CN111850157A - Molecular marker related to Chinese cabbage flower color and application thereof - Google Patents
Molecular marker related to Chinese cabbage flower color and application thereof Download PDFInfo
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Abstract
The invention relates to a molecular marker related to Chinese cabbage flower color and application thereof, and discloses an SNP locus remarkably related to a Chinese cabbage reddish orange flower gene Br-or and a KASP molecular marker for detecting the gene, wherein the SNP locus Br-or-A09-37676461(T/C) remarkably related to the reddish orange flower gene Br-or is detected by a BSA-seq (bulked segregant analysis sequencing) method, and the locus is positioned in a Br-or promoter region and an A09 chromosome 37,676,461 position. A KASP marker Br-or-KASP1 is developed aiming at the SNP locus, and can be used for auxiliary selection of a Chinese cabbage orange flower gene Br-or. The invention also discloses a primer sequence corresponding to the molecular marker and a flow for detecting the marker by using the KASP technology. The molecular marker can be applied to molecular marker assisted breeding of the red-orange flower and red-orange leaf ball centers of Chinese cabbages with high efficiency and low cost, and the disease-resistant breeding efficiency is greatly improved.
Description
Technical Field
The invention relates to the technical field of crop molecular marker assisted breeding, in particular to a high-flux detection marker for a Chinese cabbage orange flower gene Br-or and application thereof in breeding.
Background
Flower color is one of the phenotypic characters of plants adapting to the environment. The flower color generally refers to the color of petals, can be used as an important visual signal for attracting insects to pollinate by insect-borne plants, and has important significance for the survival and the propagation of the plants. The flower color can influence the aphid to feed, the breeding of different flower colors in production has the function of reducing aphid diseases, and the energy balance of the petal can be maintained, so that the damage to the flower organ is avoided. The flower color has certain influence on the aspects of plant variety breeding and growth and development, and also has important functions in the aspects of improving seed purity, removing hybrid plants, identifying natural outcrossing rate, detecting interspecific character transfer and the like. For ornamental plants, flower color not only determines the ornamental value of the plant, but also directly affects the commercial value of the plant.
Chinese cabbage (Brassica rapa L. ssp pekinensis) is one of the vegetable crops with the largest cultivation area in China, and plays an important role in the vegetable crops in China, and the flower color of the Chinese cabbage mainly comprises yellow flower, reddish orange flower and white flower. The color of Chinese cabbage can be used as the marker character of its breeding to eliminate unnecessary material in early stage, so as to speed up the breeding and breeding progress. The Chinese cabbage orange flower character is a quality character controlled by 1 pair of recessive genes, and the orange flower and the ball heart color character are completely linked, namely the ball color of the orange flower is also orange, and the orange heart material can be screened according to the flower color in the transfer process (Zhang De Shuang et al, 2003, research on genetic rules of Chinese cabbage flower color and ball color, North China agricultural science, 18 (2): 81-84.). The molecular marker linked with the reddish orange flower is developed, not only can be used for molecular marker assisted breeding of the Chinese cabbage reddish orange flower, but also can be used for molecular breeding of the Chinese cabbage reddish orange heart, and has important application value.
Previous studies show that the candidate gene of the Chinese cabbage orange safflower gene Br-or is BrCRTISO (Bra031539) for encoding carotenoid isomerase, and different types of molecular markers are developed. Feng et al (2012) obtained 5 SSR markers linked to Br-or, and the closely linked markers syau19 and syau15 flanking the Br-or gene covered a region of 4.6cM (Fengt al.,2012, Mapping of, gene comparative color on the inner leaf of Chinese chip capsule (Brassica rapa L. sp. pependensis), Molecular Breeding,29: 235-. Li pei Rong et al (2014) designed an InDel marker based on polyacrylamide gel electrophoresis detection for 6 base deletion variation on the first exon of candidate gene Bra031539 (Li pei Rong et al, 2014, Chinese cabbage orange red core carotenoid component and its gene analysis, gardening bulletin, 41 (3): 469-478). Zhang et al (2015) designed an InDel marker for 88bp variation of the promoter region of candidate gene Bra031539 (Zhang et al, 2015, molecular characterization and transfer analysis of organ head Chinese cassette (Brassica rapa L. ssp. pekinensis), Planta,241: 1381-1394). Li et al (2017) designed an InDel marker (Li et al, 2017, transcription analysis of organ head Chinese cassette (Brassica rapa L. ssp. pekinensis) and molecular marker evaluation, International Journal of Genomics,6835810:1-8.) for variation of the first intron of candidate gene Bra 031539. Lee et al (2014) designed InDel markers for variation of the first and second exons of Bra031539 (Lee et al, 2014, Association of molecular markers ordered from the BrCRTISO1 gene with prolycopen-engineered forms-vitamins in Brassica rapa, the ecological applied Genetics,127: 179-191).
Although the predecessors developed different molecular markers aiming at the variation of different positions of the orange flower gene Br-or, the developed markers are mostly based on gel electrophoresis, the time and labor consumption are high, the cost is high, and the developed markers have no universality.
Disclosure of Invention
The invention aims to solve the technical problem of identifying or assisting in identifying the flower color of the Chinese cabbage and lays a foundation for molecular marker screening and polymerization breeding of Chinese cabbage breeding with orange flower color.
In order to solve the technical problems, the invention provides a method for identifying or assisting in identifying the flower color of Chinese cabbage, which comprises the steps of detecting the genotype of the Chinese cabbage to be detected, and identifying or assisting in identifying the flower color of the Chinese cabbage according to the genotype of the Chinese cabbage to be detected; the genotype is the genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage; the Br-or-A09-37676461 site is an SNP site in a Chinese cabbage genome, the nucleotide type is C or T, and the nucleotide is the 101 th nucleotide of SEQ ID No.4 in a sequence table.
Wherein when the genotype of the Br-or-A09-37676461 locus is a CC genotype, the color of the Chinese cabbage is orange or the candidate is orange; when the genotype of the Br-or-A09-37676461 locus is a CT genotype or a TT genotype, the color of the Chinese cabbage is yellow or the candidate is yellow; wherein the CC genotype represents the homozygosity of the nucleotide type of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage, namely C; the TT genotype represents the homozygosity of the nucleotide type of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage, namely T; the CT genotype indicates that the nucleotide type of the Br-or-A09-37676461 site in the genome of the Chinese cabbage is a hybrid type of C and T.
The invention provides any one of the following applications A1-A5,
A1. the application of the substance for detecting the polymorphism or genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage in identifying or assisting in identifying the flower color of the Chinese cabbage; the Br-or-A09-37676461 site is an SNP site in a Chinese cabbage genome, the nucleotide type is C or T, and the nucleotide is the 101 th nucleotide of SEQ ID No.4 in a sequence table.
A2. The application of the substance for detecting the polymorphism or genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage in the preparation of products for identifying or assisting in identifying the flower color of the Chinese cabbage; the Br-or-A09-37676461 site is an SNP site in a Chinese cabbage genome, the nucleotide type is C or T, and the nucleotide is the 101 th nucleotide of SEQ ID No.4 in a sequence table.
A3, application of a substance for detecting polymorphism or genotype of Br-or-A09-37676461 sites in a Chinese cabbage genome in identifying or assisting in identifying the color of the leaf core of the Chinese cabbage; the Br-or-A09-37676461 site is an SNP site in a Chinese cabbage genome, the nucleotide type is C or T, and the nucleotide is the 101 th nucleotide of SEQ ID No.4 in a sequence table.
A4, application of a substance for detecting polymorphism or genotype of Br-or-A09-37676461 sites in a Chinese cabbage genome in preparation of a product for identifying or assisting in identifying the color of Chinese cabbage leaf cores; the Br-or-A09-37676461 site is an SNP site in a Chinese cabbage genome, the nucleotide type is C or T, and the nucleotide is the 101 th nucleotide of SEQ ID No.4 in a sequence table.
A5. The application of the substance for detecting the polymorphism or genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage in breeding the Chinese cabbage or preparing breeding products of the Chinese cabbage; the Br-or-A09-37676461 site is an SNP site in a Chinese cabbage genome, the nucleotide type is C or T, and the nucleotide is the 101 th nucleotide of SEQ ID No.4 in a sequence table.
The invention also provides a product containing a substance for detecting the polymorphism or genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage, which is any one of C1) -C5):
C1) detecting the product of single nucleotide polymorphism or genotype related to the Chinese cabbage flower color;
C2) identifying or assisting in identifying the products of the Chinese cabbage flower color;
C3) detecting the product of single nucleotide polymorphism or genotype related to the color of the cabbage leaf core;
C4) identifying or assisting in identifying the color of the cabbage leaf core;
C5) is used for breeding Chinese cabbage.
In the application, the method and the product, the Chinese cabbage breeding is to breed the Chinese cabbage with the color of orange red or breed the Chinese cabbage with the color of orange red. The breeding of the Chinese cabbage is to cultivate the Chinese cabbage with the leaf ball heart color of orange or breed the Chinese cabbage with the leaf ball heart color of orange.
In the application, the method and the product, the substances for detecting the polymorphism or genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage are D1), D2) or D3):
D1) the substance for detecting the polymorphism or genotype of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage contains a PCR primer for amplifying the DNA fragment of the genome of the Chinese cabbage including the Br-or-A09-37676461 locus;
D2) the substance for detecting the polymorphism or genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage is a PCR reagent containing the PCR primer;
D3) a kit containing the PCR primer described in D1) or the PCR reagent described in D2).
In the above applications, methods and products, the PCR primer is P1 or P2:
p1, the PCR primer is a primer group consisting of single-stranded DNA with the nucleotide sequence of 22 th to 39 th positions of SEQ ID No.1 in the sequence table, single-stranded DNA with the nucleotide sequence of 22 nd to 39 th positions of SEQ ID No.2 in the sequence table and single-stranded DNA with the nucleotide sequence of SEQ ID No.3 in the sequence table;
p2 and the PCR primer are primer groups of single-stranded DNA shown by SEQ ID No.1 in a sequence table, single-stranded DNA shown by SEQ ID No.2 in the sequence table and single-stranded DNA shown by SEQ ID No.3 in the sequence table.
The Chinese cabbage to be tested is the filial generation group of Chinese cabbage Y959M-5 and Chinese cabbage R16-11, such as red tangerine color Chinese cabbage Y959M-5 (P)1) As female parent, yellow flower color Chinese cabbage R16-11 (P)2) As male parent, hybridizing the two materials to obtain F1Generation of seed, F1Selfing the material to obtain F2Seed generation, selecting F2The plant obtained by seed generation culture is Y959RF2And (4) a group.
The SNP locus Br-or-A09-37676461 which is obviously related to the reddish orange flower gene Br-or is detected by the BSA-seq technology, and the KASP marker Br-or-KASP1 is developed, can detect the genotype of the Br-or at high flux, can be applied to molecular marker assisted breeding of the reddish orange flower and the reddish orange leaf core of Chinese cabbage with high efficiency and low cost, and has very important significance.
Drawings
FIG. 1 shows the result of BSA-Seq localization of gene Br-or of a red-orange flower;
FIG. 2 shows KASP genotyping of 4 red-orange flowers and 4 yellow flower material using the marker Br-or-KASP 1.
FIG. 3 shows KASP genotyping of 40 red-orange and 40 yellow-flower DH lines using the marker Br-or-KASP 1.
FIG. 4 shows the pair of Y959RF using the marker Br-or-KASP12The population was genotyped for KASP.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Experimental Material
The biological material was obtained from the applicant by the following examples of Chinese cabbage R16-11 (the parent of the Populus, et al, 2020, KASP marker development of the Gene BrFLC1 related to bolting of Chinese cabbage, journal of the Nuclear agriculture, 34(2): 0265-.
The cabbage Y510-9 (optimization and establishment of the cabbage KASP reaction system, 2018, journal of horticulture) in the following examples was publicly available from the applicant, and the biomaterial was used only for repeating the experiments related to the present invention, and was not used for other purposes.
The names of the materials referred to in the examples below and the corresponding names of the commercial products and petal colors are shown in the following table.
TABLE 1.98 Chinese cabbage Material names and corresponding commercial product names
Example 1 discovery of SNP site related to Chinese cabbage flower color
1. Location of orange flower gene Br-or
1.1 investigation of test materials and Properties
54 parts of orange flower material and 44 parts of yellow flower material were selected, and the color of petals of each material was investigated during the full bloom stage.
1.2DNA extraction
Genomic DNAs of 54 parts of orange flower material and 44 parts of yellow flower material in step 1.1 were extracted by modified CTAB method, respectively. The method comprises the following specific steps: fresh leaves were taken in a 2.0mL Eppendorf centrifuge tube, 1 bead (5 MM in diameter) was placed in each tube, 1000. mu.L of 2% CTAB extraction buffer was added, and the mixture was disrupted on a tissue disruptor (model: Retsch MM400, Germany) for 1min at a frequency of 30 cycles/sec. Bathing at 65 ℃ for 1h, cooling, adding 500 mu L of 24:1 chloroform/isoamylol extraction liquid, shaking up and down for 30 times, standing for layering, centrifuging (12000r/min) for 10min, sucking 400 mu L of supernatant into a new 1.5mL centrifuge tube, adding 400uL of isopropanol to precipitate DNA, mixing up and down, centrifuging (12000r/min) for 5min, removing supernatant, adding 750 mu L of 70% ethanol to clean DNA precipitate, centrifuging (12000r/min) for 2min, removing supernatant, drying DNA at room temperature, adding 100 mu L of ddH2O to dissolve DNA, and placing in a refrigerator at-20 ℃ for later use.
1.3 location of Gene Br-or in Citrus grandis
And (3) respectively and equivalently mixing the genomic DNAs of 54 parts of red tangerine flower flowers and 44 parts of yellow flower materials extracted in the step 1.2, constructing two extreme gene mixing pools of red tangerine flower and yellow flower, and performing whole genome re-sequencing on the two mixing pools by using a BSA-Seq technology. The genomic sequence (V1.5) of Chinese cabbage Chiifu-401-42 was used as a reference sequence, clean reads from the two pools were aligned to the reference genome by BWA (v0.7.17) software, and SNP calling was performed using Samtools (V1.9) and Bcftools (V1.9) to obtain SNP sites of each sample. Calculating SNP-index of the citrus red flower mixing pool and the yellow flower mixing pool respectively, subtracting the SNP-index of the yellow flower mixing pool from the SNP-index of the citrus red flower mixing pool to obtain delta SNP-index between the two mixing pools, and performing sliding window analysis by taking 1Mb as a window and 50kb as a step length to determine a candidate interval of the citrus red flower gene. The gene of the red-orange flower is located in the 36.75-38.25Mb interval of A09 chromosome (figure 1) by significance analysis, and the length of the interval is 1.5 Mb. The interval comprises a candidate gene BrCRTISO (Bra031539) (37,676,627-37,679,733bp) of the orange heart gene Br-or reported previously. The character concerned by previous researches is the color character of the ball center of a leaf ball of Chinese cabbage, and the used research groups are parental genetic groups, and the reddish orange flower and the color character of the ball center are completely linked. The character concerned by the invention is the color character of the Chinese cabbage petals, the used research population is a natural population, the BSA-Seq technology is utilized to quickly detect the major gene at the end of the A09 chromosome, and the major gene contains the orange heart gene Br-or researched by the previous people, so that the orange flower color gene and the orange heart gene are considered to be the same gene, and the candidate gene is BrCRTISO (Bra 031539).
2. KASP molecular marker of orange flower gene Br-or
2.1 analyzing the coding region (37,676,627-37,679,733bp) of the candidate genes Bra031539 and the SNP variation of the promoter region and the delta SNP-index between the two mixing pools of the citrus safflower mixing pool and the citron day mixing pool, finding that T/C SNP variation exists at the 37,676,461bp position of the promoter region, corresponding to the 101 th nucleotide in the sequence 1, the 101 th nucleotide can be T or C represented by Y, and naming the SNP site as Br-or-A09-37676461. The delta SNP-index value of the SNP site Br-or-A09-3767646 between the two mixing pools is 0.71, and is obviously related to the red orange flower character.
2.2 designing KASP marker Br-or-KASP1 aiming at SNP site Br-or-A09-37676461, comprising three primers: Br-or-KASP1 Fa: 5' -GAAGGTCGGAGTCAACGGATTGTAATATCCTGCACTTCT-3'；Br-or-KASP1Fb：5'-GAAGGTGACCAAGTTCATGCTGTAATATCCTGCACTTCC-3'；Br-or-KASP 1R: 5'-CAAAAAAATGTTTGGTGAAGAAC-3' are provided. Br-or-KASP1Fa and Br-or-KASP1Fb are two allele-specific forward primers, Br-or-KASP1Fa is a specific primer of a yellow flower genotype, Br-or-KASP1Fb is a specific primer of an orange flower genotype, and HEX and FAM fluorescent sequence tag sequences (underlined parts) are respectively added to the 5' end. Br-or-KASP1R is a common reverse primer.
2.3 the KASP marker Br-or-KASP1 was verified by selecting 4 parts of orange flower material Y959M-5, Y698-17, Y662-1, Y947M-13 and 4 parts of yellow flower material Yw81, Y177-12, Y510-9, R16-11. All 8 materials were DH (double Haploid) pure line materials obtained by free microspore culture.
Genomic DNAs of 4 parts of orange flower material (Y959M-5, Y698-17, Y662-1 and Y947M-13) and 4 parts of yellow flower material (Yw81, Y177-12, Y510-9 and R16-11) were extracted by the modified CTAB method, respectively. PCR amplification was carried out using 4 parts of genomic DNA of orange flower material (Y959M-5, Y698-17, Y662-1 and Y947M-13) and 4 parts of yellow flower material (Yw81, Y177-12, Y510-9 and R16-11), respectively, as templates and KASP marker Br-or-KASP1 in step 2.2 as primers under the following amplification conditions:
the KASP-PCR reaction was performed on a 96-well PCR instrument (eppendorf mastercyclerpro), with 8 μ L: mu.L of DNA (60 ng. mu.L-1), 4. mu.L of KASP Master mix (2X), 0.14. mu.L of primer mix consisting of CRs-KASP1Fa, CRs-KASP1Fb, CRs-KASP1R and ddH at a concentration of 100. mu. mol. L-12O is mixed according to the volume ratio of 12:12:30: 46), and the rest is ddH2And (4) supplementing and finishing.
The KASP-PCR amplification program was: first stage denaturation at 94 deg.C for 15 min; the second stage is denaturation at 94 ℃ for 20s and annealing at 61 ℃ for 60s for 10 cycles (from the second cycle, each cycle is reduced by 0.6 ℃); in the third stage, denaturation at 94 ℃ is carried out for 20s, annealing at 55 ℃ is carried out for 60s, and 26 cycles are carried out; fourth stage 37 deg.C for 1 min.
2.4 reading the KASP-PCR amplification product of step 2.3 by end-point method fluorescence signal using Roche fluorescent quantitative PCR Instrument LightCycler480Instrument II (LC480 II). The SNP typing results were analyzed using LC480 software v1.5.1: the genotype which is polymerized near the X axis and shows blue color is an allelic genotype connected with a FAM fluorescent label sequence, namely the homozygosity of a SNP site Br-or-A09-3767646 nucleotide type in the genome of the Chinese cabbage is C, and the genotype is named as genotype CC; the polymerized genotype which shows green near the Y axis is an allelic genotype connected with a HEX fluorescent label sequence, namely the SNP site Br-or-A09-3767646 nucleotide species in the genome of the Chinese cabbage is homozygote of T, and is named as genotype TT; the genotype with red color in the middle is a heterozygote of two alleles, namely the nucleotide type of one SNP site Br-or-A09-3767646 in the genome of the Chinese cabbage is a heterozygote of C and T, and the genotype is named as genotype CT.
Carrying out genotype identification on 4 parts of orange flower materials Y959M-5, Y698-17, Y662-1 and Y947M-13 and 4 parts of yellow flower materials Yw81, Y177-12, Y510-9 and R16-11 by using a marker Br-or-KASP1, wherein the results are shown in figure 2 and table 2, signal points of the 4 orange flower materials are blue, primers of which the 5' tail ends are connected with FAM fluorescent tag sequences are subjected to competitive amplification and polymerized near an X axis, and the Chinese cabbage with the CC genotype of the SNP site Br-or-A09-3767646 in the Chinese cabbage genome is obtained; the signal points of the 4 yellow flower materials are green, the 5' end of the yellow flower materials is connected with primer competitive amplification of a HEX fluorescent label sequence, and the signals are polymerized near the Y axis, namely the celery cabbage with the SNP site Br-or-A09-3767646 genotype TT in the genome of the celery cabbage (as shown in figure 2). The Br-or-KASP1 marker can obviously distinguish two homozygous genotypes, and the marker is successfully developed.
The relationship between the flower color and gene of the Chinese cabbage of Table 28 varieties
Example 2 KASP-tagged Br-or-KASP1 application 1
Selecting 40 parts of red tangerine flower and 40 parts of yellow flower Chinese cabbage material, and obtaining DH (double Haploid) pure line material by culturing free microspores.
Extracting genome DNA of 40 parts of red tangerine flower and 40 parts of yellow flower Chinese cabbage material according to an improved CTAB method respectively. According to the KASP reaction system, 40 parts of reddish orange flower and 40 parts of yellow flower Chinese cabbage are subjected to genotype identification by utilizing a marker Br-or-KASP 1. The results show that the marker Br-or-KASP1 can significantly divide all materials into 2 groups, the signal point of 40 homozygous orange flower materials is blue, and the polymerization is near the X axis; the signal points for 40 homozygous yellow floral materials were green and aggregated near the Y-axis, as shown in fig. 3. The relationship between the genotype and the flower color of the control was as shown in Table 3, and the genotype and the phenotype were completely consistent. The marker Br-or-KASP1 can be used for molecular marker-assisted screening of orange flower and orange leaf centroids.
TABLE 340 genotype and flower color of Citrus reticulata Blanco and 40 parts yellow Chinese cabbage material
Example 3 application of KASP-tagged Br-or-KASP1
1.Y959RF2Constructing colony with Chinese cabbage of red tangerine color Y959M-5 (P)1) As female parent, yellow flower color Chinese cabbage R16-11 (P)2) As male parent, hybridizing the two materials to obtain F1Generation of seed, F1Selfing the material to obtain F2Seed generation, selecting F2The plant obtained by seed generation culture is Y959RF2And (4) a group.
Selection of Y959RF2The genome DNA of each plant is extracted by respectively improving a CTAB method from 31 reddish orange flower single plants and 64 yellow flower single plants in the population. According to the KASP reaction system, Y959RF was coupled with a Br-or-KASP1 label2The genotype identification is carried out on 31 reddish orange flower single plants and 64 yellow flower single plants of the population. The results are shown in table 4 and fig. 4.
TABLE 4Y 959RF2Genotype and flower color of 31 reddish orange flower color single plants and 64 yellow flower color single plants in the colony
The result shows that the marker Br-or-KASP1 can remarkably divide all materials into 3 groups, the signal points of 31 single plants are blue, and the signals are polymerized near the X axis to form homozygous orange flower materials; the signal points of 18 individual plants are green, and are polymerized near the Y axis to form homozygous yellow flower materials; the signal points of 46 individuals were red and aggregated near the diagonal, as heterozygous yellow floral material. The genotypes and the phenotypes of all the individual plants are completely consistent, and the individual red tangerine flower plants are recessive homozygous genes and are gathered on an X axis; the yellow flower single plant is a dominant homozygous gene and is gathered on a Y axis, or is a heterozygous type and is gathered on a diagonal line, and the marker can be used for molecular marker assisted selection of the orange flower and the orange leaf core of the Chinese cabbage at high flux and low cost.
The SNP locus Br-or-A09-37676461 which is obviously related to the reddish orange flower gene Br-or is detected by the BSA-seq technology, and the KASP marker Br-or-KASP1 is developed, can detect the genotype of the Br-or at high flux, can be applied to molecular marker assisted breeding of the reddish orange flower and the reddish orange leaf core of Chinese cabbage with high efficiency and low cost, and has very important significance.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
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tttttaaatc tgtctgaaga a 201
Claims (9)
1. The method for identifying or assisting in identifying the flower color of the Chinese cabbage comprises the steps of detecting the genotype of the Chinese cabbage to be detected, and identifying or assisting in identifying the flower color of the Chinese cabbage to be detected according to the genotype of the Chinese cabbage to be detected; the genotype is the genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage; the Br-or-A09-37676461 site is an SNP site in a Chinese cabbage genome, the nucleotide type is C or T, and the nucleotide is the 101 th nucleotide of SEQ ID No.4 in a sequence table.
2. The method of claim 1, wherein when the genotype at the Br-or-a09-37676461 locus is CC genotype, the color of chinese cabbage is orange or candidate is orange; when the genotype of the Br-or-A09-37676461 locus is a CT genotype or a TT genotype, the color of the Chinese cabbage is yellow or the candidate is yellow; wherein the CC genotype represents the homozygosity of the nucleotide type of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage, namely C; the TT genotype represents the homozygosity of the nucleotide type of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage, namely T; the CT genotype indicates that the nucleotide type of the Br-or-A09-37676461 site in the genome of the Chinese cabbage is a hybrid type of C and T.
3. The method for identifying or assisting in identifying the color of the cabbage leaf ball center comprises the steps of detecting the genotype of a cabbage to be detected, and identifying or assisting in identifying the color of the cabbage leaf ball center according to the genotype of the cabbage to be detected; the genotype is the genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage; the Br-or-A09-37676461 site is an SNP site in a Chinese cabbage genome, the nucleotide type is C or T, and the nucleotide is the 101 th nucleotide of SEQ ID No.4 in a sequence table.
4. The method of claim 3, wherein when the genotype at the Br-or-a09-37676461 locus is CC genotype, the color of the leaf core of chinese cabbage is orange or alternatively orange; when the genotype of the Br-or-A09-37676461 locus is a CT genotype or a TT genotype, the color of the leaf core of the Chinese cabbage is white or the candidate is white; wherein the CC genotype represents the homozygosity of the nucleotide type of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage, namely C; the TT genotype represents the homozygosity of the nucleotide type of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage, namely T; the CT genotype indicates that the nucleotide type of the Br-or-A09-37676461 site in the genome of the Chinese cabbage is a hybrid type of C and T.
5. The application of a substance for detecting polymorphism or genotype of Br-or-A09-37676461 locus in a Chinese cabbage genome in A1, A2, A3, A4 or A5; the Br-or-A09-37676461 site is an SNP site in a Chinese cabbage genome, the nucleotide type of the SNP site is C or T, and the SNP site is the 101 th nucleotide of SEQ ID No.4 in a sequence table:
a1, application of a substance for detecting polymorphism or genotype of Br-or-A09-37676461 sites in a Chinese cabbage genome in identification or auxiliary identification of Chinese cabbage flower color;
a2, application of a substance for detecting polymorphism or genotype of Br-or-A09-37676461 sites in a Chinese cabbage genome in preparation of products for identifying or assisting in identifying Chinese cabbage flower color;
a3, application of a substance for detecting polymorphism or genotype of Br-or-A09-37676461 sites in a Chinese cabbage genome in identifying or assisting in identifying the color of the leaf core of the Chinese cabbage;
a4, application of a substance for detecting polymorphism or genotype of Br-or-A09-37676461 sites in a Chinese cabbage genome in preparation of a product for identifying or assisting in identifying the color of Chinese cabbage leaf cores;
p5, and the application of the substance for detecting the polymorphism or genotype of Br-or-A09-37676461 locus in the genome of Chinese cabbage in breeding Chinese cabbage or preparing breeding products of Chinese cabbage.
6. The product containing the substance for detecting the polymorphism or genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage is any one of C1) -C5):
C1) detecting the product of single nucleotide polymorphism or genotype related to the Chinese cabbage flower color;
C2) identifying or assisting in identifying the products of the Chinese cabbage flower color;
C3) detecting the product of single nucleotide polymorphism or genotype related to the color of the cabbage leaf core;
C4) identifying or assisting in identifying the color of the cabbage leaf core;
C5) is used for breeding Chinese cabbage.
7. Use according to any one of claims 5, method according to any one of claims 1 to 4 or product according to claim 6, wherein: the breeding of the Chinese cabbage is to cultivate the Chinese cabbage with orange color of flower color or breed the Chinese cabbage with orange color of leaf ball core.
8. Use according to any one of claims 5 and 7, method according to any one of claims 1 to 4 or 7 or product according to claim 7, wherein: the substances for detecting the polymorphism or genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage are D1), D2) or D3):
D1) the substance for detecting the polymorphism or genotype of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage contains a PCR primer for amplifying the DNA fragment of the genome of the Chinese cabbage including the Br-or-A09-37676461 locus;
D2) the substance for detecting the polymorphism or genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage is a PCR reagent containing the PCR primer;
D3) a kit containing the PCR primer described in D1) or the PCR reagent described in D2).
9. The use, method or product according to claim 8, wherein: the PCR primer is P1 or P2:
p1, the PCR primer is a primer group consisting of single-stranded DNA of which the nucleotide sequence is 22 th to 39 th positions of SEQ ID No.1 in the sequence table, single-stranded DNA of which the nucleotide sequence is 22 th to 39 th positions of SEQ ID No.2 in the sequence table and single-stranded DNA of which the nucleotide sequence is SEQ ID No.3 in the sequence table;
p2, the PCR primer is a primer group of single-stranded DNA shown by SEQ ID No.1 in the sequence table, single-stranded DNA shown by SEQ ID No.2 in the sequence table and single-stranded DNA shown by SEQ ID No.3 in the sequence table.
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