A SNP molecular marker for identifying hypocotyl length of brassica rapa and application thereof

Abstract

The application discloses a SNP molecular marker for identifying hypocotyl length of Brassica campestris L. var. and application thereof, and relates to the technical field of biotechnology.The molecular marker is SNP_LHY0701, which is located on a gene BraC07g017590 of the 7th chromosome of the Brassica campestris L.var., and a SNP site corresponds to a 119th base of a cDNA sequence of the gene BraC07g017590; when the base is C:C, the Brassica campestris L.var. shows long hypocotyls; and when the base is T:T, the Brassica campestris L.var. shows short hypocotyls.The KASP / PARMS technology is applied to gene typing of the SNP molecular marker, the hypocotyl length of the Brassica campestris L.var. can be quickly and accurately detected, the method has strong specificity, high-throughput and rapid detection can be achieved, the method can be simply and quickly applied to molecular-assisted breeding of the Brassica campestris L.var. suitable for mechanical harvesting, and the breeding efficiency is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage and its application. Background Technology

[0002] Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino), commonly known as bok choy, is a labor-intensive crop, especially during harvesting, highlighting the limitations of manual cultivation and harvesting. The future development trend for non-heading Chinese cabbage in my country is full mechanization. Currently, mechanized operations have been implemented in cultivation, including tilling, ditching, ridging, sowing, and pesticide application. Mechanized harvesting is also being promoted, but the limited variety and type suitable for mechanized harvesting hinder efficiency, resulting in low harvesting efficiency and poor quality, affecting the external quality of the cabbage. The length of the hypocotyl is a decisive factor in determining harvesting efficiency and quality. Only by cultivating new non-heading Chinese cabbage varieties with long hypocotyls suitable for mechanized harvesting can the problem of low harvesting efficiency be fundamentally solved.

[0003] When using conventional hybridization breeding methods to select non-heading Chinese cabbage varieties with long hypocotyls, breeders need to create a large number of new hybrid germplasm and conduct field measurements and identification of the hypocotyl length trait during the seedling stage. This process is relatively lengthy, and hypocotyl length is easily affected by environmental conditions, making it time-consuming and inefficient. With the rapid development of molecular biology techniques and the completion of genome sequencing for non-heading Chinese cabbage, molecular marker-assisted breeding technology can perform early selection of hybrid plants during the germplasm creation process, eliminating plants with non-target traits in advance. Therefore, there is an urgent need in this field to develop a molecular marker to assist in screening germplasm resources of non-heading Chinese cabbage with long hypocotyls, thereby improving breeding efficiency and saving breeding costs. Summary of the Invention

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

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: an SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage, the molecular marker being SNP_LHY0701, which is located on the gene BraC07g017590 on chromosome 7 of non-heading Chinese cabbage. The SNP site corresponds to the 119th base of the cDNA sequence of the BraC07g017590 gene. When the base at this location is C:C, non-heading Chinese cabbage exhibits a long hypocotyl; when the base at this location is T:T, non-heading Chinese cabbage exhibits a short hypocotyl.

[0006] Furthermore, 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.

[0007] Furthermore, a primer combination for amplifying the molecular marker based on KASP / PARMS technology is also provided, including forward primer F1, forward primer F2, and reverse primer R, with the primer sequences 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] Furthermore, the application of the primer combination in detecting the hypocotyl length trait of non-heading Chinese cabbage is characterized by the following detection steps:

[0014] (1) Extract genomic DNA from the non-heading Chinese cabbage to be tested;

[0015] (2) KASP / PARMS reaction; using the DNA from step (1) as a template, KASP / PARMS reaction detection was performed using the primer combination described above, and a specific KASP Primer mix and a universal PARMS Mastermix were added to it for PCR amplification to obtain PCR amplification products;

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

[0017] Preferably, the PCR reaction system in step (2) is performed according to the instructions in the following tables (Table 1 and Table 2):

[0018] Table 1 shows the PCR amplification reaction system (PARMS mix, Wuhan Jingtai Biotechnology).

[0019]

[0020] Table 2 shows the PCR amplification reaction conditions (ABI GeneAmp 9700 dual-head 384 PCR instrument).

[0021]

[0022] Preferably, after PCR in step (3), the fluorescence signal is read using a TECAN Infinite M1000 microplate reader, and then the converted fluorescence signal is analyzed using the online software snpdecoder (http: / / www.snpway.com / snpdecoder / ) to obtain a clear and intuitive genotyping diagram. The genotype results are output according to the different colors. The C:C genotype corresponds to a long hypocotyl in non-heading Chinese cabbage; the T:T genotype corresponds to a short hypocotyl in non-heading Chinese cabbage; the C:T genotype is heterozygous.

[0023] This invention also provides the application of an SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage in KASP / PARMS primers, detection reagents, and kits.

[0024] Furthermore, the SNP molecular markers, KASP / PARMS primers, detection reagents, or kits can be applied to marker-assisted breeding of hypocotyl length traits in non-heading Chinese cabbage.

[0025] Furthermore, the SNP molecular markers, KASP / PARMS primers, detection reagents, or kits can be applied to the genotyping of hypocotyl length traits in non-heading Chinese cabbage.

[0026] Furthermore, the SNP molecular markers, KASP / PARMS primers, detection reagents, or kits can be applied to the improvement of non-heading Chinese cabbage germplasm resources.

[0027] Furthermore, the SNP molecular markers, KASP / PARMS primers, reagents, or kits can be applied to the cultivation of organically harvestable, long hypocotyl-based, non-heading Chinese cabbage varieties.

[0028] Compared with existing technologies, the beneficial effects of this solution are:

[0029] (1) The above-mentioned SNP molecular markers are used in the breeding of non-heading Chinese cabbage. By using the molecular markers and detection methods, molecular markers closely linked to the hypocotyl length trait can be selected. These markers can be used for the identification of hypocotyl length trait in non-heading Chinese cabbage and for auxiliary selection of varieties. This is of great significance for promoting molecular-assisted breeding of non-heading Chinese cabbage that is suitable for mechanized harvesting. Using these markers to carry out early auxiliary selection of non-heading Chinese cabbage that is suitable for mechanized harvesting can greatly improve breeding efficiency.

[0030] (2) This invention applies KASP / PARMS technology to genotype SNP molecular markers, which can rapidly and accurately detect the hypocotyl length of non-heading Chinese cabbage, greatly improving the efficiency of germplasm detection or creation. Moreover, the detection process does not require enzyme digestion, electrophoresis, and sequencing, making it easy to operate, conducive to high-throughput rapid detection, and eliminating aerosol pollution from PCR products and environmental pollution from EB, as well as harm to the human body. Attached Figure Description

[0031] Figure 1 shows the genotyping results of SNP markers closely linked to the hypocotyl length trait of non-heading Chinese cabbage in the population in an embodiment of the present invention. The genotypes corresponding to the long hypocotyl lhy7.1 are clustered on the Y-axis, the genotypes corresponding to the short hypocotyl D612 are clustered on the X-axis, and the heterozygous genotypes are clustered on the diagonal. (A) shows the genotyping results of lhy7.1, D612, F1 and control, and (B)-(E) show the genotyping results of the (lhy7.1×D612)-F2 population. Detailed Implementation

[0032] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0033] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

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

[0035] Using the long hypocotyl non-heading Chinese cabbage inbred line lhy7.1 as the female parent and the short hypocotyl high-generation inbred line D612 as the male parent, a segregating F2 population of 311 plants was obtained through hybridization and self-pollination. The lhy7.1, D612, and F2 segregating populations were planted in an artificial climate chamber with a top-mounted LED display. Hypocotyl length of all materials was measured on day 7 after sowing. DNA was extracted from 30 plants with extremely long hypocotyls and 30 plants with extremely short hypocotyls, and the DNA was mixed to construct long hypocotyl DNA pools and short hypocotyl DNA pools, respectively. These pools, along with DNA from the long hypocotyl inbred line lhy7.1 and the short hypocotyl inbred line D612, were used for resequencing. BSA mapping analysis then located a region on chromosome 7 associated with the hypocotyl length trait in non-heading Chinese cabbage, with a total length of 3.62 Mb. Based on the parental polymorphic SNP sites in this target region, the 119th base of 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 in this invention for detecting the above-mentioned SNP molecular markers is as follows:

[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 primers were synthesized by Shanghai Sangon Biotech Co., Ltd. The non-heading Chinese cabbage samples were then analyzed using KASP / PARMS technology.

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

[0044] 1. Primers designed based on the molecular markers described in this invention are used to detect the hypocotyl length trait of non-heading Chinese cabbage in high throughput using the KASP / PARMS reaction. The primer combination designed in Example 1 is adopted.

[0045] 2. Genomic DNA was extracted from non-heading Chinese cabbage leaves using a simplified CTAB method.

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

[0047] (2) Add 750 μL of CTAB solution, shake to 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) into a fume hood and mix with an electrosurgical excision apparatus 3-4 times;

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

[0050] (5) Add an equal volume of isopropanol solution and shake gently. Precipitate at -20℃ for more than 1 hour. Centrifuge at 12000 rpm for 3 min and discard the supernatant.

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

[0052] (7) Add 300mL H2O m and dissolve for later use.

[0053] 3. KASP / PARMS reaction test:

[0054] The KKASP / PARMS reaction was performed in 384 microplates with a reaction volume of 10 μL. The reaction volume is shown in Table 1. PCR reaction conditions are shown in Table 2. After PCR, the fluorescence signal was read using a TECAN Infinite M1000 microplate reader, and then the converted fluorescence signal was analyzed using the online software snpdecoder (http: / / www.snpway.com / snpdecoder / ) to obtain a clear and intuitive genotyping diagram. The genotype results were output according to the 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 in non-heading Chinese cabbage. The C:C genotype corresponds to a long hypocotyl in non-heading Chinese cabbage; the T:T genotype corresponds to a short hypocotyl in non-heading Chinese cabbage; and the C:T genotype is heterozygous. The results in Figure 1 show good genotyping performance.

[0055] Example 3: Identification of hypocotyl length and correlation analysis of molecular markers and phenotypes in the F2 segregating population

[0056] 1. All materials were grown in a top-mounted LED artificial climate chamber. The equipment, model RDA-1000A, was purchased from Ningbo Yanghui Instrument Co., Ltd. The climate conditions of the artificial climate chamber were set as follows: photoperiod 14h / 10h; day / night temperature: 25℃ / 20℃; relative humidity: 70%; light intensity: 10000Lx. An appropriate amount of plump, non-heading Chinese cabbage seeds were selected and evenly sown in 105-cell trays filled with nutrient substrate (purchased from Dayi Agricultural Technology Co., Ltd., Wosong brand, 70L per bag), one seed per cell, with a relatively uniform sowing depth. The seeds were covered with a layer of nutrient substrate, and sufficient water was applied for germination. After germination, routine water management was implemented. The hypocotyl length of all materials was measured on the 7th day after sowing. The distance from the base of the seedling rhizome to the cotyledon attachment point was measured using a SATA digital vernier caliper and recorded as the hypocotyl length (mm).

[0057] 2. Following the method described in Example 2, the hypocotyl length phenotype of 1532 individual plants from the F2 segregating population resulting from the cross between the maternal parent lhy7.1 and the paternal parent D612 was examined. The phenotypic identification results of individual plants in the F2 segregating population highly corresponded to the genotyping results. In the F2 population, a total of 409 plants were homozygous for the long hypocotyl parent lhy7.1 with the C:C genotype; 362 plants were homozygous for the short hypocotyl parent D612 with the T:T genotype; and a total of 761 plants were C:T heterozygous (Table 3).

[0058] Table 3. Detection results of SNP_LHY0701 in the F2 segregating population.

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] 3. The χ² test was used to validate the correlation between genotype and phenotype in the F2 population. 2 Test analysis, based on phenotypic and genotypic comparisons, shows a segregation ratio consistent with the theoretical ratio of 1:2:1 (χ²). 2 =3.5626<5.99, P<0.05), indicating that the molecular marker SNP_LHY0701 is closely linked to the long hypoembryonic trait.

[0072] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. The application of a primer combination of SNP molecular markers for detecting hypocotyl length in non-heading Chinese cabbage in the detection of hypocotyl length trait in 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 Chinese cabbage. The SNP site corresponds to the 119th base of the BraC07g017590 gene cDNA sequence. When the base at this position is C:C, non-heading Chinese cabbage exhibits a long hypocotyl; when the base at this position is T:T, non-heading Chinese cabbage exhibits a short hypocotyl. 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.

2. The application of the primer combination of the SNP molecular marker for detecting hypocotyl length in non-heading Chinese cabbage as described in claim 1 in the detection of hypocotyl length trait in non-heading Chinese cabbage, characterized in that: Also available The primer combination developed based on KASP / PARMS technology for amplifying the molecular marker includes forward primer F1, forward primer F2, and reverse primer R, with the primer sequences as follows: Forward primer F1: 5'-GAAGGTGACCAAGTTCATGCTGGGGAGAAAGGGAACGAGAA-3' Forward primer F2: 5'-GAAGGTCGGAGTCAACGGATTGGGGAGAAAGGGAACGAGAG-3' Reverse primer R: 5'-TCCATCCCTATCCTTCTTCAAAA-3'.

3. The application of the primer combination of SNP molecular markers for detecting hypocotyl length in non-heading Chinese cabbage as described in claim 2 in the detection of hypocotyl length trait in non-heading Chinese cabbage, characterized in that, The testing steps are as follows: Step S1: Extract genomic DNA from the non-heading Chinese cabbage to be tested; Step S2, KASP / PARMS reaction: Using the DNA from step S1 as a template, KASP / PARMS reaction detection is performed using the primer combination described above, and a specific KASP Primer mix and a universal PARMS Master mix are added to perform PCR amplification to obtain PCR amplification products; Step S3: Analyze the PCR amplification products; use a TECAN Infinite M1000 microplate reader to read the fluorescence signal, and then use the online software snpdecoder to analyze and convert the fluorescence signal to obtain a clear and intuitive genotyping diagram, and output the genotype results according to different colors; C:C genotype corresponds to non-heading Chinese cabbage with a long hypocotyl; T:T genotype corresponds to non-heading Chinese cabbage with a short hypocotyl; C:T genotype is heterozygous.

4. The application of the primer combination of the SNP molecular marker for detecting hypocotyl length in non-heading Chinese cabbage as described in claim 3 in the detection of hypocotyl length trait in non-heading Chinese cabbage, characterized in that, The PCR amplification reaction system in step S2 is as follows: 2×PARMS reaction mixture: 5 μL volume, final concentration 1×; Primer X, 10 μM: 0.15 μL volume, final concentration 150 nM; Primer Y, 10 μM: 0.15 μL volume, final concentration 150 nM; Universal primers, 10 μM: 0.4 μL volume, final concentration 400 nM; DNA template: mass 10-100 ng, final concentration 10-100 nM; Sterile water: Add to volume 10 μL; The PCR amplification reaction conditions are as follows: Using a dual-head 384 PCR instrument, ABI Gene Amp 9700; the temperature was maintained at 94℃ for 15 minutes per cycle; the temperature was maintained at 94℃ for 20 seconds, then the temperature was changed to 65-57℃, -0.8℃ per cycle, and maintained for 1 minute, for a total of 10 cycles; the temperature was maintained at 94℃ for 20 seconds, then the temperature was changed to 57℃ and maintained for 1 minute, for a total of 32 cycles.

5. The application of a detection reagent for an SNP molecular marker used to identify the hypocotyl length of non-heading Chinese cabbage in the preparation of a kit for detecting the hypocotyl length trait in non-heading Chinese cabbage, characterized in that: The detection reagent is KASP / PARMS primers.

6. The application of the detection reagent for the SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage as described in claim 5 in the preparation of a kit for detecting the hypocotyl length trait of non-heading Chinese cabbage, characterized in that: The SNP molecular markers, KASP / PARMS primers, or detection reagents or kits are used in marker-assisted breeding of hypocotyl length trait in non-heading Chinese cabbage.

7. The application of the detection reagent for the SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage as described in claim 5 in the preparation of a kit for detecting the hypocotyl length trait of non-heading Chinese cabbage, characterized in that: The SNP molecular markers, KASP / PARMS primers, or detection reagents or kits are used in the genotyping of hypocotyl length trait in non-heading Chinese cabbage.

8. The application of the detection reagent for the SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage as described in claim 5 in the preparation of a kit for detecting the hypocotyl length trait of non-heading Chinese cabbage, characterized in that: The SNP molecular markers, KASP / PARMS primers, or detection reagents or kits are used in the improvement of non-heading Chinese cabbage germplasm resources.

9. The application of the detection reagent for the SNP molecular marker for identifying the hypocotyl length of non-heading Chinese cabbage as described in claim 5 in the preparation of a kit for detecting the hypocotyl length trait of non-heading Chinese cabbage, characterized in that: The SNP molecular markers, KASP / PARMS primers or reagents or kits are used in the cultivation of organically harvestable, long hypocotyl-based, non-heading Chinese cabbage varieties.

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