SNP molecular markers and variety identification methods for distinguishing rubber tree varieties

Abstract

The invention discloses a SNP molecular marker and a variety identification method for distinguishing rubber tree varieties. The 50 SNP molecular markers screened by the present invention have high specificity, sensitivity and resolution, are not affected by environmental conditions, and the detection results are accurate, repeatable and stable, and can fully reveal the real level of genetic diversity of rubber trees. DNA fingerprint atlas library is established by using this set of SNP marker combination, cluster analysis and variety identification can be performed on rubber tree varieties, so that the detection results are more accurate and efficient, providing a theoretical basis for the protection and utilization of rubber tree resources, effectively distinguishing 66 varieties of rubber trees for the first time, and the discrimination rate of the present invention to bred varieties can reach 100%. Detection using the single nucleotide marker site of the present invention has the advantages of being simple, fast and low cost, and can achieve large-scale application in production.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology of rubber tree varieties, and in particular relates to a SNP molecular marker for distinguishing rubber tree varieties and a variety identification method. Background Art

[0002] Rubber trees are native to the Amazon River Basin in Brazil and are important economic crops in tropical regions. The natural rubber they produce is an important strategic resource in my country and plays an irreplaceable role in the fields of military, aerospace, marine engineering, rail transportation, etc. Since the rubber tree was introduced to China in 1904, there are 6,165 germplasm materials of rubber tree cultivars, wild germplasms, and 5 species and 1 variant preserved in China. For a long time, the identification of rubber tree variety resources has been mainly based on morphological trait identification. This classic and conventional identification method generally completes its identification process after the rubber tree blooms. However, the rubber tree is a perennial cross-pollinated tall tree that generally blooms and bears fruit 5-6 years after planting. It has unfavorable factors such as a long sexual generation cycle and highly heterozygous varieties, which leads to not only a long identification cycle in the process of its trait identification, but also its agronomic traits are easily affected by environmental conditions and cultivation management measures. In addition, the traditional identification method is very difficult for some varieties with small trait differences and close genetic relationships to identify, which brings many inconveniences to the identification of rubber tree germplasm resources and varieties. Therefore, accurate molecular marker methods are urgently needed to identify rubber tree germplasm resources and varieties.

[0003] Molecular marker analysis methods are all based on DNA polymorphism, such as RFLP, RAPD, AFLP, ISSR, SSR, and SNP markers, which can all produce DNA fingerprints with high individual specificity. In the early days, RFLP, RAPD, and AFLP molecular marker technologies were used to build DNA fingerprint libraries for multiple species, but the above marker technologies often encountered problems such as poor stability, low polymorphism, and difficulty in data integration during use, and have not been widely used. Therefore, the International Union for the Protection of New Varieties recommends SSR and SNP as the preferred DNA fingerprint marker technology. Since SNP markers have the advantages of covering the entire genome, high throughput, site specificity, co-dominant inheritance, low false positive rate, sharply reduced development and testing costs, and easy data integration, they will become the main marker type for genotype identification in the future.

[0004] With the development of second generation sequencing technology, sequencing flux presents an exponential growth trend, and sequencing cost is sharply reduced, providing strong technical support for the detection of SNP markers at the whole genome level. There are abundant SNPs on each species. How to formulate SNP site screening principles, site number, site combination, judgment threshold, etc. according to different identification targets to become the key of screening SNP to build fingerprint map. The present invention is based on the SNP obtained by rubber tree resequencing, by the control of SNP sequencing quality, SNP site quality control, SNP screening judgment threshold setting, etc., to obtain the flow of standardized SNP construction fingerprint map, used to establish the molecular ID card of rubber tree. At present, there is no report of establishing complete rubber tree variety fingerprint map library to identify variety. Summary of the invention

[0005] The object of the present invention is to provide a SNP molecular marker and a variety identification method for distinguishing rubber tree varieties in view of the deficiencies in the prior art. To solve the problems of poor repeatability and low polymorphism of co-dominant DNA markers such as RAPD and ISSR in the prior art; another object of the present invention is to provide the application of polymorphic primers of SNP molecular markers of rubber tree nuclear genome in rubber tree variety identification, genetic structure and resource genetic diversity analysis, thereby providing a basis for identifying rubber tree germplasm resources and cultivars from the molecular level.

[0006] The first aspect of the present invention is to provide a SNP molecular marker combination for constructing a DNA fingerprint of a rubber tree, wherein the SNP molecular marker combination comprises LG01_54721919, and / or LG01_96036745, and / or LG02_214754, and / or LG02_8025843, and / or LG02_57849162, and / or LG03_40084932, and / or LG03_57340517, and / or LG03_96006922, and / or LG03_104230492, and / or LG04_224562, and / or L G04_16174898, and / or LG04_33465133, and / or LG04_71146652, and / or LG04_80306944, and / or LG04_96013598, and / or LG05_56475, and / or LG05_16004126, and / or LG05_88010730, and / or LG06_32377524, and / or LG07_8072524, and / or LG07_32411839, and / or LG07_57540924, and / or LG07_72003724, and / or LG07 _80004402, and / or LG08_8000585, and / or LG08_28146509, and / or LG08_35232681, and / or LG08_48608369, and / or LG09_8012416, and / or LG09_16001432, and / or LG09_46132314, and / or LG09_50708986, and / or LG09_64555870, and / or LG10_34723841, and / or LG11_54415, and / or LG11_8139610, and / or LG11_348 02586, and / or LG12_284283, and / or LG12_32322540, and / or LG12_40522222, and / or LG13_32072502, and / or LG13_40000353, and / or LG14_605466, and / or LG14_33209388, and / or LG14_56176655, and / or LG15_32001166, and / or LG15_24017141, and / or LG17_24096219, and / or LG17_32001525, and / or LG18_10766;

[0007]

[0008]

[0009]

[0010]

[0011]

[0012]

[0013] In the above table, “Sequence” is the nucleotide sequence of the SNP molecular marker; “[]” marks the position of the SNP molecular marker site, and the bases therein are the polymorphisms of the site.

[0014] It should be understood that in actual application, those skilled in the art can select appropriate SNP molecular markers for application according to actual conditions. For example, it is actually known that the rubber tree to be tested is Haiken 1 or RRIM623, then the SNP molecular markers that can be used for identification can be selected from LG01_96036745, LG03_104230492, LG04_16174898, LG04_33465133, LG04_71146652, LG04_96013598, LG07_57540924, LG07_72003724, LG09_50708986, LG09_64555870, LG11_54415, LG11_34802586, LG14_33209388, LG18_10766, etc. (refer to Table 5 below), or two or more can be selected for multiple verification and identification. For another example, actual known rubber tree to be measured is Haiken 1, RRIM623 or Dafeng 95, and only one SNP molecular marker can be used, such as LG03_104230492, LG04_33465133, LG11_34802586, LG15_32001166, etc. (refer to Table 5 below), and certainly more than two SNP molecular markers can be used. When the known rubber tree to be measured is one of four or more rubber trees, those skilled in the art can also refer to Table 5 below, select appropriate SNP molecular markers to identify. When the rubber tree to be measured is completely unknown, those skilled in the art can also identify the above-mentioned SNP molecular markers in batches.

[0015] Preferably, the SNP molecular marker combination includes LG01_54721919, LG01_96036745, LG02_214754, LG02_8025843, LG02_57849162, LG03_40084932, LG03_57340517, LG03_96006922, LG03_104230492, LG04_224562, LG04_16174898, LG04_334 65133, LG04_71146652, LG04_80306944, LG04_96013598, LG05_56475, LG05_16004126, LG05_88010730, L G06_32377524, LG07_8072524, LG07_32411839, LG07_57540924, LG07_72003724, LG07_80004402, LG08_8 000585, LG08_28146509, LG08_35232681, LG08_48608369, LG09_8012416, LG09_16001432, LG09_461323 14. LG09_50708986, LG09_64555870, LG10_34723841, LG11_54415, LG11_8139610, LG11_34802586, LG12_ at least two of LG17_24096219, LG17_32001525 and LG18_10766.

[0016] Preferably, the SNP molecular marker combination includes at least LG03_104230492, LG02_8025843, LG01_54721919, LG15_32001166, LG07_72003724, LG05_88010730 and LG02_214754.

[0017] Preferably, the SNP molecular marker combination includes LG01_54721919, LG01_96036745, LG02_214754, LG02_8025843, LG02_57849162, LG03_40084932, LG03_57340517, LG03_96006922, LG03_104230492, LG04_224562, LG04_16174898, LG04_33 465133, LG04_71146652, LG04_80306944, LG04_96013598, LG05_56475, LG05_16004126, LG05_88010730 , LG06_32377524, LG07_8072524, LG07_32411839, LG07_57540924, LG07_72003724, LG07_80004402, LG0 8_8000585, LG08_28146509, LG08_35232681, LG08_48608369, LG09_8012416, LG09_16001432, LG09_461 32314, LG09_50708986, LG09_64555870, LG10_34723841, LG11_54415, LG11_8139610, LG11_34802586, L LG17_24096219, LG17_32001525, and LG18_10766.

[0018] The second aspect of the present invention is to provide a primer set for amplifying the SNP molecular marker combination as described in the first aspect of the present invention.

[0019] Preferably, the primer pairs for each SNP molecular marker are as shown in Table 3 below.

[0020] The third aspect of the present invention is to provide a kit or a gene chip containing the primer set described in the second aspect of the present invention.

[0021] The fourth aspect of the present invention is to provide the use of the SNP molecular marker combination described in the first aspect of the present invention, or the primer set described in the second aspect of the present invention, or the kit or gene chip described in the third aspect of the present invention in the genetic structure of rubber tree variety populations, and / or the genetic diversity analysis of rubber tree resources, and / or the identification of rubber tree varieties, and / or the construction of rubber tree DNA fingerprint maps.

[0022] The fifth aspect of the present invention is to provide a method for constructing a DNA fingerprint library of rubber tree varieties: extracting total DNA of each rubber tree variety; amplifying using the primer set of the SNP molecular marker described in the second aspect of the present invention; performing SNP site polymorphism detection using the SNP molecular marker combination described in the first aspect of the present invention, and performing genotyping on each rubber tree variety according to the detection results of the SNP site, thereby constructing a DNA fingerprint library of rubber tree varieties; wherein the rubber tree variety is the rubber tree variety described in Table 2 below.

[0023] Preferably, the SNaPshot method is used to detect SNP site polymorphism, identify rubber tree varieties, and analyze population genetic structure and genetic diversity.

[0024] Preferably, the SNP molecular marker combination includes at least LG03_104230492, LG02_8025843, LG01_54721919, LG15_32001166, LG07_72003724, LG05_88010730 and LG02_214754.

[0025] Preferably, the SNP molecular marker combination includes LG01_54721919, LG01_96036745, LG02_214754, LG02_8025843, LG02_57849162, LG03_40084932, LG03_57340517, LG03_96006922, LG03_104230492, LG04_224562, LG04_16174898, LG04_33 465133, LG04_71146652, LG04_80306944, LG04_96013598, LG05_56475, LG05_16004126, LG05_88010730 , LG06_32377524, LG07_8072524, LG07_32411839, LG07_57540924, LG07_72003724, LG07_80004402, LG0 8_8000585, LG08_28146509, LG08_35232681, LG08_48608369, LG09_8012416, LG09_16001432, LG09_461 32314, LG09_50708986, LG09_64555870, LG10_34723841, LG11_54415, LG11_8139610, LG11_34802586, L LG17_24096219, LG17_32001525, and LG18_10766.

[0026] The sixth aspect of the present invention is to provide a DNA fingerprint library of rubber tree varieties constructed using the construction method described in the fifth aspect of the present invention.

[0027] The seventh aspect of the present invention is to provide a method for identifying rubber tree varieties, characterized in that the total DNA of the sample to be tested is amplified by PCR using the primer set described in the second aspect of the present invention, and then SNP site polymorphism detection is performed, and the genotyping of the SNP molecular marker is determined according to the detection result of the SNP site, and then compared with the genotyping in the rubber tree variety DNA fingerprint library described in the sixth aspect of the present invention.

[0028] Preferably, the SNaPshot method is used to detect SNP site polymorphism.

[0029] Beneficial effects of the present invention:

[0030] The 50 SNP molecular markers screened by the present invention have high specificity, sensitivity and resolution, are not affected by environmental conditions, and have accurate detection results, good repeatability and stability, and can fully reveal the real level of genetic diversity of rubber trees; a DNA fingerprint library is established by using the set of SNP marker combinations, cluster analysis and variety identification can be performed on rubber tree varieties, so that the detection results are more accurate and efficient, providing a theoretical basis for the protection and utilization of rubber tree resources, effectively distinguishing 66 varieties of rubber trees for the first time, and the discrimination rate of the present invention for bred varieties can reach 100%. Detection using the single nucleotide marker site of the present invention has the advantages of being simple, fast and low in cost, and can achieve large-scale application in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an example diagram of the typing test results of some sample amplification products in Example 2 of the present invention;

[0032] Figure 2 It is the UPGAM method cluster analysis diagram of the rubber tree varieties based on SNP in Example 2 of the present invention

[0033] Figure 3 It is the recognition rate of the core SNP for rubber tree varieties in Example 2 of the present invention. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be obtained commercially.

[0035] Example 1: Screening and determination of SNP sites in rubber trees

[0036] Genome resequencing was performed using nine rubber tree varieties, including PR107, GT1, IAN873, RRIM600, 93-114, Yunyan 77-4, Reyan 7-33-97, Reyan 8-79, and Reken 628. For specific operations, please refer to "Du, H., Yang, J., Chen, B. et al. Target sequencing reveals genetic diversity, population structure, core-SNP markers, and fruit shape-associated loci in pepper varieties. BMC Plant Biol 19, 578 (2019)".

[0037] Based on the resequencing data of the rubber tree genome, it was compared with the rubber tree reference genome (https: / / www.ncbi.nlm.nih.gov / genome / ?term=rubber+tree) to mine SNP sites, and screening and analysis of SNP marker candidate sites were performed according to the following principles: (a) minimum allele frequency MAF>0.4; (b) deletion rate <0.2; (c) heterozygosity <0.2; (d) no sequence variation within 200bp upstream and downstream; (e) two allele sites; (f) clear chromosome distribution; (g) uniform distribution on the genome.

[0038] From the genome resequencing data of the above nine representative varieties, 50 high-quality, single-copy, highly polymorphic SNP markers were finally screened out for the identification of rubber tree varieties from different sources, population genetic structure, and genetic diversity analysis of rubber tree resources. The sequence information of the 50 SNP molecular markers is shown in Table 1.

[0039] Table 1:

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] In the above table, “Sequence” is the nucleotide sequence where the SNP molecular marker is located; “[]” is marked with the position of the SNP molecular marker site, and the bases therein are the polymorphisms of the site.

[0047] Example 2: Sequencing verification and application of rubber tree SNP markers

[0048] 1 Test materials

[0049] The test materials came from the National Rubber Tree Germplasm Resource Garden of the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences. A total of 66 rubber tree varieties were selected (Table 2). Leaves from three different plants of each variety were collected and quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for extraction of rubber tree leaf DNA.

[0050] Table 266 Information on rubber tree species

[0051]

[0052]

[0053] 2 Extraction and detection of total DNA from rubber trees

[0054] The total DNA of leaves of each rubber tree variety was extracted by referring to the CTAB method, and then the total DNA quality of each variety was detected by agarose gel electrophoresis and spectrophotometer to ensure that the total DNA of each rubber tree variety extracted had good integrity and the A260 / A280 ratio was between 1.6 and 2.0.

[0055] 3SNP primer design and screening

[0056] According to the sequence information of the 50 SNP molecular marker sites screened above, the primer design software PrimerPlex2 was used to design PCR reaction primers, and 167 pairs of SNP primers were designed. After screening, a total of 50 pairs of SNP amplification primers with good specificity and high polymorphism were obtained, as shown in Table 3. The primer design software Primer3plus was used to design single base extension primers, as shown in Table 4.

[0057] Table 3 Amplification primers

[0058]

[0059]

[0060] Table 4 Extension primers

[0061]

[0062]

[0063]

[0064] In the table, in the extended primers, uppercase letters are specific sequences that bind to the SNP region; lowercase letters are extended sequences that ensure that multiple typing in one reaction can be accurately interpreted; direction represents the sequencing direction.

[0065] 4. Type experiment

[0066] 4.1 PCR amplification reaction

[0067] Using the genomic DNA of the 66 rubber tree varieties extracted above as a template, the primers in Table 3 were used to amplify the nucleotide fragment containing the SNP site: PCRmastermix was prepared in a 1.5mL EP tube and centrifuged at low speed with shaking. Using an 8-channel pipette, 9μl PCRmastermix was added to each well of the 384-well plate, and finally 1μl template DNA (20ng / uL) was added and mixed. Carefully cover the 384-well sealing film and press each well firmly to prevent evaporation during the PCR procedure. Centrifuge at 1000rpm for 1min.

[0068] The PCR amplification system was: 2×TaqPCRMasterMix 5.0μl, genomic DNA (~20ng) 1.0μl, a mixture of upstream and downstream primers (concentration 10pmol / uL, the ratio of upstream and downstream primers was 1:1) 1.0μl, ddH2O 3.0μl, and the total volume of the system was 10μL.

[0069] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 20 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; final extension at 72°C for 3 min; 16°C for 1 min; and storage at 4°C.

[0070] 4.2 Alkaline phosphatase treatment of products

[0071] After the PCR reaction is completed, the PCR product is treated with SAP (shrimpalkalinephosphatase) to remove free dNTPs in the system. Prepare the alkaline phosphatase treatment reaction solution in a new 1.5mL EP tube. The SAPmix reaction components are as follows: ddH200.75μl, SAP (1U / uL) 0.5μl, ExoI (5U / uL) 0.15μl, 10*SAPbuffer 0.6μl, PCR product 4.0μl. Then add the SAPmix reaction components to the 384-well PCR reaction plate. After the pipetting is completed, carefully cover the 384-well sealing film and press each well to prevent evaporation during the PCR program. Finally, after centrifugation, place the 384-well reaction plate on the PCR instrument and start the SAP reaction program. The SAP reaction program is as follows: 37℃40min; 85℃15min; 4℃ storage.

[0072] 4.3 Single base extension reaction

[0073] After the alkaline phosphatase treatment, a single base extension reaction was performed. The reaction system was: SNaPshotMix 0.5μl, PooledPCRProducts 3.0μl, PooledPrimers (Table 4) 1.0μl, ddH2O 0.5μl. The reaction system was added to the 384-well PCR reaction plate. After the pipetting was completed, the 384-well sealing film was carefully covered and each well was pressed firmly to prevent evaporation during the PCR program. After centrifugation, the extension reaction program was performed. The extension reaction program was: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 52℃ annealing for 5s, 60℃ extension for 5s, a total of 35 cycles; 72℃ final extension for 3min; 16℃ for 1min.

[0074] 4.4 On-the-machine

[0075] Take 2uL SNaPshot reaction product and add it to 8μl deionized formamide containing 0.8% LIZ120, denature at 95℃ for 5 minutes, then cool it at -20℃, and then sequence it on 3730XL.

[0076] 5 Primer optimization and adjustment

[0077] The raw data in .fsa format were exported from the ABI3730xl instrument, classified and archived according to the detection reaction, and then imported into the GeneMarker analysis software to read the genotype data. According to the signal value and peak condition of each site, the primer ratio of each site was adjusted to ensure that the signal value and peak condition of each site were basically consistent.

[0078] 6. Result quality control and analysis

[0079] Analyze the formal experimental results, perform statistics according to the sample and site conditions, obtain the typing results of each site of the sample, and export the PDF graph of each reaction result. Examples of typing test results of some sample amplification products are as follows: Figure 1 According to the result information, parameter analysis was performed to obtain detailed typing results. The genotypes of 50 SNP sites of 66 rubber tree varieties are shown in Table 5-9.

[0080] Table 5 Genotypes of 66 rubber tree varieties at 50 SNP loci (1)

[0081]

[0082]

[0083] Table 6 Genotypes of 666 rubber tree varieties at 50 SNP loci (2)

[0084]

[0085]

[0086] Table 7 Genotypes of 766 rubber tree varieties at 50 SNP loci (3)

[0087]

[0088]

[0089] Table 8 Genotypes of 66 rubber tree varieties at 50 SNP loci (4)

[0090]

[0091]

[0092]

[0093] Table 9 Genotypes of 66 rubber tree varieties at 50 SNP loci (5)

[0094]

[0095]

[0096] Based on the Nei genetic distance between samples, the phylogenetic tree was constructed using the phangornR package with the unweighted group average method. The UPGAM cluster analysis of 66 rubber tree varieties showed that some closely related varieties clustered together (e.g. Figure 3 As shown in the figure, the high-yield lines Reyan 879, Reyan 8-333, and Reyan 87-4-26 are clustered together; the cold-resistant lines Yunyan 77-2, Yunyan 77-4, and GT1 are clustered together, indicating that 50 SNP markers can distinguish 66 rubber tree varieties.

[0097] The present invention distinguishes 66 rubber tree varieties for the first time, and the differentiation rate of cultivated varieties can reach 100%. This shows that the detection using the single nucleotide marker site of the present invention has the advantages of being simple, rapid and low-cost, and can be applied on a large scale in production.

[0098] In order to further screen the core SNP molecular marker sites, different numbers and combinations of sites were selected from the above 50 SNP molecular marker sites to distinguish 66 varieties, and the discrimination rates of different numbers and combinations of sites for 66 rubber tree varieties were counted. The number of sites was used as the horizontal axis, and the maximum discrimination rate of different site combinations under the number of sites was used as the vertical axis. Figure 3 As shown. Figure 3It can be seen that when the number of loci is 7, the discrimination rate of 66 varieties can reach 100%. The SNP molecular marker combination that achieves 100% discrimination rate is: LG03_104230492, LG02_8025843, LG01_54721919, LG15_32001166, LG07_72003724, LG05_88010730 and LG02_214754.

[0099] 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 combination for distinguishing Haiken 1 and RRIM623, characterized in that: The SNP molecular marker combination is LG01_96036745, LG03_104230492, LG04_16174898, LG04_33465133, LG04_71146652, LG04_96013598, LG07_57540924, LG07_72003724, LG09_50708986, LG09_64555870, LG11_54415, LG11_34802586, LG14_33209388, LG18_10766, LG02_8025843, LG05_16004126, LG05_88010730, LG13_32072502, LG14_56176655, LG15_32001166, LG15_24017141, LG17_32001525, LG07_8072524, LG07_32411839, and LG09_16001432; In the above table, "Sequence" is the nucleotide sequence of the SNP molecular marker; "[]" is marked with the position of the SNP molecular marker site, and the bases therein are the polymorphisms of the site.

2. A primer pair for amplifying the SNP molecular marker combination as claimed in claim 1, or a kit or gene chip containing a primer pair for amplifying the SNP molecular marker combination as claimed in claim 1; the primer pairs for each SNP molecular marker are 。 3. Use of the SNP molecular marker combination according to claim 1, or the primer pair or kit or gene chip according to claim 2 in distinguishing Haiken 1 from RRIM623.

4. A method for distinguishing Haiken 1 from RRIM623, characterized in that: Extract the total DNA of Haiken 1 and RRIM623; use the primer pair or primer group described in claim 2 for amplification; use the SNP molecular marker combination described in claim 1 to perform SNP site polymorphism detection, and perform genotyping on Haiken 1 and RRIM623 according to the detection results of the SNP sites; use the primer pair described in claim 2 to perform PCR amplification on the total DNA of the sample to be tested, and then perform SNP site polymorphism detection, determine the genotyping of the SNP molecular marker according to the detection results of the SNP sites, and then compare it with the genotyping of Haiken 1 and RRIM623.

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