Correlation of ssr molecular markers with yield traits of fumariae radix and application
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
- Filing Date
- 2024-12-31
- Publication Date
- 2026-06-30
AI Technical Summary
Existing technologies are insufficient to efficiently increase the yield of Corydalis yanhusuo, and traditional breeding methods are time-consuming, labor-intensive, and inconvenient for trait surveys, affecting the breeding process and costs.
Using SSR molecular markers CYC2-4 and CYC8-10, gene loci associated with Corydalis yield traits were screened by PCR amplification and polyacrylamide gel electrophoresis for early prediction and screening of superior germplasm.
This method enables accurate selection of yield traits in Corydalis rhizome, shortens the breeding cycle, reduces breeding costs, and provides chromosome location information, which is helpful for high-yield breeding.
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Abstract
Description
Technical Field
[0001] This invention provides SSR molecular markers associated with the yield trait of Corydalis rhizome, 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] 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.
[0004] Yield traits are typically quantitative traits, controlled by multiple genes, but high-yield breeding of Corydalis yanhusuo is currently a blank slate. Traditional high-yield breeding methods for Corydalis yanhusuo are not only time-consuming and labor-intensive, but also inconvenient and highly destructive because the investigation of yield traits involves 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
[0005] The purpose of this invention is to provide SSR molecular markers associated with the yield trait of Corydalis yanhusuo, so as to serve as auxiliary selection markers in the high-yield breeding process of Corydalis yanhusuo, improve the accuracy of selection, and accelerate the breeding process.
[0006] To achieve the purpose of this invention, the SSR molecular marker associated with the control of Corydalis yield is designated CYC2-4. Its forward primer sequence is 5'-CAATACAAGCTCGTGAGCCA-3', and its reverse primer sequence is 5'-GGAACTGGAATTGCGTTGTT-3'.
[0007] The present invention provides amplification products of CYC2-4 sites, which are SSR molecular markers associated with the yield trait of Corydalis yanhusuo, with a size of 250 bp.
[0008] Another SSR molecular marker associated with the control of Corydalis yield in this invention is designated CYC8-10. Its forward primer sequence is 5'-CCAAGATTGGCCACGTAGAT-3', and its reverse primer sequence is 5'-CCATGCAACCATCTCATTCA-3'.
[0009] The present invention provides amplification products of CYC8-10 sites, which are SSR molecular markers associated with the yield trait of Corydalis yanhusuo, with a size of 270 bp.
[0010] This invention also provides the application of the SSR molecular marker in the early prediction and screening of Corydalis yield traits.
[0011] 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.
[0012] 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.
[0013] 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℃.
[0014] In the aforementioned application, step 3) involves detecting the PCR amplification products using polyacrylamide gel electrophoresis.
[0015] Compared with the prior art, the present invention has the following advantages: The SSR molecular markers of this invention can be directly used for screening superior germplasm, 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 controlling yield traits, and is of great significance for breeding high-yield Corydalis. Attached Figure Description
[0016] Figure 1This refers to the ΔK analysis in the Structure genetic structure analysis of this invention embodiment.
[0017] Figure 2 This is a genetic structure diagram when K=4 in an embodiment of the present invention. Detailed Implementation
[0018] Implementation Cases
[0019] Investigation and statistical analysis of the phenotypic traits of Corydalis
[0020] Ten days before the harvest of Corydalis rhizome, the yield traits of each individual plant were collected and investigated, and the maximum, minimum, range, standard deviation and coefficient of variation were statistically analyzed.
[0021] The results showed that there was a great deal of variation in all traits among individuals of Corydalis rhizome, with a high coefficient of variation of 49% (Table 1).
[0022] Table 1. Variation of yield traits of Corydalis yanhusuo
[0023]
[0024] Polymorphism analysis of SSR markers
[0025] We used the genome sequencing information of Corydalis yanhusuo reported by Xu to discover SSR sites and designed primers.
[0026] Eighty pairs of primers were selected and synthesized at Shanghai Sangon Biotech Co., Ltd., and then validated by PCR amplification.
[0027] We selected 24 SSR markers with clear bands and good polymorphism.
[0028] 24 SSR markers were used for polymorphism analysis.
[0029] Table 2. Polymorphism of 24 SSR markers
[0030]
[0031] Genetic structure analysis
[0032] 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 a final extension at 72℃ for 5 minutes. 4) Detection of PCR amplification products: 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 performed until the bromophenol blue band ran out of 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 .
[0033] Associated marker analysis of yield traits
[0034] Using the Q values of 24 individual plants obtained from the Structure analysis, regression analysis was performed on the original phenotypic values and marker variations of Corydalis yield traits in Tassel5 software using the GLM program with the Q values as covariates.
[0035] Table 3. Original phenotypic values of yield traits in individual plants of Corydalis yanhusuo in association analysis
[0036]
[0038] At a significance level of P<0.01, two markers associated with yield traits, CYC2-4 and CYC8-10, were detected.
[0039] The amplified product of the CYC2-4 site associated with yield traits was 250 bp in size, and the phenotypic variation explained was 16.72%.
[0040] The forward primer sequence for CYC2-4 is 5'-CAATACAAGCTCGTGAGCCA-3' and the reverse primer sequence is 5'-GGAACTGGAATTGCGTTGTT-3'.
[0041] The amplified product of CYC8-10 loci associated with yield traits was 270 bp in size, and the phenotypic variation explained was 14.06%.
[0042] The forward primer sequence for CYC8-10 is 5'-CCAAGATTGGCCACGTAGAT-3', and the reverse primer sequence is 5'-CCATGCAACCATCTCATTCA-3'.
[0043] Table 4. Association marker analysis of yield traits
[0044]
Claims
1. A method for the identification of molecular markers associated with fumonisin production traits in corn, characterized by: Genomic DNA of young leaves of Corydalis yanhusuo was amplified using SSR molecular markers CYC2-4 and CYC8-10 combined with PCR technology. The corresponding sites in the amplification products showed a highly significant correlation between gene polymorphism and yield traits in the tested Corydalis yanhusuo population.
2. The SSR molecular marker according to claim 1, wherein the forward primer sequence for CYC2-4 is 5'-CAATACAAGCTCGTGAGCCA-3' and the reverse primer sequence is 5'-GGAACTGGAATTGCGTTGTT-3', and the forward primer sequence for CYC8-10 is 5'-CCAAGATTGGCCACGTAGAT-3' and the reverse primer sequence is 5'-CCATGCAACCATCTCATTCA-3'.
3. The use of the SSR molecular marker according to claim 1 in screening the yield traits of Corydalis. Applications in genetic breeding.
4. The use of the SSR molecular marker according to claim 1 in screening the yield traits of Corydalis. 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 correlation analysis on yield traits.