KASP molecular marker primer group closely linked with broccoli growth period character and application of KASP molecular marker primer group

Abstract

The invention discloses a KASP molecular marker primer group closely linked with broccoli growth period characters and application of the KASP molecular marker primer group, and relates to the technical field of molecular biology. The primer group comprises an FC84287 primer group and an RC84556 primer group, the FC84287 primer group comprises three primers of which the nucleotide sequences are as shown in SEQ ID NO.1-3, and the RC84556 primer group comprises three primers of which the nucleotide sequences are as shown in SEQ ID NO.4-6. The invention further discloses a kit for detecting the content of the primer group. The molecular marker primer group provided by the invention can be efficiently and accurately used for distinguishing the growth period character of the cauliflower, can also be used for breeding cauliflower strains in different growth periods, and has an application prospect in the field of cauliflower planting.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a KASP molecular marker primer set closely linked to the growth period traits of cauliflower and its application. Background Art

[0002] Cauliflower ( Brassica oleracea var. botrytis Linnaeus) is a variety of the genus Brassica ( Brassica ) in the family Brassicaceae, and is a unique vegetable with the inflorescence meristem - composed flower head as the edible part. Its swollen flower head is rich in various anti - cancer active substances (such as glucosinolates, flavonoids, etc.) as well as nutrients such as vitamins and dietary fiber, and has high nutritional value and health care functions. Brassica oleracea With the rapid development of social economy and the continuous enhancement of residents' health awareness, consumers have put forward higher standards for the nutritional quality and functional characteristics of vegetable crops. This trend has directly driven the diversification of the demand structure for cauliflower varieties. From the perspective of industrial development, constructing a variety system with differentiated growth period characteristics plays a key role in realizing regional adaptive cultivation and annual market supply. In the practice of cauliflower genetic improvement and breeding, the growth period (the duration from transplanting to the commercial maturity of the flower head) is a key trait that determines the regional adaptability and market supply of varieties. The growth periods of existing cultivated varieties show significant differences. Early - maturing varieties (about 50 days), mid - maturing varieties (80 - 90 days) and late - maturing varieties (up to 200 days) together constitute a complete variety pedigree. Therefore, breeding cauliflower varieties with stable traits and significant differences in growth period to meet the market demand for varieties with different growth periods is one of the important goals of cauliflower breeding. However, compared with traits such as the appearance quality of the flower head, the genetic mapping research on the cauliflower growth period is relatively lagging, and there are very few molecular markers available for breeding, which severely restricts the breeding process of excellent new cauliflower varieties.

[0003] Therefore, based on the forward genetics strategy, identifying genetic loci highly associated with the cauliflower growth period traits and developing reliable molecular markers and supporting efficient detection technologies based on their relevant sequence information can provide important theoretical and practical guidance for carrying out marker - assisted selection (MAS) and accelerating the breeding of excellent cauliflower varieties with suitable growth periods.

[0004] The present invention aims to provide a KASP molecular marker primer set closely linked to the major QTL of the cauliflower growth period traits, which can be used to identify the cauliflower growth period traits and assist in the molecular breeding of cauliflower. Summary of the Invention

[0005]

[0006] ​To achieve the above object, the present invention provides a set of KASP molecular marker primers closely linked to the growth period traits of cauliflower. This set of primers consists of two groups, namely the FC84287 primer group and the RC84556 primer group. The FC84287 primer group contains 3 primers with nucleotide sequences shown in SEQ ID NO.1 - 3, and the RC84556 primer group contains 3 primers with nucleotide sequences shown in SEQ ID NO.4 - 6.

[0007] The present invention also provides a kit for identifying the growth period traits of cauliflower, which kit contains the above-mentioned primer set.

[0008] Preferably, the above-mentioned kit further contains KASP Master mix, which specifically includes a general FRET cassette fluorescent primer, ROX internal reference dye, Klear Taq DNA polymerase, dNTP, and MgCl 2 .

[0009] The primer set or kit provided by the present invention can be used in the field of cauliflower cultivation.

[0010] Preferably, the primer set or kit provided by the present invention can be used in any one of the following, including: (1) Identification of the growth period traits of cauliflower; (2) Assisting in the breeding of cauliflower at different growth periods; (3) Creating cauliflower lines with different growth periods.

[0011] Preferably, by performing PCR amplification on a cauliflower sample using the above-mentioned primer set or kit, the growth period traits of cauliflower are judged according to the genotyping of the PCR product; Among them, when the genotyping of both the FC84287 and RC84556 primer groups is A:A, the average growth period is more than 60 days; When the genotyping of the FC84287 and RC84556 primer groups is T:T and C:C respectively, the average growth period is less than 50 days.

[0012] Among them, the two primer sets provided in the present invention can be separately used for the genotyping of cauliflower and the identification of growth period traits. That is, when the genotyping of the FC84287 primer group is A:A, its average growth period is more than 60 days, and when the genotyping result is T:T, the average growth period is less than 50 days; When the genotyping of the RC84556 primer group is A:A, the average growth period is more than 60 days, and when the genotyping result is C:C, the average growth period is less than 50 days.

[0013] Preferably, the reaction system for the above PCR amplification is: PCR premix, the primer set as described in claim 1, and template DNA.

[0014] Preferably, the reaction conditions for the above PCR amplification are: pre-denaturation at 94 °C for 15 min; denaturation at 94 °C for 20 s, annealing and extension at 61 - 55 °C for 60 s, with the annealing temperature decreasing by 0.6 °C for each cycle, for a total of 10 cycles; denaturation at 94 °C for 20 s, annealing and extension at 55 °C for 60 s, for a total of 26 cycles.

[0015] The present invention has the following advantages: (1) The major QTL loci for the growth period of cauliflower provided by the present invention and its supporting molecular marker detection system are applicable to molecular marker-assisted selection for the "growth period" trait of cauliflower, and have the characteristics of simple operation, high detection throughput, and strong applicability. The method provided by the present invention can quickly identify breeding materials at the seedling stage, achieve early elimination of non-target genotype individuals, significantly shorten the breeding cycle, improve the breeding efficiency, and have good practicality and promotion prospects.

[0016] (2) As a typical quantitative trait, the "growth period" is controlled by multiple genes and is easily affected by the environment. Traditional breeding relies on field phenotypic observation for selection, with low accuracy and efficiency. By detecting the genotype of the target QTL locus at the molecular level, the present invention can effectively determine whether the sample carries the allele controlling the extension of the "growth period", thereby realizing early, efficient, and accurate identification of this trait, and is applicable to large-scale material screening and auxiliary selection in the process of molecular breeding. Description of the Drawings

[0017] Figure 1 It is the phenotypic distribution of the growth period trait of three parents and the double F 2 population constructed in the present invention.

[0018] Figure 2 It is the distribution of double-population QTLs for the growth period trait of cauliflower in the present invention.

[0019] Figure 3 It is the result of KASP genotyping of the F 2:3 population using the developed marker primers FC84287 and RC84556 in the present invention. Detailed Embodiments

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Note: Unless otherwise specified, the experimental methods in the following examples are all conventional methods, which are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0022] Experimental Example 1 This experimental example provides the development of a KASP molecular marker linked to the "growth period" trait of cauliflower, specifically as follows: 1. Genetic mapping of the "growth period" trait of cauliflower Using three high-generation inbred line parents (C4101, J1401, R06), where C4101 is a compact cauliflower material, and it takes about 50 days from transplanting the plant to the appearance of the curd; J1401 is a Chinese kale parent, and it takes about 30 days from transplanting the plant to the appearance of the bud; R06 is a local broccoli variety introduced from Italy, and it takes about 75 days from transplanting the plant to the appearance of the bud. Using C4101 as the common female parent, it was crossed and self-crossed with J1401 and R06 respectively to construct two F 2 genetic segregation populations: namely CJ-F 2 (including 220 individual plants) and CR-F 2 (including 212 individual plants). The phenotypic distributions of the growth period traits of the three parents and the double F 2 populations they constructed are shown in Figure 1 as follows. Among them, A from left to right are the growth phenotypes of the female parent cauliflower material C4101, the male parent Chinese kale material J1401, and the male parent local broccoli variety material R06; B-C are the phenotypic distributions of the growth period traits of the CJ-F 2 population and its parents; D-E are the phenotypic distributions of the growth period traits of the CR-F 2 population and its parents.

[0023] Using the developed 40K MNP liquid chip for cauliflower to detect the genotypes of the parents and individual plants of the corresponding F 2 populations. Finally, 3569 and 3685 SNPs were respectively screened out to construct the high-density genetic maps of the CJ-F 2 and CR-F 2 populations. The "growth period" traits of the parents and individual plants of the population were determined by investigating the time from transplanting the plant to the visible cauliflower head (cauliflower head diameter about 2 cm) or bud (days to curd / bud appearance, abbreviated as DCA). Combining the phenotypic and genotypic data of the individual plants of the population, using QTL IciMapping 4.0 software for QTL mapping, major QTLs were mapped on chromosome C8 in both the CJ-F 2 and CR-F 2 populations (denoted as qDCA8-1), aligned to the HDEM reference genome (https: / / www.genoscope.cns.fr / projet_BKL / cgi-bin / gbrowse / boleracea / ). The results showed that the physical positions of these two QTLs overlapped, indicating that the co-localized QTL played a stable regulatory role in the "growth period" trait of cauliflower under different genetic backgrounds. The distribution of QTLs for the growth period trait in the two populations of cauliflower is shown in Figure 2 as follows, where A: CJ-F 2 QTL mapping for the growth period trait in the population; B: CR-F 2 QTL mapping for the growth period trait in the population.

[0024] 2. Development of molecular marker primers linked to the "growth period" trait of cauliflower Within the physical interval of the HDEM reference genome corresponding to the major QTL locus obtained above, based on the SNPs in this interval from the DNA re-sequencing information of the parents, KASP primers were designed using the SNPs at both ends, denoted as primer sets FC84287 and RC84556 respectively. The specific sequences are as follows. Through further screening and verification of the genetic population, these primers can be used to quickly and accurately screen the "growth period" phenotype of plants.

[0025] The identification of the growth period trait can be achieved by using any one of the designed marker primer sets, and the other marker can be used for repeated verification.

[0026] Universal adapter sequences were added to the forward primers FAM and HEX of the primer sets respectively, as follows: FC84287 primer set: Forward primer F-FAM (SEQ ID NO.1): gaaggtgaccaagttcatgctTATGGAAATCTGCTAGTTA; Forward primer F-HEX (SEQ ID NO.2): gaaggtcggagtcaacggattTATGGAAATCTGCTAGTTT; Reverse primer R (SEQ ID NO.3): TCGTATATATCCACTCCGAACT.

[0027] RC84556 primer set: Forward primer F-FAM (SEQ ID NO.4): gaaggtgaccaagttcatgctCAGCCACGGATGTAAAGTCTTCA; Forward primer F-HEX (SEQ ID NO.5): gaaggtcggagtcaacggattCAGCCACGGATGTAAAGTCTTCC; Reverse primer R (SEQ ID NO.6): TAGAACACGCTACTTGGTTGCAGG.

[0028] In the above primer pair, the two forward primers have base differences at the 3'-end, which can competitively bind to the target site, showing the corresponding FAM or HEX fluorescence. After signal amplification, the genotype of the target site can be determined.

[0029] Experimental Example 2 This experimental example is the verification of the developed KASP molecular marker primer set in the segregating population, as follows: 1. Extraction and purification of DNA from the segregating population to be tested In the CJ-F 2-188 Among the individual plants in the population, select the individual plants with heterozygous target QTL intervals for self-crossing to construct the CJ-F 2:3 Segregating population. Randomly select 168 individual plants among them, take the leaves of the individual plants when there are 1-2 true leaves, extract the leaf DNA by the CTAB method, and detect the quality and concentration of the DNA by using a Nano-400A ultra-micro nucleic acid analyzer.

[0030] 2. Genotype identification of individual plants in the secondary segregating population using FC84287 and RC84556 Add specific KASP Primer mix (KASP primer mixture) and universal KASP Master mix to the above-extracted DNA template for PCR amplification; among them, KASP Master mix contains the following components: universal FRETcassette fluorescent primer, ROX internal reference dye, Klear Taq DNA polymerase, dNTP and MgCl 2 ; The specific conditions are as follows: The PCR reaction system for KASP detection is: 5 μL of PCR premix, 0.14 μL of KASP primer mixture (the final concentration of each primer is 5 nM), and 5 μL of 20 ng / μL template DNA; The reaction conditions of PCR are: pre-denaturation at 94 °C for 15 min; denaturation at 94 °C for 20 s, annealing and extension at 61-55 °C for 60 s, and the annealing temperature decreases by 0.6 °C for each cycle, for a total of 10 cycles; denaturation at 94 °C for 20 s, annealing and extension at 55 °C for 60 s, for a total of 26 cycles; Obtain the F using the marker primer sets FC84287 and RC845562:3 The population was genotyped by KASP, and the genotyping results are shown in Figure 3 as follows. Among them, A is the genotyping result of the FC84287 marker primer set; B is the genotyping result of the RC84556 marker primer set; C is the growth period distribution of the three genotype materials (in C, a is the C4101 genotype; b is the J1401 genotype; h is the heterozygous genotype); CJ-F 2:3 The genotype and growth period data of 168 individual plants in the CJ-F population are shown in Table 1. The results show that among the 168 individual plants, the genotyping trends of the two marker primer sets, FC84287 and RC84556, are consistent. Among them, there are 44 individual plants with the same genotype as the parent C4101 (a genotype, A:A), 52 individual plants with the same genotype as the parent J1401 (b genotype, T:T and C:C), and 92 individual plants with heterozygous genotypes (h genotype, T:A and C:A).

[0031] Table 1 CJ-F 2:3 Genotype and growth period data of 168 individual plants in the CJ-F population According to the genotyping and phenotypic results of 168 individual plants in the CJ-F 2:3 population, the lines with heterozygous genotypes were discarded because generally only homozygous ones have obvious phenotypes in marker screening, and the phenotypes of heterozygous ones vary greatly and are not indicative and selective.

[0032] By counting the time from transplanting to visible flower buds of the above 168 individual plants and combining the genotype data of each individual plant, it was found that the average growth period of a-genotype individual plants was 62.7 days; the average growth period of b-genotype was 41.3 days; the average growth period of h-genotype individual plants was 54.2 days. t - Detection showed that there were extremely significant differences between the phenotypes of the two different genotypes ( P <0.001), indicating that this marker and its molecular marker primer set can be used efficiently and accurately to distinguish the "growth period" trait of cauliflower.

[0033] 3. Genotype identification of core germplasm materials using FC84287 and RC84556 To further verify the universality of the FC84287 and RC84556 markers in identifying the "growth period" trait, a total of 31 core germplasm materials of cauliflower were selected for verification. This group of materials are homozygous parental materials created through multiple generations of self-crossing or microspore culture. Through sowing, seedling raising, and numbering, single-plant leaves were taken when there were 1-2 true leaves. Genomic DNA was extracted separately according to the conventional CTAB method, and the quality and concentration of the DNA were detected using a Nano-400A ultra-micro nucleic acid analyzer, and stored at -20°C for later use. Using the FC84287 and RC84556 primer sets and the same KASP marker PCR amplification and detection system as above, genotype identification was performed on 31 core germplasm materials. The results showed that there were 25 single plants with the same genotype as the parental C4101 (a genotype, A:A), and 6 single plants with the same genotype as the parental J1401 (b genotype, T:T and C:C). The specific phenotypic and genotype data are shown in Table 2.

[0034] Table 2 Genotype and growth period data of 31 core germplasm materials The correspondence between the growth period phenotypes and genotypes in Table 1 is relatively broad, while in Table 2 it is relatively concentrated. It is speculated that because the growth period trait is controlled by multiple target loci, and this locus in this study is a major locus with a relatively large effect. The materials in Table 1 are the segregating populations after crossing two parents with different growth periods, and the genotypes of the single plants in the population are not homozygous (possibly also affected by other minor loci and cultivation environment factors, etc.), showing a certain span of distribution in the growth period. While the core germplasm materials in Table 2 are homozygous parental materials obtained through multiple generations of self-crossing, and the growth period is relatively fixed.

[0035] Based on the growth period phenotypic determination results of 31 core germplasm materials of cauliflower, combined with the FC84287 and RC84556 genotype data of each material, the average growth period of a genotype materials is 93.8 days, and the average growth period of b genotype materials is 48.3 days. t - The detection shows that there are extremely significant differences between the phenotypes of the two different genotypes ( P <0.001), indicating the universality of this marker in distinguishing the "growth period" trait of cauliflower crops, and further indicating that this molecular marker primer set can be used efficiently and accurately to distinguish the "growth period" trait of cauliflower.

[0036] Although the content of the present invention has been introduced in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions to the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A KASP molecular marker primer set tightly linked to the growth period traits of cauliflower, characterized in that: The primer set is the FC84287 primer set or / and the RC84556 primer set, the FC84287 primer set comprises three primers whose nucleotide sequences are shown as SEQ ID NOs.1-3, and the RC84556 primer set comprises three primers whose nucleotide sequences are shown as SEQ ID NOs.4-6.

2. A kit for identifying the growth period traits of cauliflower, characterized in that: The kit comprises the primer set according to claim 1.

3. The kit according to claim 2, characterized in that The kit also contains KASP Master mix.

4. The kit according to claim 3, characterized in that The KASP Master mix contains universal FRETcassette fluorescent primers, ROX internal reference dye, Klear Taq DNA polymerase, dNTPs and MgCl2.

5. Use of the primer set according to claim 1 or the kit according to claims 2-4 in the field of cauliflower cultivation.

6. The use according to claim 5, characterized in that: The application includes any of the following: (6) Identification of growth period traits of cauliflower; (7) Assisting the cultivation of cauliflower at different growth stages; (8) Create cauliflower strains with different growth stages.

7. The use according to claim 6, characterized in that: Using the primer set according to claim 1 or the kit according to any one of claims 2 to 4 to perform PCR amplification on the cauliflower sample, and judging the growth period traits of the cauliflower according to the genotyping of the PCR product; Among them, when the genotyping of primer sets FC84287 and RC84556 were both A:A, the average growth period was more than 60 days; When the genotyping of primer sets FC84287 and RC84556 were T:T and C:C, respectively, the average growth period was less than 50 days.

8. The use according to claim 6, characterized in that: When the primer set according to claim 1 or the kit according to any one of claims 2 to 4 is used to perform PCR amplification on a cauliflower sample, the primer sets FC84287 and RC84556 can be used alone for genotyping of cauliflower and identification of growth period traits; When the genotyping result using the FC84287 primer set was A:A, the average growth period was more than 60 days, and when the typing result was T:T, the average growth period was less than 50 days; When the genotyping result using the RC84556 primer set is A:A, the average growth period is more than 60 days, and when the genotyping result is C:C, the average growth period is less than 50 days.

9. The use according to claim 7, characterized in that: The reaction system of PCR amplification comprises: PCR premix, the primer set as claimed in claim 1 and template DNA.

10. The use according to claim 7, characterized in that: The reaction conditions of the PCR amplification are as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing and extension at 61-55°C for 60 s, with the annealing temperature decreasing by 0.6°C in each cycle, for a total of 10 cycles; denaturation at 94°C for 20 s, annealing and extension at 55°C for 60 s, for a total of 26 cycles.

Images