Rubber tree A-type cultivated variety identification method based on rubber tree chloroplast SNP (Single Nucleotide Polymorphism) marker

Abstract

The invention belongs to the technical field of molecular markers, and particularly relates to a rubber tree A-type cultivated variety identification method based on a rubber tree chloroplast SNP marker, and the rubber tree chloroplast SNP marker relates to five 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 invention can be used for identifying rubber tree varieties with higher yield, and lays a foundation for further assisting rubber tree high-yield resistance breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and in particular relates to a method for identifying a rubber tree A-type cultivar based on rubber tree chloroplast SNP markers. Background Art

[0002] Natural rubber mainly comes from the Brazilian rubber tree. It is an important industrial raw material and is widely used in transportation, medical and health fields. Its excellent properties in elasticity, wear resistance, impact resistance, etc. have not been replaced by synthetic rubber so far. The Brazilian rubber tree is native to the Amazon Basin in Brazil. After many introductions, it is now widely cultivated in tropical Asia. It is cultivated in Taiwan Province, southern Fujian, Guangdong, Guangxi, Hainan and southern Yunnan, with Hainan and Yunnan being the most cultivated. There are two famous introductions of rubber trees in history. In 1876, the British Wickham collected rubber tree germplasm in Brazil, and then brought the seeds and seedlings to Kew Garden in the UK for breeding. Later, they were planted in Sri Lanka and then introduced to Malaysia, Indonesia, Thailand and other Southeast Asian countries 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 rubber tree germplasm currently available in major rubber-producing countries. After years of introduction, cultivation, hybrid breeding and screening, my country has bred a number of cold-resistant, wind-resistant and high-yield varieties, including Reyan 879, Reyan 73397 and other high-yield varieties, Reyan 917 and other wind-resistant high-yield varieties, Yunyan 774 and other cold-resistant high-yield varieties. Among them, Reyan 879 is a super high-yield variety, with an per-acre yield increased by more than 50% over the control, and is currently the variety with the highest yield in the world.

[0003] With the continuous advancement of sequencing technology, more and more rubber tree varieties, germplasms and rubber tree species genomes have been analyzed, laying a solid foundation for further genome selection and molecular-assisted breeding, and accelerating the rubber breeding process. Chloroplasts are the site of photosynthesis in green plants. They have an independent genetic system, their genetic structure is relatively stable, and gene recombination rarely occurs. Therefore, chloroplast genome sequences are widely used in plant phylogeny and species identification. Plant genome resequencing is mostly based on leaves, and mesophyll cells contain a large number of chloroplasts. In addition, the size of the chloroplast genome is also 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 are all derived from the screening of wild germplasms in the Amazon Basin of Brazil. They generally have characteristics such as high yield and stress resistance. By comparing the chloroplast genomes of cultivated varieties and wild germplasms, screening effective SNP marker sites will accelerate the breeding process of high-yield and stress-resistant rubber trees. Summary of the invention

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

[0005] The invention provides a SNP marker combination for identifying high-yield varieties of rubber trees, wherein the SNP marker combination comprises s4941-G / T, s34828-T / A, s39596-T / G, s60621-G / A and s122187-G / A; the s4941-G / T is located at SEQ ID The sequence shown in No.1 has a G / T natural variation at position 121 from the 5' end, specifically: 5'-AAAAATTTCTAATCAATTCTGAACAAAAAAAAGAAGAGATCAAAAAATATAGATAAAATAAAATTTTATAGATCACCTCTTGTCTTTTACGCTATCTATTCTTATATTCGCTATCCATTCGTATATTTTTTTATTATACATATATATACGTATATGTATACGTTTTTTATTTTCTATATTTTTATATAAATATAGAAATATAGAAAAATACTTTTTTATAACTATATTTTTTATAACTATAACAATAACA-3'; the corresponding wild-type sequence is shown in SEQ ID No.2, specifically: 5'-AAAAATTTCTAATCAATTCTGAACAAAAAAAAGAAGAGATCAAAAAATATAGATAAAATAAAATTTTATAGATCACCTCTTGTCTTTTACGCTATCTATTCTTATATTCGCTATCCATTCTTATATTTTTTTATTATACATATATATACGTATATGTATACGTTTTTTATTTTCTATATTTTTATATAAATATAGAAATATAGAAAAATACTTTTTTATAACTATATTTTTTATAACTATAACAATAACA-3'; the s34828-T / A is located in SEQ ID No.3, there is a natural T / A variation at position 118 from the 5' end, specifically: 5'-AACATTATAATGATTTATACCACATTAATACTATCATAATAATGCTATATTAAATTAATATTAATTAACTATAATATTAATTAACTATAAACTATAAACATTTAATTATAGTTTTAAATATAGTTATAAAGTTGAACATTTGTTTAGTATATTATAATGAATAATGATAAATTGGCTCTTAAATCGCCAAATTTTCTTATTTTCCACTATTCCAATCAAATCAATTCTAAAAATTA-3'; the corresponding wild-type sequence is shown in SEQ ID As shown in No.4, specifically: 5'-AACATTATAATGATTTATACCACATTAATACTATCATAATAATGCTATATTAAATTAATATTAATTATAACTATAATATTAATTATAACTATAAACTATAAAACATTTAATTATAGTTATAAAT ATAGTTATAAAAGTTGAACATTTGTTTAGTATATTATAATGAATAATGATAAATTGGCTCTTAAATCGCCAAATTTTCTTATTTTCCACTATTCCAATCAAATCAATTCTAAAAATTA-3'; the s39596-T / G is located in SEQ. ID The sequence shown in No.5 has a T / G natural variation at position 126 from the 5' end, specifically: 5'-TGACCCCTCCCCCGAATTCTTTCGGGTTGCGAAACACATTAAAATTCAATATAAGTCTAAGTTCCCAAAATGTAAAATACAAATAAAGAAAACAAAAAATTAGAGGGAGGGGTCAAACTTTTTTTTTAATTTGTAAATGTAATTAAATGTAAAATTTAGTCTAATGTGATAAAAAAATTATTATAGATGA-3'; the corresponding wild-type sequence is shown in SEQ ID No.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, and there is a natural G / A variation at this site, specifically: 5'-TATCCAATGTGGAATTAGGATACAGGTGTAGGCTAAGTAAATCAATGGATAGTTTCAGTTCTCTTGAAAATACCAGTATAGGTGAAGACCCAATTCTAAATGATACAGATAAAAACACCTATAGTTGGAGTAATAGTGACAGCTCTAGTTACAGTAATGT-3'; the corresponding wild-type sequence is 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, and there is a natural G / A variation at this site, specifically: 5'-GGTAATGCAAGGGAAGCTAGTGATAAGATACTGAATGTCGTGAATATTTTTGGCATTGAGGTAGCCATTCCACCCATTTCATCAAGGTAAACACGACGTATTCTATCATAACCCGTTCCTGCCAAGAAAAAAAGTGCGGCACCAATAAATCCATGCGATATTATTTGTAAAATGGCTCCA-3'; the corresponding wild-type sequence is shown in SEQ ID No.10, specifically: 5'-GGTAATGCAAGGGAAGCTAGTGATAAGATACTGAATGTCGTGAATATTTTTGGCATTGAGGTAGCCATTCCACCCATTTCATCAAGATAAACACGACGTATTCTATCATAACCCGTTCCTGCCAAGAAAAAAAGTGCGGCACCAATAAATCCATGCGATATTATTTGTAAAATGGCTCCA-3'; wherein the bold bases represent the sites of SNP markers; the SNP marker combination is related to the latex yield of rubber trees; when the base of the s4941-G / T site is G, the base of the s34828-T / A site is T, the base of the s39596-T / G site is T, the base of the s60621-G / A site is G and the base of the s122187-G / A site is G, it indicates that the rubber tree is a high-yield variety. .

[0006] Preferably, the high yield means latex yield>50mL / knife.

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

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

[0009] The present invention also provides the use of the SNP marker combination described in the above technical solution in the preparation of a product for identifying high-yield varieties of rubber trees.

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

[0011] The present invention also provides a kit for identifying high-yield varieties of rubber trees, wherein the kit comprises 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 constructing a rubber tree gene library.

[0013] Beneficial effects of the present invention: By searching NCBI and my country's National Genome Science Data Center, the present invention found hundreds of genome resequencing data of different varieties and wild germplasm. Among them, the project number PRJCA004986 released in September 2023 has the largest amount of data, including more than 100 cultivated varieties and more than 200 wild germplasm materials. By mining and analyzing these data, it is beneficial to screen effective SNP marker sites, laying the foundation for further assisting high-yield and resistant breeding of rubber trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0015] Figure 1 This is the evolutionary tree of 670 chloroplast genome sequences of 335 samples in Example 1; Figure 2 This is a cluster analysis heat map of 299 core SNP sites in 335 samples in Example 2; Figure 3 This is a heat map of the cluster analysis of 25 SNP sites in Example 2. DETAILED DESCRIPTION

[0016] To further illustrate the present invention, a method for identifying a rubber tree type A cultivar based on rubber tree chloroplast SNP markers provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0017] The present invention downloaded 335 genome resequencing data of rubber tree cultivars and wild germplasms from the National Genome Science Data Center, used GetOrganelle software to assemble chloroplasts of 127 cultivars and 208 wild germplasms, a total of 335 materials, and then used the chloroplast genome of the main rubber tree cultivar Reyan 73397 as a reference, used snippy software to screen and analyze core SNPs, and finally used the Heatmap analysis tool of TBtools software to cluster analyze the core SNPs, and screened out the SNP sites between the cultivars and the wild germplasms. The methods described in the following embodiments are all conventional methods unless otherwise specified.

[0018] Example 1 335 materials for chloroplast assembly The genome resequencing data of 335 rubber tree cultivars and wild germplasms were downloaded from the National Genome Science Data Center (https: / / ngdc.cncb.ac.cn / ) to the local server (Project No.: 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 -oCRR286406 -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, and named them CRR286406_1 and CRR286406_2 respectively (other samples were named in the same way). The specific commands were: ll|grepCRR|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, the cat command was used to merge the 670 sequences of 335 samples into a fasta file named CRR286-cp.fasta. The mafft software was used for multiple sequence alignment. The command was: mafft CRR286-cp.fasta>CRR286-cp.fasta.mafft. Then, the fasttree software was used to construct the evolutionary tree. The command was: fasttree CRR286-cp.fasta.mafft>CRR286-cp.fasta.mafft.tree. Transfer the obtained evolutionary tree file CRR286-cp.fasta.mafft.tree to a personal computer and use Figtree software to view and display the evolutionary tree, such as Figure 1 As shown, a total of 335 chloroplast genome sequences from the branch where CRR286406_2 was located were selected for subsequent SNP analysis.

[0019] Example 2 SNP analysis of chloroplast genomes in 335 samples According to the phylogenetic tree of the chloroplast genome sequence constructed in Example 1, a total of 335 chloroplast genome sequences in the branch where CRR286406_2 is located are selected, and core SNP analysis is performed using the software snippy. The specific command is: snippy--outdirCRR286364_1a --ref . / CRR286406_2 --ctgs CRR286364_1 --cpus 10. The command takes CRR286364_1 as an example, and the chloroplast genome sequence CRR286406_2 of the cultivated variety Reyan 73397 is used as the reference genome. The above command analysis is performed on the 335 chloroplast genome sequences respectively; then the core SNP analysis is performed, and the specific command is: snippy-core CRR286348_1a CRR286348_2a CRR286349_1a CRR286349_2a CRR286350_1a...(335 folder names in total), the analysis results are the core.tab file, which is the core SNP matrix file, including 299 core SNP sites. Use the sed command to replace A, T, C, and G with numbers 1, 2, 3, and 4. The specific command is: morecore.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 core.tab.xlsx to a personal desktop, open it with Excel, select all and selectively paste in a new table, check transpose, delete the first row to get the SNP site matrix. Use the Heatmap analysis tool of the TBtools software to draw a heat map for the transposed core.tab.xlsx file and perform cluster analysis on the columns, such as Figure 2 As shown in the figure, Cul is a high-yield resistant material selected and cultivated from wild germplasm, and cultivated on a large scale; WAC, WRO and WMG represent wild germplasm materials collected from three regions of Brazil. Through comparative analysis, it was found that 25 SNP loci can divide high-yield varieties into two complementary types, such as Figure 3As shown, the two complementary types are named A class and B class, and a SNP site covering both A and B types is named AB class SNP site, of which A class includes 5 SNP sites, B class includes 19 SNP sites, and AB class includes 1 SNP site. Among them, the 5 SNP sites of type A are represented by the structure of s-Reyan 73397 chloroplast genome physical position-cultivated variety genotype / wild germplasm genotype, and the variation information is as follows: s4941-G / T, s34828-T / A, s39596-T / G, s60621-G / A and s122187-G / A.

[0020] The five A-type SNP loci were used to identify the wild-type germplasms numbered WAC-1~WAC-33, WRO-1~WRO-53, WMG-1~WMG-29 and the cultivar samples numbered Cul-1~Cul-127. When the base of the s4941-G / T site was G, the base of the s34828-T / A site was T, the base of the s39596-T / G site was T, the base of the s60621-G / A site was G, and the base of the s122187-G / A site was G, it was indicated that the sample was the A-type of the high-yield rubber tree variety; the rest were a-type; the latex yield of the A-type rubber tree was higher than that of the a-type. The identification results are shown in Tables 1 and 2, where Cul is the cultivar material, and WAC, WRO and WMG represent wild germplasm materials collected in three regions of Brazil.

[0021] Table 1 Identification results of WAC-1~WAC-33, WRO-1~WRO-53, WMG-1~WMG-29

[0022] Table 2 Identification results of Cul-1~Cul-127

[0023] As shown in Table 1, among the 115 wild-type samples, 9 samples were detected as type A. As shown in Table 2, among the cultivated variety samples numbered Cul-1 to Cul-127, 27 samples were type A. The results are consistent with Figure 3 It can be seen that the SNP marker combination provided by the present invention can be used to identify the A-type rubber tree cultivars, and can also identify potential high-yield and resistant germplasm materials from wild germplasm, which can greatly improve the breeding process.

[0024] Test Example 1 Some varieties were extracted, and their latex yields were collected, which were expressed as the volume of latex that could be collected per cut, and then the screening results of the present invention were verified, as shown in Tables 3 and 4 below.

[0025] Table 3 Latex yield of samples WAC-1~WAC-33, WRO-1~WRO-53, WMG-1~WMG-29

[0026] Table 4 Latex yield of samples Cul-1~Cul-93

[0027] From Tables 1 to 4, it can be seen that among 242 samples, 36 A-type cultivars were obtained by screening the SNP loci of the present invention, of which 24 samples had a latex yield of >50 mL / knife, belonging to high-yield varieties.

[0028] There are certain discrepancies between the screening results and the yield results. This is because there are many factors affecting the yield, such as climatic conditions, pests and diseases, planting management methods and rubber tapping techniques, which can lead to 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 indicate 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 germplasm of rubber trees.

[0029] As can be seen from the above-described embodiments, the present invention collects and downloads resequencing data, chloroplast genome assembly and SNP screening analysis, and obtains a chloroplast genome SNP marker combination that can effectively distinguish cultivated varieties and wild germplasm. The present invention can divide cultivated varieties into three types of A type, B type and AB type by cluster analysis of the screened SNP. It can be seen from Table 1 to Table 4 that the rubber tree of cultivated variety A type has 66.7% high-yield varieties. The SNP marker combination provided by the present invention can be used for screening high-yield germplasm of rubber tree, and can assist in the breeding of high-yield varieties of rubber tree.

[0030] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A SNP marker combination for identifying high-yield varieties of rubber trees, characterized in that: 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 position 121 from the 5' end of the sequence shown in SEQ ID No. 1, and there is a natural G / T variation at this position; The s34828-T / A is located at position 118 from the 5' end of the sequence shown in SEQ ID No. 3, and there is a natural T / A variation at this position; The s39596-T / G is located at position 126 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 position; 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 position; The SNP marker combination is related to the latex yield of the rubber tree; when the base of the s4941-G / T site is G, the base of the s34828-T / A site is T, the base of the s39596-T / G site is T, the base of the s60621-G / A site is G and the base of the s122187-G / A site is G, it indicates that the rubber tree is a high-yield variety.

2. The SNP marker combination according to claim 1, wherein The high yield means that the latex yield is greater than 50 mL / knife.

3. Use of the SNP marker combination described in claim 1 in rubber tree breeding.

4. A method for identifying high-yield varieties of rubber trees based on rubber tree chloroplast SNP markers, characterized in that, The following steps are involved: The DNA of the sample to be tested is amplified, and the SNP site polymorphism detection is performed using the SNP marker combination described in claim 1. The sample to be tested is genotyped according to the detection result. When the base of the s4941-G / T site is G, the base of the s34828-T / A site is T, the base of the s39596-T / G site is T, the base of the s60621-G / A site is G, and the base of the s122187-G / A site is G, it indicates that the sample is a high-yield variety of rubber tree.

5. Use of the SNP marker combination according to claim 1 in preparing a product for identifying high-yield varieties of rubber trees.

6. The use according to claim 5, characterized in that: The product includes a kit and / or a gene chip.

7. A kit for identifying high-yield varieties of rubber trees, characterized in that: The kit comprises the SNP marker combination according to claim 1.

8. Use of the SNP marker combination according to claim 1 in constructing a rubber tree gene library.

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