SSR (Simple Sequence Repeat) molecular marker associated with fresh weight character of corydalis tuber bulbules as well as primer and application thereof

Abstract

The invention discloses an SSR (simple sequence repeat) molecular marker associated with fresh weight character of corydalis tuber bulbules as well as primers and application of the SSR molecular marker, and belongs to the technical field of molecular genetics. According to the SSR molecular marker associated with the fresh weight character of the corydalis tuber bulbous disclosed by the invention, the amplification primer sequences of the SSR molecular marker are shown as SEQ ID NO.1 and SEQ ID NO.2. The SSR molecular marker associated with the fresh weight character of the rhizoma corydalis coleobulb and the amplification primer thereof can be applied to rhizoma corydalis population structure analysis, rhizoma corydalis coleobulb fresh weight character associated marker analysis, rhizoma corydalis excellent germplasm breeding and preparation of a kit for screening rhizoma corydalis excellent germplasm with high coleobulb weight. The method can be directly used for excellent germplasm screening, gene localization and cloning and molecular marker-assisted breeding, has chromosome localization information, is beneficial to further fine localization and cloning of genes for controlling fresh weight characters of corpus balls, and has important significance in breeding of high-yield rhizoma corydalis.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular genetics, and in particular relates to an SSR molecular marker associated with the fresh weight trait of Corydalis bulblets, a primer thereof and an application thereof. Background Art

[0002] Association analysis is a method for studying the inheritance of quantitative traits and has been widely used in the mining 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 sites. SSR markers have the advantages of wide distribution, multiple allelic variations, codominance, good repeatability, and reliable results. They are widely used in association analysis, QTL positioning, marker-assisted breeding, and the construction of genetic linkage maps.

[0003] Corydalis yanhusuo WT Wang, also known as Yuanhu, is a plant of the genus Corydalis DC. of the family Papaveraceae Juss. In production, the proliferation coefficient of Corydalis yanhusuo is only about 5, and the yield of 100-150 kg of bulbs is about 500 kg in high-yield conditions. At present, there is very limited room for increasing yields through cultivation technology. Therefore, it is urgent to carry out the breeding of high-yield Corydalis yanhusuo varieties.

[0004] At present, some studies have applied SSR molecular marker technology to analyze 360 ​​samples of Corydalis yanhusuo from 19 populations, revealing its genetic diversity and genetic structure, and based on this, understanding its evolutionary potential and adaptability to the environment, providing theoretical basis and technical support for the scientific protection and utilization of Corydalis yanhusuo plant resources (Xu Hao, Zhou Tianhua, Zhang Zhiqiang, et al. Study on genetic diversity of Corydalis yanhusuo based on SSR molecular markers [J]. Northwest Botanical Journal, 2018, 38(6): 1032-1038.). However, high-yield breeding of Corydalis yanhusuo is still a blank. Improving the fresh weight of Corydalis yanhusuo bulbs is one of the goals of high-yield breeding of Corydalis yanhusuo.

[0005] Traditional high-yield breeding of Corydalis yanhusuo is not only time-consuming and labor-intensive, but also inconvenient and highly destructive because the survey of Corydalis yanhusuo yield traits is done underground. Therefore, the use of molecular marker-assisted selection not only facilitates superior line selection, but also shortens the breeding cycle and reduces breeding costs. Summary of the invention

[0006] In view of the above-mentioned problems existing in the prior art, the technical problem to be solved by the present invention is to provide an SSR molecular marker associated with the fresh weight trait of Corydalis bulblets. Another technical problem to be solved by the present invention is to provide amplification primers for SSR molecular markers associated with the fresh weight trait of Corydalis bulblets. Another problem to be solved by the present invention is to provide an application of SSR molecular markers associated with the fresh weight trait of Corydalis bulblets. Another problem to be solved by the present invention is to provide an application of amplification primers for SSR molecular markers associated with the fresh weight trait of Corydalis bulblets, for screening excellent Corydalis germplasm with high bulblet fresh weight.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

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

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

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

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

[0012] Application of the SSR molecular marker in the analysis of Corydalis yanhusuo population structure.

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

[0014] The application of the SSR molecular marker in the analysis of marker association of fresh weight traits of Corydalis bulblets.

[0015] The application of the amplification primers of the SSR molecular marker in the analysis of marker association of the fresh weight trait of Corydalis bulblets.

[0016] The application of the SSR molecular marker in the breeding of excellent germplasm of Corydalis yanhusuo.

[0017] The application of the amplification primers of the SSR molecular markers in the breeding of excellent germplasm of Corydalis yanhusuo.

[0018] The SSR molecular marker is used in preparing a kit for screening excellent germplasm of Corydalis yanhusuo with high bulb weight.

[0019] The invention discloses an application of the amplification primers of the SSR molecular markers in preparing a kit for screening excellent germplasm of Corydalis yanhusuo with high bulb weight.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention uses the sequencing information of the Corydalis yanhusuo genome (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, Issue2, 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. The individual genotypes of Corydalis yanhusuo and the fresh weight traits of the bulblets were regressed using the 24 pairs of SSR markers and their amplification primers, and the associated loci of the fresh weight of the bulblets of Corydalis yanhusuo were detected by the GLM program. Under the significance of P<0.01, a marker CYC8-10 associated with the fresh weight trait of the bulblets was detected, and the amplification product size of the CYC8-10 associated loci with the fresh weight trait of the bulblets was 270bp, and the explanation rate of phenotypic variation was 15.97%. The primers for amplifying the SSR molecular markers provided by the present invention can be applied to the screening of excellent Corydalis germplasm with high bulb weight and the positioning of key genes controlling bulb fresh weight traits and molecular marker-assisted breeding, which is of great significance for the cultivation of high-yield Corydalis varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the △K analysis diagram in Structure genetic structure analysis;

[0023] Figure 2 This is the genetic structure diagram when K=4;

[0024] Figure 3 This is the electrophoresis diagram of SSR marker CYC8-10 (M represents Marker 50bp, and the red arrow indicates the associated site). DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0026] Example 1

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

[0028] Ten days before the harvest of Corydalis yanhusuo, the fresh weight and total fresh weight of the bulblets of 41 individual plants were collected and investigated, and the maximum value, minimum value, range, standard deviation and coefficient of variation were calculated. The phenotypic data of the 41 complete individual plants are shown in Table 1. The specific collection method of the 41 individual plants is as follows: three 10 cm 2 All the Corydalis yanhusuo seedlings growing in the plots were dug out, and a total of 41 complete individual plants were obtained.

[0029] The results are shown in Table 2. There is a great variation in the fresh weight of bulblets among individuals of Corydalis yanhusuo, with a coefficient of variation of 79%.

[0030] Table 1 Phenotypic data of 41 complete plants

[0031]

[0032] Table 2 Variation of fresh weight of Corydalis bulb

[0033]

[0034] Graphpad was used to analyze the Pearson correlation between fresh weight of bulblets and total yield.

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

[0036] Table 3 Pearson coefficient of fresh weight of bulblets and total fresh weight

[0037]

[0038] Example 2

[0039] 1. Extraction of Corydalis yanhusuo genomic DNA

[0040] The DNA of fresh leaves of individual Corydalis X1-X24 plants was extracted sequentially using a new plant genomic DNA extraction kit (Shanghai Pudi Biotechnology). The extracted DNA products were detected by 1% agarose gel electrophoresis, and the DNA bands were clear and not degraded.

[0041] 2. Polymorphism analysis of SSR markers

[0042] The SSR loci were discovered using the genome sequencing information of Corydalis yanhusuo, and 80 pairs of primers were selected and synthesized. The primers were synthesized in Shanghai Bioengineering. The extracted DNA was used as a template, and the 80 pairs of primers were verified by PCR amplification. The PCR amplification products were detected by polyacrylamide gel electrophoresis.

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

[0044] The results are shown in Table 4. Finally, 24 SSR markers with clear bands and good polymorphism were screened for polymorphism analysis. The sequence information of the 24 pairs of amplification primers is shown in Table 5.

[0045] Table 4. Polymorphism of 24 SSR markers

[0046]

[0047] Table 5 24 pairs of primer sequences

[0048]

[0049] 3. Genetic structure analysis of SSR markers

[0050] The 24 pairs of SSR primers screened were used to perform PCR amplification on the DNA of 24 individual Corydalis plants.

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

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

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

[0054] Polyacrylamide gel silver staining: first fix with fixative (deionized water, 10% ethanol, 1% acetic acid) for 10 minutes, then soak in 1.5% silver nitrate solution for 10 minutes, quickly wash twice with deionized water, and then develop with color developing solution (deionized water, 1.5% sodium hydroxide, 1% formaldehyde) for 10 minutes.

[0055] Electrophoresis data analysis: The SSR primer amplification results were recorded in binary format. Bands with the same mobility at the same site were recorded as 1, and no band was recorded as 0. The genotype data of 24 Corydalis materials were obtained. The Structure 2.3.4 software was used to analyze the population structure of Corydalis in combination with the genotype data.

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

[0057] 4. Analysis of markers associated with bulblet fresh weight traits

[0058] The corresponding Q values ​​of 24 plants obtained by Structure analysis (Table 6) were used to perform regression analysis on the original phenotypic values ​​of the fresh weight of bulblets of Corydalis yanhusuo plants (Table 7) and marker variation using the GLM procedure in Tassel5 software with Q value as covariate.

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

[0060]

[0061] Table 7 The original phenotypic values ​​of fresh weight of bulblets of 24 individual plants of Corydalis yanhusuo

[0062]

[0063] The results are shown in Table 8. At a significance level of P < 0.01, a marker CYC8-10 associated with the fresh weight of bulblets was detected. The size of the amplified product of the site associated with the fresh weight of bulblets by CYC8-10 was 270 bp ( Figure 3 ), the explanation rate of phenotypic variation was 15.97%.

[0064] Table 8 Analysis of markers associated with bulb fresh weight traits

[0065]

[0066] Example 3

[0067] A kit for screening excellent germplasm of Corydalis yanhusuo with high bulb weight, the kit comprising the following molecular marker combination:

[0068] The marker CYC8-10 associated with the bulb fresh weight trait 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] Therefore, the fresh weight of Corydalis bulblets can be simply and accurately identified under different environments, different periods and different growing seasons.

[0072] In summary, the SSR molecular markers and primers developed by the present invention can be directly used for screening excellent germplasm, gene positioning and cloning, and molecular marker-assisted breeding. They also have chromosome positioning information, which is conducive to further fine positioning and cloning of genes controlling the fresh weight traits of bulblets, and are of great significance to the breeding of high-yield Corydalis yanhusuo.

Claims

1. An SSR molecular marker associated with the fresh weight trait of Corydalis bulb, characterized in that: The SSR molecular marker is CYC8-10, and its amplification primer sequence is as follows: CYC8-10-F: 5'-CCAAGATTGGCCACGTAGAT-3', CYC8-10-R: 5'-CCATGCAACCATCTCATTCA-3'.

2. Amplification primer for SSR molecular marker associated with the fresh weight trait of Corydalis bulb, characterized in that: The amplification primer sequence of the SSR molecular marker is as follows: CYC8-10-F: 5'-CCAAGATTGGCCACGTAGAT-3', CYC8-10-R: 5'-CCATGCAACCATCTCATTCA-3'.

3. Application of the SSR molecular markers described in claim 1 in the analysis of Corydalis yanhusuo population structure.

4. Use of the amplification primers of the SSR molecular markers described in claim 2 in the analysis of Corydalis population structure.

5. Application of the SSR molecular marker described in claim 1 in the analysis of marker association of fresh weight traits of Corydalis bulblets.

6. Use of the amplification primers of the SSR molecular marker described in claim 2 in the analysis of markers associated with the fresh weight trait of Corydalis bulblets.

7. Application of the SSR molecular marker described in claim 1 in the breeding of excellent germplasm of Corydalis yanhusuo.

8. Use of the amplification primers of the SSR molecular markers described in claim 2 in the breeding of excellent germplasm of Corydalis yanhusuo.

9. Use of the SSR molecular marker described in claim 1 in the preparation of a kit for screening high-quality germplasm of Corydalis yanhusuo with high bulb weight.

10. Use of the amplification primers of the SSR molecular markers according to claim 2 in the preparation of a kit for screening high-quality germplasm of Corydalis yanhusuo with high bulb weight.

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