A SNP molecular marker for rubber trees and its application in detecting the number of latex duct rows

Abstract

The invention discloses a SNP molecular marker of a rubber tree and its application in detecting the number of latex ducts. The sequence of the SNP molecular marker is shown in SEQ ID No.1, and the base at the 216 bp site from the 5' end of the sequence shown in SEQ ID No.1 is T or C. The invention identifies for the first time from a big data level that the SNP located at the 216 bp site of the nucleotide sequence of SEQ ID NO.1 is closely related to the number of latex ducts, which is conducive to detection, accurate and reliable, and convenient to operate; the SNP molecular marker is used to identify the number of latex ducts of the rubber tree, and the accuracy of screening the rubber tree with a large number of latex ducts is as high as more than 80%; the SNP molecular marker and the detection method of the invention are not affected by the age of the rubber tree, and can be selected in the seedling stage, which greatly reduces the workload of breeding, significantly shortens the rubber tree yield breeding selection cycle, improves the breeding efficiency, and can be used for rubber tree yield molecular marker assisted breeding in practice.

Description

Technical Field

[0001] The invention relates to a SNP molecular marker and an application thereof, in particular to a SNP molecular marker of a rubber tree and an application thereof in detecting the number of latex duct rows. Background Art

[0002] Natural rubber trees are an important industrial raw material, and tropical crop rubber trees are their main commercial source. Traditional sexual hybrid breeding is the main and conventional method for rubber tree yield breeding. However, the long conventional breeding cycle, narrow genetic background of rubber trees, and lack of germplasm resources have greatly restricted the breeding process of rubber tree varieties and industrial development. With the development of modern molecular biotechnology, molecular marker-assisted selection breeding can solve the above problems to a certain extent and promote the breeding process of rubber trees.

[0003] The latex tubes in the bark of rubber trees are where natural rubber is synthesized and stored. They are an important structural factor that determines the yield of natural rubber and are closely related to its yield. By exploring effective molecular markers related to the number of latex tubes in rubber trees, early yield selection and improved yield trait accuracy can be achieved, thereby shortening the rubber tree yield breeding cycle and effectively promoting the genetic improvement of rubber trees. Summary of the invention

[0004] The present invention aims to overcome the deficiencies in the prior art and provide a SNP molecular marker for rubber trees and application thereof in detecting the number of latex duct rows.

[0005] The first aspect of the present invention is to provide a SNP molecular marker for rubber trees, the sequence of the SNP molecular marker is shown in SEQ ID No.1, and the base at the 216 bp position from the 5' end of the sequence shown in SEQ ID No.1 is T or C (it should be understood that in the sequence table, the 216 bp position from the 5' end of SEQ ID No.1 is represented by "t" only for the normal submission of the nucleotide sequence table, and does not affect the relevant limitations of claim 1 of this application and the description).

[0006] The inventors found that the genotype of the site of the SNP molecular marker is TC genotype rubber tree with more latex duct rows.

[0007] Based on the SNP molecular marker, a genotype identification method for the number of latex tube rows in rubber trees can be developed. By analyzing the base sequence of the SNP site, the genotype of the SNP site is TC, and the rubber tree is preliminarily identified to have a large number of latex tube rows with high accuracy.

[0008] In order to detect the SNP molecular marker, the second aspect of the present invention is to provide a primer pair for detecting the SNP molecular marker described in the first aspect of the present invention, wherein the primer pair includes a forward primer F: 5'- AAA CTG CCAGAA GGA GGT GGT -3' and a reverse primer R: 5'- GGC AGT AGC CCT CAT AAT CTT ATC TGC -3'.

[0009] The third aspect of the present invention is to provide a kit for detecting the SNP molecular markers described in the first aspect of the present invention, which comprises the primer pair described in the second aspect of the present invention.

[0010] The fourth aspect of the present invention is to provide the use of the SNP molecular marker described in the first aspect of the present invention, or the primer pair described in the second aspect of the present invention, or the kit described in the third aspect of the present invention in the detection of latex duct numbers in rubber trees.

[0011] The fifth aspect of the present invention is to provide a method for detecting the latex duct number trait of rubber trees in the seedling stage: extracting genomic DNA of the rubber tree to be tested, using the genomic DNA of the rubber tree to be tested as a template, using the primer pair described in the second aspect of the present invention to perform PCR amplification, analyzing the sequence of the PCR amplification product, detecting the SNP molecular marker described in the first aspect of the present invention, determining the genotype of the rubber tree to be tested, and thus determining the latex duct number trait of the rubber tree to be tested.

[0012] When the genotype of the SNP site is TC, it is identified that the rubber tree has a large number of latex tube rows.

[0013] The sixth aspect of the present invention is to provide a method for breeding rubber trees: extracting genomic DNA from the rubber trees to be selected, using the obtained genomic DNA as a template, using the primer pair described in the second aspect of the present invention to perform PCR amplification, analyzing the sequence of the PCR amplification product, detecting the SNP molecular marker described in the first aspect of the present invention, determining the genotype of each rubber tree to be selected, and breeding the rubber trees.

[0014] The seventh aspect of the present invention is to provide the use of the SNP molecular marker described in the first aspect of the present invention, or the primer pair described in the second aspect of the present invention, or the kit described in the third aspect of the present invention, or the method described in the fifth aspect of the present invention, or the breeding method described in the sixth aspect of the present invention in rubber tree breeding.

[0015] The beneficial effects of the present invention are:

[0016] (1) The present invention, for the first time, identified from the big data level that the SNP located at the 216 bp site of the nucleotide sequence of SEQ ID NO.1 is closely associated with the number of laticiferous ducts, which is conducive to detection, accurate, reliable and easy to operate.

[0017] (2) The SNP molecular marker provided by the present invention is used to identify the latex duct number trait of rubber trees, and the accuracy of screening rubber trees with a large number of latex ducts is as high as over 80%.

[0018] (3) The SNP molecular marker and its detection method provided by the present invention are not affected by the age of rubber trees and can be selected in the seedling stage, which greatly reduces the breeding workload, significantly shortens the rubber tree yield breeding selection cycle, and improves the breeding efficiency. It can be used in practice for rubber tree yield molecular marker-assisted breeding. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific embodiments to provide a better understanding of the present invention.

[0020] Example 1 Acquisition of SNP molecular markers associated with the number of latex tubes in rubber trees

[0021] 1.1 Acquisition of rubber tree germplasm

[0022] The material used in this study is the rubber tree 1981'IRRDB germplasm, which was planted in the National Rubber Tree Germplasm Resource Nursery of the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences. Its genetic background mainly comes from the three states of Acre, Mato Grosso and Rondônia in the Amazon River region of Brazil. In August 2020, 40 young leaves of the germplasm were collected and frozen in liquid nitrogen and brought back to the laboratory for use.

[0023] 1.2 DNA extraction from rubber tree germplasm

[0024] Take the frozen rubber tree leaves and extract genomic DNA using the CTAB method: (1) Weigh 1g of rubber tree young leaves, freeze them in liquid nitrogen, grind them into fine powder, and transfer them to a 50ml centrifuge tube. (2) Add 10ml of 2×CTAB extraction buffer (pre-heated at 65℃) (2% β-mercaptoethanol has been added in advance), gently shake the centrifuge tube to evenly disperse the plant tissue in the extraction buffer, incubate at 65℃ for 1 hour, and gently shake the centrifuge tube from time to time. (3) After the mixture cools to room temperature, add an equal volume of Tris-phenol:chloroform:isoamyl alcohol (25:24:1), then invert the centrifuge tube to mix it. Note: do not oscillate to prevent DNA breakage. (4) Centrifuge at room temperature at 12000 rpm for 10 min to separate the phases. (5) Pipette the aqueous phase into another centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), and invert to mix it. Centrifuge at room temperature at 12000 rpm for 10 min. (6) Pipette the aqueous phase into another centrifuge tube, add an equal volume of isopropanol, mix by inversion, and place at room temperature for 20 min. (7) Centrifuge at room temperature at 14,000 rpm for 20 min. Discard the supernatant and rinse the precipitate twice with 1 ml of ice-cold 75% ethanol (centrifuge at room temperature at 14,000 rpm for 10 min each time). (8) Discard the supernatant and dry the DNA precipitate on a clean bench. Then dissolve it in 200 μl TE (pH 8.0) buffer or ddH2O, add 1 μl RNase A (10 mg / ml), incubate at 37°C in a water bath for 30 min, and store at -20°C for later use.

[0025] 1.3 Using Sanger sequencing to obtain SNP molecular markers related to the number of latex ducts in rubber trees

[0026] Based on the Sanger sequencing platform, 10 random samples in the above population were sequenced for genes related to the number of latex tubes, and their nucleotide polymorphic sites were analyzed. Tassel 3.0_standalone software was used to analyze the correlation between SNP sites and known agronomic traits, and a SNP site related to the number of latex tubes in rubber trees was found. The site is located at the 216 bp site of the sequence shown in SEQ ID NO: 1. In the SEQ ID NO: 1 sequence, "t" is used to represent the site, and the base at this site is T or C (it should be understood that the 216 bp site of SEQ ID No.1 in the sequence table is represented by "t" only for the normal submission of the nucleotide sequence table, and does not affect the relevant limitations of claim 1 and the specification of this application). The number of latex tubes in the rubber tree with a genotype of heterozygous TC at this site is higher than that of the rubber tree with a genotype of homozygous TT here. No rubber tree with a genotype of homozygous CC has been found in the tested individuals (10 materials), indicating that the SNP molecular marker is closely related to the number of latex tubes in rubber trees.

[0027] Example 2 Sequencing verification and application of SNP molecular markers related to the number of latex tubes in rubber trees

[0028] 2.1 Amplification of nucleotide fragments containing SNP sites

[0029] According to the method of "1.2 DNA extraction from rubber tree germplasm materials", genomic DNA of 40 rubber trees to be tested was extracted as templates, and the nucleotide fragment where the SNP to be tested was located was amplified using forward primer F: 5'- AAA CTG CCA GAA GGA GGT GGT -3' (SEQ ID NO: 2) and reverse primer R: 5'- GGC AGT AGC CCT CAT AAT CTT ATC TGC -3' (SEQ ID NO: 3). The PCR reaction system was 25 μl: 1 μl of 100-200 ng / μl template DNA, 1 μl of 10mM primers F and R, 2.5 μl of 10×PCR reaction buffer, 2.0 μl of 2.5 mM dNTP, 0.2 μl of 5 U / μl Tap DNA polymerase, and 17.3 μl of water; the PCR reaction conditions were: pre-denaturation at 95°C for 5 min, 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, and extension at 72°C for 5 min.

[0030] 2.2 Sequencing to identify SNP genotypes

[0031] The PCR products obtained in the above steps were detected by agarose electrophoresis, recovered and inserted into the pMD 18-T vector, and 7 monoclonal clones were selected for sequencing (Shanghai Sangon Biotech Co., Ltd.) for each sample to identify the genotype at 216 bp in the SEQ ID NO: 1 sequence (i.e., the SNP molecular marker of the present invention). The genotypes of the SNP site and the number of latex tubes of the 40 rubber tree individuals to be tested are shown in Table 1 below, among which 5 genotypes were TC, 35 genotypes were TT, and the germplasm with a large number of latex tubes (the number of latex tubes ≥ 17) among the 5 TC genotypes accounted for 80%.

[0032] 2.3 Association analysis between SNP genotype and number of laticifer rows

[0033] Based on the results in Table 1, the general linear model of Tassel 3.0_standalone software was used to analyze the association between the genotype of the SNP locus and the number of milk duct rows. It was found that the correlation between the genotype of the SNP locus and the number of milk duct rows reached a significant level ( p=0.02682). The difference between the genotype frequency of the SNP locus and the number of latex ducts was analyzed by DPS software as shown in Table 2. The mean number of latex ducts of TC heterozygous individuals was the largest, and the number of latex ducts of TT homozygous individuals was relatively small. The difference between the mean number of latex ducts of TC genotype individuals and the mean number of latex ducts of TT genotype individuals reached an extremely significant level (P<0.01). Gene homozygous CC individuals have not been found in this population (40 materials). It can be seen that the 216th base T or C from the 5' end of the nucleotide sequence shown in SEQ ID NO:1 is significantly correlated with the number of latex ducts of rubber trees, and is a SNP molecular marker related to the number of latex ducts of rubber trees. The number of latex ducts of individuals with TC genotypes marked by this SNP is significantly higher than that of individuals with TT genotypes. By selecting the SNP locus as the TC genotype, rubber tree materials with a large number of latex ducts can be quickly selected or predicted, thereby promoting the breeding process of high-yield rubber tree varieties.

[0034] Table 1

[0035] Germplasm number Number of milk duct rows (columns) genotype Germplasm number Number of milk duct rows (columns) genotype XJA00276 6.80±1.20 TT XJA04002 7.70±1.00 TT XJA00323 5.80±1.20 TT XJA04071 18.20±11.60 TT XJA00445 10.90±3.70 TT XJA04075 11.70±1.60 TT XJA01197 16.20±10.50 TT XJA04152 13.10±2.50 TT XJA01198 13.80±1.20 TT XJA04210 12.20±1.50 TT XJA01663 5.90±4.00 TT XJA04285 11.40±2.20 TT XJA01840 8.50±1.40 TT XJA04292 10.00±5.30 TT XJA02391 16.60±2.70 TT XJA04314 10.70±5.00 TC XJA02534 18.70±7.20 TT XJA04397 13.10±1.70 TT XJA02682 17.10±4.90 TT XJA04634 20.80±1.80 TC XJA02702 12.00±1.30 TT XJA04971 12.00±2.90 TT XJA02967 8.90±3.00 TT XJA04975 7.90±2.90 TT XJA02968 9.40±1.00 TT XJA04988 11.20±2.60 TT XJA02974 22.60±0.70 TC XJA05006 15.30±2.00 TT XJA03000 15.10±4.10 TT XJA05190 13.80±3.60 TT XJA03015 14.80±2.00 TT XJA05255 21.10±1.50 TC XJA03019 14.10±1.90 TT XJA05381 14.70±1.10 TT XJA03642 16.90±2.80 TT XJA05539 7.70±0.70 TT XJA03765 17.60±1.70 TC XJA05728 16.00±2.30 TT XJA03788 10.70±1.90 TT XJA05787 21.70±1.50 TT

[0036] Table 2

[0037] genotype Number of individuals Genotype frequency Allele frequency Mean number of milk duct rows (rows) Standard Deviation 5% significant level 1% extremely significant level TT 35 0.875 A: 0.9375 12.57 3.88 a A TC 5 0.125 C: 0.0625 18.56 4.76 b B

[0038] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A SNP molecular marker for rubber tree, characterized in that: The sequence of the SNP molecular marker is shown in SEQ ID No. 1, and the base at the 216 bp position from the 5' end of the sequence shown in SEQ ID No. 1 is T or C; the TC genotype rubber tree of the SNP molecular marker has a large number of latex tube rows.

2. The invention relates to a method for detecting the number of latex ducts in rubber trees by using a detection reagent or a primer pair for detecting the SNP molecular marker as claimed in claim 1, or a kit comprising the primer pair, wherein the primer pair is used for PCR amplification, the sequence of the PCR amplification product is analyzed, the SNP molecular marker as claimed in claim 1 is detected, and the genotype of the rubber tree to be tested is determined, thereby determining the number of latex ducts of the rubber tree to be tested; when the genotype of the SNP site is TC, the number of latex ducts in the rubber tree is more than that when the genotype of the SNP site is TT; the primer pair comprises a forward primer F: 5'-AAA CTG CCA GAA GGA GGT GGT -3' and a reverse primer R: 5'-GGC AGT AGC CCT CAT AAT CTT ATC TGC -3'.

3. A method for detecting the number of latex tubes in rubber trees at the seedling stage, characterized in that: The method comprises extracting genomic DNA of a rubber tree to be tested, taking the genomic DNA of the rubber tree to be tested as a template, performing PCR amplification using a primer pair, analyzing the sequence of the PCR amplification product, detecting the SNP molecular marker described in claim 1, determining the genotype of the rubber tree to be tested, and thus determining the trait of the number of latex duct rows of the rubber tree to be tested; when the genotype of the SNP site is TC, the number of latex duct rows of the rubber tree is greater than when the genotype of the SNP site is TT; and the primer pair comprises a forward primer F: 5'-AAA CTG CCA GAA GGA GGT GGT -3' and a reverse primer R: 5'-GGC AGT AGC CCT CAT AAT CTT ATC TGC -3'.

4. A breeding method for rubber trees, characterized in that, The method comprises extracting genomic DNA of each rubber tree to be selected from a group of rubber trees to be selected, taking the obtained genomic DNA as a template, adopting a primer pair to perform PCR amplification, analyzing the sequence of the PCR amplification product, detecting the SNP molecular marker described in claim 1, determining the genotype of each rubber tree to be selected, thereby determining the trait of the number of latex duct rows of each rubber tree to be selected, breeding the rubber trees, and the number of latex duct rows of the rubber tree when the genotype of the SNP site is TC is greater than that when the genotype of the SNP site is TT; the primer pair comprises a forward primer F: 5'-AAA CTG CCA GAA GGA GGTGGT -3' and a reverse primer R: 5'-GGC AGT AGC CCT CAT AAT CTT ATC TGC -3'.

5. Use of a detection reagent or a primer pair for detecting the SNP molecular marker as claimed in claim 1, or a kit containing the primer pair, or the method according to claim 3, or the breeding method according to claim 4 in rubber tree breeding, using the primer pair for PCR amplification, analyzing the sequence of the PCR amplification product, detecting the SNP molecular marker as claimed in claim 1, determining the genotype of each rubber tree to be selected, thereby determining the number of latex duct rows of each rubber tree to be selected, breeding the rubber tree, when the genotype of the SNP site is TC, the number of latex duct rows of the rubber tree is more than when the genotype of the SNP site is TT; the primer pair comprises a forward primer F: 5'-AAA CTG CCA GAA GGA GGT GGT -3' and a reverse primer R: 5'-GGC AGTAGC CCT CAT AAT CTT ATC TGC -3'.