CN111850157B - 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 a SNP locus obviously related to Chinese cabbage orange flower gene Br-or and a KASP molecular marker for detecting the gene, wherein the SNP locus Br-or-A09-37676461 (T/C) obviously related to the 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 a position of A09 chromosome 37,676,461. Aiming at the SNP locus, a KASP marker Br-or-KASP1 is developed, and can be used for auxiliary selection of the orange flower gene Br-or of the celery cabbage. 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 auxiliary breeding of the molecular markers of the orange flowers and the orange leaf centers of the cabbages with high efficiency and low cost, and greatly improves the disease-resistant breeding efficiency.
Description
Technical Field
The invention relates to the technical field of crop molecular marker assisted breeding, in particular to a high-throughput detection marker of a Chinese cabbage orange flower gene Br-or and application thereof in breeding.
Background
Flower color is one of the phenotypic traits of plants that are adapted to the environment. The flower color refers to the color of petals generally, can be used as an important visual signal for attracting insects to pollinate by insect-borne plants, and has important significance for plant survival and reproduction. The flower color can influence aphid feeding, and the crop varieties with different flower colors are bred in production, so that the effect of reducing aphid disease transmission is achieved, the energy balance of petals can be maintained, and damage to flower organs is avoided. The flower color has certain influence on plant variety breeding and growth and development, and has important effects on improving seed purity, removing hybrid plants, identifying natural outcrossing rate, checking inter-seed character transfer and the like. For ornamental plants, the flower color not only determines the ornamental value of the plant, but also directly affects its commercial value.
Chinese cabbage (Brassica rapa L. Sspfekinensis) is one of the largest vegetable crops cultivated in China, and plays an important role in the vegetable crops in China, and the color of the Chinese cabbage mainly comprises yellow flowers, orange flowers and white flowers. The color of the Chinese cabbage can be used as the marking character of the breeding so as to eliminate unnecessary materials in early stage, thereby accelerating the breeding progress. The orange flower character of the Chinese cabbage is the quality character controlled by 1 pair of recessive genes, and the orange flower and the color character of the sphere center are completely linked, namely, the color of the orange flower is orange, and the color of the orange flower is orange, so that orange heart materials can be screened according to the color in the transfer process (Zhang Deshuang and the like, 2003, research on the genetic rule of the color and the color of the Chinese cabbage, north China agricultural report, 18 (2): 81-84). The molecular marker linked with the orange flowers is developed, so that the molecular marker can be used for auxiliary breeding of the orange flowers of the Chinese cabbages, can be used for molecular breeding of orange cores of the Chinese cabbages, and has important application value.
Previous studies showed that a candidate gene for the chinese cabbage saffron gene Br-or was BrCRTISO (Bra 031539) encoding a carotenoid isomerase, and different types of molecular markers were developed. Feng et al (2012) obtained 5 SSR markers linked to Br-or, closely linked markers syau19 and syau15 on either side of the Br-or gene covered a region of 4.6cM (Feng et al, 2012,Mapping ofor,a gene conferring orange color on the inner leaf of the Chinese cabbage (Brassica rapa l. Ssp. Pekinensis), molecular Breeding, 29:235-244.). Li Peirong et al (2014) designed InDel markers based on polyacrylamide gel electrophoresis detection for deletion variation of 6 bases on the first exon of the candidate gene Bra031539 (Li Peirong et al, 2014, chinese cabbage orange-core carotenoid component and gene analysis thereof, gardening journal, 41 (3): 469-478). Zhang et al (2015) designed an InDel marker (Zhang et al 2015,Molecular characterization and transcriptome analysis of orange head Chinese cabbage (Brassica rapa L. Ssp. Pekinensis), planta, 241:1381-1394) for a 88bp variation in the promoter region of the candidate gene Bra 031539. Li et al (2017) designed InDel markers (Li et al 2017,Transcriptome analysis of orange head Chinese cabbage (Brassica rapa l. Ssp. Pekinensis) and molecular marker development, international Journal of Genomics, 6835810:1-8.) for variation of the first intron of the candidate gene Bra 031539. Lee et al (2014) designed InDel markers for variations of the first and second exons of Bra031539 (Lee et al 2014,Association of molecular markers derived from the BrCRTISO1 gene with prolycopene-enriched orange-colored leaves in Brassica rapa, theoretical Applied Genetics, 127:179-191).
Although the former develops different molecular markers aiming at the variation of different positions of the orange flower gene Br-or, most of the developed markers are gel electrophoresis-based markers, so that the time consumption, the labor consumption, the price and the cost are high, and the developed markers have no universality.
Disclosure of Invention
The invention aims to solve the technical problems that identification or auxiliary identification of the color of the Chinese cabbage lays a foundation for molecular marker screening and polymerization breeding of Chinese cabbage with orange color.
In order to solve the technical problems, the invention provides a method for identifying or assisting in identifying the flower colors of Chinese cabbages, which comprises the steps of detecting the genotype of the Chinese cabbages to be detected, and identifying or assisting in identifying the flower colors of the Chinese cabbages according to the genotype of the Chinese cabbages 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 locus is an SNP locus in a Chinese cabbage genome, the nucleotide type of the locus is C or T, and the locus is 101 nucleotide of SEQ ID No.4 in a sequence table.
Wherein, when the genotype of the Br-or-A09-37676461 locus is 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 CT genotype or TT genotype, the color of the celery cabbage is yellow or candidate yellow; wherein the CC genotype represents homozygosity of C in the nucleotide type of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage; the TT genotype represents homozygosity of T in the nucleotide type of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage; the CT genotype represents heterozygous C and T of the nucleotide variety of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage.
The present invention provides any one of the applications of A1 to A5,
A1. the application of the substance for detecting the polymorphism or genotype of Br-or-A09-37676461 locus in the genome of Chinese cabbage in identifying or assisting in identifying the flower color of Chinese cabbage; the Br-or-A09-37676461 locus is an SNP locus in a Chinese cabbage genome, the nucleotide type of the locus is C or T, and the locus is 101 nucleotide of SEQ ID No.4 in a sequence table.
A2. Application of a substance for detecting polymorphism or genotype of Br-or-A09-37676461 locus in a Chinese cabbage genome in preparing and identifying or assisting in identifying Chinese cabbage flower color products; the Br-or-A09-37676461 locus is an SNP locus in a Chinese cabbage genome, the nucleotide type of the locus is C or T, and the locus is 101 nucleotide of SEQ ID No.4 in a sequence table.
A3, detecting polymorphism or genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage, and applying the substance in identifying or assisting in identifying She Qiuxin color of the Chinese cabbage; the Br-or-A09-37676461 locus is an SNP locus in a Chinese cabbage genome, the nucleotide type of the locus is C or T, and the locus is 101 nucleotide of SEQ ID No.4 in a sequence table.
A4, detecting polymorphism or genotype of Br-or-A09-37676461 locus in the genome of the Chinese cabbage, and applying the substance in preparation and identification or auxiliary identification of She Qiuxin color products of the Chinese cabbage; the Br-or-A09-37676461 locus is an SNP locus in a Chinese cabbage genome, the nucleotide type of the locus is C or T, and the locus is 101 nucleotide of SEQ ID No.4 in a sequence table.
A5. Detecting polymorphism or genotype of Br-or-A09-37676461 locus in Chinese cabbage genome, and its application in Chinese cabbage breeding or preparing Chinese cabbage breeding products; the Br-or-A09-37676461 locus is an SNP locus in a Chinese cabbage genome, the nucleotide type of the locus is C or T, and the locus is 101 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 the Br-or-A09-37676461 locus in the genome of the Chinese cabbage, which is any one of C1) -C5):
c1 Detecting a single nucleotide polymorphism or genotype associated with chinese cabbage flower color;
c2 Identifying or assisting in identifying the product of the Chinese cabbage flower color;
c3 Detecting a single nucleotide polymorphism or genotype associated with the color of the leaf and core of the chinese cabbage;
c4 Identifying or assisting in identifying the product of the color of the celery cabbage She Qiuxin;
c5 A product for breeding Chinese cabbage.
In the application, the method and the product, the Chinese cabbage breeding is to cultivate Chinese cabbage with orange flower color or to breed Chinese cabbage with orange flower color. The breeding of the Chinese cabbage is to cultivate the Chinese cabbage with She Qiuxin orange color or to breed the Chinese cabbage with She Qiuxin orange color.
In the application, the method and the product, the substances for detecting the polymorphism or the genotype of the 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 PCR primers for amplifying the Chinese cabbage genome DNA fragments 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 D1) the PCR primer or D2) the PCR reagent.
In the above applications, methods and products, the PCR primer is P1 or P2:
the P1 PCR primer is a primer group consisting of single-stranded DNA with the nucleotide sequence of 22 th-39 th positions of SEQ ID No.1 in a sequence table, single-stranded DNA with the nucleotide sequence of 22 nd-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;
the P2 PCR primer is a primer group 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 a hybrid offspring population of Chinese cabbage Y959M-5 and Chinese cabbage R16-11, for example, chinese cabbage Y959M-5 (P) 1 ) As female parent, chinese cabbage R16-11 (P) 2 ) As male parent, two materials are hybridized to obtain F 1 Seed generation, F 1 Selfing the material to obtain F 2 Seed substitution, selecting F 2 The plant obtained by the seed culture is Y959RF 2 A population.
The SNP locus Br-or-A09-37676461 obviously related to the orange flower gene Br-or is detected by the BSA-seq technology, and the KASP marker Br-or-KASP1 is developed, so that the marker can detect the genotype of the Br-or at high flux, can be applied to molecular marker assisted breeding of the orange flowers and the orange leaf centers of the Chinese cabbages at high efficiency and low cost, and has very important significance.
Drawings
FIG. 1 shows the BSA-Seq localization result of the saffron gene Br-or;
FIG. 2 is a KASP genotyping of 4 saffron and 4 yellow flower materials using the marker Br-or-KASP1.
FIG. 3 is a KASP genotyping of 40 reddish orange and 40 yellow flower DH lines using the marker Br-or-KASP1.
FIG. 4 is a schematic diagram of a pair Y959RF using the label Br-or-KASP1 2 The population was KASP genotyped.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Experimental materials
The biological material obtained by the public in the following examples was used only for repeated experiments related to the present invention, but not as other uses, in the development of KASP markers of the chinese cabbage bolting-related gene BrFLC1, core agricultural report, 34 (2): 0265-0272).
The biological material is obtained by the public of Chinese cabbage Y510-9 (optimization and establishment of Chinese cabbage KASP reaction system, 2018, gardening report) in the following examples, and is used only for repeated experiments related to the invention, but not used for other purposes.
The material names and corresponding commercial names and petal colors referred to in the following examples are shown in the following table.
TABLE 1.98 cabbage Material names and corresponding commercially available Commodity names
Example 1 discovery of SNP loci associated with Chinese cabbage flower color
1. Positioning of saffron gene Br-or
1.1 test materials and Property investigation
54 parts of exocarpium citri rubrum flower material and 44 parts of yellow flower material are selected, and the color of each material petal is investigated in the blooming period.
1.2DNA extraction
Genomic DNA of 54 parts of the saffron material and 44 parts of the yellow flower material in step 1.1 were extracted separately using the modified CTAB method. The method comprises the following specific steps: fresh leaves were taken in 2.0mL Eppendorf centrifuge tubes, 1 shot of steel balls (5 MM diameter) was placed into each tube, after which 1000. Mu.L of 2% CTAB extraction buffer was added and the cells were broken on a tissue breaker (model: retsch MM400, germany) at a frequency of 30 times/sec for 1min. After cooling, adding 500 mu L of 24:1 chloroform/isoamyl alcohol extract, shaking up and down for 30 times, standing for delamination, centrifuging (12000 r/min) for 10min, sucking 400 mu L of supernatant into a new 1.5mL centrifuge tube, adding 400 mu L of isopropanol for precipitating DNA, centrifuging (12000 r/min) for 5min after up and down mixing, discarding supernatant, adding 70% ethanol for 750 mu L for cleaning DNA precipitation, centrifuging (12000 r/min) for 2min, discarding supernatant, airing DNA at room temperature, adding 100 mu L of ddH2O for dissolving DNA, and placing in a refrigerator at-20 ℃ for standby.
1.3 localization of the saffron Gene Br-or
And (3) respectively mixing 54 parts of exocarpium citri rubrum flower color and 44 parts of genome DNA of yellow flower materials extracted in the step (1.2) in equal quantity, constructing two extreme gene mixed pools of the exocarpium citri rubrum and the yellow flower, and carrying out whole genome re-sequencing on the two mixed pools by using a BSA-Seq technology. The genome sequence (V1.5) of Chinese cabbage Chiifu-401-42 is used as a reference sequence, clear reads of two mixed pools are respectively compared to the reference genome by BWA (v0.7.17) software, and SNP (single nucleotide polymorphism) cloning is carried out by using Samtools (V1.9) and Bcftools (V1.9) to obtain SNP loci of all samples. And respectively calculating SNP-index of the mixed pool of the orange flowers and the mixed pool of the yellow flowers, subtracting the SNP-index of the mixed pool of the yellow flowers from the SNP-index of the mixed pool of the orange flowers to obtain delta SNP-index between the two mixed pools, and carrying out sliding window analysis by taking 1Mb as a window and 50kb as a step length to determine candidate intervals of the orange flower genes. By significance analysis, the saffron gene was mapped within the 36.75-38.25Mb interval of A09 chromosome (FIG. 1), with an interval length of 1.5Mb. This region contains the candidate gene BrCRTISO (Bra 031539) for the orange heart gene Br-or reported previously (37,676,627-37, 679,733 bp). The characters of interest in previous researches are the color characters of the leaf and the core of the Chinese cabbage, and the research population used is the parental genetic population, and the color characters of the orange flowers and the core are completely linked. The character of the invention is the color character of Chinese cabbage petals, the research population is natural population, the main gene is rapidly detected at the end of A09 chromosome by utilizing BSA-Seq technology, and the main gene comprises the orange heart gene Br-or studied by the former, so that the orange flower color gene and the orange heart gene are considered to be the same gene, and the candidate genes are BrCRTISO (Bra 031539).
2. KASP molecular marker of orange flower gene Br-or
2.1 analyzing SNP variation of candidate gene Bra031539 coding region (37,676,627-37, 679,733 bp) and promoter region and delta SNP-index between two pools, and finding that T/C SNP variation exists at 37,676, 463 bp position of the promoter region, corresponding to 101 th nucleotide in sequence 1, wherein Y represents 101 th nucleotide can be T or C, and the SNP locus is named Br-or-A09-37676461. The delta SNP-index value of the SNP locus Br-or-A09-3767646 between two mixing tanks is 0.71, and the SNP locus Br-or-A09-3767646 is obviously related to the character of the reddish orange flowers.
2.2 design of the KASP marker Br-or-KASP1 for SNP locus Br-or-A09-37676461, comprising three primers: br-or-KASP1Fa:5' -GAAGGTCGGAGTCAACGGATTGTAATATCCTGCACTTCT-3'；Br-or-KASP1Fb：5'-GAAGGTGACCAAGTTCATGCTGTAATATCCTGCACTTCC-3'; br-or-KASP1R:5'-CAAAAAAATGTTTGGTGAAGAAC-3'. Br-or-KASP1Fa and Br-or-KASP1Fb are two allelesThe gene specific forward primer, br-or-KASP1Fa is a specific primer of the yellow flower genotype, br-or-KASP1Fb is a specific primer of the orange flower genotype, and HEX and FAM fluorescent sequence tag sequences (underlined parts) are respectively added at the 5' end. Br-or-KASP1R is a common reverse primer.
2.3 select 4 parts of reddish orange floral material Y959M-5, Y698-17, Y662-1, Y947M-13 and 4 parts of yellow floral material Yw81, Y177-12, Y510-9, R16-11 to verify the KASP marker Br-or-KASP1. The 8 parts of material are DH (double Haploid) pure line material obtained by culturing free microspores.
Genomic DNA of 4 parts of orange flower material (Y959M-5, Y698-17, Y662-1 and Y947M-13) and 4 parts of yellow flower material (Yw 81, Y177-12, Y510-9 and R16-11) were extracted, respectively, using the modified CTAB method. PCR amplification was performed using 4 parts of genomic DNA of the saffron materials (Y959M-5, Y698-17, Y662-1 and Y947M-13) and 4 parts of yellow flower materials (Yw 81, Y177-12, Y510-9 and R16-11) as templates, respectively, 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 apparatus (Eppendorf Mastercyclepro), 8. Mu.L of the reaction system: 1.5. Mu.L of DNA (60 ng. Mu.L-1), 4. Mu. L 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-1) 2 O is mixed according to the volume ratio of 12:12:30:46, and the rest is ddH 2 And (3) supplementing O.
The KASP-PCR amplification procedure was: denaturation at 94℃for 15min in the first stage; the second stage of denaturation at 94℃for 20s and annealing at 61℃for 60s, for a total of 10 cycles (each cycle reduced by 0.6℃from the second cycle); the third stage is denaturation at 94 ℃ for 20s and annealing at 55 ℃ for 60s, 26 cycles are carried out; the fourth stage is carried out at 37 ℃ for 1min.
2.4 the KASP-PCR amplified product of step 2.3 was read for endpoint fluorescence signal using a Roche fluorescence quantitative PCR Instrument LightCycler 480Instrument II (LC 480 II). SNP typing results were analyzed using LC480 software v 1.5.1: the genotype which is polymerized near the X axis and shows blue is the allele type connected with FAM fluorescence tag sequence, namely, the homozygous type of which one SNP locus Br-or-A09-3767646 nucleotide in the Chinese cabbage genome is C, and is named as genotype CC; the genotype which is polymerized near the Y axis and shows green is the allele type connected with HEX fluorescent tag sequence, namely, the homozygous type of one SNP locus Br-or-A09-3767646 nucleotide type in the Chinese cabbage genome is T, and the genotype is named genotype TT; the genotype with red color in the middle is heterozygous of two alleles, namely, one SNP locus Br-or-A09-3767646 nucleotide in the genome of the Chinese cabbage is heterozygous of C and T, and the genotype is named genotype CT.
4 parts of red orange flower materials Y959M-5, Y698-17, Y662-1, Y947M-13 and 4 parts of yellow flower materials Yw81, Y177-12, Y510-9 and R16-11 are subjected to genotype identification by utilizing a marker Br-or-KASP1, the signal points of the 4 red orange flower materials are blue, the 5' -end of the primer is connected with FAM fluorescent tag sequence for competitive amplification, and the primer is polymerized near an X axis, so that the Chinese cabbage with the genotype of the SNP locus Br-or-A09-3767646 in the Chinese cabbage genome being CC is obtained; the signal points of the 4 yellow flower materials are green, the 5' -end is connected with the primer of HEX fluorescent tag sequence for competitive amplification, and the primer is polymerized near the Y axis, namely the celery cabbage with the genotype TT of SNP locus Br-or-A09-3767646 in the genome of the celery cabbage (shown in figure 2). The Br-or-KASP1 marker can significantly distinguish two homozygous genotypes, and the marker development is successful.
Table 2 8 relation between the flower colors and genes of Chinese cabbage of different varieties
EXAMPLE 2 KASP-labeled Br-or-KASP1 application 1
Selecting 40 parts of exocarpium citri rubrum and 40 parts of yellow flower celery cabbage material, and culturing to obtain DH (double Haploid) pure line material by free microspore.
Extracting genome DNA of 40 parts of exocarpium citri rubrum and 40 parts of yellow flower Chinese cabbage materials according to a modified CTAB method respectively. According to the KASP reaction system, genotyping was performed on 40 parts of saffron and 40 parts of yellow flower celery cabbage material using the marker Br-or-KASP1. The result shows that the mark Br-or-KASP1 can obviously divide all materials into 2 groups, and the signal points of 40 parts of homozygous orange flower materials are blue and polymerized near the X axis; the signal points of 40 parts of homozygous yellow flower material are green and polymerized near the Y axis, as shown in fig. 3. The relationship between the control genotype and flower color is shown in Table 3, and the genotype and phenotype are completely consistent. The marker Br-or-KASP1 can be used for molecular marker assisted screening of orange flower and orange leaf sphere centers.
TABLE 3 genotype and color of 40 parts of saffron and 40 parts of yellow flower celery cabbage materials
EXAMPLE 3 KASP-labeled Br-or-KASP1 application
1.Y959RF 2 Group construction using orange flower color Chinese cabbage Y959M-5 (P 1 ) As female parent, chinese cabbage R16-11 (P) 2 ) As male parent, two materials are hybridized to obtain F 1 Seed generation, F 1 Selfing the material to obtain F 2 Seed substitution, selecting F 2 The plant obtained by the seed culture is Y959RF 2 A population.
Select Y959RF 2 And respectively extracting genome DNA of each plant by improving CTAB method of 31 orange flower single plants and 64 yellow flower single plants in the population. According to the KASP reaction system, the marker Br-or-KASP1 was used for Y959RF 2 Genotyping was performed on 31 orange flower individuals and 64 yellow flower individuals of the population. The results are shown in Table 4 and FIG. 4.
TABLE 4Y 959RF 2 Genotype and flower color of 31 orange flower individuals and 64 yellow flower individuals in the population
The result shows that the mark Br-or-KASP1 can obviously divide all materials into 3 groups, the signal points of 31 single plants are blue, and the signal points are polymerized near an X axis to form a homozygous orange red flower material; the signal points of 18 single plants are green, are polymerized near the Y axis, and are homozygous yellow flower materials; the signal points of 46 individual plants are red, and are polymerized near the diagonal line to form the heterozygous yellow flower material. The genotypes and phenotypes of all the single plants are completely consistent, the orange flower single plants are recessive homozygous genes, and are gathered on the X axis; the yellow flower single plant is either dominant homozygous gene, is gathered on the Y axis, or is heterozygous, is gathered on the diagonal, and the marker can be used for molecular marker assisted selection of orange flowers and orange leaf centers of Chinese cabbages with high throughput and low cost.
The SNP locus Br-or-A09-37676461 obviously related to the orange flower gene Br-or is detected by the BSA-seq technology, and the KASP marker Br-or-KASP1 is developed, so that the marker can detect the genotype of the Br-or at high flux, can be applied to molecular marker assisted breeding of the orange flowers and the orange leaf centers of the Chinese cabbages at high efficiency and low cost, and has very important significance.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present 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 respect to specific embodiments, it will be appreciated that the invention may 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 application of some of the basic features may be done in accordance with the scope of the claims that follow.
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gacattacgg cccaaaatta atgaataatc aacggccacg attagaagtc actaaaataa 60
agtttttttc tctttttttt tttgtaatat cctgcacttc ygagtgttct tcaccaaaca 120
tttttttgtc cttccatgtg atgaaaacca attttccaaa ttcggatgaa aggacaacac 180
tttttaaatc tgtctgaaga a 201
Claims (4)
1. The method for identifying or assisting in identifying the color of the Chinese cabbage comprises detecting the genotype of the Chinese cabbage to be detected, and identifying or assisting in identifying the 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 locus is an SNP locus in a Chinese cabbage genome, the nucleotide variety of the locus is C or T, and the locus is 101 nucleotide of SEQ ID No.4 in a sequence table; when the genotype of the Br-or-A09-37676461 locus is CC genotype, the color of the celery cabbage is orange or the color of the celery cabbage is candidate to be orange; when the genotype of the Br-or-A09-37676461 locus is CT genotype or TT genotype, the color of the celery cabbage is yellow or candidate yellow; wherein the CC genotype represents homozygosity of C in the nucleotide type of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage; the TT genotype represents homozygosity of T in the nucleotide type of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage; the CT genotype represents heterozygous C and T of the nucleotide variety of the Br-or-A09-37676461 locus in the genome of the Chinese cabbage;
the Chinese cabbage is red baby No. one, heat resistant white for 45 days, xinke red fei, chinese lion chrysanthemum red core, qin Bai No. two, beijing spring baby No.2, xia Shuai No. one, baby cabbage, qing-yan-mei-autumn, autumn pioneer, fire phoenix, hua Yangbai, beijing orange red core, chengza No. 50, red core baby cabbage, orange core mini, hongbao 70, ji Gong 65, cai Bai No.3, improved Qing-za No. 201, royal, ji Gong 308, gold, noble, oriental Xia Wang, red core Chinese cabbage, sijihuang, ji Gong, jihong doll, zaoyou No.2, beijing Xiaozhen No. 57, nongke No.1, chinese white No. 6, xiayang 40, red white No.2, shen Rongda red core, multiple resistant No. two the Chinese cabbage is one of Jiujiu, qingza No. 7, jianbai No. 5, jianchun, shijiu Xinyuqing, sichun spring, xiayang No. 303, qiu Lihuang, fengchi No. 78, ji, beijing Xin No. three, jibai No.2, qinggan No. 6, xiaqian pioneer, lujian No. 85, chunyang celery cabbage, huadu cabbage, leshanchun, defeng No.1, longxiao No. 5, susheng No. one, qinggan No. three, lu Chunbai No. one, qinggu No. 60, fuji Kuai, xiaozhen No. 56, kangchun Wang, jinqing No. 70, niu Jiu No.2, CR Xingchun, egg yolk, xuchun No. two, shen Rong hot air fast food, niu Jiuqiu and Xinke Liang No. 6.
2. Detecting polymorphism or genotype of Br-or-A09-37676461 locus in the genome of Chinese cabbage, and applying the polymorphism or genotype in A1; the Br-or-A09-37676461 locus is an SNP locus in a Chinese cabbage genome, the nucleotide variety of the locus is C or T, and the locus is 101 nucleotide of SEQ ID No.4 in a sequence table:
a1, detecting polymorphism or genotype of Br-or-A09-37676461 locus in a Chinese cabbage genome, and applying the polymorphism or genotype to Chinese cabbage breeding or preparing Chinese cabbage breeding products; the Chinese cabbage breeding is to cultivate Chinese cabbage with orange flower color;
the Chinese cabbage is red baby No. one, heat resistant white for 45 days, xinke red fei, chinese lion chrysanthemum red core, qin Bai No. two, beijing spring baby No.2, xia Shuai No. one, baby cabbage, qing-yan-mei-autumn, autumn pioneer, fire phoenix, hua Yangbai, beijing orange red core, chengza No. 50, red core baby cabbage, orange core mini, hongbao 70, ji Gong 65, cai Bai No.3, improved Qing-za No. 201, royal, ji Gong 308, gold, noble, oriental Xia Wang, red core Chinese cabbage, sijihuang, ji Gong, jihong doll, zaoyou No.2, beijing Xiaozhen No. 57, nongke No.1, chinese white No. 6, xiayang 40, red white No.2, shen Rongda red core, multiple resistant No. two the Chinese cabbage is one of Jiujiu, qingza No. 7, jianbai No. 5, jianchun, shijiu Xinyuqing, sichun spring, xiayang No. 303, qiu Lihuang, fengchi No. 78, ji, beijing Xin No. three, jibai No.2, qinggan No. 6, xiaqian pioneer, lujian No. 85, chunyang celery cabbage, huadu cabbage, leshanchun, defeng No.1, longxiao No. 5, susheng No. one, qinggan No. three, lu Chunbai No. one, qinggu No. 60, fuji Kuai, xiaozhen No. 56, kangchun Wang, jinqing No. 70, niu Jiu No.2, CR Xingchun, egg yolk, xuchun No. two, shen Rong hot air fast food, niu Jiuqiu and Xinke Liang No. 6.
3. The method according to claim 1 or the use according to claim 2, characterized in that: the substances for detecting the polymorphism or genotype of the 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 PCR primers for amplifying the Chinese cabbage genome DNA fragments 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 D1) the PCR primer or D2) the PCR reagent.
4. A method or use according to claim 3, wherein: the PCR primer is P1 or P2:
the P1 PCR primer is a primer group consisting of single-stranded DNA with the nucleotide sequence of 22 th-39 th positions of SEQ ID No.1 in a sequence table, single-stranded DNA with the nucleotide sequence of 22 nd-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;
the P2 PCR primer is a primer group 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.
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| CN113999931A (en) * | 2021-09-03 | 2022-02-01 | 泰安市农业科学研究院 | Method for auxiliary breeding by using molecular markers related to Chinese cabbage flower colors |
| CN114736980B (en) * | 2022-03-17 | 2023-06-20 | 承德市农林科学院 | Molecular marker fragment related to yellow cauliflower color and application thereof |
| CN114525363B (en) * | 2022-03-17 | 2023-05-09 | 锦州医科大学 | Molecular marker primer related to color of rape flower and method for identifying color of rape flower |
| CN115369123B (en) * | 2022-09-22 | 2023-07-25 | 河南省农业科学院园艺研究所 | KASP (KASP) mark of dominant orange gene BrOr of Chinese cabbage and application thereof |
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