Methods and applications for establishing molecular identity cards for non-heading Chinese cabbage 'Shenkangqing No. 1'

Abstract

This invention discloses a method for establishing and applying the molecular identification of the non-heading Chinese cabbage variety 'Shenkangqing No. 1'. Using a marker combination of 30 pairs of SSR primers, the DNA of 'Shenkangqing No. 1' is amplified by PCR, and detection is performed simultaneously with sequencing. Based on the relative positions of the amplified products, data from different sites are integrated to form SSR fingerprint profiles of 'Shenkangqing No. 1', its parents, and the 'Suzhou Qing' variety. Simultaneously, PCR amplification and capillary electrophoresis detection techniques are used to detect the 'Shenkangqing No. 1' variety. This invention solves the problem that existing traditional morphological and cytological identification methods are labor-intensive and difficult to accurately and reliably identify the 'Shenkangqing No. 1' variety, enabling differentiation from other non-heading Chinese cabbage varieties.

Description

Technical Field

[0001] This invention relates to the establishment of a molecular identity card, and more particularly to a method and application for establishing a molecular identity card for the non-heading Chinese cabbage 'Shenkangqing No. 1', belonging to the field of Chinese cabbage technology. Background Technology

[0002] Non-heading Chinese cabbage, also known as bok choy, baby bok choy, or rapeseed, has become the third most widely cultivated vegetable in China after chili peppers and Chinese cabbage since 2018. The cultivation area of ​​the bok choy variety of non-heading Chinese cabbage nationwide has increased from 533,300 hectares in 2005. 2 Rising to 1.3333 million hectares in 2020 2 It increased by about 2.5 times, playing an extremely important role in the year-round production and supply of vegetables.

[0003] Given the high incidence of clubroot disease in cruciferous crops, developing new and superior clubroot-resistant varieties is one of the most effective ways to control the disease. Among them, 'Shenkangqing No. 1' is a hybrid variety, of the medium-sized basal type, with good upright growth, strong vigor, green, elliptical leaves with slightly wrinkled surfaces, medium-length, flat, thick, light green petioles, and good waist-binding. It is resistant to clubroot disease and can be harvested 20-40 days after sowing. It is characterized by vigorous growth, high yield, and excellent commercial appearance.

[0004] Existing traditional morphological and cytological identification methods involve a large workload and are difficult to accurately and reliably identify the differences in the 'Shenkangqing No. 1' variety. There is an urgent need to provide a method for establishing and applying a molecular identification method for the non-heading Chinese cabbage 'Shenkangqing No. 1'. Summary of the Invention

[0005] To address the shortcomings of the aforementioned technologies, this invention provides a method and application for establishing a molecular identity for the non-heading Chinese cabbage variety 'Shenkangqing No. 1'.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: a method for establishing the molecular identity card of non-heading Chinese cabbage 'Shenkangqing No. 1', comprising the following establishment methods:

[0007] S1. Extract DNA from the 'Shenkangqing No. 1' variety and perform PCR amplification on the DNA of the 'Shenkangqing No. 1' variety using 30 pairs of specific primers from the primer set.

[0008] S2. Sequencing each amplification product;

[0009] S3. Simultaneous sequencing and detection are performed. Based on the relative positions of the amplified products, the SSR fingerprints of 'Shenkangqing No. 1' and its parents and the 'Suzhouqing' variety are formed by integrating data from different sites.

[0010] The established molecular identity card includes primer number-primer name, molecular marker, primer sequence, and fingerprint code information;

[0011] The primer sequence contains 30 designed specific primer pairs, and PCR amplification is performed based on the specific primer pairs and molecular markers;

[0012] The fingerprint codes were obtained by sequencing the PCR amplification products and contain the molecular identification fingerprint codes of 'Shenkangqing No. 1', its parents, and the control variety 'Suzhouqing'. The fingerprint information is as follows:

[0013]

[0014]

[0015] A1 refers to 17-1g-1, one of the parents of 'Shenkangqing No. 1'; B refers to 'Shenkangqing No. 1'; A2 refers to 17-1g-10, one of the parents of 'Shenkangqing No. 1'; and C refers to 'Suzhou Qing'.

[0016] Preferably, the primer numbers and primer names are SA000189-cnu_m139a, SA000190-nia_m086a, SA000191-nia_m098a, SA000192-nia_m138a, SA000193-cnu_m046a, SA000194-nia_m121a, and SA000195-cnu_ m073a, SA000196-cnu_m288a, SA000197-cnu_m316a, SA000198-cnu_m327a, SA000199-n ia_m101a, SA000200-cnu_m252a, SA000201-Na10-D09, SA000202-cnu_m289a, SA000203 -cnu_m442a, SA000204-cnu_m050a, SA000205-cnu_m149a, SA000206-nia_m037a, SA00 0207-nia_m049a, SA000208-cnu_m182a, SA000209-cnu_m295a, SA000210-cnu_m090a, S A000211-cnu_m537a, SA000212-cnu_m016a, SA000213-cnu_m530a, SA000214-cnu_m534 a, SA000215-nia_m022a, SA000216-nia_m038a, SA000217-ENA2, SA000218-nia_m035a.

[0017] Preferably, the molecular markers added to the primers are amplified using the following primer pairs: SEQ ID NO. 1-2, SEQ ID NO. 3-4, SEQ ID NO. 5-6, SEQ ID NO. 7-8, SEQ ID NO. 9-10, SEQ ID NO. 11-12, SEQ ID NO. 13-14, SEQ ID NO. 15-16, SEQ ID NO. 17-18, SEQ ID NO. 19-20, SEQ ID NO. 21-22, SEQ ID NO. 23-24, SEQ ID NO. 25-26, SEQ ID NO. 27-28, SEQ ID NO. 29-30, SEQ ID NO. 31-32, SEQ ID NO. 33-34, SEQ ID NO. 35-36, SEQ ID NO. 37-38, SEQ ID NO. 39-40, SEQ ID NO. 41-42, SEQ NO. 43-44, SEQ ID NO. 45-46, SEQ ID NO. 1-2, SEQ ID NO. 3- ... Primer pairs shown in NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52, SEQ ID NO.53-54, SEQ ID NO.55-56, SEQ ID NO.57-58, and SEQ ID NO.59-60.

[0018] Preferably, the 30 pairs of specific primers are: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO.33-34, SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42, SEQ ID NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, SEQ ID NO. NO.49-50, SEQ ID NO.51-52, SEQ ID NO.53-54, SEQ ID NO.55-56, SEQ ID NO.57-58, SEQ ID NO.59-60.

[0019] Preferably, the fingerprint information is formed by integrating data from different loci based on the relative positions of the amplified products. Specifically: when the amplified product read band is absent, it is recorded as 0 for dominant markers; when amplification conditions are present, the dominant marker is recorded as 1; when the band is weak or the genotype cannot be determined, it is recorded as ?; invalid bands are recorded as --; the allelic variation data of homozygous loci in co-dominant markers is recorded as X / X, and the amplified product read band is homozygous parent 1, where X is the allelic variation size at that locus; the allelic variation data of homozygous loci is recorded as Y / Y, and the amplified product read band is homozygous parent 2, where Y is the allelic variation size at that locus; the allelic variation data of heterozygous loci is recorded as X / Y, and the amplified product is biparental banding, where X and Y are two different allelic variations at that locus; the part before the " / " is a small fragment, and the part after the " / " is a large fragment.

[0020] As a preferred method, fingerprint QR codes are generated for 'Shenkangqing No. 1' and its parent and control variety 'Suzhouqing'.

[0021] A method for applying the molecular identification of non-heading Chinese cabbage 'Shenkangqing No. 1' includes the following steps: extracting DNA from the sample to be identified, performing PCR amplification, and detecting the PCR products using capillary electrophoresis to identify the variety of the sample to be tested.

[0022] This invention discloses a method for establishing and applying the molecular identification of the non-heading Chinese cabbage variety 'Shenkangqing No. 1'. It utilizes a marker combination composed of 30 pairs of SSR primers and employs PCR amplification and capillary electrophoresis detection techniques to detect the 'Shenkangqing No. 1' variety. This method can complete the identification and genetic diversity evaluation of the 'Shenkangqing No. 1' variety in a short time, offering advantages such as time-saving, high efficiency, speed, accuracy, and ease of operation. This method for establishing the molecular identification of the non-heading Chinese cabbage variety 'Shenkangqing No. 1' can effectively monitor the authenticity of the 'Shenkangqing No. 1' variety, revealing the genetic variation and genetic relationships of the variety at the DNA level, protecting crop varieties, and preventing counterfeit and substandard products from entering the market. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments.

[0024] This invention provides a method for establishing the molecular identification of the non-heading Chinese cabbage 'Shenkangqing No. 1', which solves the problem that existing traditional morphological and cytological identification methods are labor-intensive and difficult to accurately and truly identify the variety type of 'Shenkangqing No. 1', and enables its differentiation from other non-heading Chinese cabbage varieties.

[0025] The embodiments of the present invention provide a method for establishing the fingerprint spectrum of non-heading Chinese cabbage 'Shenkangqing No. 1'. The established molecular identity card includes information such as material name, material characteristic properties, primer sequence, fingerprint spectrum code and QR code.

[0026] First, primers were designed with the primer numbers and names as follows: SA000189-cnu_m139a, SA000190-nia_m086a, SA000191-nia_m098a, SA000192-nia_m138a, SA000193-cnu_m046a, SA000194-nia_m121a, and SA0001... 95-cnu_m073a, SA000196-cnu_m288a, SA000197-cnu_m316a, SA000198-cnu_m327a, SA00 0199-nia_m101a, SA000200-cnu_m252a, SA000201-Na10-D09, SA000202-cnu_m289a, SA0 00203-cnu_m442a, SA000204-cnu_m050a, SA000205-cnu_m149a, SA000206-nia_m037a, S A000207-nia_m049a, SA000208-cnu_m182a, SA000209-cnu_m295a, SA000210-cnu_m090a , SA000211-cnu_m537a, SA000212-cnu_m016a, SA000213-cnu_m530a, SA000214-cnu_m53 4a, SA000215-nia_m022a, SA000216-nia_m038a, SA000217-ENA2, SA000218-nia_m035a;

[0027] The molecular markers were amplified using the following primer pairs: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO.33-34, SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42, SEQ NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, SEQ ID NO. Using the primer pairs shown in NO.49-50, SEQ ID NO.51-52, SEQ ID NO.53-54, SEQ ID NO.55-56, SEQ ID NO.57-58, and SEQ ID NO.59-60, molecular markers of 'Shenkangqing No. 1' and its parents were obtained.

[0028] The designed primer combination consists of the following 30 pairs of specific primers: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO.33-34, SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42, SEQ ID NO.43-44, SEQ ID NO.45-46, SEQ ID NO. SEQ IDs NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52, SEQ ID NO.53-54, SEQ ID NO.55-56, SEQ ID NO.57-58, and SEQ ID NO.59-60 provide detailed primer combination information.

[0029] Example 1

[0030] A kit for combining molecular markers and primers, used to detect PCR amplification products, is shown below:

[0031] When the amplified product read band is absent, it is recorded as 0 for dominant markers; when amplification conditions are present, it is recorded as 1 for dominant markers; when the band is weak or the genotype cannot be determined, it is recorded as ?; invalid bands are recorded as --; the allelic variation data of homozygous loci in co-dominant markers is recorded as X / X, and the amplified product read band is homozygous parent 1, where X is the allelic variation size at that locus; the allelic variation data of homozygous loci is recorded as Y / Y, and the amplified product read band is homozygous parent 2, where Y is the allelic variation size at that locus; the allelic variation data of heterozygous loci is recorded as X / Y, and the amplified product is biparental banding, where X and Y are two different allelic variations at that locus; the part before the " / " is a small fragment, and the part after the " / " is a large fragment; by integrating the data from different loci, the SSR fingerprint codes of 'Shenkangqing No. 1' and its parents and the control variety 'Suzhouqing' are formed.

[0032] The fingerprint information of 'Shenkangqing No. 1' and its parents and 'Suzhouqing' is as follows:

[0033]

[0034]

[0035] In the above, A refers to one of the parent lines of 'Shenkangqing No. 1', 17-1g-1; B refers to 'Suzhouqing'; A2 refers to another parent line of 'Shenkangqing No. 1', 17-1g-10; and C refers to 'Shenkangqing No. 1'.

[0036] The application of molecular markers and primer combinations in the identification of the Chinese cabbage variety 'Shenkangqing No. 1', including the PCR amplification method:

[0037] The reaction program was as follows: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 45 s, annealing at 65℃ for 40 s, extension at 72℃ for 45 s, decreasing by 1℃ per cycle, for a total of 15 cycles; denaturation at 94℃ for 45 s, annealing at 50℃ for 30 s, extension at 72℃ for 45 s, for a total of 30 cycles; extension at 72℃ for 10 min, and holding at 4℃.

[0038] Reaction system: 25 μL reaction system volume, containing 0.25 mmol / L of each dNTP, 0.4 μmol / L of each forward and reverse primer, 1.0 U of Taq DNA polymerase, 1×PCR buffer (without Mg2+), 1.5 mmol / L of MgCl2, and 10-40 ng of sample DNA.

[0039] Application of molecular markers and primer combinations in the identification of 'Shenkangqing No. 1' and its parent 'Shenkangqing No. 1'.

[0040] Primer combinations were used to amplify the DNA of 'Shenkangqing No. 1' and its parents by PCR, and the PCR products were detected by capillary electrophoresis. Fluorescent capillary electrophoresis was also used for detection. Fluorescent labels were added to the primers. Details are as follows:

[0041]

[0042]

[0043] In Example 1, fingerprint sequences of 'Shenkangqing No. 1' and its parents were obtained, and different varieties of Chinese cabbage were compared and analyzed to identify their differences. The significant differences in band size between the SSR primer amplification products formed a unique DNA fingerprint, which effectively distinguished different varieties. This molecular identification method of the present invention can effectively identify the 'Shenkangqing No. 1' variety, offering advantages such as high efficiency, accuracy, low cost, and simple operation.

[0044] Furthermore, for ease of use, fingerprint spectrum QR codes were generated for 'Shenkangqing No. 1' and its parent and control variety 'Suzhouqing'.

[0045] Example 2

[0046] A molecular identification method for the 'Shenkangqing No. 1' variety includes the following steps:

[0047] S1. Extract DNA from the 'Shenkangqing No. 1' variety and perform PCR amplification on the DNA of the 'Shenkangqing No. 1' variety using 30 pairs of SSR primers from the primer set.

[0048] S2. Perform first-generation sequencing on the amplification products;

[0049] S3. Simultaneous sequencing and detection are performed. Based on the relative positions of the amplified products, data from different loci are integrated to form SSR fingerprints of 'Shenkangqing No. 1', its parents, and the 'Suzhou Qing' variety. When the amplified product read band is absent, it is recorded as 0 for dominant markers; when amplification conditions are present, it is recorded as 1 for dominant markers; when the band is weak or the genotype cannot be determined, it is recorded as ?; invalid bands are recorded as --. The allelic variation data for homozygous loci in co-dominant markers is recorded as X / X, with the amplified product read band representing homozygous parent 1, where X represents the allelic variation size at that locus; the allelic variation data for homozygous loci is recorded as Y / Y, with the amplified product read band representing homozygous parent 2, where Y represents the allelic variation size at that locus; the allelic variation data for heterozygous loci is recorded as X / Y, with the amplified product showing both parental bands, where X and Y represent two different allelic variations at that locus; the part before the " / " indicates a small fragment, and the part after the " / " indicates a large fragment.

[0050] In this second embodiment, the molecular identification card of the present invention can effectively monitor the authenticity of 'Shenkangqing No. 1' and its parent seeds, protect the species, and prevent counterfeit and inferior varieties from entering the market.

[0051] The above embodiments are not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the technical solution of the present invention are also within the protection scope of the present invention.

Claims

1. A method for establishing the molecular identity of non-heading Chinese cabbage 'Shenkangqing No. 1', characterized in that: The method comprises the following steps: S1, extracting the DNA of the 'Shinkangqing No.1' variety, and performing PCR amplification on the DNA of the 'Shinkangqing No.1' variety with 30 pairs of specific primer pairs in the primer combination; S2, sequencing the amplification products one by one; S3, detecting while sequencing, and forming the 'Shinkangqing No.1' SSR fingerprint map by integrating the data of different sites according to the relative positions of the amplification products; The established molecular identity card comprises primer number-primer name, molecular marker, primer sequence and fingerprint code information; The primer number-primer name is respectively SA000189-cnu_m139a, SA000190-nia_m086a, SA000191-nia_m098a, SA000192-nia_m138a, SA000193-cnu_m046a, SA000194-nia_m121a, SA000195-cnu_m073a, SA000196-cnu_m288a, SA000197-cnu_m316a, SA000198-cnu_m327a, SA000199-nia_m101a, SA000200-cnu_m252a, SA000201-Na10-D09, SA000202-cnu_m289a, SA000203-cnu_m442a, SA000204-cnu_m050a, SA000205-cnu_m149a, SA000206-nia_m037a, SA000207-nia_m049a, SA000208-cnu_m182a, SA000209-cnu_m295a, SA000210-cnu_m090a, SA000211-cnu_m537a, SA000212-cnu_m016a, SA000213-cnu_m530a, SA000214-cnu_m534a, SA000215-nia_m022a, SA000216-nia_m038a, SA000217-ENA2, SA000218-nia_m035a; The primer sequence comprises 30 pairs of specific primer pairs designed, and the PCR amplification is performed based on the specific primer pairs and the molecular marker, and the 30 pairs of specific primer pairs are as follows: The SSR fingerprint map is obtained by sequencing the PCR amplification products, and the map information is as follows: The above B refers to 'Shinkangqing No.1'.

2. The method for establishing the molecular identity certificate of Brassica campestris L. var. parachinensis Bailey 'Shikangqing No. 1' according to claim 1, characterized in that: The two-dimensional code of the SSR fingerprint map of 'Shinkangqing No.1' is generated.

3. The application method of the molecular identification card of Brassica campestris L. var. parachinensis Bailey 'Shikangqing No. 1' obtained by the establishment method according to claim 1, characterized in that: The method comprises the following steps: extracting the DNA of the sample to be identified, performing PCR amplification and detecting the PCR products by capillary electrophoresis to identify the variety of the sample to be detected.

Images