SSR (Simple Sequence Repeat) molecular marker primer associated with cordobulb number character of corydalis tuber and application thereof

Abstract

The invention discloses SSR (simple sequence repeat) molecular marker primers associated with the cordobulb number character of corydalis tuber and application of the SSR molecular marker primers, and belongs to the technical field of molecular genetics. The nucleotide sequences of the SSR molecular marker primer associated with the corpuscle number character of corydalis tuber disclosed by the invention are shown as SEQ ID NO.1 and SEQ ID NO.2. The invention also discloses a kit for detecting the corpuscle number character of corydalis tuber. The SSR molecular marker associated with the corydalis tuber coglobule number character and the amplification primer thereof can be applied to corydalis tuber population structure analysis, corydalis tuber coglobule number character associated marker analysis, corydalis tuber excellent germplasm breeding and preparation of a kit for identifying the corydalis tuber coglobule number. 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 the character of the number of the corpuscles, 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 SSR molecular marker primers associated with the number traits of Corydalis bulblets 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 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. It is widely distributed in Heilongjiang, Zhejiang, Jiangsu, Anhui, Hubei, Henan and other places in China. Corydalis yanhusuo is included in the pharmacopoeia, and its dried tubers are used as medicine, which has the effects of promoting blood circulation, promoting qi and relieving pain. 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 under high yield conditions. At present, the cultivation and management level of Corydalis yanhusuo is very mature, and there is very limited room for increasing yield through cultivation technology. Therefore, it is urgent to carry out the breeding of high-yield Corydalis yanhusuo varieties.

[0004] There is currently no research on high-yield breeding of Corydalis yanhusuo. The yield of Corydalis yanhusuo is mainly determined by two components: the fresh weight of the mother bulb and the fresh weight of the newly formed bulblets. In production, Corydalis yanhusuo is sown through bulbs, that is, mother bulbs. During the subsequent growth and development, some mother bulbs will continue to grow, while some mother bulbs will be shriveled due to nutrient consumption, and the changes in the mother bulbs are relatively limited. The number and fresh weight of the newly formed bulblets in the subsequent growth directly affect the total yield. Through field trait surveys, it was found that when the bulb size was consistent, the number of bulblets produced between the materials was significantly different, and correlation analysis showed that there was a very significant positive correlation between the number of bulblets and the final yield. Therefore, increasing the number of Corydalis yanhusuo bulblets 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 problems existing in the prior art, the technical problem to be solved by the present invention is to provide SSR molecular marker primers associated with the number of bulblets of Corydalis. Another problem to be solved by the present invention is to provide the application of SSR molecular marker primers associated with the number of bulblets of Corydalis for screening excellent germplasm of Corydalis with high bulblet number.

[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 number trait of Corydalis bulblets is CYC4-8.

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

[0010] CYC4-8-F: 5'-CAGTGCATGCTTGATCACCT-3',

[0011] CYC4-8-R: 5'-TAACTCCTCCAGAACCACGG-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 associated markers of the number traits of Corydalis bulblets.

[0015] The application of the amplification primers of the SSR molecular markers in the analysis of marker associations of the number traits 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 a high number of bulbils.

[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 number of bulbils.

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

[0021] The present invention uses the genome sequencing information of Corydalis yanhusuo (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 discover and finally screen 24 SSR markers with clear bands and good polymorphism for polymorphism analysis, and synthesizes 24 pairs of amplification primers. The genotype of individual Corydalis yanhusuo and the number of bulbs trait were regressed using the 24 pairs of SSR markers and their amplification primers, and the associated loci of the number of bulbs of Corydalis yanhusuo were detected by the GLM program. Under the significance of P<0.01, a marker CYC4-8 associated with the number of bulbs trait was detected, and the amplification product size of the site associated with the number of bulbs trait of CYC4-8 was 130bp, and the explanation rate of phenotypic variation was 23.23%. The SSR molecular marker of the present invention can be directly used for screening high-quality germplasm with high bulb number, gene positioning and cloning, and molecular marker-assisted breeding. It also has chromosome positioning information, which is conducive to further fine positioning and cloning of genes controlling the bulb number trait, and is of great significance in the breeding of high-yield Corydalis yanhusuo. 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 CYC4-8 (M represents Marker 2000bp, 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 number of bulbils among individuals of Corydalis yanhusuo, with a coefficient of variation of 60%.

[0030] Table 1 Phenotypic data of 41 complete plants

[0031]

[0032] Table 2 Variation of the number of bulblets in Corydalis yanhusuo

[0033]

[0034] Graphpad was used to analyze the Pearson correlation between the number of daughter balls and the total yield.

[0035] The results are shown in Table 3. There is a very significant positive correlation between the number of bulblets and the total fresh weight, with a correlation coefficient of 0.673.

[0036] Table 3 Pearson coefficient between the number 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+10× PCR reaction buffer 1.0 μL, 2.5 mM dNTP 0.2 μL each, 10 μM upstream and downstream primers 0.5 μL each, 40 ng / μL DNA template 0.5 μL, ddHO 2 The PCR reaction program was as follows: 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, for a total of 32 cycles; 72°C extension for 10 min, and 4°C incubation.

[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] 10 μL PCR amplification system: 20 ng genomic DNA, 2.5 mM MgCl 2 , 0.5 mM dNTPs, 20 ng primers, 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, extension at 72°C for 30 seconds, 32 cycles; and final 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 lowest end 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 the number of bulblets

[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 number 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 the number 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 CYC4-8 associated with the number of daughter bulbs was detected. The size of the amplified product of the site associated with the number of daughter bulbs was 130 bp ( Figure 3 ), the explanation rate of phenotypic variation is 23.23%. The sequence of the CYC4-8 amplification primer is as follows:

[0064] CYC4-8-F: 5'-CAGTGCATGCTTGATCACCT-3',

[0065] CYC4-8-R: 5'-TAACTCCTCCAGAACCACGG-3'.

[0066] Table 8 Analysis of markers associated with the number of bulblets

[0067]

[0068] Example 3

[0069] A kit for screening excellent germplasm of Corydalis yanhusuo with high bulb number, the kit comprising the following molecular marker combination:

[0070] The marker CYC4-8 associated with the bulb number trait and its amplification primers, the amplification primer sequences are as follows:

[0071] CYC4-8-F: 5'-CAGTGCATGCTTGATCACCT-3',

[0072] CYC4-8-R: 5'-TAACTCCTCCAGAACCACGG-3'.

[0073] Therefore, the number of Corydalis bulblets can be simply and accurately identified under different environments, different periods and different growing seasons.

[0074] In summary, the SSR molecular markers and primers developed by the present invention can be directly used for screening high-quality germplasm with high bulb number, 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 bulb number trait, and is of great significance in the breeding of high-yield Corydalis yanhusuo.

Claims

1. An SSR molecular marker associated with the number of bulblets of Corydalis yanhusuo, characterized in that: The SSR molecular marker is CYC4-8.

2. Amplification primer for SSR molecular markers associated with the number of bulblets of Corydalis yanhusuo, characterized in that: The amplification primer sequence of the SSR molecular marker is as follows: CYC4-8-F: 5'-CAGTGCATGCTTGATCACCT-3', CYC4-8-R: 5'-TAACTCCTCCAGAACCACGG-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 associations of the number 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 number traits 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 preparing a kit for screening excellent germplasm of Corydalis yanhusuo with a high number of bulbils.

10. Use of the amplification primers of the SSR molecular marker according to claim 2 in the preparation of a kit for screening excellent germplasm of Corydalis yanhusuo with a high number of bulbils.

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