CN113817865B

CN113817865B - SNP molecular marker related to leaf low-temperature yellowing on Chinese cabbage A09 chromosome and application thereof

Info

Publication number: CN113817865B
Application number: CN202111287487.2A
Authority: CN
Inventors: 汪维红; 张凤兰; 于拴仓; 苏同兵; 温常龙; 张建; 李佩荣; 辛晓云; 余阳俊; 张德双; 赵岫云
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2021-11-02
Filing date: 2021-11-02
Publication date: 2022-03-22
Anticipated expiration: 2041-11-02
Also published as: CN113817865A

Abstract

The invention relates to the field of molecular marker assisted breeding, in particular to an SNP molecular marker related to leaf low-temperature yellowing on a Chinese cabbage A09 chromosome and application thereof. The invention discloses an application of a substance for detecting polymorphism or genotype of site A0945007157 in identification or auxiliary identification of cabbage low temperature resistance, wherein the site A0945007157 is an SNP site in a cabbage genome, and is the 101 th nucleotide of SEQ ID No.4, and the nucleotide type is C or G. By making a pair F₂The accuracy rate of the marker can reach 100% through verification of 187 single plants in the population, and the marker can be used for low-temperature-resistant molecular marker-assisted breeding of Chinese cabbages. The molecular marker obtained by the invention has low cost, high flux and high specificity in practical application, and provides an efficient auxiliary breeding method and technology for accelerating the low-temperature resistant breeding of the Chinese cabbage.

Description

SNP molecular marker related to leaf low-temperature yellowing on Chinese cabbage A09 chromosome and application thereof

Technical Field

The invention relates to the field of molecular marker assisted breeding, in particular to an SNP molecular marker related to leaf low-temperature yellowing on a Chinese cabbage A09 chromosome and application thereof.

Background

Chlorophyll is one of the most important pigments in the process of plant photosynthesis and plays an important role in the growth and development of plants. During the evolution process of the plants, a series of complex mechanisms are formed to accurately regulate and control the biosynthesis of chlorophyll, and finally, leaf color mutants such as albino, etiolated, evergreen, dark green, light green, stripes and the like are generated. The functional gene and the molecular regulation mechanism of the leaf color mutation are deeply excavated, which has important significance for improving the photosynthetic efficiency and increasing the yield of crops.

Brassica species crops of Brassicaceae Brassica include Brassica rapa subspecies, Brassica rapa subspecies and Brassica rapa subspecies. Leaf color mutants generally exist in brassica crops, but few reports are made about temperature-induced leaf color mutants, particularly low-temperature-induced leaf color mutants. The photosynthesis of the etiolation mutant under low-temperature induction is quickly weakened, the growth of the plant is slowed down and even stopped, and the serious yield reduction is caused. With the continuous expansion of the cultivation area of Chinese cabbage vegetables in winter and spring, the breeding of low temperature resistant varieties is particularly urgent. So far, a large number of important turnip trait genes such as clubroot resistance, bolting resistance and the like are widely applied to the breeding of white vegetables, so that the disease resistance and stress resistance breeding process of the white vegetables is accelerated, and the important contribution is made to the development of the cabbage industry. At the same time, turnip also has some undesirable traits, and these undesirable traits are often introduced into other brassica vegetables as well as excellent traits. Therefore, the retention of excellent traits and the elimination of unfavorable traits will be the focus in the future.

The marker-assisted selection using molecular markers linked to target traits is a very effective method in genetic breeding. The SNP marker belongs to a new generation of molecular marker, and has the characteristics of high abundance, easy realization of automation of detection and the like. The SNP (single nucleotide polymorphism) genotyping detection based on KASP (competitive allele specific PCR) is a high-throughput SNP genotyping technology developed by LGC (laboratory of the great chemistry) company in the United kingdom, has the characteristics of accuracy, flexibility, low cost and high throughput, is one of the mainstream methods for SNP analysis internationally at present, and is widely applied to auxiliary breeding of high-throughput molecular markers of crops.

Disclosure of Invention

The invention aims to provide an SNP molecular marker related to leaf low-temperature yellowing on a Chinese cabbage A09 chromosome and application thereof.

To achieve the above object, the present invention provides, in a first aspect, the use of a substance for detecting a polymorphism or genotype at the A0945007157 site as defined in A1) to A6) below,

A1) identifying or assisting in identifying the low-temperature resistance of the Chinese cabbage;

A2) preparing a product for identifying or assisting in identifying the low-temperature resistance of the Chinese cabbage;

A3) screening or auxiliary screening of low-temperature-resistant Chinese cabbages;

A4) preparing and screening or auxiliary screening products of the low-temperature resistant Chinese cabbage;

A5) auxiliary breeding of Chinese cabbage;

A6) preparing a product for assisting in breeding the Chinese cabbage;

the site A0945007157 is a SNP site in the genome of Chinese cabbage, and is the 101 th nucleotide of SEQ ID No.4, and the nucleotide type is C or G.

Further, in the application, the substance for detecting the polymorphism or genotype at the A0945007157 site can be D1), D2) or D3) as follows:

D1) the substance for detecting the polymorphism or genotype of the site A0945007157 contains a PCR primer for amplifying the Chinese cabbage genome DNA fragment including the site A0945007157;

D2) the substance for detecting the polymorphism or genotype of the A0945007157 site can be a PCR reagent containing the PCR primer;

D3) a kit containing the PCR primer described in D1) or the PCR reagent described in D2).

Further, in the above application, the PCR primer may be P1 or P2:

p1, wherein the PCR primer is a primer group consisting of single-stranded DNA with the nucleotide sequence of 22 th to 44 th sites of a sequence 1 in a sequence table, single-stranded DNA with the nucleotide sequence of 22 th to 44 th sites of a sequence 2 in the sequence table and single-stranded DNA with the nucleotide sequence of a sequence 3 in the sequence table;

and P2, wherein the PCR primer is a primer group consisting of a single-stranded DNA shown in a sequence 1 in a sequence table, a single-stranded DNA shown in a sequence 2 in the sequence table and a single-stranded DNA shown in a sequence 3 in the sequence table.

In a second aspect, the invention provides a method for identifying or assisting in identifying the low-temperature resistance of a Chinese cabbage, which comprises the steps of detecting the genotype of the A0945007157 site of the Chinese cabbage to be detected, identifying or assisting in identifying the low-temperature resistance of the Chinese cabbage according to the genotype of the Chinese cabbage to be detected, wherein the A0945007157 site is an SNP site in the genome of the Chinese cabbage, is the 101 th nucleotide of SEQ ID No.4, and the nucleotide type is C or G; the genotype is CC genotype, GG genotype or CG genotype; the CC genotype represents that the nucleotide type of the A0945007157 site in the genome of the Chinese cabbage is homozygote C, and the GG genotype represents that the nucleotide type of the A0945007157 site in the genome of the Chinese cabbage is homozygote G; the CG genotype indicates that the nucleotide type of the A0945007157 site in the Chinese cabbage genome is a heterozygous type of C and G;

the resistance of the Chinese cabbage to be detected with the CC genotype to low temperature is lower than that of the Chinese cabbage to be detected with the GG genotype or the CG genotype or is candidate to be lower than that of the Chinese cabbage to be detected with the CC genotype.

In a third aspect, the invention provides a method for breeding Chinese cabbage, which comprises the steps of selecting the Chinese cabbage with the genotype of the A0945007157 site of GG or CG as a parent to breed, wherein the A0945007157 site is an SNP site in the genome of the Chinese cabbage, is the 101 th nucleotide of SEQ ID No.4, and the nucleotide type is C or G, and the GG genotype represents the homozygous type of the nucleotide type of the A0945007157 site of G in the genome of the Chinese cabbage; the CG genotype indicates that the nucleotide type of the A0945007157 site in the Chinese cabbage genome is a heterozygous type of C and G.

In a fourth aspect, the present invention provides a product containing the substance for detecting a polymorphism or genotype at the a0945007157 site described above, which may be any of products C1) -C3):

C1) detecting single nucleotide polymorphism or genotype products related to the low-temperature resistance of the Chinese cabbage;

C2) identifying or assisting in identifying the products of the low-temperature resistance of the Chinese cabbage;

C3) is used for the auxiliary breeding of Chinese cabbage.

Further, in the product, the substance for detecting the polymorphism or genotype at the A0945007157 site can be D1), D2) or D3) as follows:

D1) the substance for detecting the polymorphism or genotype of the site A0945007157 contains a PCR primer for amplifying the DNA fragment of the Chinese cabbage and/or turnip genome including the site A0945007157;

D2) the substance for detecting the polymorphism or genotype of the site A0945007157 is a PCR reagent containing the PCR primer;

Further, in the product, the PCR primer can be a primer group consisting of single-stranded DNA with a nucleotide sequence of 22 th to 44 th positions of SEQ ID No.1 in the sequence table, single-stranded DNA with a nucleotide sequence of 22 nd to 44 th positions of SEQ ID No.2 in the sequence table and single-stranded DNA with a nucleotide sequence of SEQ ID No.3 in the sequence table.

Further, in the above product, the PCR primer may be a primer set consisting of a single-stranded DNA represented by SEQ ID No.1 of the sequence Listing, a single-stranded DNA represented by SEQ ID No.2 of the sequence Listing, and a single-stranded DNA represented by SEQ ID No.3 of the sequence Listing.

In the invention, the Chinese cabbage can be obtained by hybridizing Chinese cabbage and turnip.

Further, the Chinese cabbage may be filial generation of Chinese cabbage SD and turnip MRT (Y), such as F₁Generation, F₂And (4) generation.

Wherein, the Chinese cabbage SD is disclosed in the literature "Tongbing S, Shuancang Y, Zhenping G, et al, evaluating multiple resistance to major or diseases in a core set of the interrupted lines of Brassica rapa at a seed stage [ J ]. Journal of Plant Pathology 2018.100: 457-465."; the turnip MRT (Y) is obtained by selfing 4 generations of turnip material Melon Red Top and separating, and the turnip material Melon Red Top (No. 11017) is derived from a Beijing vegetable research germplasm resource library.

Any one of the A0945007157C/G sites is 101 th nucleotide from 5' end of a sequence 4 in a sequence table in a Chinese cabbage genome, and the A0945007157C/G site is C/G polymorphism.

Any one of the low temperature resistant Chinese cabbages may be a Chinese cabbage satisfying the following description: sowing the Chinese cabbage seeds in a sunlight greenhouse (greenhouse), controlling the temperature at 4-15 ℃ in the daytime and the temperature at-2-4 ℃ at night, and investigating true leaf color when the seedlings grow to the stage of 2 true leaves, wherein the true leaf color is green.

Any one of the above-mentioned low temperature intolerant cabbages may specifically be a cabbage satisfying the following description: sowing the Chinese cabbage seeds in a sunlight greenhouse (greenhouse), controlling the temperature at 4-15 ℃ in the daytime and the temperature at-2-4 ℃ at night, and investigating true leaf color when the seedlings grow to 2 true leaf stages, wherein the true leaf color is yellow.

In the present invention, the product may be a reagent or a kit.

The breeding aim comprises breeding or selecting the low temperature resistant Chinese cabbage.

The low temperature may be-2 ℃ to 4 ℃.

In the above applications, methods and products, the PCR primers may or may not be labeled with a label. The label refers to any atom or molecule that can be used to provide a detectable effect and that can be attached to a nucleic acid. Labels include, but are not limited to, dyes; a radioactive label, such as 32 p; binding moieties such as biotin (biotin); haptens such as Digoxin (DIG); a luminescent, phosphorescent, or fluorescent moiety; and a fluorescent dye alone or in combination with a portion of the emission spectrum that can be suppressed or shifted by Fluorescence Resonance Energy Transfer (FRET). Labels can provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Labels can be charged moieties (positive or negative) or alternatively, can be charge neutral. The label may comprise or be combined with a nucleic acid or protein sequence, provided that the sequence comprising the label is detectable. In some embodiments, the nucleic acid is detected directly (e.g., direct sequence read) without a label.

Experiments prove that the selection accuracy of the molecular marker (SNP locus) A0945007157C/G locus and the corresponding primer after low-temperature induction is 100%. Therefore, the molecular marker (SNP locus) A0945007157C/G locus and the corresponding primer provided by the invention are beneficial to screening of low-temperature-resistant Chinese cabbage germplasm resources, can be used for molecular marker-assisted selective breeding, and provide material storage and technical support for genetic improvement of the low-temperature-resistant Chinese cabbage.

The invention discloses a BSA-Seq-based main effect QTL which is located on an A09 chromosome and is tightly linked with cabbage low-temperature yellowing. Developing 1 SNP marker closely linked with the low-temperature yellowing of the Chinese cabbage by pairing F₂The accuracy rate of the marker can reach 100% through verification of 187 single plants in the population, and the marker can be used for low-temperature-resistant molecular marker-assisted breeding of Chinese cabbages. The molecular marker obtained by the invention has low cost, high flux and high specificity in practical application, and provides an efficient auxiliary breeding method and technology for accelerating the low-temperature resistant breeding of the Chinese cabbage.

Drawings

FIG. 1 is a diagram of the genotyping result of the A0945007157C/G locus in the Chinese cabbage material to be tested.

FIG. 2 is a major QTL obtained based on BSA-Seq and located on chromosome A09 and closely linked with cabbage low-temperature yellowing.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Chinese cabbage SD is disclosed in the document "Tongbing S, Shuancang Y, Zhenping G, et al. evaluating multiple reactions to major diseases in a core set of organized lines of Brassica rapa at a seed stage [ J ]. Journal of Plant Pathology 2018.100: 457-.

In the invention, the turnip MRT (Y) is obtained by self-crossing 4 generations of turnip material Melon Red Top, and the turnip material Melon Red Top (number 11017) is derived from Beijing vegetable research germplasm resource library (the website is http://123.127.162.62:9999/Web/index. html).

In the embodiment of the invention, the method for detecting whether the plant to be detected is low temperature resistant (namely whether the plant to be detected is yellowed after low temperature treatment) comprises the following steps: sowing seeds of a plant to be detected in a sunlight greenhouse (greenhouse), controlling the temperature at 4-15 ℃ in the daytime and the temperature at-2-4 ℃ at night, investigating true leaf color when seedlings grow to the stage of 2 true leaves, and if the true leaf color is yellow, the plant to be detected is not resistant to low temperature (low-temperature yellowing), and if the true leaf color is green, the plant to be detected is resistant to low temperature (low-temperature non-yellowing).

Examples 1 and F₂Preparation of the Generation population

The Chinese cabbage SD is not yellow at low temperature. Turnip MRT (Y) is obtained by self-crossing 4 generations of turnip material Melon Red Top (No. 11017) and is detected as a material which is not resistant to low temperature (low temperature yellowing).

Hybridizing with Chinese cabbage material SD as female parent and turnip material MRT (Y) as male parent to obtain F₁Generation of seed, F₁The plant grown by the seed generation is F₁And (5) plant generation. F₁Selfing the plant to obtain F₂Generation of seed, F₂The plant grown by the seed generation is F₂And (5) plant generation.

Example 2 Low-temperature yellowing trait Gene mapping and SNP molecular marker development

2.1、F₂Population (F from example 1)₂Generation plant composition), counting the yellowing and non-yellowing plants of leaves after low-temperature treatment, respectively selecting single plants of 30 low-temperature yellowing plants and 30 low-temperature non-yellowing plants to construct a low-temperature yellowing pool and a low-temperature non-yellowing pool for re-sequencing.

2.2, obtaining 1 main effect QTL which is tightly linked with the low-temperature yellowing of the Chinese cabbage on an A09 chromosome by adopting a BSA-Seq method, further developing an SNP molecular marker according to an SNP locus obtained by resequencing in the interval, and finally obtaining an SNP locus A0945007157 (the 101 th nucleotide in a sequence 4 of a sequence table) which is tightly linked with the low-temperature yellowing character and is a C/G polymorphism, and naming the SNP locus as a A0945007157 locus as shown in figure 2.

Based on the competitive allele-specific PCR principle, primers were designed for the A0945007157 site as follows:

an upstream primer A0945007157C/G-FF (sequence 1 of the sequence table):

5'-GAAGGTGACCAAGTTCATGCTACTTGAGGAAGAAGAAGCCAAGC-3' (where the FAM fluorescent tag sequence is underlined);

upstream primer A0945007157C/G-FV (sequence 2 in sequence table):

5'-GAAGGTCGGAGTCAACGGATTACTTGAGGAAGAAGAAGCCAAGG-3' (where the HEX fluorescent tag sequence is underlined);

the downstream primer A0945007157C/G-R (sequence 3 of the sequence table): 5'-GTTAATTATCAAATAGGCTATGAGCAATAAAAGG-3' are provided.

The single-stranded DNA molecule shown in SEQ ID No.1 and SEQ ID No.3 amplifies the fragment of which the 101 st nucleotide in the sequence 4 of the sequence table is C, and an instrument can read a fluorescent signal of a fluorescent group combined with the FAM sequence;

the single-stranded DNA molecules shown in SEQ ID No.2 and SEQ ID No.3 amplify the fragment with the 101 st nucleotide G in the sequence 4 of the sequence table, and an instrument can read the fluorescent signal of the fluorescent group combined with the HEX sequence.

The last nucleotide C/G of the upstream primer corresponds to the SNP site of 45007157 th (C/G) on A09 chromosome, namely the 101 th nucleotide in the sequence 4 of the sequence table.

Example 3, application of A0945007157C/G locus in identifying yellowing and non-yellowing materials of leaves after low-temperature induction of Chinese cabbage

3.1 DNA extraction

The genome DNA of 190 Chinese cabbage materials to be detected in the table 1 is respectively extracted by a conventional CTAB method.

190 parts of material as parent SD (P in Table 1)₂) Parent MRT (Y) (P in Table 1)₁)、F₁And F₂Passage 187 individuals.

Agarose electrophoresis and Nanodrop2100 are used for respectively detecting the quality of the extracted DNA, and the extracted genomic DNA meets the related quality requirements, namely, the agarose electrophoresis shows that the DNA band is single and is not obviously dispersed; the Nanodrop2100 detects A260/280 between 1.8 and 2.0 (DNA sample is not polluted by protein); a260/230 is between 1.8 and 2.0 (the concentration of salt ions in the DNA sample is low); no significant light absorption at 270nm (no phenol contamination of the DNA sample); the amount of DNA used for detection by the competitive allele-specific PCR technique is 4-10ng per sample. The concentration of the diluted DNA was 10 ng/. mu.l for use, to obtain a test DNA.

3.2 competitive allele-specific PCR

This was done according to the standard experimental protocol (based on competitive allele-specific PCR technology) provided by LGC (Laboratory of the Goverment Chemist Government Chemist Laboratory) Inc., of the United kingdom. The reagents used were all provided as kits by LGC company, except for the specific instructions, and the amounts, usage, and overall experimental procedures were in accordance with LGC company's operating guidelines GenetypingAssay, Manual Part #15004070 Rev.B. The reaction was carried out in 1536 microwell plates.

The method comprises the following specific steps: first, 1.5. mu.l of the template solution was added to a 1536-well reaction plate using a K-pette dispensing station, and dried at 60 ℃ for 30min (dry box, LGC Co.). Then under a Kraken operating system, a Meridian sample adding workstation is used for respectively adding 1 microliter of reaction system into each reaction hole, and after subpackaging is finished, the microporous plates are sequentially placed on a Kube heat sealing instrument and a Fusion laser film sealing instrument for sealing films.

Reaction system: mixing 10 mu M A0945007157A1 solution, 10 mu M A0945007157A2 solution and 10 mu M A0945007157C solution in sequence according to the volume ratio of 6:6:15 to obtain primer premix; 2 XMASTER mix (Part No. KBS-1016-Asonic 011), primer premix and water are mixed evenly according to the volume ratio of 36:1: 36. Wherein, the composition of the 10 mu M A0945007157A1 solution is as follows: 4.82nmol of the primer shown in sequence 1 in the sequence Listing was added to 482ul of ddH₂O, uniformly mixing (the primer is synthesized by actual companies, and the finished product is dry powder); the composition of the 10 μ M A0945007157A2 solution was as follows: 4.79nmol of the primer shown in sequence 2 of the sequence Listing was added to 479ul of ddH₂O, mixing uniformly; the composition of the 10 μ M A0945007157C solution was as follows: 6.25nmol of the primer shown in sequence 1 of the sequence Listing was added to 625ul of ddH₂And O, and mixing uniformly. PCR reaction in a high-flux water bath system Hydrocycleer, the specific procedure is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20 seconds, renaturation extension for 1min (first cycle 61 ℃, 0.6 ℃ reduction per cycle, last cycle 55 ℃); denaturation at 94 ℃ for 20 seconds, extension at 55 ℃ for 60 seconds, 26 cycles. After the amplification is finished, a BMG PHERAStar instrument is used for detecting a fluorescence signal and checking the typing condition.

The results of the reaction system are shown in FIG. 1. In FIG. 1, A represents CC genotype (red fluorescence), B represents CG genotype (green fluorescence), and C represents GG genotype (blue fluorescence).

The CC genotype indicates that the nucleotide type of the A0945007157 site in the Chinese cabbage genome is homozygote C; the GG genotype represents that the nucleotide type of the A0945007157 site in the Chinese cabbage genome is homozygote G; the CG genotype indicates that the nucleotide type of the A0945007157 site in the Chinese cabbage genome is a heterozygous type of C and G.

The CC genotype is provided with FAM fluorescent markers and shows red fluorescence; GG genotype has HEX fluorescent label and shows blue fluorescence, and CG genotype shows green fluorescence.

The result shows that the typing effect is good, and the primer can effectively identify whether the genotype of a tested plant based on the SNP locus is CC genotype, GG genotype or CG genotype.

Chinese cabbage SD (shown as "P" in Table 1)₁") the genotypes based on the A0945007157 sites were GG, turnip MRT (Y) (indicated as" P "in Table 1)₂") genotype based on A0945007157 was CC, F₁Representative (shown as "F" in Table 1)₁") the genotype based on A0945007157 was CG. 187 Strain F₂The genotype results at the A0945007157 site of the progeny plants (denoted "1-187" in Table 1) are shown in Table 1.

3.3 detection of yellowing after Low temperature Induction

According to the following method, whether the 190 Chinese cabbage materials to be detected in the table 1 are low temperature resistant (namely whether the Chinese cabbage materials are yellowed after low temperature treatment) is detected. Sowing seeds of a plant to be detected in a sunlight greenhouse (greenhouse), controlling the temperature at 4-15 ℃ in the daytime and the temperature at-2-4 ℃ at night, investigating true leaf color when seedlings grow to the stage of 2 true leaves, and if the true leaf color is yellow, the plant to be detected is not resistant to low temperature (low-temperature yellowing), and if the true leaf color is green, the plant to be detected is resistant to low temperature (low-temperature non-yellowing).

The results are shown in Table 1.

Combining the results in table 1, the following conclusions can be drawn: if the genotype of the site A0945007157 of the Chinese cabbage to be detected is CC genotype (C: C in the table 1), the Chinese cabbage to be detected is not resistant to low temperature (leaf yellowing after low-temperature induction); if the genotype of the A0945007157 site of the Chinese cabbage to be detected is GG genotype (shown as G: G in table 1) or CG genotype (shown as C: G in table 1), the Chinese cabbage to be detected is resistant to low temperature (leaves are not yellowed after low-temperature induction).

Among 144 parts of materials with phenotype of non-yellowing leaves after low-temperature induction, 37 parts of materials with molecular marker identification A0945007157C/G loci being homozygous GG and 107 parts of heterozygous C/G loci, and the identification accuracy rate is 100%. In 43 materials with phenotype of leaf yellowing after low-temperature induction, all A0945007157C/G sites are CC homozygous, and the identification accuracy rate is 100%. The average identification accuracy of the method of the invention is 100%.

TABLE 1 187 parts of F₂Genotyping and phenotype statistical table of population material at site A0945007157

The results show that the method and the molecular marker can be used for detecting whether the leaves of the Chinese cabbage to be detected are yellow after low-temperature induction.

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

<110> agriculture and forestry academy of sciences of Beijing City

SNP molecular marker related to leaf low-temperature yellowing on chromosome of <120> Chinese cabbage A09 and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gaaggtgacc aagttcatgc tacttgagga agaagaagcc aagc 44

<210> 2

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gaaggtcgga gtcaacggat tacttgagga agaagaagcc aagg 44

<210> 3

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gttaattatc aaataggcta tgagcaataa aagg 34

<210> 4

<211> 201

<212> DNA

<213> cabbage (Brassica rapa)

<400> 4

tacaatttat gttgtttcaa tggcaaggta gggatataag cctacatgga cactataaaa 60

ttataataga agactctgac ttgaggaaga agaagccaag saccatgcta ccttttattg 120

ctcatagcct atttgataat taactattct aattaaggtg ttgattggtt aaaacctgtc 180

caagtacatc tcaagctctt t 201

Claims

1. The use of a substance for detecting polymorphism or genotype at the A0945007157 site in A1) to A6) described below,

A5) auxiliary breeding of Chinese cabbage;

A6) preparing a product for assisting in breeding the Chinese cabbage,

the site A0945007157 is a SNP site in the genome of Chinese cabbage, is the 101 th nucleotide of SEQ ID No.4, and the nucleotide type is C or G,

the Chinese cabbage is obtained by hybridizing Chinese cabbage and turnip.

2. Use according to claim 1, characterized in that: the substance for detecting the polymorphism or genotype of the A0945007157 site is D1), D2) or D3) as follows:

D2) the substance for detecting the polymorphism or genotype of the site A0945007157 is a PCR reagent containing the PCR primer D1);

3. Use according to claim 2, characterized in that: the PCR primer is P1 or P2:

p1, wherein the PCR primer is a primer group consisting of a primer with a nucleotide sequence of 22 th to 44 th sites of a sequence 1 in a sequence table, a primer with a nucleotide sequence of 22 th to 44 th sites of a sequence 2 in the sequence table and a primer with a nucleotide sequence of 3 in the sequence table;

p2, the PCR primer is a primer group consisting of a primer with a nucleotide sequence shown as a sequence 1 in the sequence table, a primer with a nucleotide sequence shown as a sequence 2 in the sequence table and a primer with a nucleotide sequence shown as a sequence 3 in the sequence table.

4. The method for identifying or assisting in identifying the low-temperature resistance of the Chinese cabbage is characterized by comprising the following steps of: the method comprises the steps of detecting the genotype of a A0945007157 site of a Chinese cabbage to be detected, and identifying or assisting in identifying the low-temperature resistance of the Chinese cabbage according to the genotype of the Chinese cabbage to be detected, wherein the A0945007157 site is an SNP site in the genome of the Chinese cabbage, is the 101 th nucleotide of SEQ ID No.4, and has the nucleotide type of C or G; the genotype is CC genotype, GG genotype or CG genotype; the CC genotype represents that the nucleotide type of the A0945007157 site in the genome of the Chinese cabbage is homozygote C, and the GG genotype represents that the nucleotide type of the A0945007157 site in the genome of the Chinese cabbage is homozygote G; the CG genotype indicates that the nucleotide type of the A0945007157 site in the Chinese cabbage genome is a heterozygous type of C and G;

the resistance of the Chinese cabbage to be detected with the CC genotype to low temperature is lower than or is lower than the candidate of the Chinese cabbage to be detected with the GG genotype or the CG genotype;

the Chinese cabbage is obtained by hybridizing Chinese cabbage and turnip.

5. The Chinese cabbage breeding method is characterized by comprising the following steps: the method comprises the steps of selecting a Chinese cabbage with an A0945007157 locus genotype of GG or CG as a parent to breed, wherein the A0945007157 locus is an SNP locus in the genome of the Chinese cabbage, is the 101 th nucleotide of SEQ ID No.4, and has the nucleotide type of C or G, and the GG genotype represents the homozygosity of the A0945007157 locus in the genome of the Chinese cabbage, wherein the nucleotide type of the A0945007157 locus is G; the CG genotype indicates that the nucleotide type of the A0945007157 site in the Chinese cabbage genome is a heterozygous type of C and G;

the Chinese cabbage is obtained by hybridizing Chinese cabbage and turnip.

6. The product containing the substance for detecting polymorphism or genotype at site A0945007157 as claimed in claim 1 or 2, which is any one of products C1) -C3):

C3) a product for auxiliary breeding of Chinese cabbage;

the Chinese cabbage is obtained by hybridizing Chinese cabbage and turnip.

7. The product of claim 6, wherein: the substance for detecting the polymorphism or genotype of the A0945007157 site is D1), D2) or D3) as follows:

8. The product of claim 7, wherein: the PCR primer is a primer group consisting of a primer with a nucleotide sequence of 22 th to 44 th sites of a sequence 1 in a sequence table, a primer with a nucleotide sequence of 22 th to 44 th sites of a sequence 2 in the sequence table and a primer with a nucleotide sequence of 3 in the sequence table.

9. The product according to claim 7 or 8, characterized in that: the PCR primer is a primer group consisting of a primer with a nucleotide sequence shown as a sequence 1 in a sequence table, a primer with a nucleotide sequence shown as a sequence 2 in the sequence table and a primer with a nucleotide sequence shown as a sequence 3 in the sequence table.