SSR Molecular Markers Associated with the Fresh Weight Trait of Corydalis bungeana Turcz. Tubers, Their Primers and Applications

Abstract

The present invention discloses SSR molecular markers associated with the fresh weight trait of Corydalis bulbocapniodes sub-bulbs, their primers and applications, belonging to the technical field of molecular genetics. The SSR molecular markers associated with the fresh weight trait of Corydalis bulbocapniodes sub-bulbs disclosed by the present invention have amplification primer sequences as shown in SEQ ID NO.1 and 2. The SSR molecular markers associated with the fresh weight trait of Corydalis bulbocapniodes sub-bulbs and their amplification primers can be applied to the population structure analysis of Corydalis bulbocapniodes, the analysis of markers associated with the fresh weight trait of Corydalis bulbocapniodes sub-bulbs, the selection of excellent germplasms of Corydalis bulbocapniodes and the preparation of a kit for screening excellent germplasms of Corydalis bulbocapniodes with high sub-bulb weight, and can be directly used for screening excellent germplasms, gene mapping and cloning, and molecular marker-assisted breeding. Moreover, they have chromosome mapping information, which is beneficial to further fine mapping and cloning of genes controlling the fresh weight trait of sub-bulbs, and is of great significance in the breeding of high-yield Corydalis bulbocapniodes.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular genetics, and particularly relates to SSR molecular markers associated with the fresh weight trait of Corydalis yanhusuo tubers, primers thereof, and applications thereof. Background Art

[0002] Association analysis is a method for studying the inheritance of quantitative traits and has been widely used in the discovery of beneficial genes in crops. Association analysis can use natural populations as materials, and can simultaneously examine all alleles of the same locus in a large number of germplasm materials to find associated loci. SSR markers have the advantages of wide distribution, many allelic variations, co-dominance, good repeatability, reliable results, etc., and are widely used in association analysis, QTL mapping, marker-assisted breeding, and the construction of genetic linkage maps, etc.

[0003] Corydalis yanhusuo W.T. Wang, also known as Yuanhu, is a plant of the genus Corydalis DC. in the family Papaveraceae Juss. In production, the multiplication coefficient of Corydalis yanhusuo is only about 5. For 100 - 150 kg of seed tubers, the high-yield situation is about 500 kg of yield. At present, the space for increasing yield through cultivation techniques is already very limited. Therefore, it is urgent to carry out the breeding work of high-yield Corydalis yanhusuo varieties.

[0004] At present, there has been a study applying SSR molecular marker technology to analyze 360 Corydalis yanhusuo samples from 19 populations, revealing their genetic diversity and genetic structure, and thereby understanding their evolutionary potential and adaptability to the environment, providing a theoretical basis and technical support for the scientific protection and utilization of Corydalis yanhusuo plant resources (Xu Hao, Zhou Tianhua, Zhang Zhiqiang, etc. Study on Genetic Diversity of Corydalis yanhusuo Based on SSR Molecular Markers [J]. Acta Botanica Boreali-Occidentalia Sinica, 2018, 38(6): 1032 - 1038.). However, the high-yield breeding of Corydalis yanhusuo is currently blank. Improving the fresh weight of Corydalis yanhusuo tubers is one of the goals of high-yield breeding of Corydalis yanhusuo.

[0005] Traditional high-yield breeding of Corydalis yanhusuo not only has a long cycle, is time-consuming and laborious, but also because the investigation of the yield traits of Corydalis yanhusuo is for the underground part, it makes the trait investigation inconvenient and extremely destructive. Therefore, using molecular marker-assisted selection is not only convenient for the selection of excellent lines, but also can shorten the breeding cycle and reduce the breeding cost. Summary of the Invention

[0006] In view of the above problems existing in the prior art, the technical problems to be solved by the present invention are to provide SSR molecular markers associated with the fresh weight trait of Corydalis bulbocapniodes bulblets. Another technical problem to be solved by the present invention is to provide amplification primers for the SSR molecular markers associated with the fresh weight trait of Corydalis bulbocapniodes bulblets. Another problem to be solved by the present invention is to provide the application of the SSR molecular markers associated with the fresh weight trait of Corydalis bulbocapniodes bulblets. Another problem to be solved by the present invention is to provide the application of the amplification primers for the SSR molecular markers associated with the fresh weight trait of Corydalis bulbocapniodes bulblets, which is used to screen excellent germplasms of Corydalis bulbocapniodes with high bulblet fresh weight.

[0007] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] SSR molecular markers associated with the fresh weight trait of Corydalis bulbocapniodes bulblets, and the SSR molecular marker is CYC8-10.

[0009] Amplification primers for the SSR molecular markers associated with the fresh weight trait of Corydalis bulbocapniodes bulblets, and the sequences of the amplification primers for the SSR molecular markers are as follows:

[0010] CYC8-10-F: 5’-CCAAGATTGGCCACGTAGAT-3’,

[0011] CYC8-10-R: 5’-CCATGCAACCATCTCATTCA-3’.

[0012] The application of the SSR molecular markers in the population structure analysis of Corydalis bulbocapniodes.

[0013] The application of the amplification primers for the SSR molecular markers in the population structure analysis of Corydalis bulbocapniodes.

[0014] The application of the SSR molecular markers in the analysis of markers associated with the fresh weight trait of Corydalis bulbocapniodes bulblets.

[0015] The application of the amplification primers for the SSR molecular markers in the analysis of markers associated with the fresh weight trait of Corydalis bulbocapniodes bulblets.

[0016] The application of the SSR molecular markers in the selection and breeding of excellent germplasms of Corydalis bulbocapniodes.

[0017] The application of the amplification primers for the SSR molecular markers in the selection and breeding of excellent germplasms of Corydalis bulbocapniodes.

[0018] The application of the SSR molecular markers in the preparation of a kit for screening excellent germplasms of Corydalis bulbocapniodes with high bulblet weight.

[0019] The application of the amplification primers for the SSR molecular markers in the preparation of a kit for screening excellent germplasms of Corydalis bulbocapniodes with high bulblet weight.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The present invention utilizes the Corydalis yanhusuo genome sequencing information (Dingqiao Xu, Ziqi Ye. et al. Haplotype-resolved genome assembly of Corydalis yanhusuo, a traditional Chinese medicine with unusual telomere motif, Horticulture Research, Volume 11, Issue 2, February 2024, uhad296) to explore SSR loci, and finally screens 24 SSR markers with clear bands and good polymorphism for polymorphism analysis, and synthesizes 24 pairs of amplification primers. Using 24 pairs of SSR markers and their amplification primers, a regression analysis is performed on the individual genotypes of Corydalis yanhusuo and the fresh weight trait of daughter bulbs. Through the GLM program, the associated loci of the fresh weight of Corydalis yanhusuo daughter bulbs are detected. At a significance level of P<0.01, 1 marker CYC8-10 associated with the fresh weight trait of daughter bulbs is detected. The amplification product size of the associated locus between CYC8-10 and the fresh weight trait of daughter bulbs is 270bp, and the phenotypic variation explanation rate is 15.97%. The primers for amplifying the SSR molecular markers provided by the present invention can be applied to screening excellent germplasms of Corydalis yanhusuo with high daughter bulb weight, localizing key genes controlling the fresh weight trait of daughter bulbs, and molecular marker-assisted breeding, which is of great significance for cultivating high-yield Corydalis yanhusuo varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a ΔK analysis diagram in the Structure genetic structure analysis;

[0023] Figure 2 It is a genetic structure diagram when K = 4;

[0024] Figure 3 It is an electrophoresis diagram of the SSR marker CYC8-10 (M represents Marker 50bp, and the red arrow represents the associated locus). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified in detail, the technical means used are all conventional means well known to those skilled in the art.

[0026] Example 1

[0027] 1. Investigation and statistical analysis of the phenotypic traits of Corydalis yanhusuo

[0028] Ten days before the harvest of Corydalis yanhusuo, the fresh weight of daughter bulbs and the total fresh weight data of 41 individual plants were collected and investigated, and their maximum value, minimum value, range, standard deviation and coefficient of variation were statistically analyzed. The phenotypic data of 41 complete individual plants are shown in Table 1. The specific collection method of 41 individual plants is as follows: Randomly divide three plots of 10 cm in the middle of the field, and dig out all the Corydalis yanhusuo seedlings growing in the plots, and a total of 41 complete individual plants are obtained. 2 The results are shown in Table 2. There is a great variation in the fresh weight of daughter bulbs among Corydalis yanhusuo individuals, and the coefficient of variation is 79%.

[0029] Table 1 Phenotypic data of 41 complete individual plants

[0030] Table 2 Variation of fresh weight traits of daughter bulbs of Corydalis yanhusuo

[0031]

[0032] Table 3 Pearson coefficient between fresh weight of daughter bulbs and total fresh weight

[0033]

[0034] Use Graphpad to analyze the Pearson correlation between the fresh weight of daughter bulbs and the total yield.

[0035] The results are shown in Table 3. There is a very significant positive correlation between the fresh weight of daughter bulbs and the total fresh weight, and the correlation coefficient is 0.957.

[0036] Table 3 Pearson coefficient between fresh weight of daughter bulbs and total fresh weight

[0037]

[0038] Example 2

[0039] 1. Extract the genomic DNA of Corydalis yanhusuo

[0040] Use a new plant genomic DNA extraction kit (Shanghai Pudi Biology) to extract the DNA of fresh leaves of Corydalis yanhusuo individual plants X1-X24 in sequence. The extracted DNA products are detected by 1% agarose gel electrophoresis, and the DNA bands are clear and undegraded.

[0041] 2. Polymorphism analysis of SSR markers

[0042] Use the genomic sequencing information of Corydalis yanhusuo to discover SSR loci, select and synthesize 80 pairs of primers, and the primers are synthesized by Shanghai Sangon; using the extracted DNA as a template, 80 pairs of primers are used for PCR amplification verification, and the PCR amplification products are detected by polyacrylamide gel electrophoresis.

[0043] The PCR amplification system is: 0.2 μL of 2.5 U / μL Taq DNA polymerase, containing 10 mM Mg 2+1.0 μL of 10× PCR reaction buffer, 0.2 μL each of 2.5 mM dNTPs, 0.5 μL each of 10 μM upstream and downstream primers, 0.5 μL of 40 ng / μL DNA template, and made up to 10 μL with ddH2O. The PCR reaction program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, for a total of 32 cycles; extension at 72°C for 10 min, and incubation at 4°C.

[0044] As shown in Table 4, a total of 24 SSR markers with clear bands and good polymorphism were finally screened for polymorphism analysis. The sequence information of 24 pairs of amplification primers is shown in Table 5.

[0045] Table 4. Polymorphism of 24 SSR markers

[0046]

[0047] Table 5. Sequences of 24 pairs of primers

[0048]

[0049] 3. Genetic structure analysis of SSR markers

[0050] The DNA of 24 individual Corydalis yanhusuo plants was subjected to PCR amplification using the 24 pairs of SSR primers screened out.

[0051] The 10 μL PCR amplification system was: 20 ng genomic DNA, 2.5 mM MgCl2, 0.5 mM dNTPs, 20 ng primer, and 0.5 U Taq DNA polymerase.

[0052] The PCR reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 20 s, extension at 72°C for 30 s, for 32 cycles; finally, extension at 72°C for 5 min.

[0053] PCR products were detected using 10% polyacrylamide gel electrophoresis. The sample loading volume was 1.5 μL, the electrophoresis buffer was 1× TBE, the voltage was set at 220 V, and electrophoresis was carried out until the bromophenol blue band ran out of the bottom of the gel.

[0054] Silver staining of polyacrylamide gel: First, fix with a fixing solution (deionized water, 10% ethanol, 1% acetic acid) for 10 min, then soak in 1.5% silver nitrate solution for 10 min. After quickly washing twice with deionized water, develop with a developing solution (deionized water, 1.5% sodium hydroxide, 1% formaldehyde) for 10 min.

[0055] Electrophoresis data analysis: The amplification results of SSR primers were recorded in binary. Bands with the same mobility at the same locus were recorded as 1, and bands without were recorded as 0 to obtain the genotype data of 24 Corydalis yanhusuo materials; the population structure of Corydalis yanhusuo was analyzed using Structure 2.3.4 software in combination with the genotype data.

[0056] The results are as Figure 1 and Figure 2 shown. When K = 4, the △K value is the largest, indicating that it is most reasonable to divide the 24 Corydalis yanhusuo materials into 4 groups ( Figure 1 ); when K = 4, the genetic structure of the 24 Corydalis yanhusuo populations is as Figure 2 shown.

[0057] 4. Analysis of association markers for the fresh weight trait of daughter bulbs

[0058] Using the corresponding Q values of the 24 individual plants obtained from the Structure analysis (Table 6), the GLM program in Tassel5 software was used with the Q value as the covariate to perform a regression analysis on the original phenotypic values (Table 7) of the fresh weight of the daughter bulbs of Corydalis yanhusuo individual plants and the marker variations.

[0059] Table 6 Q value matrix of 24 individual plants

[0060]

[0061] Table 7 Original phenotypic values of the fresh weight of the daughter bulbs of 24 Corydalis yanhusuo individual plants

[0062]

[0063] The results are shown in Table 8. At a significance level of P < 0.01, 1 marker CYC8 - 10 associated with the fresh weight trait of the daughter bulbs was detected. The amplification product size of the locus where CYC8 - 10 is associated with the fresh weight trait of the daughter bulbs is 270 bp ( Figure 3 ), and the phenotypic variation explanation rate is 15.97%.

[0064] Table 8 Analysis of association markers for the fresh weight trait of the daughter bulbs

[0065]

[0066] Example 3

[0067] A kit for screening excellent germplasms of Corydalis yanhusuo with high daughter bulb weight, the kit includes the following molecular marker combinations:

[0068] The marker CYC8 - 10 associated with the fresh weight trait of the daughter bulbs and its amplification primers. The amplification primer sequences are as follows:

[0069] CYC8-10-F: 5’-CCAAGATTGGCCACGTAGAT-3’,

[0070] CYC8-10-R: 5’-CCATGCAACCATCTCATTCA-3’.

[0071] Thus, it is possible to simply and accurately identify the fresh weight of Corydalis yanhusuo tubers under different environments, different periods, and different growing season conditions.

[0072] In summary, the SSR molecular markers and their primers developed in the present invention can be directly used for screening excellent germplasms, gene mapping and cloning, and molecular marker-assisted breeding. Moreover, they have chromosome mapping information, which is beneficial to further fine mapping and cloning of the genes controlling the fresh weight trait of tubers, and is of great significance in the breeding of high-yield Corydalis yanhusuo.

Claims

1. Amplification primers of SSR molecular markers associated with the fresh weight trait of Corydalis bulbocapnoides, characterized in that, The amplification primer sequences of the SSR molecular markers are as follows: CYC8-10-F: 5’-CCAAGATTGGCCACGTAGAT-3’, CYC8-10-R: 5’-CCATGCAACCATCTCATTCA-3’.

2. Application of the amplification primer of the SSR molecular marker associated with the fresh weight trait of Corydalis bulbocapnioides in the analysis of the marker associated with the fresh weight trait of Corydalis bulbocapnioides as described in claim 1.

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