SSR primer combination for genetic diversity analysis of soapberry germplasm resources and application of SSR primer combination

Abstract

The invention discloses an SSR primer combination for genetic diversity analysis of soapberry germplasm resources and application of the SSR primer combination, and belongs to the technical field of biology. The SSR primer combination for genetic diversity analysis of soapberry germplasm resources, provided by the invention, is a combination of 21 pairs of primers as shown in SEQ ID NO: 1-SEQ ID NO: 42, and can be used for interspecific genetic diversity analysis of soapberry, interspecific and intraspecific UPGMA clustering analysis of the soapberry, intraspecific population structure analysis of the soapberry and / or genetic diversity analysis of soapberries from different producing areas; the method has important guiding significance for constructing different utilization target core germplasms of the sapindus mukorossi, improving the improved variety breeding efficiency, accurately identifying important germplasms and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an SSR primer combination for genetic diversity analysis of soapberry germplasm resources and an application thereof. Background Art

[0002] Sapindus mukorossi has attracted widespread attention from researchers for its outstanding ecological, economic, and social value. Domestically, research is underway on the collection and conservation of germplasm resources, the selection of improved varieties, and efficient cultivation, targeting diverse utilization objectives, such as afforestation and increasing fruit yield. With the continued advancement of extensive targeted breeding efforts, problems such as the loss of Sapindus mukorossi germplasm and the disorganization of its resources have become increasingly prominent, severely hindering its genetic conservation and resource utilization. Although some regions have established Sapindus mukorossi resource banks to conduct research such as phenotypic evaluation of germplasm, genetic evaluation of germplasm resources using molecular markers derived from the Sapindus mukorossi genome has been reported rarely, and progress in this area has been slow. The vast number of Sapindus mukorossi germplasm resources collected and conserved across various regions not only presents difficulties and challenges for breeders in conducting in-depth research and their rational utilization, but also presents challenges such as mixed germplasm, low conservation efficiency, and time-consuming and labor-intensive conservation. Therefore, research on the genetic diversity of Sapindus mukorossi germplasm resources, as well as the analysis and evaluation of these resources, is crucial for establishing core collections for diverse utilization objectives, improving the efficiency of improved variety selection, and accurately identifying important germplasm. Summary of the Invention

[0003] In response to one or more problems existing in the prior art, one aspect of the present invention provides an SSR primer combination for genetic diversity analysis of Sapindus germplasm resources, which is a combination of 21 pairs of primers shown in SEQ ID NO: 1-SEQ ID NO: 42.

[0004] The application of the SSR primer combination provided by the present invention in the genetic diversity analysis of Sapindus germplasm resources, such as the genetic diversity analysis among Sapindus species, the UPGMA cluster analysis among and within Sapindus species, the population structure analysis within Sapindus species, and / or the genetic diversity analysis of Sapindus from different origins, also falls within the scope of the present invention.

[0005] In some embodiments, the genus Sapindus includes Sapindus mukorossi, Sapindus yunnanensis, and Sapindus mukorossi.

[0006] Another aspect of the present invention provides a method for analyzing genetic diversity of Sapindus mukorossi germplasm resources, comprising the following steps:

[0007] 1) Extracting genomic DNA from Sapindus germplasm materials;

[0008] 2) performing PCR amplification on the genomic DNA of each Sapindus germplasm material extracted in step 1) using the SSR primer combination of claim 1;

[0009] 3) detecting the PCR amplified bands obtained in step 2) and reading the data using GeneMarker software; and

[0010] 4) The number of alleles, effective number of alleles, observed heterozygosity, expected heterozygosity, and Shannon diversity index for each primer pair were calculated using the GenAlEx plug-in. The polymorphism information content of the primers was calculated using Power Marker software. The genetic similarity coefficient and genetic distance between Sapindus mukorossi samples were obtained according to Nei's method, and cluster diagrams were constructed using the UPGMA method based on the genetic distance using MEGA software. The population structure was analyzed using Structure software, and the Q value was calculated.

[0011] The SSR primer combination for genetic diversity analysis of Sapindus germplasm resources provided based on the above technical solution can be used to reveal the genetic diversity level and population structure of Sapindus germplasm resources, and provide a theoretical basis for exploring the genetic diversity, population structure and genetic differentiation of Sapindus germplasm resources, as well as for the subsequent collection, organization and hybrid breeding of Sapindus germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The agarose gel electrophoresis images of primers S11-4, S11-5 and S11-6 for 8 Sapindus mukorossi samples.

[0013] Figure 2 The capillary electrophoresis results of primer S3-2 for four Sapindus mukorossi samples (AD) were read.

[0014] Figure 3 This is a UPGMA cluster analysis diagram based on Nei's genetic distance.

[0015] Figure 4 The optimal population number is determined based on the Structure software, where a is the change in Lnp(D) value and b is the change in Delta K value.

[0016] Figure 5 The analysis results of Structure software when K=3.

[0017] Figure 6 This is the UPGMA cluster analysis diagram of Sapindus mukorossi from different production areas. DETAILED DESCRIPTION

[0018] The present invention will be further described below with reference to specific examples. The methods used in the following examples are all conventional methods unless otherwise specified. The ways of obtaining the various biological materials described in the examples only provide a way of obtaining them through experiments to achieve the specific disclosed purposes, and should not be a limitation on the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the examples. The examples are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are given. The examples will help to understand the present invention, but should not be used as limitations on the contents of the present invention. The primers involved in the following examples can all be synthesized by existing technologies.

[0019] The research method used in the following examples includes: using ROX, TAMRA, FAM, HEX four-fluorescence capillary electrophoresis technology to perform amplified band detection, using GeneMarker v2.2 software to read data. GenAlEx 6.503 plug-in is used to calculate the number of alleles (Number of alleles, Na), effective number of alleles (Effective number of alleles, Ne), observed heterozygosity (Observed heterozygosity, Ho), expected heterozygosity (Expected heterozygosity, He) and Shannon's diversity index (Shannon's information index, I) for each pair of primers; PowerMarker software is used to calculate primer polymorphic information content (Polymorphic information content, PIC); the genetic similarity coefficient and genetic distance between soapberry samples are obtained according to Nei's method, and cluster diagrams are constructed using MEGA 11 software in UPGMA mode based on genetic distance; Structure software is used to analyze population structure, and Q value is calculated. All of the above software uses default parameter settings.

[0020] Example 1: Sample collection and DNA extraction

[0021] A total of 478 accessions were obtained from the National Forest Germplasm Bank of Sapindus mukorossi in Anji County, Zhejiang Province. These included 412 accessions of Sapindus mukorossi (33 from high-suitability areas, 284 from moderate-suitability areas, and 91 from low-suitability areas), 38 accessions of Sichuan-Yunnan Sapindus mukorossi, and 28 accessions of Sapindus mukorossi from Zhejiang, Hunan, Jiangxi, Fujian, and Yunnan (Table 1). Fresh young leaves were collected from individual plants that were healthy and pest-free. Genome sequencing samples were selected from adult Sapindus mukorossi plants grown in Zhejiang. These samples were washed with distilled water and 75% ethanol, dried, placed in liquid nitrogen, brought back to the laboratory, and stored at -80°C. DNA was extracted using a modified CTAB method, and DNA concentration and quality were determined using a microspectrophotometer and 1.5% agarose gel electrophoresis, respectively.

[0022] Table 1: Collection information of soapberry germplasm resources

[0023]

[0024] Example 2: Primer design

[0025] This example uses Primer 3.0 software to scan the entire genome sequence of Sapindus mukorossi and design primers. Only 2-3 base repeat SSR sites with at least 8 repeats are identified. Primer lengths are set to 18-25 bp, amplification product lengths are 100-500 bp, and the annealing temperature is 50-65°C. Ten primer pairs were randomly selected for each chromosome, for a total of 140 primer pairs. The primers were named after the chromosome, for example, the 10 primer pairs for chromosome 1 were named S1-1 to S1-10. These primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0026] Eight samples of Sapindus mukorossi (AHCZ424, JXYF304, ZJTT63, ZJFY143, ZJTT37, ZJTT9, ZJHZ66, and ZJTL108) with distinct geographical locations and significant differences in seed and plant type (see Table 1) were selected for screening SSR primers with clear bands and stable amplification. A 25 μL PCR reaction system was used, consisting of 22 μL 1.1× Gold Mix ver. 2 enzyme (Beijing Qingke Biotechnology Co., Ltd.), 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), and 1 μL DNA template. The PCR reaction procedure is shown in Table 2. PCR product amplification bands were detected by 1.2% agarose gel electrophoresis at a constant voltage of 120 V to screen for primers that exhibited good amplification and clear bands. Capillary electrophoresis was performed using the Qsep100 fully automated nucleic acid and protein analysis system, and primer polymorphisms were analyzed using GeneMarker v2.2 software.

[0027] Table 2: PCR reaction program

[0028]

[0029]

[0030] The effectiveness of 140 pairs of SSR primers was verified using 8 samples of soapberry. The agarose gel electrophoresis diagram of some primers is shown in the figure below. Figure 1 As shown, lanes 1-8 are test samples, and from left to right are the gel electrophoresis results of primers S11-4, S11-5, and S11-6 for 8 test samples. The results of the first round of primer screening showed that: 33 pairs of primers all amplified clear bands; 36 pairs of primers had clear bands, but 1 to 2 samples failed to amplify; the remaining 71 pairs of primers had poor amplification effects, with problems such as dragging or messy bands, and these primers were discarded in the first round of screening. In the second round of primer screening, the 36 pairs of primers with missing bands were re-amplified, and the reaction system was optimized (changing the annealing temperature, number of cycles, reaction time and electrophoresis parameters, etc.). A total of 17 pairs of primers were able to amplify clear bands. According to the above experiments, after two rounds of primer amplification screening, a total of 50 pairs of SSR primers with good amplification effects and stability were obtained. The primer sequence information is shown in Table 3 below.

[0031] Table 3: Sequence information of 50 pairs of SSR primers

[0032]

[0033]

[0034] The 50 pairs of primers screened were used to detect 268 germplasm materials of Sapindus mukorossi (including 266 Sapindus mukorossi and 2 Sapindus mukorossi from Sichuan and Yunnan) using the four-fluorescence capillary electrophoresis method. Figure 2 As shown, it exemplifies the capillary electrophoresis result reading diagram of primer S3-2 for 4 Sapindus mukorossi samples (AD) (data was read using GeneMarker v2.2 software) to verify the polymorphism and stability of these 50 pairs of primers. The results showed that the PIC values ​​of the 50 pairs of SSR primers were between 0.00 and 0.88, and an average of 8.42 alleles (Na) and 2.75 effective alleles (Ne) were detected per primer pair. Taking PIC>0.5 as the standard, combined with primer amplification bands, genetic parameters, etc., 21 pairs of high-quality primers with clear bands, high polymorphism and good repeatability were finally screened out. The sequence information is shown in SEQ ID NO: 1-SEQ ID NO: 42 in Table 3 (i.e., SSR primers numbered 1-21).

[0035] Example 3: Analysis of Genetic Diversity among Sapindus species

[0036] Using the 21 pairs of primers (SEQ ID NO: 1 - SEQ ID NO: 42) screened and obtained in Example 2, PCR amplification was performed on the genomic DNA of 412 Sapindus mukorossi germplasm materials, 38 Sapindus delavayi germplasm materials, and 28 Sapindus rarak var. villosus germplasm materials (the PCR reaction system and PCR reaction program are as shown in Example 2), and the PCR amplification results were analyzed. The results are shown in Table 4 below. As can be seen from Table 4, the average number of detected alleles (Na) of the 21 pairs of SSR primers is 20.29, with the primer S12 - 9 having the most, which is 39, and the primer S12 - 2 having the least, which is 8; the effective number of alleles (Ne) ranges from 3.30 to 11.50, with an average of 6.21; the observed heterozygosity (Ho) and expected heterozygosity (He) are 0.35 and 0.82 on average, respectively, and Ho < He, indicating that there is a certain phenomenon of heterozygote deficiency among the tested germplasm materials; the Shannon information index (I) ranges from 1.55 to 2.77, with an average of 2.13; the polymorphism information content (PIC) ranges from 0.71 to 0.91, with an average of 0.80, and PIC is greater than 0.5 for all, indicating that the 21 pairs of SSR primers have high polymorphism and the genetic diversity of Sapindus resources is rich.

[0037] Table 4: Genetic diversity analysis of 21 SSR loci

[0038]

[0039] This example also analyzed the genetic diversity of Sapindus mukorossi, Sapindus delavayi, and Sapindus rarak var. villosus germplasm materials respectively. The results are shown in Table 5 below. It can be seen that the average Na, Ne, I, Ho, He, and PIC of the Sapindus mukorossi samples are 18.33, 5.56, 2.00, 0.36, 0.79, and 0.77 respectively, and each genetic parameter is higher than that of Sapindus delavayi and Sapindus rarak var. villosus; except for the F value, each genetic parameter of the Sapindus delavayi samples is relatively higher than that of Sapindus rarak var. villosus, and the genetic diversity of the two is relatively low. The above differences may be mainly caused by the sample size and the sample distribution range. The genetic differentiation coefficient, genetic distance, and genetic identity among different species are shown in Table 6. It can be seen that the genetic differentiation coefficients between Sapindus mukorossi and Sapindus delavayi, Sapindus rarak var. villosus are 0.20 and 0.30 respectively, and the genetic differentiation coefficient between Sapindus delavayi and Sapindus rarak var. villosus is the largest (0.41); the genetic relationships among different species are relatively distant, among which the genetic relationship between Sapindus mukorossi and Sapindus rarak var. villosus is the farthest, and the genetic distance and genetic identity are 2.17 and 0.11 respectively; indicating that there is great genetic differentiation among different species of Sapindus, and the genetic relationship between Sapindus mukorossi and Sapindus rarak var. villosus is farther.

[0040] Table 5: Analysis of genetic diversity among species of Sapindus

[0041]

[0042] Table 6: Genetic relationships among different species of Sapindus

[0043]

[0044] Note: SM is Sapindus mukorossi, SD is Sapindus mukorossi, SR is Sapindus mukorossi.

[0045] Example 4: UPGMA cluster analysis between and within Sapindus species

[0046] The genomic DNA of 412 Sapindus mukorossi, 38 Sapindus mukorossi, and 28 Sapindus mukorossi germplasm materials were amplified by PCR using the 21 pairs of primers (SEQ ID NO: 1-SEQ ID NO: 42) obtained by screening in Example 2 (the PCR reaction system and PCR reaction procedure are as shown in Example 2), and the PCR amplification results were analyzed. The genetic distance between individuals was calculated according to Nei's method, and UPGMA cluster analysis was performed using MEGA 11 software. Figure 3 It can be clearly seen that soapberry, hairy-petaled soapberry, and Sichuan-Yunnan soapberry are divided into three groups. Within the hairy-petaled soapberry group, materials from Xishuangbanna, Molianzhai, and Kunming in Yunnan are divided into three small groups, among which the germplasm materials from Kunming are relatively distantly related. Among the Sichuan-Yunnan soapberry germplasm materials, materials from different production areas are also distinguished, among which the materials from Chengdu, Sichuan are the most distantly related. Among the soapberry germplasm materials, materials from Rongjiang, Guizhou are separately divided into a group, which is clearly different from other production areas; secondly, small groups from Nanjing, Jiangsu, Tongbai, Henan, Zhongshan, Guangdong, and Guangxi are divided into one group; materials from Yifeng, Jiangxi, and Jianou, Fujian are divided into one group; and the other large group is mainly composed of Zhejiang germplasm materials, and includes some materials from Hunan, Guangdong, Anhui, Henan and other places, existing in the form of single individuals or small groups, which may be related to the transportation and utilization of germplasm materials.

[0047] Example 5: Analysis of population structure within the Sapindus mukorossi species

[0048] The 21 pairs of primers (SEQ ID NO: 1-SEQ ID NO: 42) obtained from Example 2 were used to perform PCR amplification on the genomic DNA of 412 Sapindus mukorossi, 38 Sapindus mukorossi, and 28 Sapindus mukorossi germplasm materials (the PCR reaction system and PCR reaction procedure are as shown in Example 2), and the PCR amplification results were analyzed. The population structure of the Sapindus mukorossi germplasm resources was further analyzed using Structure 2.3.4 software. The number of subpopulations K was preset to 1 to 10 and repeated 10 times. Figure 4 As shown in Figure a, the Lnp(D) value increases gradually with the increase of K value, first fast and then slow, and the number of soapberry subpopulations cannot be clearly determined; when K = 3, Delta K reaches the maximum value, indicating that the soapberry germplasm resources are most appropriately divided into three subpopulations, such as Figure 4 Zhongb and Figure 5 shown.

[0049] When the soapberry germplasm resources were divided into three subgroups, the Q values ​​for the three subgroups were 0.93, 0.93, and 0.98, respectively. Subgroup I included all germplasm materials from Fujian, Guangdong, Guizhou, and Jiangsu, as well as most of the germplasm materials from Jiangxi and a small number of samples from Zhejiang, Anhui, Hunan, and Henan, totaling 112 samples. Subgroup II included most of the materials from Zhejiang and Anhui, as well as some samples from Jiangxi, Henan, Hunan, and Guangxi, totaling 231 samples. Subgroup III consisted primarily of 65 germplasm materials from Zhejiang, with very few samples from Anhui and Jiangxi, totaling 69 samples, as shown in Table 7. These results indicate that the genetic background of the soapberry germplasm resources is relatively uniform, with a distinct genetic structure and obvious geographical differentiation. There is some gene exchange between the germplasm materials from Zhejiang, Anhui, Jiangxi, and Hunan.

[0050] Table 7: Distribution of Sapindus mukorossi samples from different origins in the three subpopulations

[0051]

[0052] Example 6: Genetic diversity analysis of soapberry from different origins

[0053] PCR amplification was performed on the genomic DNA of 412 Sapindus mukorossi, 38 Sichuan and Yunnan Sapindus mukorossi, and 28 hairy petal Sapindus mukorossi germplasm materials using the 21 pairs of primers (SEQ ID NO: 1-SEQ ID NO: 42) screened in Example 2 (the PCR reaction system and PCR reaction procedure are as described in Example 2), and the PCR amplification results were analyzed. The genetic diversity analysis of Sapindus mukorossi germplasm resources from different production areas is shown in Table 8. The results showed that the number of alleles (Na) among the production areas ranged from 2.14 to 14.57, the Shannon information index ranged from 0.60 to 1.81, and the PIC value ranged from 0.43 to 0.74. Among them, the Zhejiang materials all had the highest value, followed by Anhui, indicating that the materials from these two areas are rich in genetic diversity; while the genetic diversity levels of Guizhou and Guangxi germplasm materials are relatively low.

[0054] Table 8: Genetic diversity analysis of soapberry from different production areas

[0055]

[0056]

[0057] Further analysis of the genetic differentiation among the production areas yielded the results shown in Table 9. The average genetic differentiation coefficient among the ten production areas was 0.27, indicating that 27% of the genetic variation came from between production areas and 73% of the genetic variation came from within a production area. The average gene flow value was 0.79, which was less than 1. Therefore, the genetic differentiation level of soapberry germplasm materials among different production areas was high, but the main genetic variation came from within a production area, and there was little gene exchange among different production areas.

[0058] The genetic distance and genetic consistency between different origins of soapberry were obtained by Nei's method to characterize the relationship between populations from different origins. The larger the genetic distance or the smaller the consistency, the more distant the relationship between the populations. Figure 6 It can be seen that the soapberry materials from Zhejiang and Anhui are most closely related, with genetic distances and genetic consistency of 0.13 and 0.88 respectively. They are also relatively closely related to samples from Jiangxi and Henan, with genetic distances of 0.23 and 0.31 respectively. The soapberry materials from Guizhou are the most distantly related to those from other places of origin, followed by Jiangsu and Fujian.

[0059] Table 9: Genetic differentiation of soapberries from different production areas

[0060]

[0061] Table 10: Nei's genetic identity and genetic distance of Sapindus mukorossi population

[0062]

[0063] Note: The lower left part is genetic distance, and the upper right part is genetic identity.

[0064] From the above results, it can be seen that the present invention has developed and screened 21 pairs of SSR primers with clear bands, high polymorphism and good repeatability based on the whole genome sequence of Sapindus mukorossi. The 21 pairs of high-quality SSR primers were used to PCR amplify the number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), Shannon information index and polymorphic information content of 412 Sapindus mukorossi, 38 Sichuan and Yunnan Sapindus mukorossi and 28 hairy petal Sapindus mukorossi germplasm materials, and the average values ​​were 20.29, 6.21, 0.35, 0.82, 2.13 and 0.80, respectively. The genetic diversity of Sapindus mukorossi germplasm resources is richer, with obvious geographical differentiation. The main genetic variation comes from the production area. The resources in Guizhou, Jiangsu, Fujian and other places are more distantly related to other production areas, while the genetic relationship between Zhejiang and Anhui is the closest.

[0065] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An SSR primer combination for genetic diversity analysis of Sapindus germplasm resources, which is a combination of 21 pairs of primers shown in SEQ ID NO: 1-SEQ ID NO:

42.

2. Application of the SSR primer combination described in claim 1 in genetic diversity analysis of Sapindus mukorossi germplasm resources.

3. Application of the SSR primer combination described in claim 1 in genetic diversity analysis among species of Sapindus, UPGMA cluster analysis among and within species of Sapindus, population structure analysis within Sapindus, and / or genetic diversity analysis of Sapindus from different origins.

4. The SSR primer combination according to claim 1, or the use according to claim 2 or 3, wherein the genus Sapindus includes Sapindus mukorossi, Sapindus yunnanensis, and Sapindus mukorossi.

5. A method for analyzing genetic diversity of Sapindus mukorossi germplasm resources, comprising the following steps: 1) Extracting genomic DNA from Sapindus germplasm materials; 2) using the SSR primer combination of claim 1 to perform PCR amplification on the genomic DNA of each Sapindus germplasm material extracted in step 1); 3) Detecting the PCR amplified bands obtained in step 2) and reading the data using GeneMarker software; 4) The number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He) and Shannon's information index (I) of each primer pair were calculated using the GenAlEx plug-in; the polymorphic information content (PIC) of primers was calculated using the Power Marker software; the genetic similarity coefficient and genetic distance between Sapindus mukorossi samples were obtained according to the Nei's method, and a cluster diagram was constructed based on the genetic distance using the MEGA software in the UPGMA method; the population structure was analyzed using the Structure software, and the Q value was calculated.

6. The method according to claim 5, wherein the 25 μL PCR reaction system for PCR amplification comprises: 22 μL 1.1× Gold Mix ver.2 reaction enzyme (Beijing Qingke Biotechnology Co., Ltd.), 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 1 μL DNA template.

7. The method according to claim 5 or 6, wherein the reaction procedure of the PCR amplification is: pre-denaturation at 98°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at Tm+5°C for 15 seconds, extension at 72°C for 10 seconds, for a total of 35 cycles; and finally extension at 72°C for 4 minutes.

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