SNP (Single Nucleotide Polymorphism) molecular marker for identifying hypocotyl length of non-heading Chinese cabbage and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker for identifying the length of a hypocotyl of a non-heading Chinese cabbage and application of the SNP molecular marker, and relates to the technical field of biology.The SNP molecular marker is technically characterized in that the molecular marker is SNPLHY0701, the SNPLHY0701 is located on a No.7 chromosome gene BraC07g017590 of the non-heading Chinese cabbage, an SNP site corresponds to the 119th basic group of a BraC07g017590 gene cDNA sequence, when the basic group is C, the non-heading Chinese cabbage shows a long hypocotyl, and when the basic group is C, the non-heading Chinese cabbage shows a long hypocotyl; when the basic group is T: T, the non-heading Chinese cabbage shows a short hypocotyl. The SNP molecular marker is subjected to genetic typing by applying a KASP / PARMS technology, the hypocotyl length of the non-heading Chinese cabbage can be rapidly and accurately detected, high specificity is shown, high-throughput rapid detection is facilitated, the SNP molecular marker can be simply, conveniently and rapidly applied to non-heading Chinese cabbage variety improvement molecule-assisted breeding suitable for mechanical harvesting, and the breeding efficiency is greatly improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a SNP molecular marker for identifying the length of hypocotyls of non-heading Chinese cabbage and an application thereof. Background Art

[0002] Non-heading cabbage (Brassica campestris ssp.chinensis Makino) is commonly known as green cabbage, Chinese cabbage, and rapeseed in the north. The production of non-heading cabbage, especially the harvesting, has always been a labor-intensive process, and the shortcomings of manual planting and harvesting are prominent. The future development trend of non-heading cabbage in my country is full mechanization. At present, the cultivation of non-heading cabbage has achieved mechanized operations such as ploughing, ditching, ridge making, sowing and pesticide application. Mechanized harvesting has begun to be promoted and applied, but the efficiency of mechanized harvesting is restricted by the small number of varieties suitable for mechanical harvesting and the single type, resulting in low mechanical harvesting efficiency and poor harvesting quality, which affects the external quality of non-heading cabbage. The length of the hypocotyl is the decisive factor that determines the harvesting efficiency and quality. Only by cultivating new varieties of non-heading cabbage with long hypocotyls suitable for mechanized harvesting can the problem of low efficiency of non-heading harvesting be fundamentally solved.

[0003] When conventional hybrid breeding methods are used to breed non-heading cabbage varieties with long hypocotyls, breeders need to create a large number of new hybrid germplasms and conduct field measurement and identification of hypocotyl length traits at the seedling stage. The cycle is relatively long, and the hypocotyl length is easily affected by environmental conditions, which is time-consuming and has low selection efficiency. With the rapid development of molecular biology technology and the completion of genome sequencing of non-heading cabbage, molecular marker-assisted breeding technology can be used to select hybrid individual plants in the process of non-heading cabbage germplasm creation at an early stage, and eliminate non-target trait individual plants in advance. Therefore, this field is in urgent need of developing a molecular marker for assisting the screening of non-heading cabbage long hypocotyl germplasm resources, improving breeding efficiency, and saving breeding costs. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a SNP molecular marker for identifying the length of hypocotyl of non-heading cabbage and its application.

[0005] In order to achieve the above-mentioned object, the technical scheme of the present invention is as follows: a SNP molecular marker for identifying the length of the hypocotyl of non-heading cabbage, the molecular marker is SNP_LHY0701, the SNP_LHY0701 is located on the gene BraC07g017590 on chromosome 7 of the non-heading cabbage, the SNP site corresponds to the 119th base of the cDNA sequence of the BraC07g017590 gene, when the base is C:C, the non-heading cabbage exhibits a long hypocotyl, and when the base is T:T, the non-heading cabbage exhibits a short hypocotyl.

[0006] Furthermore, the cDNA sequence of the non-heading cabbage BraC07g017590 gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2; the 119th base of the sequence shown in SEQ ID NO: 1 is C, and the 119th base of the sequence shown in SEQ ID NO: 2 is T.

[0007] Furthermore, a primer combination for amplifying the molecular marker developed based on KASP / PARMS technology is also provided, including a forward primer F1, a forward primer F2 and a reverse primer R, and the primer sequences are as follows:

[0008] Forward primer F1:

[0009] 5'-GAAGGTGACCAAGTTCATGCTGGGGAGAAAGGGAACGA GAA-3'

[0010] Forward primer F2:

[0011] 5'-GAAGGTCGGAGTCAACGGATTGGGGAGAAAGGGAACG AGAG-3'

[0012] Reverse primer R: 5'-TCCATCCCTATCCTTCTTCAAAA-3'.

[0013] Further, the application of the primer combination in detecting the length trait of the hypocotyl of non-heading Chinese cabbage is characterized in that the detection steps are as follows:

[0014] (1) extracting genomic DNA of non-heading cabbage to be tested;

[0015] (2) KASP / PARMS reaction: using the DNA from step (1) as a template, performing KASP / PARMS reaction detection using the primer combination, and adding a specific KASP Primer mix and a universal PARMS Mastermix thereto to perform PCR amplification to obtain a PCR amplification product;

[0016] (3) Analyze the PCR amplification products.

[0017] Preferably, the PCR reaction system in step (2) is carried out according to the following table (Table 1, Table 2):

[0018] Table 1 PCR amplification reaction system is as follows (PARMS mix Wuhan Jingpeptide Biology)

[0019]

[0020] Table 2 PCR amplification reaction conditions are as follows (dual-head 384 PCR instrument ABI GeneAmp 9700)

[0021]

[0022] Preferably, after the PCR in step (3) is completed, the fluorescence signal is read using a TECAN infinite M1000 microplate reader, and then the online software snpdecoder (http: / / www.snpway.com / snpdecoder / ) is used to parse and convert the fluorescence signal to obtain a clear and intuitive typing diagram, and the genotype results are output according to different colors. The C:C genotype corresponds to non-heading cabbage with long hypocotyls; the T:T genotype corresponds to non-heading cabbage with short hypocotyls; the C:T genotype is a heterozygous type.

[0023] The present invention also provides an application of a SNP molecular marker for identifying the length of the hypocotyl of non-heading Chinese cabbage in a KASP / PARMS primer, a detection reagent and a kit.

[0024] Furthermore, the SNP molecular marker, KASP / PARMS primer or detection reagent or kit can be applied to molecular marker-assisted breeding of hypocotyl length trait of non-heading Chinese cabbage.

[0025] Furthermore, the SNP molecular marker, KASP / PARMS primer or detection reagent or kit can be used in the genotyping of the hypocotyl length trait of non-heading Chinese cabbage.

[0026] Furthermore, the SNP molecular marker, KASP / PARMS primer or detection reagent or kit can be applied to the improvement of non-heading cabbage germplasm resources.

[0027] Furthermore, the SNP molecular marker, KASP / PARMS primer or reagent or kit can be used in cultivating non-heading cabbage varieties with long hypocotyls suitable for mechanized harvesting.

[0028] Compared with the prior art, this solution has the following beneficial effects:

[0029] (1) The application of the above-mentioned SNP molecular marker in the breeding of non-heading cabbage. By using the molecular marker and the detection method, a molecular marker closely linked to the hypocotyl length trait is selected, which can be used for the identification of the hypocotyl length trait of non-heading cabbage and auxiliary selection of varieties, which is of great significance for promoting the molecular assisted breeding of non-heading cabbage for mechanized harvesting. Using this marker to carry out early auxiliary selection of the trait of non-heading cabbage for mechanized harvesting can greatly improve the breeding efficiency.

[0030] (2) The present invention uses KASP / PARMS technology to perform genotyping on SNP molecular markers, which can quickly and accurately detect the hypocotyl length of non-heading cabbage, greatly improving the efficiency of germplasm detection or creation. In addition, the detection process does not require enzyme cutting, electrophoresis, sequencing, etc., which is easy to operate and conducive to high-throughput rapid detection, eliminating aerosol pollution of PCR products and EB pollution to the environment, as well as harm to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG1 is a diagram showing the genotyping results of SNP markers closely linked to the hypocotyl length trait of non-heading Chinese cabbage in a population according to an embodiment of the present invention, wherein the genotypes corresponding to the long hypocotyl lhy7.1 are clustered to the Y axis, the genotypes corresponding to the short hypocotyl D612 are clustered to the X axis, the heterozygous genotypes are clustered to the diagonal, and (A) is the genotypes of lhy7.1, D612 and F 1 and control genotyping results, (B)-(E) are (lhy7.1×D612)-F 2 Genotyping results of the population. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0033] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0034] Example 1. Development of SNP molecular markers for detecting hypocotyl length of non-heading Chinese cabbage

[0035] The long hypocotyl non-heading Chinese cabbage high-generation inbred line lhy7.1 was used as the female parent and the short hypocotyl high-generation inbred line D612 was used as the male parent. The F 2 There were 311 isolates in the population. 2The segregating population was planted in an artificial climate box with overhead LEDs, and the hypocotyl length of all materials was measured on the 7th day after sowing. Thirty extremely long hypocotyls and 30 extremely short hypocotyls were selected and DNA was extracted and mixed to construct long hypocotyl DNA pools and short hypocotyl DNA pools, respectively, and used for resequencing together with DNA from the long hypocotyl inbred line lhy7.1 and the short hypocotyl inbred line D612. Then BSA positioning analysis was performed, and a region associated with the hypocotyl length trait of non-heading cabbage on chromosome 7 was located, with a total length of 3.62Mb. In this target region, based on the parental polymorphic SNP sites, the 119th base of the gene BraC07g017590 was found to be a C / T mutation site, and specific primers that can be used for KASP / PARMS technology were designed.

[0036] The primer combination designed by the present invention for detecting the above SNP molecular markers is:

[0037] Forward primer F1:

[0038] 5'-GAAGGTGACCAAGTTCATGCTGGGGAGAAAGGGAACGA GAA-3'

[0039] Forward primer F2:

[0040] 5'-GAAGGTCGGAGTCAACGGATTGGGGAGAAAGGGAACG AGAG-3'

[0041] Reverse primer R: 5'-TCCATCCCTATCCTTCTTCAAAA-3'

[0042] The above primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. KASP / PARMS technology was used to detect the non-heading cabbage to be tested.

[0043] Example 2: Primers for amplifying SNP molecular markers based on KASP / PARMS technology and their application

[0044] 1. Primers were designed based on the molecular markers described in the present invention. The KASP / PARMS reaction was used to detect the hypocotyl length trait of non-heading cabbage with high throughput. The primer combination designed in Example 1 was used.

[0045] 2. Use the simplified CTAB method to extract genomic DNA from non-heading cabbage leaves.

[0046] (1) Take an appropriate amount of fresh or frozen non-heading cabbage leaf sample, put it into a 2 ml centrifuge tube, add two steel balls, and grind it on a tissue grinder;

[0047] (2) Add 750 μL of CTAB solution, shake and homogenize, and incubate at 65°C for 0.5-1 h;

[0048] (3) Cool to room temperature, add 750 μL of chloroform:isoamyl alcohol (24:1) in a fume hood and mix by electrosurgery 3-4 times;

[0049] (4) Centrifuge at 12000 rpm for 10 min and transfer 500 μL of the supernatant into a new 1.5 mL centrifuge tube;

[0050] (5) Add an equal volume of isopropanol solution and shake gently, precipitate at -20°C for more than 1 hour, centrifuge at 12000 rpm for 3 min, and discard the supernatant;

[0051] (6) Add 1 mL of 70% ethanol, flick the precipitate, centrifuge at 1000 rpm for 3 min, and discard the supernatant;

[0052] (7) Add 300 mL H 2 O m, dissolve and set aside.

[0053] 3. KASP / PARMS reaction test:

[0054] The KKASP / PARMS reaction was carried out in a 384-well microplate, and the reaction system was 10 μL. The reaction system is shown in Table 1. The PCR reaction conditions are shown in Table 2. After the PCR was completed, the fluorescence signal was read using a TECAN infinite M1000 microplate reader, and then the online software snpdecoder (http: / / www.snpway.com / snpdecoder / ) was used to parse and convert the fluorescence signal to obtain a clear and intuitive typing diagram, and the genotype results were output according to different colors. The results are shown in Figure 1, which is a genotyping test diagram of SNP markers closely linked to the hypocotyl length trait of non-heading cabbage in the population; among them, the C:C genotype corresponds to non-heading cabbage with long hypocotyls; the T:T genotype corresponds to non-heading cabbage with short hypocotyls; the C:T genotype is a heterozygous type, and the results in Figure 1 show that the typing effect is good.

[0055] Example 3, F 2 Identification of hypocotyl length in segregating populations and analysis of correlation between molecular markers and phenotypes

[0056] 1. All materials were planted in an overhead LED artificial climate box, and the equipment was purchased from Ningbo Yanghui Instrument Co., Ltd., model RDA-1000A. The climate conditions of the artificial climate box are: photoperiod 14h / 10h; day / night temperature: 25℃ / 20℃; relative humidity: 70%; light intensity 10000Lx. Select an appropriate amount of non-heading cabbage seeds with full grains and evenly sow them in a 105-hole tray filled with nutrient matrix (purchased from Dayi Agricultural Technology Co., Ltd., Wosong brand, 70L per bag), 1 seed per hole, and the sowing depth is relatively consistent. The surface of the seeds is covered with a layer of nutrient matrix, and the germination water is poured. After the seeds emerge, the conventional water management is carried out, and the hypocotyl length of all materials is measured on the 7th day after sowing. The distance from the base of the seedling rhizome to the cotyledon attachment point is measured using a Star brand digital display vernier caliper as the hypocotyl length (mm).

[0057] 2. Referring to the method in Example 2, the hybrid F1 of the female parent lhy7.1 and the male parent D612 was detected. 2 Hypocotyl length phenotype of 1532 plants in the segregating population. 2 The phenotypic identification results of the isolated population were highly consistent with the genotyping results. 2 There were 409 plants with the homozygous genotype C:C consistent with the long hypocotyl parent lhy7.1; 362 plants with the homozygous genotype T:T consistent with the short hypocotyl parent D612; and 761 plants with heterozygous C:T (Table 3).

[0058] Table 3 SNP_LHY0701 in F 2 Detection results in segregating populations

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] 3. For F 2 The results of the genotype-phenotype correlation test were verified by χ 2 The test analysis showed that the phenotype and genotype comparison was consistent with the theoretical separation ratio of 1:2:1 (χ 2 =3.5626<5.99, P<0.05), indicating that the molecular marker SNP_LHY0701 is closely linked to the long hypoembryo trait.

[0072] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A SNP molecular marker for identifying the length of hypocotyl of non-heading Chinese cabbage, characterized by: The molecular marker is SNP_LHY0701, which is located on the gene BraC07g017590 on chromosome 7 of non-heading cabbage. The SNP site corresponds to the 119th base of the cDNA sequence of the BraC07g017590 gene. When the base at this position is C:C, the non-heading cabbage exhibits a long hypocotyl, and when the base at this position is T:T, the non-heading cabbage exhibits a short hypocotyl.

2. The SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage according to claim 1, characterized in that: The cDNA sequence of the non-heading Chinese cabbage BraC07g017590 gene is shown in SEQ ID NO:1 or SEQ ID NO:2; the 119th base of the sequence shown in SEQ ID NO:1 is C, and the 119th base of the sequence shown in SEQ ID NO:2 is T.

3. The SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage according to claim 1, characterized in that: Also available The primer combination for amplifying the molecular marker developed based on KASP / PARMS technology includes a forward primer F1, a forward primer F2 and a reverse primer R, and the primer sequences are as follows: Forward primer F1: 5'-GAAGGTGACCAAGTTCATGCTGGGGAGAAAGGGAACGA GAA-3' Forward primer F2: 5'-GAAGGTCGGAGTCAACGGATTGGGGAGAAAGGGAACG AGAG-3' Reverse primer R: 5'-TCCATCCCTATCCTTCTTCAAAA-3'.

4. Use of the primer combination as claimed in claim 3 in detecting the length trait of hypocotyls of non-heading Chinese cabbage, characterized in that: The detection steps are as follows: Step S1, extracting genomic DNA of the non-heading Chinese cabbage to be tested; Step S2, KASP / PARMS reaction: using the DNA in step S1 as a template, performing KASP / PARMS reaction detection using the primer combination, and adding a specific KASP Primer mix and a universal PARMS Master mix thereto, performing PCR amplification, and obtaining a PCR amplification product; Step S3, analyzing the PCR amplification product; using a TECAN infinite M1000 microplate reader to read the fluorescence signal, and then using the online software snpdecoder to parse and convert the fluorescence signal to obtain a clear and intuitive typing diagram, and output the genotype result according to different colors; C: C genotype corresponds to non-heading cabbage with long hypocotyls; T: T genotype corresponds to non-heading cabbage with short hypocotyls; C: T genotype is a heterozygous type.

5. Use of the primer combination according to claim 4 in detecting the length trait of hypocotyls of non-heading Chinese cabbage, characterized in that: The PCR amplification reaction system in step S2 is as follows: 2×PARMS reaction mixture: volume 5 μL, final concentration 1×; Primer X, 10 μM: volume 0.15 μL, final concentration 150 nM; Primer Y, 10 μM: volume 0.15 μL, final concentration 150 nM; Universal primer, 10 μM: volume 0.4 μL, final concentration 400 nM; DNA template: mass 10-100ng, final concentration 10-100nM; Sterile water: make up to 10 μL; The PCR amplification reaction conditions are as follows: A dual-head 384 PCR instrument ABI GeneAmp 9700 was used; the temperature was maintained at 94°C for 15 minutes for one cycle; the temperature was maintained at 94°C for 20 seconds and then the temperature was changed to 65-57°C (-0.8°C / per cycle) and maintained for 1 minute, and the whole process was repeated 10 times; the temperature was maintained at 94°C for 20 seconds and then the temperature was changed to 57°C and maintained for 1 minute, and the whole process was repeated 32 times.

6. Application of a SNP molecular marker for identifying the length of the hypocotyl of non-heading cabbage in KASP / PARMS primers, detection reagents and kits.

7. The use of a SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage as claimed in claim 6 in KASP / PARMS primers, detection reagents and kits, characterized in that: The SNP molecular marker, KASP / PARMS primer or detection reagent or kit is applied in molecular marker-assisted breeding of hypocotyl length trait of non-heading Chinese cabbage.

8. The use of a SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage as claimed in claim 6 in KASP / PARMS primers, detection reagents and kits, characterized in that: The SNP molecular marker, KASP / PARMS primer or detection reagent or kit is used in the genotyping of the hypocotyl length trait of non-heading Chinese cabbage.

9. The use of a SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage in KASP / PARMS primers, detection reagents and kits as claimed in claim 6, characterized in that: The SNP molecular marker, KASP / PARMS primer or detection reagent or kit is applied in the improvement of non-heading Chinese cabbage germplasm resources.

10. The use of a SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage as claimed in claim 6 in KASP / PARMS primers, detection reagents and kits, characterized in that: The SNP molecular marker, KASP / PARMS primer or reagent or kit is used in cultivating non-heading Chinese cabbage varieties with long hypocotyls suitable for mechanized harvesting.

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