A method for identifying rubber tree cultivar A based on chloroplast SNP markers of rubber tree leaves

Abstract

The present invention belongs to the technical field of molecular markers, and particularly relates to a method for identifying rubber tree cultivar A based on chloroplast SNP markers of rubber tree. The present invention involves a total of 5 SNP loci: s4941-G / T, s34828-T / A, s39596-T / G, s60621-G / A, and s122187-G / A. The SNP marker combination provided by the present invention can be used to identify rubber tree cultivars with higher yields, laying a foundation for further assisting the high-yield and disease-resistant breeding of rubber trees.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular markers, and particularly relates to a method for identifying Hevea brasiliensis cultivar A based on chloroplast SNP markers of rubber tree leaves. Background Art

[0002] Natural rubber mainly comes from Hevea brasiliensis, which is an important industrial raw material and is widely used in fields such as transportation, medical and health. Due to its excellent properties in elasticity, abrasion resistance, impact resistance, etc., it still cannot be replaced by synthetic rubber so far. Hevea brasiliensis is native to the Amazon Basin in Brazil. After multiple introductions, it is now widely cultivated in tropical regions of Asia. Historically, there were two well-known introductions of rubber trees. In 1876, the British Wickham collected rubber tree germplasm in Brazil, then brought the seeds and seedlings to Kew Garden in the UK for breeding, and then planted them in Sri Lanka, and subsequently introduced them to Southeast Asian countries such as Malaysia, Indonesia, and Thailand for planting. The second time was in 1981 when the International Rubber Research and Development Board (IRRDB) organized the collection and introduction of rubber tree germplasm. In addition, it also includes other germplasms of the genus Hevea that are available in the main rubber-producing countries. After years of introduction, cultivation, and screening of crossbred varieties, China has bred multiple cold-resistant, wind-resistant, and high-yield varieties, including high-yield varieties such as Reyan 879 and Reyan 73397, wind-resistant varieties such as Reyan 917, and cold-resistant varieties such as Yunyan 774. Among them, the super-high-yield variety Reyan 879 has a yield per mu that is more than 50% higher than the control, and it is currently the variety with the highest yield per unit area in the world.

[0003] With the continuous progress of sequencing technology, the genomes of more and more rubber tree varieties, germplasms, and rubber tree species have been analyzed, laying a solid foundation for further carrying out genomic selection and molecular-assisted breeding to accelerate the rubber breeding process. Chloroplast is the place where green plants carry out photosynthesis, and it has an independent genetic system. Its genetic structure is relatively stable and gene recombination occurs less frequently. Therefore, chloroplast genome sequences are widely used in plant phylogeny and species identification. Plant genome resequencing mostly uses leaves as materials, and mesophyll cells contain a large number of chloroplasts. In addition, the chloroplast genome size is very small, only more than 100 kb. Therefore, compared with the nuclear genome, only a small amount of leaf resequencing data can be used for chloroplast genome assembly and SNP analysis. Rubber tree cultivars all originate from the screening of wild germplasms in the Amazon Basin of Brazil and generally have characteristics such as high yield and stress resistance. By comparing and analyzing the chloroplast genomes of cultivated varieties and wild germplasms and screening effective SNP marker sites, the process of high-yield and stress-resistant breeding of rubber trees will be accelerated. Summary of the Invention

[0004] The object of the present invention is to provide an SNP marker combination that can be used for high-yielding varieties of rubber trees, enrich molecular markers available for breeding excellent rubber tree varieties, and provide new ideas for high-yield and stress-resistant breeding of rubber trees.

[0005] The present invention provides a SNP marker combination for identifying high-yielding varieties of rubber trees. The SNP marker combination includes s4941-G / T, s34828-T / A, s39596-T / G, s60621-G / A, and s122187-G / A. The s4941-G / T is located at the 121st position from the 5'-end of the sequence shown in SEQ ID No. 1, and there is a natural variation of G / T at this site, specifically: 5'-AAAAATTTCTAATCAATTCTGAACAAAAAAAAGAAGAGATCAAAAAATATAGATAAAATAAAATTTTATAGATCACCTCTTGTCTTTTACGCTATCTATTCTTATATTCGCTATCCATTCGTATATTTTTTTATTATACATATATATACGTATATGTATACGTTTTTTATTTTCTATATTTTTATATAAATATAGAAATATAGAAAAATACTTTTTTATAACTATATTTTTTATAACTATAACAATAACA-3'; its corresponding wild-type sequence is as shown in SEQ ID No. 2, specifically: 5'-AAAAATTTCTAATCAATTCTGAACAAAAAAAAGAAGAGATCAAAAAATATAGATAAAATAAAATTTTATAGATCACCTCTTGTCTTTTACGCTATCTATTCTTATATTCGCTATCCATTCTTATATTTTTTTATTATACATATATATACGTATATGTATACGTTTTTTATTTTCTATATTTTTATATAAATATAGAAATATAGAAAAATACTTTTTTATAACTATATTTTTTATAACTATAACAATAACA-3'; The s34828-T / A is located at SEQ ID No.At the 118th position from the 5'-end of the sequence shown in Figure 3, there is a natural T / A variation at this site, specifically: 5'-AACATTATAATGATTTATACCACATTAATACTATCATAATAATGCTATATTAAATTAATATTAATTATAACTATAATATTAATTATAACTATAAACTATAAACATTTAATTATAGTTTTAAATATAGTTATAAAGTTGAACATTTGTTTAGTATATTATAATGAATAATGATAAATTGGCTCTTAAATCGCCAAATTTTCTTATTTTCCACTATTCCAATCAAATCAATTCTAAAAATTA-3'; The corresponding wild-type sequence is as shown in SEQ ID No. 4, specifically: 5'-AACATTATAATGATTTATACCACATTAATACTATCATAATAATGCTATATTAAATTAATATTAATTATAACTATAATATTAATTATAACTATAAACTATAAACATTTAATTATAGTTATAAATATAGTTATAAAGTTGAACATTTGTTTAGTATATTATAATGAATAATGATAAATTGGCTCTTAAATCGCCAAATTTTCTTATTTTCCACTATTCCAATCAAATCAATTCTAAAAATTA-3'; The s39596-T / G is located at the 126th position from the 5'-end of the sequence shown in SEQ ID No. 5, and there is a natural T / G variation at this site, specifically: 5'-TGACCCCTCCCCCGAATTCTTTCGGGTTGCGAAACACATTAAAATTCAATATAAGTCTAAGTTCCCAAAATGTAAAATACAAATAAAGAAAACAAAAAATTAGAGGGAGGGGTCAAACTTTTTTTTTAATTTGTAAATGTAATTAAATGTAAAATTTAGTCTAATGTGATAAAAAAATTATTATAGATGA-3'; The corresponding wild-type sequence is as shown in SEQ ID No.As shown in Figure 6, specifically: 5'-TGACCCCTCCCCCGAATTCTTTCGGGTTGCGAAACACATTAAAATTCAATATAAGTCTAAGTTCCCAAAATGTAAAATACAAATAAAGAAAACAAAAAATTAGAGGGAGGGGTCAAACTTTTTTTGTAATTTGTAAATGTAATTAAATGTAAAATTTAGTCTAATGTGATAAAAAAATTATTATAGATGA-3'; The s60621-G / A is located at the 81st position from the 5' end of the sequence shown in SEQ ID No. 7. There is a natural G / A variation at this site, specifically: 5'-TATCCAATGTGGAATTAGGATACAGGTGTAGGCTAAGTAAATCAATGGATAGTTTCAGTTCTCTTGAAAATACCAGTATAGGTGAAGACCCAATTCTAAATGATACAGATAAAAACACCTATAGTTGGAGTAATAGTGACAGCTCTAGTTACAGTAATGT-3'; Its corresponding wild-type sequence is as shown in SEQ ID No. 8, specifically: 5'-TATCCAATGTGGAATTAGGATACAGGTGTAGGCTAAGTAAATCAATGGATAGTTTCAGTTCTCTTGAAAATACCAGTATAAGTGAAGACCCAATTCTAAATGATACAGATAAAAACACCTATAGTTGGAGTAATAGTGACAGCTCTAGTTACAGTAATGT-3'; The s122187-G / A is located at the 87th position from the 5' end of the sequence shown in SEQ ID No. 9. There is a natural G / A variation at this site, specifically: 5'-GGTAATGCAAGGGAAGCTAGTGATAAGATACTGAATGTCGTGAATATTTTTGGCATTGAGGTAGCCATTCCACCCATTTCATCAAGGTAAACACGACGTATTCTATCATAACCCGTTCCTGCCAAGAAAAAAAGTGCGGCACCAATAAATCCATGCGATATTATTTGTAAAATGGCTCCA-3'; Its corresponding wild-type sequence is as shown in SEQ ID No.As shown in SEQ ID NO: 10, specifically: 5'-GGTAATGCAAGGGAAGCTAGTGATAAGATACTGAATGTCGTGAATATTTTTGGCATTGAGGTAGCCATTCCACCCATTTCATCAAGATAAACACGACGTATTCTATCATAACCCGTTCCTGCCAAGAAAAAAAGTGCGGCACCAATAAATCCATGCGATATTATTTGTAAAATGGCTCCA-3'; the bolded bases represent the SNP marker sites; the SNP marker combination is related to the latex yield of rubber trees; when the base at the s4941-G / T site is G, the base at the s34828-T / A site is T, the base at the s39596-T / G site is T, the base at the s60621-G / A site is G, and the base at the s122187-G / A site is G, it indicates that the rubber tree is a high-yield variety.

[0006] Preferably, the high yield means the latex yield > 50 mL per tapping.

[0007] The present invention also provides the application of the SNP marker combination described in the above technical solution in the breeding of rubber trees.

[0008] The present invention also provides a method for identifying high-yield rubber tree varieties based on chloroplast SNP markers of rubber trees, comprising the following steps: amplifying the DNA of the sample to be tested, detecting the SNP site polymorphism using the SNP marker combination described in the above technical solution, and performing genotyping on the sample to be tested according to the detection results. When the base at the s4941-G / T site is G, the base at the s34828-T / A site is T, the base at the s39596-T / G site is T, the base at the s60621-G / A site is G, and the base at the s122187-G / A site is G, it indicates that the sample is a high-yield rubber tree variety.

[0009] The present invention also provides the application of the SNP marker combination described in the above technical solution in the preparation of products for identifying high-yield rubber tree varieties.

[0010] Preferably, the product includes a kit and / or a gene chip.

[0011] The present invention also provides a kit for identifying high-yield rubber tree varieties, which includes the SNP marker combination described in the above technical solution.

[0012] The present invention also provides the application of the SNP marker combination described in the above technical solution in the construction of a rubber tree gene library.

[0013] The beneficial effects of the present invention are as follows:

[0014] By searching the NCBI and the National Genomics Data Center of China, the inventors found that there were hundreds of genome resequencing data of different varieties and wild germplasms. Among them, the data with the project number PRJCA004986 released in September 2023 had the largest amount, including more than 100 cultivated varieties and more than 200 wild germplasm materials. By mining and analyzing these data, it was beneficial to screen effective SNP marker sites, laying a foundation for further assisting the high-yield and disease-resistant breeding of rubber trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0016] Figure 1 It is an evolutionary tree of 670 chloroplast genome sequences of 335 samples in Example 1;

[0017] Figure 2 It is a heatmap of cluster analysis of 299 core SNP sites of 335 samples in Example 2;

[0018] Figure 3 It is a heatmap of cluster analysis of 25 SNP sites in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] In order to further illustrate the present invention, the following will describe in detail a method for identifying rubber tree cultivar A based on rubber tree chloroplast SNP markers provided by the present invention in combination with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0020] The inventors downloaded the genome resequencing data of 335 rubber tree cultivars and wild germplasms from the National Genomics Data Center, used the GetOrganelle software to assemble the chloroplasts of a total of 335 materials, including 127 cultivated varieties and 208 wild germplasms. Then, taking the chloroplast genome of the main rubber tree cultivar Reyan 73397 as a reference, the snippy software was used for core SNP screening and analysis. Finally, the Heatmap analysis tool of the TBtools software was used to perform cluster analysis on the core SNPs to screen out the SNP sites between cultivated varieties and wild germplasms. The methods described in the following embodiments are all conventional methods unless otherwise specified.

[0021] Example 1

[0022] Chloroplast assembly of 335 materials

[0023] Downloaded the genome resequencing data of 335 cultivated varieties and wild germplasms of rubber tree from the National Genomic Data Center (https: / / ngdc.cncb.ac.cn / ) to the local server (Project ID: PRJCA004986). The chloroplast genomes of each sample were assembled using the GetOrganelle software. The specific command was: get_organelle_from_reads.py -1 CRR286406_f1.fastq.gz -2 CRR286406_r2.fastq.gz -F embplant_pt -o CRR286406 -R 15 -t 20 --reverse-lsc. The chloroplast genomes of 335 samples were obtained. The chloroplast genome of each sample included two configurations, namely embplant_pt.K85.complete.graph1.1.path_sequence.fasta and embplant_pt.K85.complete.graph1.2.path_sequence.fasta, which were named CRR286406_1 and CRR286406_2 respectively (other samples were named in the same way). The specific commands were: ll|grep CRR|awk '{print "cat "$NF" / *complete.graph1.1.path_sequence.fasta>Result / "$NF"_1"}'|sh and ll|grep CRR|awk '{print "cat "$NF" / *complete.graph1.2.path_sequence.fasta>Result / "$NF"_2"}'|sh. Then, 670 sequences of 335 samples were merged into a single fasta file named CRR286-cp.fasta using the cat command. Multiple sequence alignment was performed using the mafft software. The command was: mafft CRR286-cp.fasta>CRR286-cp.fasta.mafft. Then, a phylogenetic tree was constructed using the fasttree software. The command was: fasttree CRR286-cp.fasta.mafft>CRR286-cp.fasta.mafft.tree. The obtained phylogenetic tree file CRR286-cp.fasta.mafft.tree was transferred to a personal computer, and the phylogenetic tree was viewed and displayed using the Figtree software. As Figure 1 shown, 335 chloroplast genome sequences in the branch where CRR286406_2 was located were selected for subsequent SNP analysis.

[0024] Example 2

[0025] SNP Analysis of Chloroplast Genomes of 335 Materials

[0026] Based on the phylogenetic tree of the chloroplast whole-genome sequences constructed in Example 1, 335 chloroplast genome sequences in the branch where CRR286406_2 is located were selected, and the core SNP analysis was performed using the software snippy. The specific command is: snippy --outdir CRR286364_1a --ref. / CRR286406_2 --ctgs CRR286364_1 --cpus 10. Taking the command with CRR286364_1 as an example, the chloroplast genome sequence CRR286406_2 of the cultivated variety Reyan 73397 was used as the reference genome, and the above command analysis was performed on 335 chloroplast genome sequences respectively; then the core SNP analysis was carried out, and the specific command is: snippy-core CRR286348_1a CRR286348_2a CRR286349_1a CRR286349_2a CRR286350_1a... (a total of 335 folder names). The analysis result obtained the core.tab file, which is the core SNP matrix file, including a total of 299 core SNP sites. Using the sed command, A, T, C, and G were replaced with the numbers 1, 2, 3, and 4 respectively. The specific command is: more core.tab|sed -e's / A / 1 / g' -e's / T / 2 / g' -e's / C / 3 / g' -e's / G / 4 / g'>core.tab.xlsx. Transfer the core.tab.xlsx to a personal desktop computer, open it with Excel, select all and perform selective paste in a new table, check the transpose, and delete the first row to obtain the SNP site matrix. Using the Heatmap analysis tool of TBtools software, a heatmap was drawn for the core.tab.xlsx file after transposition processing, and column clustering analysis was performed. As Figure 2 shown, where Cul is a cultivated variety material obtained by large-scale cultivation of high-yield and resistant materials selected and cultivated from wild germplasms; WAC, WRO, and WMG represent wild germplasm materials collected from three regions in Brazil. Through comparative analysis, it was found that 25 SNP sites could divide the high-yield varieties into two complementary types, as Figure 3As shown in the figure, two complementary types are named A type (A class) and B type (B class) respectively. An SNP locus covering both A and B types is named AB type (AB class) SNP locus. Among them, the A type includes 5 SNP loci, the B type includes 19 SNP loci, and the AB type includes 1 SNP locus. Among the 5 SNP loci of the A type, the structure of s-phyical position of Reyan 73397 chloroplast genome - cultivated variety genotype / wild germplasm genotype is used for representation, and the variation information is as follows: s4941-G / T, s34828-T / A, s39596-T / G, s60621-G / A and s122187-G / A.

[0027] Using the 5 SNP loci of the A type respectively to identify wild germplasm numbered WAC-1~WAC-33, WRO-1~WRO-53, WMG-1~WMG-29 and cultivated variety samples numbered Cul-1~Cul-127. When the base of the s4941-G / T locus is G, the base of the s34828-T / A locus is T, the base of the s39596-T / G locus is T, the base of the s60621-G / A locus is G, and the base of the s122187-G / A locus is G, it indicates that the sample is the high-yield variety A type of rubber tree; the rest are a type; the latex yield of rubber trees of the A type is higher than that of the a type. The identification results are shown in Tables 1~2, where Cul is the cultivated variety material, and WAC, WRO and WMG represent wild germplasm materials collected from three regions in Brazil.

[0028] Table 1 Identification results of WAC-1~WAC-33, WRO-1~WRO-53, WMG-1~WMG-29

[0029]

[0030] Table 2 Identification results of Cul-1~Cul-127

[0031]

[0032] As can be seen from Table 1, among the 115 wild-type samples, a total of 9 samples were detected as the A type. As can be seen from Table 2, among the cultivated variety samples numbered Cul-1~Cul-127, 27 samples are of the A type, and the results are Figure 3 matched. It can be seen that by using the SNP marker combination provided by the present invention, the cultivated variety of rubber tree of the A type can be identified, and potential high-yield and disease-resistant germplasm materials can also be identified from wild germplasms, which can greatly improve the breeding process.

[0033] Test Example 1

[0034] Extract some varieties, collect their latex yields, which are expressed as the volume of latex that can be collected per cut, and then verify the screening results of the present invention, as shown in Tables 3 to 4 below.

[0035] Table 3 Latex yields of samples of WAC-1 to WAC-33, WRO-1 to WRO-53, and WMG-1 to WMG-29

[0036]

[0037] Table 4 Latex yields of samples of Cul-1 to Cul-93

[0038]

[0039] Combining Tables 1 to 4, it can be seen that among 242 samples, 36 cultivated varieties of type A were screened out using the SNP loci of the present invention. Among them, there were 24 samples with a latex yield > 50 mL per cut, belonging to high-yield varieties.

[0040] There are certain discrepancies between the screening results and the yield results. This is because there are many factors affecting the yield. For example, climatic conditions, pests and diseases, planting management methods, and tapping techniques can all cause deviations in the yield results. Among the germplasms screened using the SNP markers of the present invention, 66.7% are high-yield varieties, which can show that the SNP marker combination provided by the present invention is related to the latex yield of rubber trees, and the SNP marker combination provided by the present invention can be used to screen high-yield germplasms of rubber trees.

[0041] From the above embodiments, it can be seen that the present invention obtained a chloroplast genome SNP marker combination that can effectively distinguish cultivated varieties and wild germplasms through resequencing data collection, download, chloroplast genome assembly, and SNP screening and analysis. By performing cluster analysis on the screened SNPs, the cultivated varieties can be divided into three types: type A, type B, and type AB. Combining Tables 1 to 4, it can be seen that for rubber trees of type A cultivated varieties, 66.7% are high-yield varieties. The SNP marker combination provided by the present invention can be used to screen high-yield germplasms of rubber trees and can assist in the breeding of high-yield varieties of rubber trees.

[0042] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting SNP marker combinations in identifying high-yielding rubber tree varieties, characterized in that, The SNP marker combination is s4941-G / T, s34828-T / A, s39596-T / G, s60621-G / A, and s122187-G / A; The s4941-G / T is located at the 121st position from the 5'-end of the sequence shown in SEQ ID No. 1, and there is a natural G / T variation at this site; The s34828-T / A is located at the 118th position from the 5'-end of the sequence shown in SEQ ID No. 3, and there is a natural T / A variation at this site; The s39596-T / G is located at the 126th position from the 5'-end of the sequence shown in SEQ ID No. 5, and there is a natural T / G variation at this site; The s60621-G / A is located at the 81st position from the 5'-end of the sequence shown in SEQ ID No. 7, and there is a natural G / A variation at this site; The s122187-G / A is located at the 87th position from the 5'-end of the sequence shown in SEQ ID No. 9, and there is a natural G / A variation at this site; The SNP marker combination is related to the latex yield of rubber trees; when the base at the s4941-G / T site is G, the base at the s34828-T / A site is T, the base at the s39596-T / G site is T, the base at the s60621-G / A site is G, and the base at the s122187-G / A site is G, it indicates that the rubber tree is a high-yield variety, and the high yield means the latex yield > 50 mL per cut.

2. A method for identifying high-yielding rubber tree varieties based on chloroplast SNP markers of rubber tree leaves, characterized in that, It includes the following steps: Amplify the DNA of the sample to be tested, perform SNP site polymorphism detection on the SNP marker combination described in claim 1, and perform gene typing on the sample to be tested according to the detection results. When the base at the s4941-G / T site is G, the base at the s34828-T / A site is T, the base at the s39596-T / G site is T, the base at the s60621-G / A site is G, and the base at the s122187-G / A site is G, it indicates that the sample is a high-yield variety of rubber tree, and the high yield means the latex yield > 50 mL per cut.

3. Use of a reagent for detecting the SNP marker combination according to claim 1 in the preparation of a product for identifying high-yielding varieties of rubber trees, characterized in that, The application includes: Amplify the DNA of the sample to be tested, perform SNP site polymorphism detection on the SNP marker combination described in claim 1, and perform gene typing on the sample to be tested according to the detection results. When the base at the s4941-G / T site is G, the base at the s34828-T / A site is T, the base at the s39596-T / G site is T, the base at the s60621-G / A site is G, and the base at the s122187-G / A site is G, it indicates that the sample is a high-yield variety of rubber tree, and the high yield means the latex yield > 50 mL per cut.

4. The application according to claim 3, wherein The product includes a kit and / or a gene chip.

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