CN117701760A - SNP molecular marker related to bran heart shape of fruit radish meat quality and application thereof - Google Patents
SNP molecular marker related to bran heart shape of fruit radish meat quality and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular markers, and particularly relates to an SNP molecular marker related to the bran centrality of the pulp root of a fruit radish and application thereof. The invention uses radish XYB36-2 as a reference genome to find a first SNP molecular marker and a second SNP molecular marker which are closely linked with the bran heart shape of the radish meat quality root; the first SNP molecular marker corresponds to 22562437bp of chromosome 8 of the XYB36-2 radish genome, and the base is G/A; the second SNP molecular marker corresponds to 24150267bp of chromosome 8 of the XYB36-2 radish genome, and the base is C/T. Based on the SNP locus, the bran heart shape of the fleshy root of the fruit radish can be identified rapidly, stably, efficiently and with low cost, a foundation is laid for breeding of the fleshy root bran heart-resistant fruit radish, and the accuracy rate of identifying the bran heart-resistant phenotype of the fleshy root by using the SNP molecular marker provided by the invention is 85.1%.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to an SNP molecular marker related to the bran centrality of the pulp root of a fruit radish and application thereof.
Background
Radish (Raphanus sativus L.) is one of the large vegetables in our country, and the annual cultivation area reaches 1800 ten thousand mu. In recent years, green radish variety, namely fruit radish, is favored by the vegetable consumer market in China due to the characteristics of crisp and sweet taste, refreshing taste, juiciness and other fresh eating characteristics. However, in the agricultural production process, most fruit radishes are found to have bran heart diseases in the development period, the harvest period and the shelf life of the fleshy root, so that the water and nutrient substances of the fleshy root are seriously lost, and the fleshy root cannot be eaten. This physiological disease severely affects the formation of fruit radish yield and quality, limiting further development of the radish industry.
Traditional bran heart phenotype investigation means rely on destructive sampling investigation, the survival rate of the investigated materials is extremely low, and breeders are limited to cultivate bran heart-resistant fruit radish varieties by using a traditional breeding method. The development of a nondestructive phenotype investigation means has great significance for cultivating bran-heart-resistant radish varieties.
SNPs (single nucleotide polymorphisms) are the smallest unit of genomic variation, and the development of SNPs as molecular markers can identify a certain genetic trait with pertinence. The bran-heart-resistant phenotype is excavated and highly correlated with SNP, and an effective molecular marker is developed, which is important to realize the nondestructive identification of the bran-heart phenotype of the fruit radish and culture of new bran-heart-resistant fruit radish varieties.
Disclosure of Invention
The invention aims to provide the development of SNP molecular markers related to the bran heart shape of the fleshy root of the fruit radish, and the rapid, stable, efficient and low-cost identification of the bran heart shape of the fleshy root of the fruit radish lays a foundation for breeding of the bran heart-resistant fruit radish of the fleshy root on the premise of not damaging the fleshy root.
The invention provides an SNP molecular marker related to the bran centrality of the pulp root of a fruit radish, wherein the SNP molecular marker comprises a first SNP molecular marker and a second SNP molecular marker;
the first SNP molecular marker corresponds to 22562437bp of chromosome 8 of the XYB36-2 radish genome, and the base is G/A;
the second SNP molecular marker corresponds to 24150267bp of chromosome 8 of the XYB36-2 radish genome, and the base is C/T.
The invention also provides a DNA fragment related to the bran centrality of the fruit radish fleshy root, wherein the DNA fragment comprises a first DNA fragment and a second DNA fragment;
the first DNA fragment comprises a nucleotide sequence shown as SEQ ID NO.1, wherein Y is G/A;
the second DNA fragment comprises a nucleotide sequence shown as SEQ ID NO.2, wherein Y is C/T.
The invention also provides a primer combination for amplifying the SNP molecular marker or the DNA fragment in the technical scheme, wherein the primer combination comprises a first primer pair and a second primer pair;
the first primer pair comprises a first forward primer with a nucleotide sequence shown as SEQ ID NO.3, a first upstream primer with a nucleotide sequence shown as SEQ ID NO.4 and a first reverse primer with a nucleotide sequence shown as SEQ ID NO. 5;
the second primer pair comprises a second forward primer with a nucleotide sequence shown as SEQ ID NO.6, a second upstream primer with a nucleotide sequence shown as SEQ ID NO.7 and a second reverse primer with a nucleotide sequence shown as SEQ ID NO. 8.
Preferably, the 5' ends of the first forward primer and the first upstream primer are respectively connected with a fluorescent group, and the fluorescent groups connected with the first forward primer and the first upstream primer are different;
the 5 '-end of the second forward primer and the 5' -end of the second upstream primer are respectively connected with fluorescent groups, and the fluorescent groups connected with the second forward primer and the second upstream primer are different.
Preferably, the 5' end of the first forward primer is linked to a fluorescent group FAM; the 5' end of the first upstream primer is connected with a fluorescent group HEX;
the 5' -end of the second forward primer is connected with a fluorescent group FAM; the 5' end of the second upstream primer is linked to a fluorescent group HEX.
The invention also provides application of the primer combination in the technical scheme in preparation of the SNP molecular marker KASP parting product for detection.
The invention also provides a kit for detecting the SNP molecular marker according to the technical scheme, and the kit comprises the primer combination according to the technical scheme.
The invention also provides application of the SNP molecular marker or the DNA fragment or the primer combination or the kit in identifying the fleshy root bran-heart-resistant fruit radish.
The invention also provides application of the SNP molecular marker or the DNA fragment or the primer combination or the kit in auxiliary breeding of the bran-heart-resistant fruit radish of the fleshy root.
The invention also provides a method for identifying the bran-heart-resistant fruit radish with the fleshy root, which comprises the following steps:
taking DNA of a fruit radish sample to be detected as a template, and carrying out PCR amplification by using the first primer pair in the primer combination in the technical scheme to obtain a first PCR amplification product;
taking DNA of a fruit radish sample to be detected as a template, and carrying out PCR amplification by using a second primer pair in the primer combination in the technical scheme to obtain a second PCR amplification product;
if the first PCR amplification product corresponds to the 22562437-bit deoxyribonucleotide pair of the XYB36-2 radish genome chromosome 8 and is A and the second PCR amplification product corresponds to the 24150267-bit deoxyribonucleotide pair of the XYB36-2 radish genome chromosome 8 and is T, the fruit radish sample to be detected is bran-heart-resistant fruit radish.
The beneficial effects are that:
the invention takes Beijing-research green-Xiu radish and green-like jade radish as parents and utilizes an Illumina Hiseq 2500 sequencing platform to pair parent materials and F 2 The population extreme phenotype pool materials were individually subjected to whole genome re-sequencing. Analyzing the polymorphism of the amphipathic SNP locus by taking radish XYB36-2 as a reference genome (http:// brassica db. Cn /), and finding out the SNP locus closely linked with the bran heart shape of the radish fleshy root, wherein the SNP locus comprises a first SNP molecular marker and a second SNP molecular marker; the first SNP molecular marker corresponds to 22562437bp of chromosome 8 of the XYB36-2 radish genome, and the base is G/A; the second SNP molecular markerThe nucleotide is C/T, which corresponds to 24150267bp of chromosome 8 of XYB36-2 radish genome. Based on the SNP locus, the bran heart shape of the fleshy root of the fruit radish can be identified rapidly, stably, efficiently and with low cost, and a foundation is laid for breeding of the bran heart-resistant fruit radish of the fleshy root. The results of the examples show that the accuracy of identifying the bran-heart-resistant phenotype of the fleshy root by using the SNP molecular marker provided by the invention is 85.1%.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a radish fleshy root bran heart grading standard; wherein, the red square frame indicates the bran center part, and the scale is 5cm;
FIG. 2 shows the results of identifying the bran heart phenotype of green, e.g., yu-radish, beijing-developed Luxiu radish and their progeny; wherein A is green radix Raphani, and F is radix Raphani 1 The selfing line has bran heart phenotype, P74 is Beijing-research green-Xiu radish, P75 is green-like Yu radish, and the scale is 5cm; b is F of green radix Raphani and Jing-Shang green radix Raphani 2 Separating bran core grade distribution results of genetic groups;
FIG. 3 is a graph showing the results of BSA pool sequencing mining control of the correspondence of the bran-heart-resistant key regulatory sites of the fruit radish fleshy root in example 2;
FIG. 4 shows genotyping results of KASP genotyping PCR method on different genetic materials; wherein, A is the P08SNP3 parting result, the polymerized Y-axis red mark sample is A homozygous, the polymerized X-axis blue mark sample is G homozygous, the polymerized middle green sample is A G heterozygous; b is the P08SNP4 typing result, the polymerized Y-axis red marked sample is T:T homozygous, the polymerized X-axis blue marked sample is C:C homozygous, and the polymerized middle green sample is G:T heterozygous.
Detailed Description
The invention provides an SNP molecular marker related to the bran centrality of the pulp root of a fruit radish, wherein the SNP molecular marker comprises a first SNP molecular marker and a second SNP molecular marker;
the first SNP molecular marker corresponds to 22562437bp of chromosome 8 of the XYB36-2 radish genome, and the base is G/A;
the second SNP molecular marker corresponds to 24150267bp of chromosome 8 of the XYB36-2 radish genome, and the base is C/T.
The invention also provides a DNA fragment related to the bran centrality of the fruit radish fleshy root, wherein the DNA fragment comprises a first DNA fragment and a second DNA fragment;
the first DNA fragment comprises a nucleotide sequence shown as SEQ ID NO.1, wherein Y is G/A;
the second DNA fragment comprises a nucleotide sequence shown as SEQ ID NO.2, wherein Y is C/T.
The invention also provides a primer combination for amplifying the SNP molecular marker or the DNA fragment in the technical scheme, wherein the primer combination comprises a first primer pair and a second primer pair;
the first primer pair comprises a first forward primer with a nucleotide sequence shown as SEQ ID NO.3, a first upstream primer with a nucleotide sequence shown as SEQ ID NO.4 and a first reverse primer with a nucleotide sequence shown as SEQ ID NO. 5;
the second primer pair comprises a second forward primer with a nucleotide sequence shown as SEQ ID NO.6, a second upstream primer with a nucleotide sequence shown as SEQ ID NO.7 and a second reverse primer with a nucleotide sequence shown as SEQ ID NO. 8.
In the present invention, the 5' -ends of the first forward primer and the first upstream primer are preferably linked to fluorescent groups, respectively, and the fluorescent groups to which the first forward primer and the first upstream primer are linked are different. The 5' end of the first forward primer is preferably connected with a fluorescent group FAM; the 5' end of the first upstream primer is preferably linked to a fluorescent group HEX. The invention connects the fluorescent group at the 5' end of the first forward primer and the first upstream primer, and can be used for KASP typing detection.
In the present invention, the 5' -ends of the second forward primer and the second upstream primer are preferably linked to fluorescent groups, respectively, and the fluorescent groups to which the second forward primer and the second upstream primer are linked are different. The 5' end of the second forward primer is preferably connected with a fluorescent group FAM; the 5' end of the second upstream primer is preferably linked to a fluorescent group HEX. The invention connects the fluorescent groups at the 5' terminal of the second forward primer and the second upstream primer respectively, and can be used for KASP typing detection.
The nucleotide sequence of the fluorescent group FAM is preferably shown as SEQ ID NO. 9; the nucleotide sequence of the fluorescent group HEX is preferably shown in SEQ ID NO. 10.
The nucleotide sequence information of SEQ ID NO. 1-10 of the present invention is specifically as follows:
SEQ ID NO.1:5'-ATTCTCAACCCGTTTTTCTCATATATCTTATTTTATCTT TTCAACATTCTCTAGCTAAAAATGCCTTTAGAGTCTGTTGTATACCCGCAAGATCCACTCGYTTACCTCTCCACTTGCAAAGATTTCACGTTCCAAGATCTGTACTATCAAGAAGAGGTAGTAGTAGCTCAAGACACGAAGAACAACATTAACAAGTTAGGG-3'; wherein Y is G/A;
SEQ ID NO.2:5'-TCGATCATCACCGGTCGTCTCTCCACGTGTACCGGA GGTGTGAGAGAGGATCCTTAAAAACATACCAAAAGAAGCCGAGACACCATTTTCAAATCAGATTYGATGGCGACGATCCCTCCCCAATTCCCCTTGGAGATCCGCTCTGCTCTACGTCGGGCGGCGGCTTCCTCCACCGTCTACTTCCTCCGCCCAATCACCACC-3'; wherein Y is C/T;
SEQ ID NO.3：5'-AAATCTTTGCAAGTGGAGAGGTAAC-3'；
SEQ ID NO.4：5'-GAAATCTTTGCAAGTGGAGAGGTAAT-3'；
SEQ ID NO.5：5'-CTTTAGAGTCTGTTGTATACCCGCAAG-3'；
SEQ ID NO.6：5'-CGAGACACCATTTTCAAATCAGATTC-3'；
SEQ ID NO.7：5'-CCGAGACACCATTTTCAAATCAGATTT-3'；
SEQ ID NO.8：5'-AAGGGGAATTGGGGAGGGATCG-3'；
SEQ ID NO.9：5'-GAAGGTGACCAAGTTCATGCT-3'；
SEQ ID NO.10：5'-GAAGGTCGGAGTCAACGGATT-3'；
the invention also provides application of the primer combination in the technical scheme in preparation of the SNP molecular marker KASP parting product for detection. In the present invention, the KASP typing product preferably comprises a kit.
The invention also provides a kit for detecting the SNP molecular marker according to the technical scheme, and the kit comprises the primer combination according to the technical scheme. In the present invention, the kit further preferably includes a fluorophore probe, a fluorophore-quenching probe, a high-fidelity DNA synthetase, and dNTPs.
The SNP molecular marker or the DNA fragment provided by the invention is closely related to the bran heart shape of the fleshy root of the fruit radish, and in view of the effect of the SNP molecular marker or the DNA fragment provided by the invention, the application of the SNP molecular marker or the DNA fragment or the primer combination or the kit in the identification of the fleshy root bran-heart-resistant fruit radish and the application of the SNP molecular marker or the DNA fragment or the primer combination or the kit in the auxiliary breeding of the fleshy root bran-heart-resistant fruit radish belong to the protection scope of the invention
The invention also provides a method for identifying the bran-heart-resistant fruit radish with the fleshy root, which comprises the following steps:
taking DNA of a fruit radish sample to be detected as a template, and carrying out PCR amplification by using the first primer pair in the primer combination in the technical scheme to obtain a first PCR amplification product;
taking DNA of a fruit radish sample to be detected as a template, and carrying out PCR amplification by using a second primer pair in the primer combination in the technical scheme to obtain a second PCR amplification product;
if the first PCR amplification product corresponds to the 22562437-bit deoxyribonucleotide pair of the XYB36-2 radish genome chromosome 8 and is A and the second PCR amplification product corresponds to the 24150267-bit deoxyribonucleotide pair of the XYB36-2 radish genome chromosome 8 and is T, the fruit radish sample to be detected is bran-heart-resistant fruit radish.
The invention uses the DNA of the fruit radish sample to be detected as a template, and uses the first primer pair in the primer combination to carry out PCR amplification to obtain a first PCR amplification product. In the present invention, when the 5' -end of the first forward primer and the first upstream primer are respectively ligated to a fluorescent group, the reaction system of PCR amplification is preferably 10. Mu.L in terms of 50ng of radish genomic DNA, 5. Mu.L of KASP V4.02X Master mix, 0.14. Mu.L of KASP 72X assaymix and the balance of ddH 2 O. The inventionThe KASP V4.02XMasterMix preferably includes a fluorophore probe, a fluorescence quenching probe, a high fidelity DNA synthetase, and dNTPs. The KASP 72X assay mix of the present invention preferably comprises 100. Mu. Mol.L -1 Forward primer, 100. Mu. Mol.L -1 Upstream primer, 100. Mu. Mol.L -1 Reverse primer and ddH 2 O, preferably in a volume ratio of 12:12:30:46; the reaction procedure for PCR amplification preferably comprises: pre-denaturation at 95℃for 15min; denaturation at 94℃for 20s, annealing at 61℃for 60s (0.6℃per cycle), 10 cycles; denaturation at 94℃for 20s, annealing at 55℃for 60s,26 cycles.
The invention uses the DNA of the fruit radish sample to be detected as a template, and uses the second primer pair in the primer combination to carry out PCR amplification to obtain a second PCR amplification product. The reaction system and procedure for PCR amplification according to the present invention are described above and will not be described here.
The invention can judge the bran heart phenotype of the fleshy root of the fruit radish sample to be detected based on the first PCR amplification product and the second PCR amplification product, and specifically: if the first PCR amplification product corresponds to the XYB36-2 radish genome chromosome 8 and the 22562437-bit deoxyribonucleotide pair is A: A, and the second PCR amplification product corresponds to the XYB36-2 radish genome chromosome 8 and the 24150267-bit deoxyribonucleotide pair is T: T, the fruit radish sample to be detected is bran-heart-resistant fruit radish, and the rest results are bran-heart-resistant fruit radish. The invention preferably makes a determination based on the fluorescence results, in particular: if the fluorescent signal of the first PCR amplification product is red and the fluorescent signal of the second PCR amplification product is blue, the fruit radish sample to be detected is a bran-heart-resistant fruit radish, and the rest results are all bran-heart fruit radishes. In the specific implementation of the invention, the description of radish germplasm resources is published by the national agricultural crop germplasm resources platform (http:// www.cgris.net) preferably, and the center of radish bran is divided into 5 grades. Level 0: no bran core; stage 1: the center of the fleshy root has a few scattered, punctiform or strip-shaped whitish tissues; 3 stages: tissue of the fleshy root, about 1/3 of which is outwards from the center, is in a whitened state; 5 stages: tissue of the fleshy root, about 2/3 of the center outwards, is in a whitened state; 7 stages: the whole fleshy root is completely bran-centered. Of these, the bran heart grade 0 and grade 1 phenotypes are considered bran heart resistant phenotypes, and bran heart grade 3 to grade 7 are considered bran heart phenotypes.
For further explanation of the present invention, the following describes in detail a SNP molecular marker related to the bran-heart shape of the pulp root of fruit radish and its application, with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Evaluation standard for bran heart characteristics of meat quality root of fruit radish
Bran phenotype investigation was performed on 57 fruit radish planting resources stored in the institute of vegetable research at the institute of agriculture and forestry, beijing city. The bran core levels of the fruit radishes were divided into 5 grades according to the reference specification (fig. 1):
bran core 0 grade: bran-free heart tissue;
bran core 1 grade: the center of the fleshy root has a few scattered, punctiform or strip-shaped whitish tissues;
bran core 3 grade: tissue of the fleshy root, about 1/3 of which is outwards from the center, is in a whitened state;
bran core 5 grade: tissue of the fleshy root, about 2/3 of the center outwards, is in a whitened state;
bran core 7 grade: the whole fleshy root is completely bran-cored;
of these, the bran heart grade 0 and grade 1 phenotypes are considered bran heart resistant phenotypes, and bran heart grade 3 to grade 7 are considered bran heart phenotypes.
2. Based on the evaluation criteria of step 1, an isolated genetic population was constructed with jingyangxiu radish (bran heart variety, bran heart grade 7) and green like Yu radish (bran heart resistant variety, bran heart grade 0) as parents, wherein jingyangxiu radish (hereinafter this material will be denoted as P74) and green like Yu radish (hereinafter this material will be denoted as P75) were both purchased from jingyangyun (beijing) seed company. By the method of F 2 Bran heart phenotype investigation of isolated population found F 1 The plant of the generation presents bran heart 3-level phenotype, F 2 The bran heart phenotype of the individual plants of the segregating genetic population exhibited different grade distributions (figure 2). Therefore, the bran center of the radish fleshy root is the quantitative genetic character controlled by the dominant gene.
Example 2
Control of digging key QTL (quantitative trait locus) site of bran-heart-resistant radish fleshy root
1. To explore the genetic mechanism of bran heart controlling radish meat quality, F in example 1 2 The genetic population was isolated for BSA pool sequencing analysis. From 354 parts F 2 In the segregation genetic population, 20 single plants with extreme bran heart (7 grade) and extreme bran heart (0 grade) phenotypes are selected respectively, equal amounts of genome DNA are taken and evenly mixed, and an extreme bran heart phenotype mixed pool pp1 and an extreme bran heart phenotype mixed pool pp2 are constructed;
2. extraction of different radish genetic Material leaf genomic DNA Using CTAB method and ddH 2 O dissolves whole genome DNA; p74, P75, pp1 and pp2 genomic DNA samples were resequenced using Illumina Hiseq 2500 sequencing platform. And analyzing the polymorphism of the amphipathic SNP locus by taking radish XYB36-2 as a reference genome (http:// brassica db. Cn /), and respectively obtaining 4.82, 5.03, 6.56 and 6.82Gb clean reads, wherein the sequencing depth reaches 12.6×, 13.1×, 17.1×and17.8×, and the data volume meets the subsequent analysis requirements.
3. The frequency of variation of SNP sites between genomes of various genetic materials was analyzed by GATK (v 3.8) software. First, the SNP variant sites were filtered according to the following criteria: 1. filtering SNP loci with multiple genotypes, and only reserving two-level genotype loci; 2. filtering SNP loci with mixed pool reads support less than 4; 3. filtering out SNP loci with homozygous and consistent genotypes among the mixed pools; 4. SNP sites that are homozygous and inconsistent for non-two parents are filtered out. Finally, 641047 high-quality SNP loci are obtained. Subsequently, the high quality SNP site variation frequency between pools was analyzed by SNP-index algorithm, and it was found that the delta SNP-index value was significantly higher than 0.5 within 22090000 ～ 25400000 interval (3.31 Mb) of radish No.8 chromosome (corresponding to XYB36-2 radish gene) alone (P <0.001; FIG. 3). Therefore, a main QTL interval that controls the bran-resistant heart of the fleshy root exists in chromosome 8 of radish.
Example 3
SNP locus for rapidly identifying bran-resistant heart phenotype of fruit radish fleshy root by excavation
1. Based on the conclusion of example 2, it was further found that 18538 SNP sites coexist in the bran-resistant interval of the control radish fleshy root, in which the number of SNP sites having a ΔSNP-index of more than 0.5 and a P value of less than 0.001 is 6551. In order to develop reliable SNP molecular markers for detecting bran-heart-resistant phenotype, 22562437 and 24150267 high-quality SNP loci are selected on two sides of a main-effect regulatory region of a radish 8 chromosome according to a radish reference genome (radish XYB36-2 genome) sequence for cloning verification. Specific primers were designed 100bp upstream and downstream of each SNP site, and specific primer information is shown in Table 1.
TABLE 1 primers designed based on SNP loci
Note that: the underlined part of the primer is FAM fluorescent tag sequence, and the bolded part is HEX fluorescent tag sequence.
2. The genome DNA of the amphiphilic material is used as a template, and DNA fragments containing specific SNP are amplified by using high-fidelity polymerase. The PCR products were sequenced and analyzed, and the results are shown in Table 2.
TABLE 2 amplification results for different materials
As can be seen from Table 2, the 22562437 nucleotide pair of chromosome 8 is G: G homozygote in the P74 material, A: A homozygote in the P75 material, and the SNP locus is named as P08SNP3 (the first SNP molecular marker corresponding locus); the 24150267 nucleotide pair of chromosome 8 is C:C homozygous in the P74 material, T:T homozygous in the P75 material, and the SNP locus is named as P08SNP4 (the second SNP molecular marker corresponding locus).
3. To examine whether the SNP locus can be developed into a reliable molecular marker, the KASP genotyping PCR method was used for the detection of P74, P75 and F 1 Generation (2 strains) and F 2 Extreme phenotype individual (extreme resistance 4 strainAnd extreme bran 4 strains) material for genotyping analysis;
KASP genotyping PCR reaction system: 50ng of radish genomic DNA, 5. Mu.L of KASP V4.02 XMastermix, 0.14. Mu.L of KASP 72 Xassaymix, add ddH 2 O to 10 μl (suitable for 96-well plates). Wherein KASPV 4.02XMasterMix is LGC company product, and comprises FAM fluorescent group probe (excitation wavelength is 485nm, emission wavelength is 520 nm), HEX fluorescent group probe (excitation wavelength is 528nm, emission wavelength is 560 nm), FAM fluorescence quenching probe, HEX fluorescence quenching probe, high-fidelity DNA synthetase, dNTP, etc. KASP 72X assay mix is prepared from a concentration of 100. Mu. Mol.L -1 100. Mu. Mol.L -1 Upstream primer, 100. Mu. Mol.L -1 Reverse primer and ddH 2 O, according to the volume ratio of 12:12:30:46, mixing and preparing; setting 1 blank negative control for each batch of PCR reaction;
KASP genotyping PCR reaction procedure: pre-denaturation at 95℃for 15min; denaturation at 94℃for 20s, annealing at 61℃for 60s (0.6℃per cycle), 10 cycles; denaturation at 94℃for 20s, annealing at 55℃for 60s,26 cycles;
and collecting fluorescent signals of KASP genotyping PCR products under different excitation wavelength conditions by using a two-way single excitation plate reader PHERAstar. PHERAstar was scanned by Kraken (TM) software and analyzed for specific SNP site polymorphisms. Wherein, the homozygous allele type sample connected with the FAM fluorescence label sequence presents blue mark, and the detection sample is polymerized near the X axis; the homozygous allele type sample connected with the HEX fluorescent tag sequence presents a red mark, and the detection sample is polymerized near the Y axis; the heterozygous allele type sample presents a green mark, and the detection sample is polymerized between the X axis and the Y axis; pink marks represent samples in which the amplified products are not clearly typed; black marks represent negative control groups.
The KASP genotyping PCR results showed that the SNP site P08SNP3 and P08SNP4 detection primers were able to accurately genotype different genetic materials (FIG. 4). G homozygosity or A G heterozygosity of the P08SNP3 locus G, and the phenotype of the plant is bran heart grade 7; the locus is A, A is homozygous, and the phenotype of the fleshy root is bran heart grade 0. P08SNP4 locus C is C homozygous or T is C heterozygous, the phenotype of the fleshy root is bran heart 7 grade, the locus is T homozygous, and the phenotype of the plant is bran heart 0 grade. The SNP locus P08SNP3 locus A: A homozygote and the P08SNP4 locus T: T homozygote can be used as molecular markers for identifying the bran-heart resistant phenotype of the succulent root of the fruit radish, so that the molecular markers are respectively named as SNP molecular markers P08SNP3[ A: A ] and P08SNP4[ T: T ];
if the P08SNP3 locus is G:G homozygote and the P08SNP4 locus is C:C homozygote, the material is bran heart phenotype; if the P08SNP3 locus is G: A heterozygote, the P08SNP4 locus is C: C homozygous or C: T heterozygote or G: G homozygous, the material is bran heart phenotype; if the P08SNP3 locus is A: A homozygous and the P08SNP4 locus is T: T homozygous, the material is bran-heart resistant.
Example 4
Application evaluation of SNP molecular markers P08SNP3[ A: A ] and P08SNP4[ T: T ] for detecting bran-resistant heart phenotype of fruit radish
For defining SNP molecular marker P08SNP3[ A: A]And P08SNP4[ T: T ]]The accuracy of the bran-heart resistant phenotype of the fleshy root of the fruit radish is identified, and F is identified 2 The individuals of the segregating population (354) were subjected to genotype and bran heart phenotype association analysis. At F 2 In the segregating population, 74 single plants with P08SNP3 locus and P08SNP4 locus being homozygous are 74 plants, the fleshy root presents bran-resistant phenotype as 63 plants, the fleshy root presents bran-resistant phenotype as 11 plants, P08SNP3[ A]And P08SNP4[ T: T ]]The accuracy of the dual-marker phenotype identification was 85.1% (Table 3).
TABLE 3 fruit radish F 2 Analysis of bran heart phenotype and genotype of meat quality root of segregating population
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According to the above, the molecular markers P08SNP3[ A: A ] and P08SNP4[ T: T ] developed by the invention are closely related to the bran heart phenotype of the radish fleshy root, and the detection of the bran heart resistant phenotype of the radish fleshy root based on the molecular markers P08SNP3[ A: A ] and P08SNP4[ T: T ] has high success rate of identification, can be applied to large-scale screening of the bran heart resistant phenotype of the fruit radish under the non-damage condition, can also be applied to molecular marker assisted selection, and can be used for rapidly screening excellent breeding materials from backcross and isolated groups.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. A SNP molecular marker related to the bran heart shape of the fleshy root of a fruit radish, which is characterized in that the SNP molecular marker comprises a first SNP molecular marker and a second SNP molecular marker;
the first SNP molecular marker corresponds to 22562437bp of chromosome 8 of the XYB36-2 radish genome, and the base is G/A;
the second SNP molecular marker corresponds to 24150267bp of chromosome 8 of the XYB36-2 radish genome, and the base is C/T.
2. A DNA segment associated with the bran-heart shape of the pulp of fruit radishes, characterized in that said DNA segment comprises a first DNA segment and a second DNA segment;
the first DNA fragment comprises a nucleotide sequence shown as SEQ ID NO.1, wherein Y is G/A;
the second DNA fragment comprises a nucleotide sequence shown as SEQ ID NO.2, wherein Y is C/T.
3. Amplifying the SNP molecular marker of claim 1 or the primer combination of the DNA fragment of claim 2, the primer combination comprising a first primer pair and a second primer pair;
the first primer pair comprises a first forward primer with a nucleotide sequence shown as SEQ ID NO.3, a first upstream primer with a nucleotide sequence shown as SEQ ID NO.4 and a first reverse primer with a nucleotide sequence shown as SEQ ID NO. 5;
the second primer pair comprises a second forward primer with a nucleotide sequence shown as SEQ ID NO.6, a second upstream primer with a nucleotide sequence shown as SEQ ID NO.7 and a second reverse primer with a nucleotide sequence shown as SEQ ID NO. 8.
4. The primer combination of claim 3, wherein the 5' ends of the first forward primer and the first upstream primer are each attached to a fluorescent group, and wherein the fluorescent groups to which the first forward primer and the first upstream primer are attached excite different fluorescent colors;
the 5 '-end of the second forward primer and the 5' -end of the second upstream primer are respectively connected with a fluorescent group, and the fluorescent groups connected with the second forward primer and the second upstream primer are different in fluorescence color.
5. The primer combination of claim 4 wherein the 5' end of the first forward primer is attached to a fluorescent group FAM; the 5' end of the first upstream primer is connected with a fluorescent group HEX;
the 5' -end of the second forward primer is connected with a fluorescent group FAM; the 5' end of the second upstream primer is linked to a fluorescent group HEX.
6. Use of the primer combination according to claim 4 or 5 for the preparation of a product for detecting the SNP molecular marker KASP typing according to claim 1.
7. A kit for detecting the SNP molecular marker as set forth in claim 1, characterized in that the kit comprises the primer combination as set forth in any one of claims 3 to 5.
8. Use of the SNP molecular marker of claim 1 or the DNA fragment of claim 2 or the primer combination of any one of claims 3-5 or the kit of claim 7 for identifying a fleshy root resistant bran-heart fruit radish.
9. Use of the SNP molecular marker of claim 1 or the DNA fragment of claim 2 or the primer combination of any one of claims 3-5 or the kit of claim 7 in auxiliary breeding of fleshy root bran-heart-resistant fruit radishes.
10. A method for identifying a fleshy root bran-heart-resistant fruit radish, which is characterized by comprising the following steps:
carrying out PCR amplification by using the DNA of the fruit radish sample to be detected as a template and using the first primer pair in the primer combination of any one of claims 3-5 to obtain a first PCR amplification product;
carrying out PCR amplification by using the DNA of the fruit radish sample to be detected as a template and using the second primer pair in the primer combination according to any one of claims 3-5 to obtain a second PCR amplification product;
if the first PCR amplification product corresponds to the 22562437-bit deoxyribonucleotide pair of the XYB36-2 radish genome chromosome 8 and is A and the second PCR amplification product corresponds to the 24150267-bit deoxyribonucleotide pair of the XYB36-2 radish genome chromosome 8 and is T, the fruit radish sample to be detected is bran-heart-resistant fruit radish.
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