SSR molecular markers associated with the proliferation trait of Corydalis yanhusuo mother bulbs and their applications

Abstract

This invention provides SSR molecular markers associated with the proliferation trait of Corydalis rhizomes, belonging to the field of molecular genetics. Trait correlation analysis shows that Corydalis rhizomes proliferation is a key factor affecting its yield. This invention utilizes 24 pairs of SSR markers to perform regression analysis on the individual genotype and rhizome proliferation trait of Corydalis rhizomes. The association loci for Corydalis rhizomes proliferation were detected using the GLM program, explaining 18.73% of phenotypic variation, with the SSR molecular marker CYC2-8 showing a highly significant correlation. The primers provided by this invention for amplifying the aforementioned SSR molecular markers can be used to screen for superior Corydalis rhizomes with high rhizome proliferation, locate key genes controlling the rhizome proliferation trait, and for marker-assisted breeding, which is of great significance for cultivating high-yielding Corydalis rhizomes varieties.

Description

Technical Field

[0001] This invention provides SSR molecular markers associated with the proliferation trait of Corydalis yanhusuo mother bulbs, belonging to the field of molecular genetics. Background Technology

[0002] Corydalis yanhusuo WT Wang, also known as Yuanhu, is a plant belonging to the genus Corydalis (Corydalis DC.) of the family Papaveraceae (Juss.). It is widely distributed in Heilongjiang, Zhejiang, Jiangsu, Anhui, Hubei, and Henan provinces of China. Corydalis is listed in the Chinese Pharmacopoeia, and its dried tubers are used medicinally, possessing properties of promoting blood circulation, regulating qi, and relieving pain. In production, the propagation coefficient of Corydalis is only about 5, with a yield of approximately 500 kg from 100-150 kg of bulbs under high-yield conditions. Currently, the cultivation and management of Corydalis is quite mature, leaving limited room for further yield increases through cultivation techniques. Therefore, there is an urgent need to develop high-yield Corydalis varieties.

[0003] High-yield breeding of Corydalis yanhusuo is currently a blank area. The yield of Corydalis yanhusuo is mainly determined by two components: the fresh weight of the mother bulb and the fresh weight of newly formed daughter bulbs. In production, Corydalis yanhusuo is sown as seed bulbs (mother bulbs). During subsequent growth and development, some mother bulbs continue to enlarge, while others become empty and shriveled due to nutrient consumption. Therefore, the proliferation of mother bulbs during subsequent growth directly affects the yield. Field trait surveys have revealed significant differences in mother bulb proliferation among different materials when the mother bulbs are of uniform size. Correlation analysis shows a highly significant positive correlation between mother bulb proliferation and final yield. Therefore, improving the mother bulb proliferation rate of Corydalis yanhusuo is one of the goals of high-yield breeding of Corydalis yanhusuo.

[0004] 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. It utilizes natural populations as materials, allowing for the simultaneous examination of all alleles at the same locus across numerous germplasm materials to identify associated loci. SSR markers, with their advantages of wide distribution, numerous allelic variations, co-dominance, good reproducibility, and reliable results, are widely used in association analysis, QTL mapping, marker-assisted breeding, and the construction of genetic linkage maps.

[0005] Traditional high-yield breeding of Corydalis yanhusuo is not only time-consuming and labor-intensive, but also inconvenient and highly destructive because the investigation of yield traits involves the underground parts of Corydalis yanhusuo. Therefore, using molecular marker-assisted selection not only facilitates the selection of superior lines, but also shortens the breeding cycle and reduces breeding costs. Summary of the Invention

[0006] The purpose of this invention is to provide SSR molecular markers associated with the proliferation traits of Corydalis yanhusuo mother bulbs, which can be used as auxiliary selection markers for the proliferation traits of mother bulbs in the process of high-yield breeding of Corydalis yanhusuo, thereby improving the accuracy of selection and accelerating the breeding process.

[0007] To achieve the purpose of this invention, the present invention relates to an SSR molecular marker associated with the control of the proliferation trait of Corydalis yanhusuo mother bulb, which is numbered CYC2-8, with a forward primer sequence of 5'-AGTTACGGAGCCATTGGTG-3' and a reverse primer sequence of 5'-TGTGAACGCCATCAGAGAAG-3'.

[0008] The present invention provides an amplification product of SSR molecular marker sites associated with the proliferation trait of Corydalis yanhusuo mother bulbs with a size of 280 bp.

[0009] This invention also provides the application of the SSR molecular marker in the early prediction and screening of the proliferative traits of Corydalis yanhusuo mother bulbs.

[0010] The application includes the following steps: 1) Extract genomic DNA from the Corydalis rhizome to be tested; 2) Using the extracted DNA as a template, a PCR reaction was performed using primers with the SSR molecular marker; 3) Analyze the PCR amplification products.

[0011] Step 2) The PCR amplification system is as follows: 0.2 μL of 2.5 U / μL Taq DNA polymerase, 1.0 μL of 10×PCR reaction buffer containing 10 mM Mg2+, 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 ddH2O to make up to 10 μL.

[0012] The PCR reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 32 cycles; 72℃ extension for 10 min, and incubation at 4℃.

[0013] In the aforementioned application, step 3) involves detecting the PCR amplification products using polyacrylamide gel electrophoresis.

[0014] Compared with the prior art, the present invention has the following advantages: The SSR molecular markers of this invention can be directly used to screen superior germplasm with high mother bulb proliferation, gene mapping and cloning, and molecular marker-assisted breeding. Moreover, they have chromosome mapping information, which is beneficial for further fine mapping and cloning of genes that control mother bulb proliferation traits, and is of great significance for high-yield Corydalis yanhusuo breeding. Attached Figure Description

[0015] Figure 1 This refers to the ΔK analysis in the Structure genetic structure analysis of this invention embodiment.

[0016] Figure 2 This is a genetic structure diagram when K=4 in an embodiment of the present invention. Detailed Implementation

[0017] Implementation Cases

[0018] Investigation and statistical analysis of the phenotypic traits of Corydalis

[0019] For sowing, the weight of the mother bulbs of Corydalis was selected to be 0.8-1.0 grams. Ten days before the harvest of Corydalis, the fresh weight of the mother bulbs and the total fresh weight of each individual plant were collected and investigated. The maximum value, minimum value, range, standard deviation and coefficient of variation were calculated.

[0020] The results showed that there was a large variation in the fresh weight of the mother bulbs among individual Corydalis yanhusuo plants before harvest, with a high coefficient of variation of 39% (Table 1).

[0021] Table 1. Variation of agronomic traits in fresh weight of Corydalis rhizomes

[0022]

[0023] Pearson correlation analysis was used to analyze the correlation between mother bulb proliferation and yield.

[0024] Analysis showed that there was a highly significant positive correlation between mother bulb proliferation and total fresh weight, with a correlation coefficient of 0.638.

[0025] Polymorphism analysis of SSR markers

[0026] We used the genome sequencing information of Corydalis yanhusuo reported by Xu to discover SSR sites and designed primers.

[0027] Eighty pairs of primers were selected and synthesized at Shanghai Sangon Biotech Co., Ltd., and then validated by PCR amplification.

[0028] We selected 24 SSR markers with clear bands and good polymorphism.

[0029] 24 SSR markers were used for polymorphism analysis.

[0030] Table 2. Polymorphism of 24 SSR markers

[0031]

[0032] Genetic structure analysis

[0033] Includes the following steps: 1) DNA was extracted from 24 individual Corydalis plants; 2) PCR amplification was performed using 24 pairs of SSR primers. The amplification system consisted of 10 μL containing 20 ng of genomic DNA, 2.5 mM MgCl2, 0.5 mM dNTPs, 20 ng of primers, and 0.5 U Taq DNA polymerase. 3) The PCR reaction program is as follows: 94℃ pre-denaturation for 5 minutes, 94℃ denaturation for 30 seconds, 58℃ annealing for 20 seconds, 72℃ extension for 30 seconds, 32 cycles, and finally 72℃ extension for 5 minutes. 4) PCR product detection: PCR products were detected using 10% polyacrylamide gel electrophoresis. The sample loading volume was 1.5µL, the electrophoresis buffer was 1×TBE, the voltage was set to 220V, and electrophoresis was continued until the bromophenol blue band appeared at the bottom of the gel. 5) Polyacrylamide gel silver staining: First fix with fixative (deionized water, 10% ethanol, 1% acetic acid) for 10 min, then soak in 1.5% silver nitrate solution for 10 min, wash quickly twice with deionized water, and then develop with colorant (deionized water, 1.5% sodium hydroxide, 1% formaldehyde) for 10 min. 6) Electrophoresis data analysis: The SSR primer amplification results were recorded in binary format. Bands with the same migration rate at the same locus were marked as 1, and no band was marked as 0. Genotype data of 24 Corydalis materials were obtained. 7) The population structure of Corydalis rhizome was analyzed using Structure 2.3.4 software combined with genotype data, and the ΔK ( Figure 1 ); 8) When K=4, the genetic structure of the 24 Corydalis populations is as follows: Figure 2 .

[0034] Association marker analysis of mother bulb proliferation trait

[0035] Using the Q values ​​of 24 individual plants obtained from Structure analysis, regression analysis was performed in Tassel5 software using the GLM program with Q values ​​as covariates to analyze the original phenotypic values ​​and marker variations of Corydalis mother bulb proliferation.

[0036] Table 3. Original phenotypic values ​​of mother bulb fresh weight of individual Corydalis plants in association analysis

[0037]

[0038] At a significance level of P<0.01, one marker associated with the mother bulb proliferation trait, CYC2-8, was detected.

[0039] The amplification product of the CYC2-8 site associated with the mother bulb proliferation trait was 280 bp in size, and the phenotypic variation explained 18.73%.

[0040] The forward primer sequence for CYC2-8 is 5'-ATGTTAACGGAGCCATTGGTG-3', and the reverse primer sequence is 5'-TGTGAACGCCATCAGAGAAG-3'.

[0041] Table 4. Association marker analysis of maternal bulb proliferation traits

[0042]

Claims

1. A molecular marker method for associating the proliferation trait of Corydalis yanhusuo mother bulbs, characterized by: Genomic DNA of young leaves of Corydalis yanhusuo was amplified using the SSR molecular marker CYC2-8 and PCR technology. The 280 bp site in the amplified product was significantly correlated with gene polymorphism and mother bulb proliferation in the tested Corydalis yanhusuo population.

2. The forward primer sequence 5'-AGTTAACGGAGCCATTGGTG-3' and the reverse primer sequence 5'-TGTGAACGCCATCAGAGAAG-3' of the SSR molecular marker CYC2-8 according to claim 1.

3. The application of the SSR molecular marker according to claim 1 in screening for the proliferative traits of Corydalis rhizomes, characterized in that: Applications in genetic breeding.

4. The application of the SSR molecular marker according to claim 1 in screening for the proliferative traits of Corydalis rhizomes, characterized in that: The preparation method of SSR molecular markers includes the following steps: 1) Extract genomic DNA from the Corydalis rhizome to be tested; 2) PCR amplification; 3) Use the GLM model in the Tassel software to perform association analysis on traits.

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