Primer for cauliflower hybrid purity identification and application thereof

Abstract

The invention belongs to the technical field of biology, and provides a primer and a kit for cauliflower hybrid purity identification and a cauliflower hybrid purity identification method. The primer for identifying the purity of the cauliflower hybrid, provided by the invention, is clear in amplification band pattern, good in stability and relatively high in polymorphism, and is very suitable for identifying the purity of the cauliflower hybrid.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to primers for identifying the purity of cauliflower hybrids and their applications. Background Technology

[0002] Cauliflower (Brassica oleracea L. var. botrytis L.), also known as broccoli or flower florets, belongs to the Brassicaceae family and the Brassica genus. It evolved from wild cabbage, and its edible part is the flower head. It has a delicious taste and is rich in nutrients. Studies have shown that cauliflower has disease-preventing and anti-cancer effects, making it very popular. The cauliflower industry has become one of China's important vegetable industries, and it is now widely cultivated in Fujian, Taiwan, Guangdong, Zhejiang, and other provinces and cities, with more than 170 main varieties.

[0003] Cauliflower originated in Europe. my country has limited cauliflower varietal resources and relatively lagging genetic research. Due to the dependence on and blindness in the selection of parents during the breeding process, it is becoming increasingly difficult to breed new cauliflower varieties. In addition, the mixing of varieties during seed production leads to the frequent occurrence of the same species with different names and the same name with different varieties in the market. Therefore, it is urgent to carry out the identification of cauliflower germplasm resources and genetic diversity analysis, and to strengthen the preservation and utilization of cauliflower germplasm resources. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, this invention provides an application of SSR primers in the evaluation of cauliflower resources.

[0005] The first aspect of the present invention provides a kit for identifying cauliflower varieties, the kit containing 29 pairs of SSR primers numbered 1-58 in the sequence listing.

[0006] A second aspect of this invention provides a method for identifying cauliflower varieties, the method comprising:

[0007] SSR molecular markers were detected in cauliflower varieties with known varietal information using 29 pairs of SSR primers numbered 1-58 in the sequence listing, and the SSR fingerprint information of cauliflower varieties with known varietal information was obtained.

[0008] 29 pairs of SSR primers numbered 1-58 in the sequence listing were used to detect SSR molecular markers in the cauliflower varieties to be tested, and the SSR fingerprint information of the cauliflower varieties to be tested was obtained.

[0009] The SSR fingerprint information of the cauliflower variety to be tested is compared with the SSR fingerprint information of the cauliflower varieties of the known varieties. If the similarity between the SSR fingerprint information of the cauliflower variety to be tested and the SSR fingerprint information of the cauliflower varieties of the known varieties reaches 98% or more, it can be determined that the cauliflower variety to be tested is consistent with the corresponding cauliflower variety of the known varieties.

[0010] A third aspect of the present invention provides a kit for evaluating the kinship of cauliflower, the kit containing 29 pairs of SSR primers numbered 1-58 in the sequence listing.

[0011] A fourth aspect of the present invention provides a method for evaluating the kinship of cauliflower, the method comprising:

[0012] SSR molecular markers were detected in multiple cauliflower varieties using 29 pairs of SSR primers numbered 1-58 in the sequence listing, and SSR fingerprint information of multiple cauliflower varieties was obtained.

[0013] Based on the SSR fingerprint information of multiple cauliflower varieties, the genetic distance between the multiple cauliflower varieties was calculated;

[0014] Based on the calculated genetic distance, the kinship between multiple cauliflower varieties can be determined.

[0015] The fifth aspect of the present invention provides a method for cultivating new cauliflower germplasm, the method comprising determining the kinship relationship between multiple cauliflower varieties using the cauliflower kinship evaluation method provided in the fourth aspect of the present invention, and then selecting the two cauliflower varieties with the most distant kinship relationship as parent materials.

[0016] The sixth aspect of the present invention provides a kit for identifying the purity of the "Jingyan Sijiqing 110" cauliflower hybrid, the kit containing at least one SSR primer pair numbered 1, 3, 15, 24, 25, 28, 29 in the sequence listing.

[0017] The seventh aspect of the present invention provides a kit for identifying the purity of the "Jingyan Sijiqing 100" cauliflower hybrid, the kit containing at least one SSR primer pair numbered 3, 6, 15, 24 in the sequence listing.

[0018] The 29 pairs of SSR primers screened in this invention have clear amplification bands, good stability, and high polymorphism, making them very suitable for cauliflower variety identification, phylogenetic evaluation, and breeding of new cauliflower germplasm. Attached Figure Description

[0019] Figure 1The electrophoresis results of the eight primer pairs in Example 1 are shown; where: the eight results on the left of row A are the amplification results using primer number 7 of primer pair in Table 2; the eight results on the right of row A are the amplification results using primer number 17 of primer pair in Table 2; the eight results on the left of row B are the amplification results using primer number 25 of primer pair in Table 2; the eight results on the right of row B are the amplification results using primer number 26 of primer pair in Table 2; the numbers in the figure are the numbers of cauliflower in Table 1; the letter "M" is a 500bp DNA marker.

[0020] Figure 2 The amplification results of primer pair number 15 from Table 2, used in Example 1, are shown; the numbers in the figure are the numbers of cauliflower in Table 1; the letter "M" represents a 500bp DNA marker.

[0021] Figure 3 Cluster diagram of 84 cauliflower varieties in Example 2 is shown.

[0022] Figure 4 A bar chart showing the genetic distance frequency of 84 cauliflower varietal materials in Example 2 is shown.

[0023] Figure 5 Electrophoresis diagram of the codominant markers of the "Jingyan Sijiqing 110" cauliflower hybrid in Example 3 is shown.

[0024] Figure 6 Electrophoresis diagram of codominant markers in the "Jingyan Sijiqing 100" cauliflower hybrid in Example 3 is shown.

[0025] Figure 7 The image shows an electrophoresis diagram of the purity detection of 118 "Jingyan Sijiqing 110" cauliflower hybrid materials in Example 3 using primer pair number 1 (primer pair name CB-2) in Table 2.

[0026] Figure 8 The image shows an electrophoresis diagram of the purity detection of 118 “Jingyan Sijiqing 100” cauliflower hybrid materials in Example 3 using primer pair number 6 (primer pair name CB-21) in Table 2.

[0027] Figure 9 The image shows an electrophoresis diagram of the purity detection of 118 "Jingyan Sijiqing 110" cauliflower hybrid materials in Example 3 using primer pair number 24 (primer pair name CH-54) in Table 2.

[0028] Figure 10The image shows an electrophoresis diagram of the purity detection of 118 “Jingyan Sijiqing 100” cauliflower hybrid materials in Example 3 using primer pair number 24 (primer pair name CH-54) in Table 2. Detailed Implementation

[0029] To make the technical solution, objectives, and advantages of the present invention clearer, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0030] The first aspect of the present invention provides a kit for identifying cauliflower varieties, the kit containing 29 pairs of SSR primers numbered 1-58 in the sequence listing.

[0031] According to a first aspect of the present invention, the kit may further contain at least one of PCR amplification buffer, dNTPs, Taq enzyme, and DNA chromogenic dye.

[0032] A second aspect of this invention provides a method for identifying cauliflower varieties, the method comprising:

[0033] SSR molecular markers were detected in cauliflower varieties with known varietal information using 29 pairs of SSR primers numbered 1-58 in the sequence listing, and the SSR fingerprint information of cauliflower varieties with known varietal information was obtained.

[0034] 29 pairs of SSR primers numbered 1-58 in the sequence listing were used to detect SSR molecular markers in the cauliflower varieties to be tested, and the SSR fingerprint information of the cauliflower varieties to be tested was obtained.

[0035] The SSR fingerprint information of the cauliflower variety to be tested is compared with the SSR fingerprint information of the cauliflower varieties of the known varieties. If the similarity between the SSR fingerprint information of the cauliflower variety to be tested and the SSR fingerprint information of the cauliflower varieties of the known varieties reaches 98% or more (preferably 100%), it can be determined that the cauliflower variety to be tested is consistent with the corresponding cauliflower variety of the known varieties.

[0036] According to a second aspect of the present invention, the cauliflower varieties for which the known variety information is provided are shown in Table 1.

[0037] According to a second aspect of the present invention, the SSR molecular marker detection includes:

[0038] The following 24 μL PCR reaction system was used:

[0039] 6 μL DNA template, 2 μL 10X PCR buffer, 2 μL 10X dye, 0.4 μL 10 mM dNTPs, 0.3 μL 2.5 U Taq enzyme, 2 μL each of forward and reverse primers, and 9.3 μL ddH2O.

[0040] The PCR reaction procedure is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 4 min; store at 16℃.

[0041] A third aspect of the present invention provides a kit for evaluating the kinship of cauliflower, the kit containing 29 pairs of SSR primers numbered 1-58 in the sequence listing.

[0042] According to a third aspect of the present invention, the kit may further contain at least one of PCR amplification buffer, dNTPs, Taq enzyme, and DNA chromogenic dye.

[0043] A fourth aspect of the present invention provides a method for evaluating the kinship of cauliflower, the method comprising:

[0044] SSR molecular markers were detected in multiple cauliflower varieties using 29 pairs of SSR primers numbered 1-58 in the sequence listing, and SSR fingerprint information of multiple cauliflower varieties was obtained.

[0045] Based on the SSR fingerprint information of multiple cauliflower varieties, the genetic distance between the multiple cauliflower varieties was calculated;

[0046] Based on the calculated genetic distance, the kinship between multiple cauliflower varieties can be determined.

[0047] The fifth aspect of the present invention provides a method for cultivating new cauliflower germplasm, the method comprising determining the kinship relationship between multiple cauliflower varieties using the cauliflower kinship evaluation method provided in the fourth aspect of the present invention, and then selecting the two cauliflower varieties with the most distant kinship relationship as parent materials.

[0048] The sixth aspect of the present invention provides a kit for identifying the purity of the "Jingyan Sijiqing 110" cauliflower hybrid, the kit containing at least one SSR primer pair numbered 1, 3, 15, 24, 25, 28, 29 in the sequence listing.

[0049] The method for identifying the purity of the "Jingyan Sijiqing 110" cauliflower hybrid, using the reagent kit provided in the sixth aspect of this invention, is also within the scope of protection of this invention.

[0050] The seventh aspect of the present invention provides a kit for identifying the purity of the "Jingyan Sijiqing 100" cauliflower hybrid, the kit containing at least one SSR primer pair numbered 3, 6, 15, 24 in the sequence listing.

[0051] The method for identifying the purity of the "Jingyan Sijiqing 100" cauliflower hybrid using the kit provided in the seventh aspect of this invention is also within the scope of protection of this invention.

[0052] In this invention, the "Jingyan Sijiqing 110" and "Jingyan Sijiqing 100" cauliflower varieties are from the Biotechnology Research Laboratory of the Vegetable Research Institute of Beijing Academy of Agricultural and Forestry Sciences. You can contact the inventor of this invention to obtain them.

[0053] Unless otherwise specified, all reagents and materials used in the following examples are products that can be obtained from commercial channels; unless otherwise specified, all testing and detection methods used in the following examples are conventional testing and detection methods in the field and can be obtained from textbooks, reference books or academic journals.

[0054] The cauliflower varieties used in this invention totaled 84, all from the Vegetable Research Institute of the Beijing Academy of Agricultural and Forestry Sciences. Among them, 66 were DH pure lines and 18 were high-generation inbred lines. Specific information on the 84 cauliflower varieties is shown in Table 1.

[0055] Table 1. Cauliflower Variety and Material Information

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] During the cauliflower head maturation stage, a suitable amount of tender leaf tissue was collected for DNA extraction. The tender cauliflower leaf tissue was directly placed into 1.5 mL centrifuge tubes and labeled. It was then stored at -20℃ for later use.

[0062] Genomic DNA was extracted from cauliflower using the SDS extraction method. The specific steps are as follows:

[0063] Add 150 μL of SDS-DNA extraction buffer to a 1.5 mL centrifuge tube containing cauliflower leaf tissue, add 3–4 small steel balls, and homogenize in a high-throughput tissue homogenizer for 2.5 min. After homogenization, add 450 μL of SDS-DNA extraction buffer. Heat in a 65°C water bath for 15 min, mixing the contents at least 3 times during heating. After the water bath, add 300 μL of 5M potassium acetate solution, mix well, and incubate on ice for 15 min. Centrifuge at 13000 rpm for 20 min at room temperature. Transfer 650 μL of the supernatant to another centrifuge tube containing 650 μL of isopropanol (1.5 mL), invert to mix, and precipitate at 4°C for 10–20 min. Centrifuge at 13000 rpm for 10 min at room temperature and discard the supernatant. Wash the precipitate with 500 μL of 75% ethanol, repeating once. Dry the DNA in a vacuum dryer for 10–20 min, then add 300 μL of 5M potassium acetate solution. μL of sterile distilled water was used; finally, the integrity, concentration, and quality of the DNA were detected using a gel imaging system, and the DNA was stored at -20℃ for later use.

[0064] Example 1

[0065] Using the PCR reaction system and procedure described below, and with the extracted cauliflower DNA as a template, 695 pairs of SSR primers were screened.

[0066] 24 μL PCR reaction system: 6 μL DNA template, 2 μL 10X PCR buffer, 2 μL 10X dye, 0.4 μL 10 mM dNTPs, 0.3 μL 2.5 U Taq enzyme, 2 μL each of forward and reverse primers, and 9.3 μL ddH2O.

[0067] PCR reaction procedure: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 4 min; store at 16℃.

[0068] The PCR amplification products were collected and electrophoresed on a 2.6% high-resolution agarose gel at 200–250 V for 60–80 min. The raw data were then collected by photographing under UV light using a G:BOX Chemi-XRQ image analysis system.

[0069] First, based on the variety origin, maturity, and head tightness of the tested materials, DNA from 8 cauliflower varieties (variety numbers 1, 10, 22, 39, 42, 63, 66, and 81 in Table 1) out of the 84 cauliflower samples shown in Table 1 was selected as templates. Using 695 pairs of SSR primers, PCR amplification was performed according to the PCR reaction system and procedure described above. The PCR amplification products were then subjected to electrophoresis and observation as described above, from which 63 pairs of primers with significant polymorphism and stable amplification were selected. Partial results of these 63 primer pairs are shown below. Figure 1 As shown, the results are as follows: the eight results on the left side of row A are amplification results using primer pair number 7 (primer pair name CB-28) in Table 2; the eight results on the right side of row A (excluding column M, where column M represents the electrophoresis results of DNA molecular weight standards) are amplification results using primer pair number 17 (primer pair name CB-139) in Table 2; the eight results on the left side of row B are amplification results using primer pair number 25 (primer pair name CH-56) in Table 2; the eight results on the right side of row B (excluding column M, where column M represents the electrophoresis results of DNA molecular weight standards) are amplification results using primer pair number 26 (primer pair name CH-79) in Table 2.

[0070] Then, using these 63 primer pairs, and with the DNA from the 84 cauliflower samples shown in Table 1 as templates, PCR amplification was performed according to the above PCR reaction system and procedure. The PCR amplification products were then subjected to electrophoresis and observation as described above. Primers with significant band differences, good stability, and high polymorphism were selected, thus determining the 29 SSR primer pairs shown in Table 2. Figure 2 The amplification results using primer pair number 15 (primer pair name CB-124) from Table 2 are shown; Figure 2 The numbers in the table are the cauliflower numbers in Table 1; the letter "M" represents a 500bp DNA marker.

[0071] Table 2 29 pairs of SSR primer sequences

[0072]

[0073]

[0074] Using the 29 selected SSR primer pairs and the extracted cauliflower DNA from Table 1 as a template, PCR amplification was performed using the aforementioned PCR reaction system and procedure. The PCR amplification products were then subjected to electrophoresis as described above. Band patterns were statistically analyzed based on the electrophoresis results, with bands marked as 1 and no bands as 0. This data was recorded using Excel 2019 software, constructing a 0 / 1 data matrix (as shown in Table 3). This data matrix was then used for further analysis. Data transformations were performed using DataFormater 2.7 software.

[0075] Table 3. Amplification results of 29 SSR primer pairs in 84 cauliflower varieties.

[0076]

[0077]

[0078]

[0079]

[0080] Continued table

[0081]

[0082]

[0083]

[0084]

[0085] Continued table

[0086]

[0087]

[0088]

[0089]

[0090] The effective allele count (Ne), average Nei's diversity index (H), and Shannon's information index (I) of the population were calculated using Popgene 32 software. The polymorphism index (PIC) of each SSR primer pair was calculated using PowerMarker V3.25 software. The results are shown in Table 4.

[0091] Table 4. Analysis data of 29 SSR primer pairs in 84 cauliflower varietal materials

[0092]

[0093] Genetic distances and genetic similarity coefficients among the cauliflower varieties shown in Table 1 were calculated using NTSYS-pc 2.10e software. Cluster analysis among the varieties was then performed using the unweighted pair-group method with arithmetic means (UPGMA) in NTSYS-pc 2.10e software. The results are as follows: Figure 3 , Figure 4 As shown in Table 5.

[0094] Table 5. Genetic distances among some cauliflower varieties.

[0095]

[0096] The results showed that, following the above-described procedure, the 29 SSR core primer pairs shown in Table 2 detected a total of 81 alleles in the 84 cauliflower varieties shown in Table 1. The number of alleles detected by each primer pair ranged from 2 to 4. As shown in Table 3, the average number of alleles detected per primer pair (Na) was 2.79, and 72.4% of the primers detected 3 or more alleles. Among them, primer pairs CB-58 (primer pair number 10 in Table 2) and CB-157 (primer pair number 21 in Table 2) had the highest number of alleles, with 4. The effective number of alleles (Ne) ranged from 1.50 to 2.82, with an average of 2.09. The Nei's diversity index (H) ranged from 0.33 to 0.65, with an average of 0.51.

[0097] As shown in Table 4, the Shannon Information Index (I) ranged from 0.58 to 1.08, with an average of 0.80. The expected heterozygosity (He) averaged 0.12. The Polymorphism Information Content Index (PIC value) ranged from 0.29 to 0.57, with an average of 0.42. Among them, primer pairs CB-8 (primer pair number 2 in Table 2), CB-10 (primer pair number 3 in Table 2), CB-21 (primer pair number 6 in Table 2), CB-28 (primer pair number 7 in Table 2), CB-48 (primer pair number 9 in Table 2), CB-157 (primer pair number 21 in Table 2), and CH-6 (primer pair number 23 in Table 2) had PIC values ​​≥ 0.50, indicating high polymorphism and good discrimination ability, capable of identifying the vast majority of materials. 22 pairs of moderately polymorphic SSR primers (0.25 ≤ PIC < 0.5) were used, with an average PIC value of 0.38. No low-polymorphic SSR primers were used (PIC < 0.25). This indicates that the 29 pairs of SSR primers screened in this invention exhibit high polymorphism and good identification ability. The 29 pairs of primers screened in this invention were used to amplify the 84 cauliflower varietals shown in Table 1. On average, each primer pair identified 2.79 alleles, with an effective allele count of 2.09. The difference between the two was small, indicating a relatively even distribution of alleles in the population. The average expected heterozygosity was 0.12, and the average Nei's diversity index was 0.51, indicating that the genetic diversity of cauliflower germplasm is relatively narrow, and the diversity of cauliflower germplasm resources needs to be developed.

[0098] In summary, these parameters indicate that the 29 pairs of SSR core primers shown in Table 2 have high polymorphism and good overall identification ability, enabling them to effectively identify cauliflower varieties and analyze genetic diversity.

[0099] Example 2

[0100] This embodiment illustrates the application of the 29 pairs of SSR primers selected in Example 1 in the evaluation of cauliflower phylogenetic relationships and the identification of cauliflower germplasm resources.

[0101] The assessment of genetic distance can serve as a basis for the breeding of new varieties, and cluster analysis helps breeders understand the genetic diversity and kinship of parents in order to obtain ideal parent combinations.

[0102] As shown in Table 5 and Figure 4As shown in Table 1, the genetic distance among the 84 cauliflower varieties ranged from 0.0345 to 0.8507, with an average of 0.5638 and a variation range of 0.8162. The genetic distances were mainly concentrated between 0.3 and 0.6, with the highest range being 0.4 to 0.5 (29.9%), followed by 0.5 to 0.6 (28.1%). The genetic distance between 0.7 and 0.9 accounted for 2.0%. According to the genetic distance matrix, the varieties with the closest genetic relationship were materials 31 and 32, and 34 and 35, with a genetic distance of 0.0345; the varieties with the furthest genetic relationship were materials 67 and 45, with a genetic distance of 0.8507. These two varieties could be prioritized as parental materials for breeding new cauliflower germplasm.

[0103] like Figure 4 As shown, the genetic similarity coefficients of the 84 cauliflower materials ranged from 0.50 to 0.97, with an average of 0.57. When the genetic similarity coefficient was 0.55, the 84 cauliflower materials could be clustered into the following group IV.

[0104] Group I contains 61 cauliflower materials numbered 1, 15, 3, 76, 13, and 60, accounting for 72.6% of the tested materials. It consists of 55 DH pure lines and 6 inbred lines, including 12 medium-maturing varieties, 44 late-maturing varieties, and 5 very late-maturing varieties. They originated from Taiwan (21), Tianjin (18), Zhejiang (6), Xiamen (8), Guangdong (6), Beijing (1), and Hebei (1), respectively. At a genetic similarity coefficient of 0.60, the 61 cauliflower materials in Group I can be further divided into 4 subgroups. Subgroup I-1 contains 17 varieties with material numbers 1, 3, 4, and 15, accounting for 27.9% of the materials in Group I, mainly late-maturing varieties; Subgroup I-2 contains 22 varieties with material numbers 5, 7, and 18, accounting for 36.1% of the materials in Group I, mainly late-maturing varieties; Subgroup I-3 contains 10 varieties, mainly late-maturing varieties; and Subgroup I-4 contains 12 varieties, mainly mid-maturing varieties.

[0105] Group II contains cauliflower materials numbered 2, 77, 73, 78, 80, 75, and 79. Material 2 is a pure DH mid-maturing variety, while the other materials are all inbred late-maturing varieties. They are from Taiwan (2), Zhejiang (2), Xiamen (2), and Beijing (1), respectively, and the geographical distribution of germplasm resources is relatively scattered.

[0106] Group III contains 10 cauliflower materials numbered 6, 8, 29, and 30, consisting of one mid-maturing variety and nine late-maturing varieties. All are pure DH lines, originating from Taiwan (3), Zhejiang (6), and Guangdong (1), respectively. At a genetic similarity coefficient of 0.86, the cauliflower materials in Group III can be divided into two subclasses: III-1 includes materials 6, 8, and 30, all from Changsheng, Taiwan; III-2 includes materials 29, 33, 34, 35, 38, 39, and 40. Except for material 29, which is distributed in Xiamen, Guangdong, the rest are from Shenliang, Zhejiang.

[0107] Group IV includes cauliflower materials numbered 63, 82, 66, 67, 81, and 84, all of which are inbred lines, mainly mid-to-late maturing varieties, originating from Germany (2 accessions), Hebei (1 accession), and Beijing (3 accessions), respectively. In summary, the clustering of cauliflower germplasm materials is not closely related to geographical origin; cauliflower materials from the same origin did not cluster together, while most cauliflowers with similar maturity and germplasm types were clustered together.

[0108] SSR primer screening is a prerequisite for germplasm resource identification and genetic diversity analysis. When screening SSR primers, it is required to select primers with high polymorphism, good repeatability, and uniform chromosome distribution. The Polymorphism Information Content Index (PIC) is an important indicator for measuring polymorphism. In the 29 pairs of SSR primers screened in this invention, the average number of alleles per primer pair was 2.79, and the effective number of alleles was 2.09. The difference between the two was small, indicating that the alleles were relatively evenly distributed in the population. The average expected heterozygosity was 0.12, the average Nei's diversity index was 0.51, and the PIC values ​​ranged from 0.29 to 0.57, with an average PIC value of 0.42. This is higher than the PIC value of 0.316 found by Zhu Shiyang (2019) in 165 cauliflower inbred lines, similar to the PIC value of 0.43 obtained by Ding Yan et al. (2021) in 70 cauliflower varieties, and lower than the PIC value of 0.60 found by Iniguez-Luy et al. (2008) in 4 cauliflower materials. This indicates that the tested cauliflower varieties have low genetic diversity, which may be related to artificial directional selection.

[0109] The assessment of genetic distance and genetic similarity coefficient can serve as the basis for the breeding of new varieties. Cluster analysis helps breeders understand the genetic diversity and kinship of parents in order to obtain ideal parent combinations. Zhu et al. (2018) used SSR markers to find that the genetic distance of 165 cauliflower varieties in southeastern China ranged from 0 to 0.67, with an average genetic distance of 0.30. Liu et al. (2018) detected that the genetic similarity coefficient of SSR markers of cauliflower varieties in the Yangtze River Delta region ranged from 0.59 to 0.99, with an average of 0.77. Zhao et al. (2014) detected that the similarity coefficient of SSR markers of cauliflower ranged from 0.56 to 1.00, with an average of 0.74. In this invention, the genetic diversity analysis of 84 cauliflower varieties showed that the genetic distance between the varieties ranged from 0.0345 to 0.8507, with an average genetic distance of 0.5638. The genetic similarity coefficients ranged from 0.50 to 0.97, with an average of 0.57. This is higher than the genetic diversity of cauliflower varieties detected by previous studies using SSR molecular markers (Liu et al. 2018; Zhao et al. 2014; Zhu et al. 2018). The genetic distance between some cauliflower varieties was 0.0345, close to 0, and the genetic similarity coefficient was 0.97, close to 1, suggesting that these varieties may have very similar genetic backgrounds.

[0110] Maturity is an important indicator for identifying cauliflower germplasm resources and a key factor affecting cluster analysis results, showing a certain correlation with genetic kinship. Cauliflower breeding practice has proven that the key to cultivating new varieties lies in selecting hybrid parents with complementary comprehensive traits and distant kinship. Meanwhile, head compactness is also an important agronomic trait that breeders need to consider. In this invention, SSR molecular marker technology was used to conduct genetic diversity analysis and cluster analysis on 84 tested cauliflower materials, revealing the genetic kinship of these 84 cauliflower germplasm resources at the molecular level. The results showed that the maturity and germplasm type of cauliflower varieties are related to genetic kinship. For example, Group I contained 61 cauliflower materials, of which inbred lines accounted for only 9.8%, mainly late-maturing and mid-maturing varieties; Group III consisted entirely of DH pure lines, with material 6 being a mid-maturing variety and the rest being late-maturing varieties; in Groups II and IV, except for material 2, which was a DH pure line, the rest were inbred lines, mainly late-maturing varieties. However, there was no significant correlation between the geographical origin and phylogenetic relationships of cauliflower varieties. For example, the cauliflower varieties in Groups I and II originated from Taiwan, Tianjin, Zhejiang, Xiamen, Guangdong, and other regions in China. This result is consistent with the findings of Zhu et al. (2018), suggesting that the dependence of each breeding unit on a few key parents is the main reason for the lack of significant phylogenetic relationships.

[0111] Example 3

[0112] This example illustrates the purity identification of cauliflower hybrids.

[0113] Using the 29 pairs of SSR primers in Table 2, the PCR reaction system and PCR reaction procedure in Example 1, DNA from "Jingyan Sijiqing 110" cauliflower (F1 generation) and its paternal parent W23-22 and maternal parent W23-43; and "Jingyan Sijiqing 100" cauliflower (F1 generation) and its paternal parent W23-28 and maternal parent W23-50 were amplified as templates. High-resolution agarose gel electrophoresis was used for detection. Primers that amplified polymorphic, stable banding patterns and complementary banding patterns of the parents in the F1 generation were selected. These primers can distinguish both the maternal parent and the parents and can be used to identify seed purity.

[0114] like Figure 5 As shown in Table 2, for the cauliflower hybrid "Jingyan Sijiqing 110", the seven primer pairs numbered 1 (primer pair name CB-2), 3 (primer pair name CB-10), 15 (primer pair name CB-124), 24 (primer pair name CH-54), 25 (primer pair name CH-56), 28 (primer pair name CH-170), and 29 (primer pair name QT-14) showed co-dominance and complementary banding between the parents, indicating that these seven primer pairs can be used for seed purity identification of the "Jingyan Sijiqing 110" cauliflower variety.

[0115] Then, using primer pair number 1 (primer pair name CB-2) from Table 2, 118 cauliflower hybrids of "Jingyan Sijiqing 110" and their parents were tested, and the results are as follows: Figure 7 As shown, it can be seen that the 36 materials numbered 3, 5, 7, 9, 10, 11, 14, 19, 25, 27, 28, 29, 31, 32, 34, 36, 37, 46, 49, 53, 95, 96, 98, 99, 100, 101, 102, 103, 107, 109, 110, 113, 114, 115, 116, and 118 can only amplify the maternal type band, indicating that they are maternal inbred lines. Therefore, the purity of the "Jingyan Sijiqing 110" cauliflower hybrid is (118-36) / 118 = 69.5%. Replacing primer pair 1 in Table 2 with primer pair 24 (primer pair name CH-54) while keeping everything else unchanged yields the following results: Figure 9 As shown, the identification results are completely consistent with those obtained using primer pair number 1 (primer pair name CB-2) in Table 2.

[0116] like Figure 6As shown in Table 2, for the cauliflower hybrid "Jingyan Sijiqing 100", the four primer pairs numbered 3 (primer pair name CB-10), 6 (primer pair name CB-21), 15 (primer pair name CB-124), and 24 (primer pair name CH-54) showed co-dominance and complementary banding between the parents, indicating that these seven primer pairs can be used for seed purity identification of the "Jingyan Sijiqing 110" cauliflower variety.

[0117] Then, using primer pair number 6 (primer pair name CB-21) from Table 2, 118 cauliflower hybrids of "Jingyan Sijiqing 100" and their parents were tested, and the results are as follows: Figure 8 As shown, only material numbered 108 can amplify the maternal type band, indicating it is a maternal inbred line. Therefore, the purity of the "Jingyan Sijiqing 100" cauliflower hybrid is (118-1) / 118 = 99.2%. Replacing primer pair number 6 in Table 2 with primer pair number 24 (primer pair name CH-54) while keeping everything else unchanged yields the following results: Figure 10 As shown, the identification results are completely consistent with those obtained using primer pair number 6 (primer pair name CB-21) in Table 2.

[0118] The purity P of a hybrid is expressed as a percentage and is calculated using the following formula:

[0119]

[0120] Where: P - variety purity; N - total number of seeds tested; N1 - number of individual plants identified as different varieties.

[0121] Cauliflower varieties mainly exist in the form of hybrids. Cauliflower hybrids have advantages such as high viability, strong adaptability, and excellent quality, but impurities in the hybrids are inevitable. This invention uses the "Jingyan Sijiqing 110" and "Jingyan Sijiqing 100" cauliflower hybrids as materials. It utilizes 29 pairs of SSR primers screened in this invention for purity identification of cauliflower hybrids. Based on the primer characteristics of complementary banding patterns required by the first-generation hybrids and their parents, primers with complementary banding patterns from both parents are screened for purity identification. Among them, the "Jingyan Sijiqing 110" cauliflower hybrid has 7 pairs of specific primers, and the "Jingyan Sijiqing 100" cauliflower hybrid has 4 pairs of specific primers. Primers CB-10, CB-124, and CH-54 all show specificity in both "Jingyan Sijiqing 110" cauliflower and hybrid No. 2, and can be used as the preferred core primers for purity identification. Other primers can be used as alternative primers. Two primer pairs were randomly selected from the specific primers for purity identification. The results were consistent, demonstrating the accuracy and reliability of SSR markers and saving time in field planting verification, thus improving the efficiency of seed purity detection. In this example, the purity of the "Jingyan Sijiqing 110" cauliflower hybrid was low, possibly due to the male sterility and self-incompatibility of cauliflower. Insufficient pollination led to some cauliflower materials exhibiting maternal characteristics during self-pollination, which, when mixed with the hybrid, reduced the purity of the cauliflower.

[0122] In summary, the 29 pairs of SSR primers screened in this invention exhibit clear amplification bands, good stability, and high polymorphism, making them highly suitable for applications such as cauliflower variety identification, phylogenetic evaluation, cultivation of new cauliflower germplasm, and purity identification of cauliflower hybrids.

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Primers for identifying the purity of cauliflower hybrids, wherein the primers are SSR primer pairs numbered 15 or 24 in the sequence listing; in, The cauliflower hybrid variety is "Jingyan Sijiqing 110".

2. The primers for identifying the purity of cauliflower hybrids according to claim 1, wherein the primers further include at least one SSR primer pair numbered 1, 3, 25, 28, 29 in the sequence listing.

3. A kit for identifying the purity of cauliflower hybrids, wherein the kit contains SSR primer pairs numbered 15 or 24 in the sequence listing; in, The cauliflower hybrid variety is "Jingyan Sijiqing 110".

4. The kit according to claim 3, wherein the kit further comprises at least one SSR primer pair numbered 1, 3, 25, 28, 29 in the sequence listing.

5. A method for identifying the purity of cauliflower hybrids, the method comprising using the primers described in claim 1 or 2, or using the kit described in claim 3 or 4; in, The cauliflower hybrid variety is "Jingyan Sijiqing 110".

6. Primers for identifying the purity of cauliflower hybrids, wherein the primers are SSR primer pairs numbered 15 or 24 in the sequence listing; in, The cauliflower hybrid variety is "Jingyan Sijiqing 100".

7. The primers for identifying the purity of cauliflower hybrids according to claim 1, wherein the primers further include SSR primer pairs numbered 3 and / or 6 in the sequence listing.

8. A kit for identifying the purity of cauliflower hybrids, the kit containing SSR primer pairs numbered 15 or 24 in the sequence listing; in, The cauliflower hybrid variety is "Jingyan Sijiqing 100".

9. The kit according to claim 3, wherein the kit further comprises SSR primer pairs numbered 3 and / or 6 in the sequence listing.

10. A method for identifying the purity of cauliflower hybrids, the method comprising using the primers of claim 6 or 7, or the kit of claim 8 or 9; in, The cauliflower hybrid variety is "Jingyan Sijiqing 100".

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