CN110656195B

CN110656195B - SNP locus for identifying flesh color of Xinlimei radish and application thereof

Info

Publication number: CN110656195B
Application number: CN201910874248.3A
Authority: CN
Inventors: 张丽; 王庆彪; 王艳萍
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2019-09-17
Filing date: 2019-09-17
Publication date: 2021-10-01
Anticipated expiration: 2039-09-17
Also published as: CN110656195A

Abstract

The invention relates to SNP loci for identifying the flesh color of American radish in mind and application thereof, wherein the SNP loci for identifying the flesh color of American radish in mind are Rs _ scan 158-73, Rs _ scan 158-87 and/or Rs _ scan 158-94, and the Rs _ scan 158-73 is located at the 101 th nucleotide of SEQ ID No.1 on the Rs _ scan 158 of radish genome; the Rs _ Scaf158-87 is located at the 101 st nucleotide of SEQ ID No.2 on the radish genome Rs _ Scaf 158; the Rs _ Scaf158-94 is located at nucleotide 101 of SEQ ID No.3 on the radish genome Rs _ Scaf 158. The locus can be applied to identification or auxiliary identification of the flesh color of the Xinlimei radish or breeding of the Xinlimei radish.

Description

SNP locus for identifying flesh color of Xinlimei radish and application thereof

Technical Field

The invention relates to a radish identification method in the field of biotechnology, in particular to an SNP locus for identifying the flesh color of a Xinlimei radish and application thereof.

Background

The Xinlimei radish is also called red heart radish, belongs to radish seeds (Raphanus sativus var L) of the genus Raphanus of the family Brassicaceae, is a special radish variety, and is characterized in that the outer skin is light green, the leaves are dark green, the inner lining is blood red, and the radish variety is crisp in taste, sweet in taste and tender in meat quality. Beijing Xinlimei is reputed to be Jinghua by the long planting history and excellent quality and flavor of Beijing Xinlimei and rich in anti-cancer substance anthocyanin. The flesh color of the heart is an important index for identifying the quality of the variety, and the 100% red blood flesh rate is an ideal variety expected by breeders and growers. However, in the process of variety breeding, white meat plants appear in the self-bred or hybridized progeny, which affects the purity and commodity of the varieties. Therefore, the meat color identification is the key for breeding the Xinlimei variety with high blood red flesh rate. Traditional flesh color identification needs to cut or make holes on fleshy roots of plants, so that the survival rate of breeding materials is reduced, and the normal propagation of seeds is influenced. Therefore, there is a need to develop a method for rapidly identifying red and white meat materials of Beijing Xinlimei radish.

Single Nucleotide Polymorphism (SNP) refers to a variation of a single nucleotide in a genome, which is the most minute unit of variation and is a variation formed by substitution, inversion, insertion or deletion of a single nucleotide pair.

Disclosure of Invention

The invention solves the technical problem of providing a method for quickly identifying red meat and white meat materials of Beijing Xinlimei radish on the premise of not damaging fleshy roots of the radish.

The invention claims the application of Rs _ Scaf158-73, Rs _ Scaf158-87 and/or Rs _ Scaf158-94 in identifying or assisting in identifying the color of the heart beauty radish; the Rs _ Scaf158-73 is a SNP locus of the radish genome, and the Rs _ Scaf158-73 is located at the 101 st nucleotide of SEQ ID No.1 on the Rs _ Scaf158 of the radish genome, and is T or C; the Rs _ Scaf158-87 is an SNP locus of the radish genome, and the Rs _ Scaf158-87 is located at the 101 st nucleotide of SEQ ID No.2 on the Rs _ Scaf158 of the radish genome, and is T or C; the Rs _ Scaf158-94 is a SNP site of the radish genome, and the Rs _ Scaf158-94 is located at the 101 st nucleotide of SEQ ID No.3 on the Rs _ Scaf158 of the radish genome, and is T or C.

The invention also claims the application of the substance for detecting the polymorphism or genotype of the Rs _ Scaf158-73 in the genome of the Xinlimei radish, the substance for detecting the polymorphism or genotype of the Rs _ Scaf158-87 in the genome of the Xinlimei radish and/or the substance for detecting the polymorphism or genotype of the Rs _ Scaf158-94 in the genome of the Xinlimei radish in preparing products for identifying or assisting in identifying the flesh color of the Xinlimei radish; the Rs _ Scaf158-73 is a SNP locus of the radish genome, and the Rs _ Scaf158-73 is located at the 101 st nucleotide of SEQ ID No.1 on the Rs _ Scaf158 of the radish genome, and is T or C; the Rs _ Scaf158-87 is an SNP locus of the radish genome, and the Rs _ Scaf158-87 is located at the 101 st nucleotide of SEQ ID No.2 on the Rs _ Scaf158 of the radish genome, and is T or C; the Rs _ Scaf158-94 is a SNP site of the radish genome, and the Rs _ Scaf158-94 is located at the 101 st nucleotide of SEQ ID No.3 on the Rs _ Scaf158 of the radish genome, and is T or C.

Further, the substance for detecting the polymorphism of the Rs _ Scaf158-73 in the genome of the California radish is a primer pair for amplifying DNA fragments of the California radish genome including the Rs _ Scaf158-73, wherein the primer pair comprises single-stranded DNA consisting of 22 th to 48 th nucleotides in SEQ ID No.4, single-stranded DNA consisting of 22 th to 48 th nucleotides in SEQ ID No.5 and single-stranded DNA consisting of nucleotides in SEQ ID No. 6;

the substance for detecting the polymorphism of the Rs _ Scaf158-87 in the genome of the Xinlimei radish is a primer pair for amplifying DNA fragments of the Xinlimei radish genome including the Rs _ Scaf158-87, wherein the primer pair comprises single-stranded DNA consisting of 22 th to 48 th nucleotides in SEQ ID No.7, single-stranded DNA consisting of 22 th to 48 th nucleotides in SEQ ID No.8 and single-stranded DNA consisting of nucleotides in SEQ ID No. 9;

the substance for detecting the polymorphism of the Rs _ Scaf158-94 in the genome of the California radish is a primer pair for amplifying DNA fragments of the California radish genome including the Rs _ Scaf158-94, wherein the primer pair comprises single-stranded DNA consisting of 22 th to 48 th nucleotides in SEQ ID No.10, single-stranded DNA consisting of 22 th to 48 th nucleotides in SEQ ID No.11 and single-stranded DNA consisting of nucleotides in SEQ ID No. 12.

The invention also provides a method for identifying or assisting in identifying the flesh color of the Xinlimei radish, which comprises the following steps of detecting the genotype of the Rs _ Scaf158-73, the genotype of the Rs _ Scaf158-87 and/or the genotype of the Rs _ Scaf158-94 in the Xinlimei radish to be detected, and identifying or assisting in identifying the flesh color of the Xinlimei radish according to the genotype of the Xinlimei radish to be detected:

the genotype of the Rs _ scaf158-73 is homozygous for TT, and the flesh color of the Xinlimei radish is red or red as a candidate; the genotype of the Rs _ scaf158-73 is CC homozygote, and the flesh color of the Xinli radish is white or candidate white; the genotype of the Rs _ scaf158-73 is a hybrid type of CT, and the flesh color of the Xinlimei radish is red or red as a candidate;

the genotype of the Rs _ scaf158-87 is homozygous for TT, and the flesh color of the Xinlimei radish is red or red as a candidate; the genotype of the Rs _ scaf158-87 is CC homozygote, and the flesh color of the Xinli radish is white or candidate white; the genotype of the Rs _ scaf158-87 is a hybrid type of CT, and the flesh color of the Xinli American radish is red or is red as a candidate;

the genotype of the Rs _ scaf158-94 is homozygous for TT, and the flesh color of the Xinlimei radish is red or red as a candidate; the genotype of the Rs _ scaf158-94 is CC homozygote, and the flesh color of the Xinli radish is white or candidate white; the Rs _ scaf158-94 genotype is a hybrid type of CT, and the flesh color of the Xinlimei radish is red or is red as a candidate.

The application of the Rs _ Scaf158-73, Rs _ Scaf158-87 and/or Rs _ Scaf158-94 in breeding American radish in mind is also within the protection scope of the patent.

Further, the breeding of the Xinlimei radish is to cultivate a green skin red meat variety.

The application of the method for identifying or assisting in identifying the flesh color of the American heart radish in breeding the American heart radish also belongs to the protection scope of the invention.

The invention also provides a breeding method of the Xinlimei radish, which comprises the following steps: detecting polymorphism of Rs _ Scaf158-73, Rs _ Scaf158-87 and/or Rs _ Scaf158-94 in claim 1 in a California radish genome, and selecting the California radish with the genotype of Rs _ Scaf158-73, Rs _ Scaf158-87 and/or Rs _ Scaf158-94 in claim 1 as a parent for breeding.

Any of the following products 1) to 6) should also be within the scope of the present invention:

1) a primer pair for amplifying a DNA fragment including the 101 st nucleotide of SEQ ID No.1 on the radish genome scaf 158;

2) a primer composition consisting of the single-stranded DNA shown in SEQ ID No.4, the single-stranded DNA shown in SEQ ID No.5 and the single-stranded DNA shown in SEQ ID No. 6;

3) a primer pair for amplifying a DNA fragment including the 101 st nucleotide of SEQ ID No.2 on the radish genome scaf 158;

4) a primer composition consisting of the single-stranded DNA shown in SEQ ID No.7, the single-stranded DNA shown in SEQ ID No.8 and the single-stranded DNA shown in SEQ ID No. 9;

5) a primer pair for amplifying a DNA fragment including the 101 st nucleotide of SEQ ID No.3 on the radish genome scaf 158;

6) a primer composition consisting of the single-stranded DNA shown in SEQ ID No.10, the single-stranded DNA shown in SEQ ID No.11 and the single-stranded DNA shown in SEQ ID No. 12.

By identifying the genotype of the SNP locus of the radish to be detected, the flesh color of the fleshy root of the radish can be quickly identified. For 94F at the same time₂The detection experiment results of the Xinlimei radishes show that the accuracy rate of detecting the flesh color of the fleshy root through the Rs _ Scaf158-73 and/or the Rs _ Scaf158-87 can reach 99%, and the accuracy rate of detecting the flesh color of the fleshy root through the Rs _ Scaf158-94 can reach 98%.

Drawings

FIG. 1 is a graph showing the results of KASP detection of the Rs _ Scaf158-73 genotype of 96 varieties of Cardimei radish;

FIG. 2 is a graph showing the results of KASP detection of the Rs _ Scaf158-87 genotype of 96 varieties of Cardimei radish;

FIG. 3 is a graph showing the results of KASP detection of the Rs _ Scaf158-94 genotype of 96 varieties of Cardimei radish.

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 are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

1. Materials and methods

Radish full red (leaf of chinese radish) hereinafter referred to as full red, expressed as green skin red meat, purchased from Beijing research and welfare agriculture (Beijing) Seiko science and technology Limited.

Radish, Jingcui No.1, hereinafter abbreviated as Jingcui No.1, is expressed as green skin white meat and is purchased from Jingyan Yinong (Beijing) Seiko science and technology Limited.

The invention constructs F by taking Mantang red (MTH-01) as a female parent and Jingcui No.1 (JC-01) as a male parent₂And (4) a group. The genome DNA of the parents and the F2 population individual is extracted by using an improved CTAB method, and the quality and the concentration of the DNA are measured. Selecting 20 individual plants with mutant character (green skin and white meat), respectively taking equal amount of DNA, and uniformly mixing to construct mutant 'super pool' F₂Mut _ type _ Bulk. Respectively carrying out sequencing on parent MTH-01, JC-01 and F by utilizing Illumina HiSeq2000 sequencing system₂Mut type Bulk was subjected to whole gene resequencing.

2. Discovery of SNP (single nucleotide polymorphism) sites closely linked with accumulation of anthocyanin in fleshy root of radish

Re-sequencing genomes of red meat and white meat materials of Xinlimei radish: respectively carrying out sequencing on parent MTH-01, JC-01 and F by utilizing Illumina HiSeq2000 sequencing system₂Mut type Bulk was subjected to whole gene resequencing.

Using the GATK analysis software, MTH-01, JC-01 and F were mapped to radish reference genomic sequences, respectively₂And carrying out SNP typing on the _Mut _ type _ Bulk to obtain 3,242,800 SNPs. The SNP locus data obtained in the typing results are more, and in order to obtain candidate loci causing the offspring differential traits, the following filtering criteria are implemented:

(1) the parental genotype is homozygous, and the SNP positions in the three samples are covered; (2) sequencing depth of parent is more than or equal to 10; (3) the sequencing depth of the filial generation is more than or equal to 10.

37,638 SNPs were obtained by co-screening in the chromosome of Yuimei radish, and the SNP-index value of each SNP was calculated. SNP-Index is the proportion of each SNP site aligned to the mutant base at the position in F2_ Mut _ type _ Bulk.

Wherein the reference genome sequence of radish is (NCBI BioProject: PRJDB707) and comprises radish genome Rs _ Scaf158 sequence (GenBank: DF 196983.1).

Screening candidate SNP loci: because the assembly of the radish reference genome is in a scaffold level, the number is large, and some scaffolds are short in length and cannot perform window sliding on SNP sites meeting the conditions. We select the highest 1% of SNP-index as the threshold for screening candidate regions of characters. And (3) respectively extending the front and the back by 10kp by taking the candidate SNP locus as a center to serve as candidate regions where the candidate SNP locus is located, and if one candidate region has overlap, combining the two candidate regions to form a larger candidate region. Based on the screening criteria, 61 candidate regions were obtained initially covering 785,125bp and containing 388 SNP markers (as shown in Table 1).

TABLE 1 statistical results for SNPs in candidate regions

To verify the availability of SNP markers within the candidate region, 150 pairs of primers specific for KASP genotyping were co-synthesized using MTH-01 (strain 3), JC-01 (strain 3) and F₂The primary screening of the single plant (5 plants) material for the marker showed that 65 primer pairs were counted in parents and F₂The single plant typing is better. Further screening the relation between the SNP loci and the fleshy root flesh color, and finding that the KASP genotyping results of the 3 SNP loci on the Rs _ Scaf158 are co-separated from the fleshy root flesh color in the F2 population. The three SNP sites were designated Rs _ Scaf158-73, Rs _ Scaf158-87, and Rs _ Scaf158-94, respectively.

The Rs _ Scaf158-73 is located at the 248277 th nucleotide on the Rs _ Scaf158 of the radish genome and is C or T, the Rs _ Scaf158-73 corresponds to the 101 th nucleotide of SEQ ID No.1 on the Rs _ Scaf158 of the radish genome, and the Y at the 101 th nucleotide in SEQ ID No.1 represents that the Rs _ Scaf can be C or T; the Rs _ Scaf158-73 genotypes were three as follows: TT (T: T), CC (C: C) and TC (T: C), wherein TT is homozygous for T at position 101 (Rs _ scan 158-73) of SEQ ID No.1, CC is homozygous for C at position 101 (Rs _ scan 158-73) of SEQ ID No.1, and TC is heterozygous for T and C at position 101 (Rs _ scan 158-73) of SEQ ID No. 1.

The Rs _ Scaf158-87 is located at the 257194 th nucleotide on the Rs _ Scaf158 of the radish genome and is T or C, the Rs _ Scaf158-87 corresponds to the 101 th nucleotide of the SEQ ID No.2 on the Rs _ Scaf158 of the radish genome, and the Y at the 101 th nucleotide in the SEQ ID No.2 represents that the Rs _ Scaf can be C or T; the genotype of Rs _ Scaf158-87 is as follows: TT (T: T), CC (C: C) and TC (T: C), wherein TT is homozygous for T at position 101 (Rs _ scan 158-87) of SEQ ID No.2, CC is homozygous for C at position 101 (Rs _ scan 158-87) of SEQ ID No.2, and TC is heterozygous for T and C at position 101 (Rs _ scan 158-87) of SEQ ID No. 2.

The Rs _ Scaf158-94 is located at the 266436 th nucleotide on the Rs _ Scaf158 of the radish genome and is C or T, the Rs _ Scaf158-94 corresponds to the 101 th nucleotide of SEQ ID No.3 on the Rs _ Scaf158 of the radish genome, and Y at the 101 th nucleotide in SEQ ID No.3 represents that the Rs _ Scaf can be C or T; the Rs _ Scaf158-20 genotypes were three as follows: TT (T: T), CC (C: C) and TC (T: C), wherein TT is homozygous for T at position 101 (Rs _ scan 158-94) of SEQ ID No.3, CC is homozygous for C at position 101 (Rs _ scan 158-94) of SEQ ID No.3, and TC is heterozygous for T and C at position 101 (Rs _ scan 158-94) of SEQ ID No. 3.

The KASP genotyping primers for Rs _ Scaf158-73, Rs _ Scaf158-87, and Rs _ Scaf158-94 are shown in Table 2, in which 5' -end of the primer is locatedGAAGGTGACCAAGTTCATGCT-3' is a FAM fluorescent tag sequence; 5 'located at the 5' end of the primerGAAGGTCGGAGTCAACGGATT-3' is a HEX fluorescent tag sequence.

Table 2 SNP loci and KASP genotyping primers closely linked to fleshy root flesh color of Xinlimei radish

3. The genotypes of the 96 Cardiosis cardinalis Rs _ Scaf158-73, Rs _ Scaf158-87 and Rs _ Scaf158-94 of Table 3 were examined

1) Detection of Rs _ Scaf158-73 genotypes of 96 Cardios radishes

96 Cardimes from Table 3 (including MTH01 and JC01, and alsoF constructed by taking MTH-01 as female parent and JC-01 as male parent₂94 individuals in the population), and performing PCR amplification and detection on the genomic DNA from leaves by using KASP genotyping primers 73-F1, 73-F2 and 73-R to determine the type of nucleotide 248277 (Rs _ Scaf158-73) of the Rs _ Scaf158 in the genome of the Xinlima radish. The Rs _ scan 158-73 genotype of each California radish was determined based on the nucleotide type at position 248277 (Rs _ scan 158-73) of each California radish genome Rs _ scan 158.

Wherein, KASP gene typing PCR reaction system: 10ng of genomic DNA, 5. mu.LKASPV 4.02 XMasterMix, 0.14. mu.LKASPP 72 Xassay mix, plus ddH₂O to 10. mu.L. The KASPV4.02 multiplied by Master Mix is a product of LGC company and consists of a fluorescent probe A, a fluorescent probe B, a quenching probe A, a quenching probe B, high-fidelity Taq enzyme, dNTP and the like. KASP 72 × assay mix was prepared at a concentration of 100. mu. mol. L^-173-F1, 73-F2 and 73-R with ddH₂Mixing O at a volume ratio of 12: 30: 46. Blank controls without adding template DNA in the reaction system are set at the same time of the test, and each PCR plate is provided with 1 blank control.

The gradient PCR reaction procedure included: denaturation at 95 deg.C for 15 min; 10 cycles of denaturation at 94 ℃ for 20s, and annealing at 61 ℃ (0.6 ℃ per cycle) for 60 s; 26 cycles, denaturation at 94 ℃ for 20s, and annealing at 55 ℃ for 60 s.

And scanning the PCR amplification product by adopting a bidirectional single-excitation plate reader PHERAStar, wherein the FAM excitation wavelength is 485nm, the emission wavelength is 520nm, the HEX excitation wavelength is 528nm, the emission wavelength is 560nm, the system reference fluorescence ROX excitation wavelength is 575nm, and the emission wavelength is 610 nm.

Analyzing the scanning data of the bidirectional single-excitation plate reader PHERAStar by adopting Kraken (TM) software: the genotype of the sample showing blue color close to the X axis is the allele linked to the FAM fluorescent tag sequence, the genotype of the sample showing red color close to the Y axis is the allele linked to the HEX fluorescent tag sequence, and the genotype of the sample showing green color in the middle is a heterozygous of both alleles. Correspondingly, if the result of the detection is that red dots close to the Y axis indicate that the Rs _ scan 158-73 genotype is T: T; if the detection result is that the blue point close to the X axis indicates that the Rs _ scan 158-73 genotype is C: C; if the result of the detection is that the green dot located in the middle region indicates that the genotype is C: T. In addition, samples that show pink color may not be clearly typed due to poor DNA quality or too low a concentration, and samples that show black color in the lower left corner are blank controls. FIG. 1 is a graph showing the results of KASP detection of the Rs _ scan 158-73 genotype of 96 Cardioma radish, whose Rs _ scan 158-73 genotype is compared with the flesh color of fleshy roots as shown in Table 3.

Genomic DNA was extracted from leaves of 96 Cardiococcus cardinalis (including parental MTH01 and JC01, and plants in 94F 2 populations) in Table 3, PCR-amplified and tested using pairs of KASP genotyping primers 87-F1, 87-F2 and 87-R, to determine the identity of nucleotide 257194 (Rs _ Scaf158-87) in the Cardiococcus cardinalis genome Rs _ Scaf 158. The Rs _ scan 158-87 genotype of each California radish was determined according to the nucleotide type at position 257194 (Rs _ scan 158-87) of each California radish genome Rs _ scan 158. Detecting by KASP method (the detection method and the determination principle refer to the records in the detection of Rs _ scan 158-73 genotypes of 96 Xinlimei radishes), and if the detection result is that red dots close to the Y axis indicate that the Rs _ scan 158-87 genotypes are C: C; if the detection result is that the blue point close to the X axis indicates that the Rs _ scan 158-87 genotype is T: T; if the result of the detection is that the green dot located in the middle region indicates that the genotype is C: T. FIG. 2 is a graph showing the results of KASP detection of the Rs _ Scaf158-87 genotype of 96 Cardioamericans, whose Rs _ Scaf158-87 genotype is compared with the flesh color of fleshy roots as shown in Table 3.

Genomic DNA was extracted from leaves of 96 Cardiococcus cardinalis (including parental MTH01 and JC01, and plants in 94F 2 population) of Table 3, PCR-amplified with pairs of KASP genotyping primers 94-F1, 94-F2 and 94-R, and the PCR products were examined by the KASP method to determine the identity of nucleotide 266436 (Rs _ Scaf158-94) of the Cardiococcus cardinalis genome Rs _ Scaf 158. The Rs scaffold 158-94 genotype of each California radish was determined based on the nucleotide type at position 266436 (Rs scaffold 158-94) of each California radish genome Rs _ scaffold 158. Detecting by KASP method (the detection method and the determination principle refer to the records in the detection of Rs _ scan 158-73 genotypes of 96 Xinlimei radishes), and if the detection result is that red dots close to the Y axis indicate that the Rs _ scan 158-94 genotypes are C: C; if the detection result is that the blue point close to the X axis indicates that the Rs _ scan 158-94 genotype is T: T; if the result of the detection is that the green dot located in the middle region indicates that the genotype is C: T. FIG. 3 is a graph showing the results of KASP detection of the Rs _ Scaf158-94 genotype of 96 Cardioma radish, whose Rs _ Scaf158-94 genotype is compared with the flesh color of fleshy roots as shown in Table 3.

The correlation between the genotype test results and the flesh color of the 96 cardioverter radishes is shown in table 3. As can be seen from Table 3, when the Rs _ scan 158-73 genotype of the Xinlimei radish is TT, the corresponding Xinlimei radish has the character of green skin and red meat; when the Rs _ scan 158-73 genotype of the Xinlimei radish is CT, the corresponding Xinlimei radish has the character of red skin and red meat; when the Rs _ scan 158-73 genotype of the Xinlimei radish is CC, the corresponding Xinlimei radish has the character of green skin and white meat. The flesh color of the fleshy root of the radish is detected by identifying the genotype of the Rs _ scaf158-73 in the radish, and the detection accuracy of the flesh color is 99 percent (except 12-96) if the factors of the skin color are not considered.

When the Rs _ Scaf158-87 genotype of the Xinlimei radish is TT, the corresponding Xinlimei radish has the character of green skin and red meat; when the Rs _ scan 158-87 genotype of the Xinlimei radish is CT, the corresponding Xinlimei radish has the character of red skin and red meat; when the Rs _ scan 158-87 genotype of the Xinlimei radish is CC, the corresponding Xinlimei radish has the character of green skin and white meat. The flesh color of the fleshy root of the radish is detected by identifying the genotype of the Rs _ scaf158-87 in the radish, and the detection accuracy of the flesh color is 99 percent (except 12-96) if the factors of the skin color are not considered.

When the Rs _ Scaf158-94 genotype of the Xinlimei radish is TT, the corresponding Xinlimei radish has the character of green skin and red meat; when the Rs _ Scaf158-94 genotype of the Xinlimei radish is TC, the corresponding Xinlimei radish has the character of red skin and red meat; when the Rs _ scan 158-94 genotype of the Xinlimei radish is CC, the corresponding Xinlimei radish has the character of green skin and white meat. The flesh color of the fleshy root of the radish is detected by identifying the genotype of Rs _ scaf158-94 in the radish, and the detection accuracy of the flesh color is 98% if the skin color factor is not considered (the genotype does not accord with the flesh color, and the genotypes are 12-96 and 12-110 in the table 3).

TABLE 3 KASP genotyping assay results for 3 SNP loci Using 96 plants

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.

Sequence listing

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Claims

The application of the Rs _ Scaf158-73 in identifying or assisting in identifying the flesh color of the Xinlimei radish; the Rs _ Scaf158-73 is a SNP locus of the radish genome, and the Rs _ Scaf158-73 is located at the 101 st nucleotide of SEQ ID No.1 on the Rs _ Scaf158 of the radish genome, and is T or C.
2. The application of the substance for detecting the polymorphism or genotype of Rs _ Scaf158-73 in the genome of the Xinli American radish in preparing and identifying or assisting in identifying the flesh color product of the Xinli American radish; the Rs _ Scaf158-73 is a SNP locus of the radish genome, and the Rs _ Scaf158-73 is located at the 101 st nucleotide of SEQ ID No.1 on the Rs _ Scaf158 of the radish genome, and is T or C:

the substance for detecting the polymorphism of the Rs _ Scaf158-73 in the genome of the Xinlimei radish is a primer pair for amplifying a radish genome DNA fragment comprising the Rs _ Scaf 158-73; the primer pair is a primer composition consisting of single-stranded DNA shown in SEQ ID No.4, single-stranded DNA shown in SEQ ID No.5 and single-stranded DNA shown in SEQ ID No. 6.
3. The method for identifying or assisting in identifying the flesh color of the Xinlimei radish comprises the steps of detecting the genotype of the Rs _ Scaf158-73 to be detected in claim 1 of the Xinlimei radish, identifying or assisting in identifying the flesh color of the Xinlimei radish according to the genotype of the Xinlimei radish to be detected:

the Rs _ scaf158-73 of claim 1, having genotype homozygous for TT, red for heart radish or red as a candidate; the Rs _ scaf158-73 of claim 1, genotype is homozygous for CC, white for heart radish or candidate white; the Rs _ craft 158-73 genotype of claim 1 is a hybrid of CT, red for heart radish or red candidate.
The application of the Rs _ Scaf158-73 in breeding of the American radish in mind, wherein the Rs _ Scaf158-73 is an SNP locus of a radish genome, and the Rs _ Scaf158-73 is located at the 101 th nucleotide of SEQ ID No.1 on the Rs _ Scaf158 of the radish genome, and is T or C.
5. Use according to claim 4, characterized in that: the breeding of the Xinlimei radish is to cultivate a green skin red meat variety.
6. The method for identifying or assisting in identifying the flesh color of the American radish according to claim 3 is applied to breeding of the American radish.
7. Use according to claim 6, characterized in that: the breeding of the Xinlimei radish is to cultivate a green skin red meat variety.
8. The breeding method of the Xinlimei radish comprises the following steps: detecting polymorphism of Rs _ Scaf158-73, Rs _ Scaf158-87 and/or Rs _ Scaf158-94 in claim 1 in the California radish genome, and selecting the California radish with the genotype of Rs _ Scaf158-73, the genotype of Rs _ Scaf158-87 and/or the genotype of Rs _ Scaf158-94 in claim 1 in the California radish genome as a parent for breeding.
9. A product is a primer composition consisting of single-stranded DNA shown in SEQ ID No.4, single-stranded DNA shown in SEQ ID No.5 and single-stranded DNA shown in SEQ ID No. 6.