCR amplification products of some plants. DETAILED DESCRIPTION

[0058] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0059] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples were all repeated three times, and the results were averaged. Unless otherwise specified, the field crops in the examples are farmlands located in the suburbs of Beijing. The sowing time of the plants was the beginning of autumn, and differences in root properties could be observed 35 days after sowing.

[0060] The turnip inbred line "MM" (i.e., "European turnip line MM" in the literature) and the Chinese cabbage inbred line "Bai Yang" are both recorded in the following literature (i.e., "Chinese cabbage line BY" in the literature): 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.

[0061] Example 1. Acquisition of molecular markers

[0062] 1. Acquisition of genetic populations

[0063] A hybridization was conducted using the turnip inbred line "MM" as the female parent and the cabbage inbred line "Bai Yang" as the male parent to obtain F1 generation seeds, which were then grown into plants. The F1 generation plants were then self-pollinated to obtain F2 generation seeds, which were then grown into plants, creating the F2 generation plants. The F2 generation plants formed the F2 generation segregating population, referred to as the F2 population. The F2 population consisted of 830 individual plants.

[0064] The roots of the turnip inbred line "MM" are enlarged fleshy roots. Figure 1 The roots of the Chinese cabbage inbred line "Bai Yang" are ordinary roots that do not swell. Figure 1 The root phenotype of the F1 generation plants is between the two parents (see Figure 1 (middle picture).

[0065] 2. Preliminary positioning of genes related to root enlargement

[0066] Plants of the turnip inbred line "MM", plants of the cabbage inbred line "Bai Yang", the F1 generation plants obtained in step 1, and individual plants of the F2 population obtained in step 1 were planted in the field.

[0067] During the mature stage of fleshy root development, 30 plants with fleshy roots and 30 plants with ordinary roots were selected from the F2 population to construct a large root pool (L-pool) and a small root pool (S-pool). DNA from parent MM, parent Baiyang, and two mixed pools was extracted using the CTAB method and resequenced. Based on the results of BSA-seq sequencing analysis, the interval controlling the root expansion trait was located within the 2699001bp-17149000 interval of chromosome A01, with an interval size of 14.45MB. The positioning results based on BSA-seq sequencing are shown in the figure. Figure 2 .

[0068] 3. Fine Mapping of Root Enlargement-Related Genes

[0069] To further validate and narrow down the candidate intervals associated with root enlargement, fine mapping was performed using an expanded F2 population. Based on the results of parental resequencing, SNPs within the candidate interval were screened and KASP molecular markers were developed. These KASP markers were then used to genotype individual F2 plants and select crossover plants. Combining genotypic and phenotypic data from the extreme crossover plants, the gene regulating root enlargement was ultimately localized to a region of approximately 227 kb between markers A01-4680161 and A01-4906217 on chromosome A01.

[0070] 4. Acquisition of molecular markers and design of primers

[0071] Based on the reference genome sequence information, the resequencing data of the parents MM and Baiyang were analyzed, and the sequence variation sites of the two parents within the positioning interval were screened. The screened sequence variations were associated with the root expansion phenotype, and an indel was found that was closely linked to the root expansion trait. The nucleotide sequence of this indel is shown in SEQ ID NO: 1.

[0072] A specific primer pair was designed based on this Indel, consisting of IndelBrrRSN1-F and IndelBrrRSN1-R.

[0073] IndelBrrRSN1-F (SEQ ID NO: 2): 5'-GGAGATGCCCTAACCATA-3';

[0074] IndelBrrRSN1-R (SEQ ID NO: 3): 5'-TGGCAGCATGAGTAAGACAAA-3'.

[0075] Example 2: Application of Indel Markers in Identifying Root Expansion Traits in Brassica Plants

[0076] 198 individual plants were randomly selected from the F2 population obtained in Example 1 and used as test plants. Plants of the turnip inbred line "MM" (denoted by P1) and plants of the Chinese cabbage inbred line "Bai Yang" (denoted by P2) were used as control plants for the test plants. The test plants and control plants were cultured in parallel in the field for 56 days (counting days from sowing). Root traits of the test plants and control plants were then tested and genotyped.

[0077] The method for genotyping is as follows:

[0078] 1. Take young leaves of the test plants and extract genomic DNA.

[0079] 2. Using the genomic DNA obtained in step 1 as a template, PCR amplification was performed using the primer pair consisting of IndelBrrRSN1-F and IndelBrrRSN1-R.

[0080] 3. Take the PCR amplification product obtained in step 2 and perform agarose gel electrophoresis.

[0081] An exemplary electrophoresis diagram of some plants is shown in Figure 3 (The lanes other than P1 and P2 represent different individual test plants.) Electrophoresis of the amplified product of the Brassica rapa inbred line "MM" showed only one band, sized between the 1500 bp marker band and the 2000 bp marker band. Sequencing revealed that the size of this band was 1909 bp, as shown in SEQ ID NO: 4. Electrophoresis of the amplified product of the Brassica rapa inbred line "Bai Yang" showed only one band, sized between the 1000 bp marker band and the 1500 bp marker band. Sequencing revealed that the size of this band was 1206 bp, as shown in SEQ ID NO: 5. If the electrophoretic band pattern of the amplified product of the test plant was consistent with that of the amplified product of the Brassica rapa inbred line "MM" (i.e., it showed only one band, sized between the 1500 bp marker band and the 2000 bp marker band), the genotype of the plant was defined as genotype A. If the electrophoretic pattern of the amplified product of the test plant is consistent with that of the amplified product of the Chinese cabbage inbred line "Bai Yang" (i.e., only one band is shown and its size is located between the 1000 bp marker band and the 1500 bp marker band), the genotype of the plant is defined as genotype B. If the electrophoretic pattern of the amplified product of the test plant shows two bands, and their sizes are located between the 1500 bp marker band and the 2000 bp marker band and between the 1000 bp marker band and the 1500 bp marker band, respectively, the genotype of the plant is defined as genotype H.

[0082] The root trait and genotype results for each plant are shown in Table 1. Of the 198 individual plants in the F2 population, 55 were of genotype A and 98 were of genotype H, all exhibiting fleshy root phenotypes; 45 were of genotype B, exhibiting non-fleshy root phenotypes (normal roots without enlargement). The identification accuracy was 100%. The results demonstrate that the indel marker is tightly linked to the root enlargement trait and that the indel marker discovered in this invention can be used to assist in the identification of root enlargement.

[0083] The Indel marker discovered in the present invention was named Indel-BrrRSN1.

[0084] Table 1 Genotyping and phenotype of Indel markers in 198 F2 population materials

[0085]

[0086]

[0087]

[0088] Example 3: Application of Indel Markers in Assisted Identification of Root Expansion Traits in Brassica Plants

[0089] The plant materials used for the test are as follows:

[0090] Fleshy root varieties (all of which are turnip germplasm resources disclosed in the prior art): Shenlu Wenzhou Pancai, Dongsheng Small Turnip, Huangchang Turnip, Ziyuan Turnip, Huangyuan Turnip, Jinlong Bliuke, Lvling Kewu No. 1, Wenzhou Pancai, Ziding Turnip, Jinqiu Turnip, Changbai Ginseng Turnip, Yuan Turnip, Xinjiang Qiamagu, Changbai Turnip, and Disease-resistant Chang Turnip, a total of 15 accessions;

[0091] Non-fleshy root varieties (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, Jingyan Yellow Leaf, Beijing Chunlv, Beijing No. 68, Beijing Daniuxin, and Beijing Autumn 701, a total of 15 samples.

[0092] The turnip inbred line "MM" plants and the cabbage inbred line "Bai Yang" plants were used as control plants for the test plants.

[0093] The test plants and control plants were cultured in parallel in the field for 80-90 days (counting the days from sowing the plants). The root traits of the test plants and control plants were then tested and their genotypes were identified.

[0094] The method for genotype identification is the same as in Example 2.

[0095] The results are shown in Table 2. Of the 15 germplasms with a fleshy root phenotype, 14 were genotype A and 1 was genotype H, with an identification accuracy of 100%. All 15 germplasms with a non-fleshy root phenotype (normal roots without swelling) were genotype B, with an identification accuracy of 100%. These results demonstrate that the indel molecular markers discovered in this invention can be used for marker-assisted breeding of root swelling traits.

[0096] Table 2 Genotyping and phenotype of Indel markers in 30 natural population materials

[0097]

[0098] 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 practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A DNA molecule as shown in SEQ ID NO:

1.

2. Use of the DNA molecule according to claim 1 as a molecular marker for the root enlargement trait of Brassica rapa plants.

3. Use of the DNA molecule according to claim 1 as a molecular marker for root traits of Brassica plants; the root trait is fleshy root or non-fleshy root.

4. Use of the DNA molecule of claim 1 in Brassica plant breeding; the breeding goal is to select plant germplasm with fleshy root traits from Brassica plants.

5. A primer pair, consisting of primer F and primer R; the primer F is shown in SEQ ID NO: 2, and the primer R is shown in SEQ ID NO:

3.

6. The use of the primer pair according to claim 5, wherein: (a1) Use in identifying or assisting in identifying root traits of test plants; (a2) Use in breeding or assisting in breeding plants with different root traits from test plants; The root trait is fleshy root or non-fleshy root; The test plants are Brassica plants.

7. Use of the primer pair according to claim 5 in preparing a kit; the function of the kit is as follows (b1) or (b2): (b1) Identify or assist in identifying the root characteristics of test plants; (b2) breeding or assisting in the breeding of plants with different root traits from the test plants; The root trait is fleshy root or non-fleshy root; The test plants are Brassica plants.

8. A kit comprising the primer pair according to claim 5; The functions of the kit are as follows (b1) or (b2): (b1) Identify or assist in identifying the root characteristics of test plants; (b2) breeding or assisting in the breeding of plants with different root traits from the test plants; The root trait is fleshy root or non-fleshy root; The test plants are Brassica plants.

9. A method for screening or assisting in screening plants having different root traits, comprising the following steps: Using the genomic DNA of the test plant as a template, PCR amplification is performed using the primer pair described in claim 5; If the PCR amplification yields only one amplification product with a size between 1500-2000 bp, the genotype of the test plant is genotype A; if the PCR amplification yields only one amplification product with a size between 1000-1500 bp, the genotype of the test plant is genotype B; if the PCR amplification yields two amplification products with sizes between 1500-2000 bp and 1000-1500 bp, respectively, the genotype of the test plant is genotype H; Plants of genotype A are plants with a fleshy root phenotype; Plants of genotype H are plants with a fleshy root phenotype; Plants of genotype B are plants that do not have a fleshy root phenotype; The test plants are Brassica plants.

10. A method for identifying or assisting in identifying plant root traits, comprising the following steps: Using the genomic DNA of the test plant as a template, PCR amplification is performed using the primer pair described in claim 5; If the PCR amplification yields only one amplification product with a size between 1500-2000 bp, the genotype of the test plant is genotype A; if the PCR amplification yields only one amplification product with a size between 1000-1500 bp, the genotype of the test plant is genotype B; if the PCR amplification yields two amplification products with sizes between 1500-2000 bp and 1000-1500 bp, respectively, the genotype of the test plant is genotype H; The root characteristics of plants with genotype A are fleshy roots; The root traits of plants with genotype H are fleshy roots; The root characteristics of plants with genotype B are non-fleshy roots; The test plants are Brassica plants.

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