CN112831587A - Molecular marker and application thereof, and method for identifying sapindus mukorossi and sapindus mukorossi germplasm - Google Patents
Molecular marker and application thereof, and method for identifying sapindus mukorossi and sapindus mukorossi germplasm Download PDFInfo
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Abstract
The invention discloses a molecular marker, application thereof and a method for identifying the germ plasm of sapindus muk and sapindus muk, wherein the molecular marker comprises primers Samuk10G0092500, Samuk04G0084900, Samuk07G0006800, Samuk12G0105900 and/or Samuk03G 0235800: used for identifying the germ plasm of the soapberry and the soapberry with the hair lobe; extracting DNA of a sapindus mukorossi or sapindus mukorossi young leaf sample to be detected; performing PCR amplification according to the SSR molecular marker; detecting on a capillary electrophoresis machine; analyzing the original data of the sample to construct a phylogenetic tree; and determining the germ plasm amplification product as soapberry or soapberry with hairy petals. The fluorescence labeling SSR capillary electrophoresis detection method established based on the gene sequencer is effective combination of a fluorescence sequencing technology and an SSR molecular labeling technology, realizes high-efficiency and accurate acquisition of the length of an SSR molecular labeling amplification product, can distinguish difference fragments within 2bp, accurately acquires the length of the amplification product, and has more stable, accurate and high-efficiency detection results.
Description
Technical Field
The invention belongs to the field of soapberry germplasm identification, and relates to a molecular marker for identifying soapberry and soapberry hairy petal germplasm, application of the molecular marker and a method for identifying soapberry and soapberry hairy petal germplasm by using the molecular marker.
Background
Sapindus mukorossi Gaertn and Sapindus mukorossi DC are trees of Sapindus (Sapindaceae) genus Sapindus (sapindacae L.), are called Soapberry (Soapberry) abroad, are also called hand washing fruit, soapbark tree, delavay Soapberry and the like at home, are distributed only in Yunnan province of China, and are widely distributed in southern regions of China. The oil content of the kernels of the sapindus mukorossi and the sapindus mukorossi is as high as about 40 percent, the oleic acid and the linoleic acid in the oil are as high as 62.5 percent, and the fatty acid of C16-C20 accounts for 98.2 percent, thereby meeting various standards of modern biomass diesel oil and being refined into high-grade lubricating oil; the pulp contains 5-28% of saponin, is an excellent natural washing cosmetic raw material, and has an elution rate of over 90%; the seed shell can be made into high-grade active carbon; the research at home and abroad finds that the kernel, the pulp and the root system of the Chinese medicinal composition have functional active ingredients with great development potential for resisting cancer, reducing blood fat and lowering blood pressure. Therefore, the sapindus mukorossi and the sapindus mukorossi are multifunctional raw material tree species which integrates biological energy, biochemical engineering and biological medicine into a whole in China.
Chinese soapberry and soapberry belong to the related species of soapberry, are distributed in Yunnan province of China, have rich germplasm resources, have similar phenotypic characters and are easily influenced by development stages, cultivation measures and environmental conditions, and are difficult to distinguish the soapberry and the soapberry from the soapberry in the contemporary phenotypic staggered distribution area of the soapberry and the soapberry in the Yunnan province, which brings great difficulty to germplasm identification, cross breeding, new variety breeding and the like of the sapindus of the soapberry and the soapberry. Therefore, the research team develops molecular biology SSR molecular markers to assist in identifying the germ plasm resources of the soapberry and the soapberry with the hair petals, promotes the fine variety breeding of the soapberry and the soapberry with the hair petals, accelerates the development and utilization of the germ plasm resources, and protects the germ plasm resource diversity of the soapberry and the soapberry with the hair petals.
The prior art has not disclosed the literature data related to identifying the germplasm of the soapberry and the soapberry with the SSR molecular marker of transcriptome sequencing.
Disclosure of Invention
In view of the above, the present invention aims to provide a molecular marker capable of rapidly and accurately identifying the germplasm of sapindus mukorossi and sapindus mukorossi.
The invention also aims to provide application of the molecular marker for identifying the germplasm of the soapberry and the soapberry with the hair flap.
Still another object of the present invention is to provide a method for identifying sapindus mukorossi and sapindus mukorossi germplasm.
The inventor provides a molecular marker for identifying sapindus mukorossi and sapindus mukorossi germplasm through long-term exploration and attempt, and multiple experiments and efforts, and continuous innovation, and in order to solve the technical problems, the molecular marker is one or more pairs of sequences in the following primers:
Samuk10G0092500：
an upstream primer: 5'-TTCTTCCGATTGAGCGCCAT-3', respectively;
a downstream primer: 5'-CGAATCCAGTGGCAGTAGCA-3', respectively;
Samuk04G0084900：
an upstream primer: 5'-CTAGCTGTGGGGGCACATAC-3', respectively;
a downstream primer: 5'-GCATATTAGCACCGACCGGA-3', respectively;
Samuk07G0006800：
an upstream primer: 5'-GAAGCCGGATCTAATGGGCA-3', respectively;
a downstream primer: 5'-TCACTCCAACAGCCTTGTCC-3', respectively;
Samuk12G0105900：
an upstream primer: 5'-AGGAGATTCAAGTGGTGGCG-3', respectively;
a downstream primer: 5'-GACGACGTACACTGCTCCAT-3', respectively;
Samuk03G0235800：
an upstream primer: 5'-CAGTCCGATCTCAGCAGCAT-3', respectively;
a downstream primer: 5'-TTGCCCTAGGGTGTTCTTGG-3' are provided.
The invention also provides an application of the molecular marker, which is used for identifying the germplasm of the soapberry and the soapberry with the hair lobe, and comprises the following steps:
1) extracting DNA of a sapindus mukorossi or sapindus mukorossi young leaf sample to be detected;
2) performing PCR amplification according to SSR molecular markers by using the DNA extracted in the step 1) as a template;
3) detecting on a capillary electrophoresis machine;
taking 0.3 mu L of PCR product obtained in the step 2), 0.5 mu L of molecular weight internal standard and 9.5 mu L of deionized formamide, mixing, adding into a PCR plate, denaturing at 95 ℃ for 5min, cooling at 4 ℃, centrifuging, and performing on-machine detection on 1 Xbuffer Buffer solution;
4) analyzing the original data of the sample to obtain the lengths of amplified fragments of different samples; further analyzing data to obtain primer polymorphism, assigning alleles obtained by each pair of primers by using Arabic numerals 0/1, establishing a soapberry and hairball soapberry germplasm 0/1 matrix, calculating a genetic distance, and constructing a phylogenetic tree;
5) judging the soapberry or the soapberry with the hair lobe according to the result of detecting the germ plasm amplification product.
According to one embodiment of the application of the molecular marker of the present invention, in step 2), the SSR molecular marker is one or more pairs of sequences in the following primers:
Samuk10G0092500：
an upstream primer: 5'-TTCTTCCGATTGAGCGCCAT-3', respectively;
a downstream primer: 5'-CGAATCCAGTGGCAGTAGCA-3', respectively;
Samuk04G0084900：
an upstream primer: 5'-CTAGCTGTGGGGGCACATAC-3', respectively;
a downstream primer: 5'-GCATATTAGCACCGACCGGA-3', respectively;
Samuk07G0006800：
an upstream primer: 5'-GAAGCCGGATCTAATGGGCA-3', respectively;
a downstream primer: 5'-TCACTCCAACAGCCTTGTCC-3', respectively;
Samuk12G0105900：
an upstream primer: 5'-AGGAGATTCAAGTGGTGGCG-3', respectively;
a downstream primer: 5'-GACGACGTACACTGCTCCAT-3', respectively;
Samuk03G0235800：
an upstream primer: 5'-CAGTCCGATCTCAGCAGCAT-3', respectively;
a downstream primer: 5'-TTGCCCTAGGGTGTTCTTGG-3' are provided.
According to one embodiment of the use of the molecular marker of the present invention, in said step 2),
the PCR amplification adopts a 20 mu L reaction system: comprises 10-50ng template DNA 1-2 μ L, forward and reverse primers 0.2 μ L and 0.2 μ L, respectively, 2 XTaq Plus PCR reaction mixture 10 μ L of Rui Boxing Ke, and appropriate amount of ddH2O；
The PCR reaction program is: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 30s, and extension at 72 ℃ for 10min after 20 cycles; storing at 4 ℃.
According to one embodiment of the application of the molecular marker of the present invention, in the step 3), the apparatus used for the on-machine detection is a gene analyzer ABI 3730XL DNA analyzer, a capillary tube ABI 96X 50cm 4331246.
According to one embodiment of the application of the molecular marker of the present invention, the gene analyzer parameters are set as follows:
Module File:GeneMapper50_POP7_1；
Run Voltage:15.0kv；
Injection Voltage:1.6kv；
Injection Duration:15sec；
Temperature:63DegC；
Current_Stability:30.0uA。
according to one embodiment of the application of the molecular marker of the invention, the PCR reaction mixture is MgCl2：3mM；dNTPs：0.4mM。
According to an embodiment of the application of the molecular marker of the present invention, the step 4) is specifically: analyzing the original data of the sample by using GeneMarker V2.2.0 software to obtain the lengths of amplified fragments of different samples; analyzing data by using software PopGen32 to obtain primer polymorphism, assigning alleles obtained by each pair of primers by using Arabic numerals 0/1, establishing a sapindus and sapindus hirsutus germ plasm 0/1 matrix, calculating genetic distance by using Nysts software, and constructing a phylogenetic tree by using a Clustering module SAHN function.
The invention also provides a method for identifying the germplasm of the soapberry and the soapberry with the hair petals based on the molecular marker, which comprises the following steps:
1) extracting DNA of a sapindus mukorossi or sapindus mukorossi young leaf sample to be detected;
2) performing PCR amplification according to the molecular marker by using the DNA extracted in the step 1) as a template;
the PCR adopts a 20 mu L reaction system: comprises 10-50ng template DNA 1-2 μ L, forward and reverse primers 0.2 μ L and 0.2 μ L, respectively, 2 XTaq Plus PCR reaction mixture 10 μ L of Rui Boxing Ke, and appropriate amount of ddH2O; the reaction mixture is MgCl2：3mM；dNTPs：0.4mM；
The PCR reaction program is: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 30s, and extension at 72 ℃ for 10min after 20 cycles; storing at 4 ℃;
3) detecting on a capillary electrophoresis machine;
taking 0.3 mu L of PCR product in the step 2), 0.5 mu L of molecular weight internal standard and 9.5 mu L of deionized formamide, mixing, adding into a PCR plate, denaturing at 95 ℃ for 5min, cooling at 4 ℃, centrifuging, and performing on-machine detection by using 1 Xbuffer Buffer solution; and (3) loading a detection instrument: gene analyzer ABI 3730XL DNA analyzer, capillary ABI 96X 50cm 4331246;
4) after electrophoresis is finished, the original data of the samples are analyzed by GeneMarker V2.2.0 software, and the lengths of the amplified fragments of different samples are obtained. Analyzing data by using software PopGen32 to obtain primer polymorphism, assigning alleles obtained by each pair of primers by using Arabic numerals 0/1, establishing a soapberry and hairball soapberry germplasm 0/1 matrix, calculating genetic distance by using Nysts software, and constructing a phylogenetic tree by using a Clustering module SAHN function;
5) judging the soapberry or the soapberry with the hair lobe according to the result of detecting the germ plasm amplification product.
According to one embodiment of the method for identifying sapindus mukorossi and sapindus mukorossi germplasm of the present invention, in the step 2), the molecular marker is one or more pairs of sequences among the following primers:
Samuk10G0092500：
an upstream primer: 5'-TTCTTCCGATTGAGCGCCAT-3', respectively;
a downstream primer: 5'-CGAATCCAGTGGCAGTAGCA-3', respectively;
Samuk04G0084900：
an upstream primer: 5'-CTAGCTGTGGGGGCACATAC-3', respectively;
a downstream primer: 5'-GCATATTAGCACCGACCGGA-3', respectively;
Samuk07G0006800：
an upstream primer: 5'-GAAGCCGGATCTAATGGGCA-3', respectively;
a downstream primer: 5'-TCACTCCAACAGCCTTGTCC-3', respectively;
Samuk12G0105900：
an upstream primer: 5'-AGGAGATTCAAGTGGTGGCG-3', respectively;
a downstream primer: 5'-GACGACGTACACTGCTCCAT-3', respectively;
Samuk03G0235800：
an upstream primer: 5'-CAGTCCGATCTCAGCAGCAT-3', respectively;
a downstream primer: 5'-TTGCCCTAGGGTGTTCTTGG-3' are provided.
Compared with the prior art, one of the technical solutions has the following advantages:
compared with the traditional morphological, sporopollenic and biochemical identification, the SSR specific primer identification technology has the following obvious advantages:
(1) the identification is accurate, and the germplasm of the soapberry and the soapberry with the hair petals can be effectively judged;
(2) the biological agent can be detected in different growth and development periods and tissues, and is hardly influenced by the environment;
(3) the detection is convenient, only trace leaves or other tissues of seeds are needed for detecting samples, and the detection cost is extremely low;
(4) the SSR molecular marker has high allelic variation, co-dominant inheritance, good stability and simple and quick operation.
The fluorescence labeling SSR capillary electrophoresis detection method established based on the gene sequencer is effective combination of a fluorescence sequencing technology and an SSR molecular labeling technology, realizes high-efficiency and accurate acquisition of the length of an SSR molecular labeling amplification product, can distinguish difference fragments within 2bp, accurately acquires the length of the amplification product, and has more stable, accurate and high-efficiency detection results.
Drawings
FIG. 1 is an electrophoresis picture of Samuk10G0092500 primer amplification of soapberry and Sapindus mukurossi germplasm.
FIG. 2 is a group A SSR primer construction phylogenetic tree.
FIG. 3 is a group B SSR primers to construct phylogenetic trees.
Detailed Description
The following description will be given with reference to specific examples.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the embodiments of the present invention will be described in detail and completely with reference to the accompanying drawings. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Thus, the following detailed description of embodiments of the invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention.
Example 1
In this example, the inventors screened 5 pairs of SSR specific primers through a large number of experimental studies. In the process of identifying the germ plasm of the sapindus mukorossi and the sapindus mukorossi, 1 pair, 2 pairs, 3 pairs, 4 pairs or 5 pairs of the germ plasm can be selected as SSR molecular markers. The 5 pairs of SSR specific primers are shown in a sequence number 1-10 in a table 1 or a sequence table.
Table 1 information of 5 pairs of SSR specific primers obtained by screening
Example 2
The present example is based on the application of the SSR molecular marker described in example 1 in the method for identifying sapindus mukorossi and sapindus mukorossi germplasm.
The steps of identifying the soapberry and the soapberry cotyledon germplasm by utilizing the SSR molecular marker of transcriptome sequencing are as follows.
1) Extracting DNA of a sapindus mukorossi or sapindus mukorossi young leaf sample to be detected;
2) performing PCR amplification according to SSR molecular markers by using the DNA extracted in the step 1) as a template;
the PCR adopts a 20 mu L reaction system: comprises 10-50ng template DNA 1-2 μ L, forward and reverse primers 0.2 μ L and 0.2 μ L, respectively, Rui Boxing Ke 2 × Taq Plus PCR MasterMix (MgCl)2:3 mM; dNTPs (dATP, dCTP, dGTP, dTTP): 0.4mM) 10. mu.L, and an appropriate amount of ddH2O；
The PCR reaction program is: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 30s, and extension at 72 ℃ for 10min after 20 cycles; storing at 4 ℃.
The SSR marker primers are selected from 1, 2 or 3 pairs of the 3 pairs of SSR specific primers described in example 1.
3) Detecting on a capillary electrophoresis machine;
in step 2), taking 0.3 μ L of PCR product, 0.5 μ L of molecular weight internal standard and 9.5 μ L of deionized formamide, mixing, adding into a PCR plate, denaturing at 95 ℃ for 5min, cooling at 4 ℃, centrifuging, and detecting on a 1 × Buffer solution machine (gene analyzer ABI 3730XL DNA analyzer, capillary ABI 96 × 50cm 4331246), wherein the specific parameters are shown in the following table:
TABLE 2 Gene Analyzer parameter settings
4) After electrophoresis is finished, the original data of the samples are analyzed by GeneMarker V2.2.0 software, and the lengths of the amplified fragments of different samples are obtained. Analyzing data by using software PopGen32 to obtain primer polymorphism, assigning alleles obtained by each pair of primers by using Arabic numerals 0/1, establishing a sapindus and sapindus hirsutus germ plasm 0/1 matrix, calculating genetic distance by using Nysts software, and constructing a phylogenetic tree by using a Clustering module SAHN function.
5) Judging the soapberry or the soapberry with the hair lobe according to the result of detecting the germ plasm amplification product.
Example 3
This example further describes in detail the method for identifying Sapindus mukurossi or Sapindus mukurossi using the SSR molecular markers described in example 1, and the specific steps of the method are as follows.
1) 20 parts of DNA of the germ plasm tender leaf samples of Chinese soapberry and soapberry with different provinces (shown in table 3) are extracted by using a high-efficiency plant genome DNA extraction kit (DP320-03) for the Tiangen.
TABLE 3 Sapindus mukorossi and Sapindus mukorossi germplasm information
In this example, two sets of SSR-specific primer controls were set. The experimental group A is 5 pairs of SSR specific primers described in example 1 of the present invention, namely S78, S129, S105, S449 and S38 (sequence information is shown in Table 1), the control group B is other 5 pairs of SSR specific primers randomly extracted from a large amount of experimental data in the process of completing the present invention, namely S704, S36, S63, S140 and S449 (sequence information is shown in sequence No. 11-20 or Table 4 in the sequence list), and the two groups of specific SSR forward and reverse primers are synthesized by Begoni Boxing Biotech Co., Ltd.
TABLE 4 group B SSR-specific primer information
2) Using the DNA extracted in the step 1) as a template, and respectively using the A, B two groups of SSR specific primers to carry out PCR amplification, wherein the model of the PCR instrument is a Bio-rad T100 thermal cycle PCR instrument.
The PCR system adopts a 20 mu L reaction system: comprises 10-50ng template DNA 1-2 μ L, forward and reverse primers 0.2 μ L and 0.2 μ L, respectively, Rui Boxing Ke 2 × Taq Plus PCR MasterMix (MgCl)2:3 mM; dNTPs (dATP, dCTP, dGTP, dTTP): 0.4mM) 10. mu.L, and an appropriate amount of ddH2O；
The PCR reaction program is: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 30s, and extension at 72 ℃ for 10min after 20 cycles; storing at 4 ℃.
3) Detecting on a capillary electrophoresis machine;
in step 2), taking 0.3 μ L of PCR product, 0.5 μ L of molecular weight internal standard and 9.5 μ L of deionized formamide, mixing, adding into a PCR plate, denaturing at 95 ℃ for 5min, cooling at 4 ℃, centrifuging, and detecting on a 1 × Buffer solution machine (gene analyzer ABI 3730XL DNA analyzer, capillary ABI 96 × 50cm 4331246), wherein the specific parameters are shown in the following table:
4) after electrophoresis is finished, the raw data of the two groups of samples are respectively analyzed by GeneMarker V2.2.0 software, and the lengths of amplified fragments of different samples are obtained. Analyzing data by using software PopGen32 to obtain primer polymorphism, assigning alleles obtained by each pair of primers by using Arabic numerals 0/1, establishing a sapindus and sapindus hirsutus germ plasm 0/1 matrix, calculating genetic distance by using Nysts software, and constructing a phylogenetic tree by using a Clustering module SAHN function.
TABLE 5 Sapindus and Sapindus mukurossi germplasm A group primer amplification product lengths
Indicates no bands.
TABLE 6 Sapindus and Sapindus mukurossi germplasm B group primer amplification product lengths
Indicates no bands.
The experimental results are shown in tables 5 and 6, and the differences of the amplification products of the sapindus mukorossi and the sapindus mukorossi germplasm in the two groups of specific SSR primers are obvious. In group A (Table 5), the products of sapindus pinkistrocarpus inds 14-16, 18-20 amplified by S129 specific primers were 180bp homozygote or 183bp and 189bp heterozygote; products amplified by the specific primer of S78 are all 180bp, and are homozygotes; products obtained by amplification of the S105 specific primer are 231bp and 234bp, and are heterozygotes; products obtained by amplification of the S449 specific primer are 168bp and are homozygotes; under the amplification of the S38 specific primer, the length of the soapberry cotyledon germplasm product is different, but is obviously different from that of the soapberry cotyledon. In group B (Table 6), the 5 pairs of SSR specific primer amplification products are different, and the differences among the germplasm are obvious, but the germplasm of the soapberry and the soapberry with the hair lobe cannot be effectively identified. Converting the lengths of amplification products of various germplasms into 0/1 matrixes, calculating a genetic distance matrix in Nysts software, constructing phylogenetic trees by utilizing a Clustering module SAHN function, and dividing the sapindus and the sapindus tomentosa germplasms into two groups at the position with the coefficient of 0.04 in the phylogenetic trees constructed by 5 pairs of SSR specific primers in the group A (shown in figure 2) and the sapindus tomentosa germplasms in the position with the coefficient of 0.04, wherein the upper group comprises ind1, 2, 5, 3, 8, 17, 11, 12, 13, 6, 4, 7, 10 and 9 germplasms which are sapindus, the lower group comprises ind14, 15, 18, 19, 20 and 16 germplasms which are sapindus, and the 5 pairs of SSR specific primers can completely identify the sapindus and the sapindus tomentosa. In group B (figure 3), 5 pairs of SSR specific primers construct a phylogenetic tree, but the germplasm of soapberry and soapberry with a hair flap cannot be effectively identified, and the germplasm of soapberry with a hair flap are mixed together, which indicates that the group B3 pairs of SSR primers cannot effectively identify the germplasm of soapberry and the germplasm of soapberry with a hair flap.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
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FUJIAN YUANHUA FORESTRY BIOTECHNOLOGY Co.,Ltd.
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cagtccgatc tcagcagcat 20
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence (DNA)
<400> 10
ttgccctagg gtgttcttgg 20
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (DNA)
<400> 11
acaactggca agagatcgca 20
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (DNA)
<400> 12
cacacctcca tttggctcct 20
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence (DNA)
<400> 13
gtcacagctc aggtgttcct 20
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence (DNA)
<400> 14
tcgccactcc tttaggcttt 20
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence (DNA)
<400> 15
ttgctttctc gttggcctca 20
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence (DNA)
<400> 16
acagattgtg gttggacgca 20
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence (DNA)
<400> 17
gctacccaca gctcacaagt 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (DNA)
<400> 18
actctgtgag gagggtcaga 20
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence (DNA)
<400> 19
agcaatcacc gggaacaaca 20
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence (DNA)
<400> 20
ccctgagccg agacaacatt 20
Claims (10)
1. A molecular marker for identifying sapindus mukorossi and sapindus mukorossi germplasm, said molecular marker comprising one or more pairs of sequences of the following primers:
Samuk10G0092500：
an upstream primer: 5'-TTCTTCCGATTGAGCGCCAT-3', respectively;
a downstream primer: 5'-CGAATCCAGTGGCAGTAGCA-3', respectively;
Samuk04G0084900：
an upstream primer: 5'-CTAGCTGTGGGGGCACATAC-3', respectively;
a downstream primer: 5'-GCATATTAGCACCGACCGGA-3', respectively;
Samuk07G0006800：
an upstream primer: 5'-GAAGCCGGATCTAATGGGCA-3', respectively;
a downstream primer: 5'-TCACTCCAACAGCCTTGTCC-3', respectively;
Samuk12G0105900：
an upstream primer: 5'-AGGAGATTCAAGTGGTGGCG-3', respectively;
a downstream primer: 5'-GACGACGTACACTGCTCCAT-3', respectively;
Samuk03G0235800：
an upstream primer: 5'-CAGTCCGATCTCAGCAGCAT-3', respectively;
a downstream primer: 5'-TTGCCCTAGGGTGTTCTTGG-3' are provided.
2. Use of a molecular marker according to claim 1 for identifying sapindus mukorossi and sapindus mukorossi germplasm, comprising the steps of:
1) extracting DNA of a sapindus mukorossi or sapindus mukorossi young leaf sample to be detected;
2) performing PCR amplification according to SSR molecular markers by using the DNA extracted in the step 1) as a template;
3) detecting on a capillary electrophoresis machine;
taking 0.3 mu L of PCR product obtained in the step 2), 0.5 mu L of molecular weight internal standard and 9.5 mu L of deionized formamide, mixing, adding into a PCR plate, denaturing at 95 ℃ for 5min, cooling at 4 ℃, centrifuging, and performing on-machine detection on 1 Xbuffer Buffer solution;
4) analyzing the original data of the sample to obtain the lengths of amplified fragments of different samples; further analyzing data to obtain primer polymorphism, assigning alleles obtained by each pair of primers by using Arabic numerals 0/1, establishing a soapberry and hairball soapberry germplasm 0/1 matrix, calculating a genetic distance, and constructing a phylogenetic tree;
5) judging the soapberry or the soapberry with the hair lobe according to the result of detecting the germ plasm amplification product.
3. The use of the molecular marker for identifying sapindus mukorossi and sapindus mukorossi germplasm according to claim 2, wherein in step 2), the molecular marker is one or more of the following primers:
Samuk10G0092500：
an upstream primer: 5'-TTCTTCCGATTGAGCGCCAT-3', respectively;
a downstream primer: 5'-CGAATCCAGTGGCAGTAGCA-3', respectively;
Samuk04G0084900：
an upstream primer: 5'-CTAGCTGTGGGGGCACATAC-3', respectively;
a downstream primer: 5'-GCATATTAGCACCGACCGGA-3', respectively;
Samuk07G0006800：
an upstream primer: 5'-GAAGCCGGATCTAATGGGCA-3', respectively;
a downstream primer: 5'-TCACTCCAACAGCCTTGTCC-3', respectively;
Samuk12G0105900：
an upstream primer: 5'-AGGAGATTCAAGTGGTGGCG-3', respectively;
a downstream primer: 5'-GACGACGTACACTGCTCCAT-3', respectively;
Samuk03G0235800：
an upstream primer: 5'-CAGTCCGATCTCAGCAGCAT-3', respectively;
4. a downstream primer: 5'-TTGCCCTAGGGTGTTCTTGG-3' are provided. The use according to claim 2, wherein, in the step 2),
the PCR amplification adopts a 20 mu L reaction system: comprises 10-50ng template DNA 1-2 μ L, forward and reverse primers 0.2 μ L and 0.2 μ L, respectively, 2 XTaq Plus PCR reaction mixture 10 μ L of Rui Boxing Ke, and appropriate amount of ddH2O；
The PCR reaction program is: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 30s, and extension at 72 ℃ for 10min after 20 cycles; storing at 4 ℃.
5. The use of claim 2, wherein in step 3), the apparatus used in the on-machine detection is a gene analyzer ABI 3730XL DNA analyzer, capillary ABI 96X 50cm 4331246.
6. The use of claim 5, wherein the gene analyzer parameters are set as follows:
module file GeneMapper50_ POP7_ 1;
the operating voltage is 15.0 kv;
the sample injection voltage is 1.6 kv;
the sample injection duration is 15 sec;
the temperature is 63 DegC;
current stability 30.0uA
7. The use according to claim 4, wherein the PCR reaction mixture is MgCl 2: 3 mM; dNTPs: 0.4 mM.
8. The application according to claim 2, wherein the step 4) is specifically: analyzing the original data of the sample by using GeneMarker V2.2.0 software to obtain the lengths of amplified fragments of different samples; analyzing data by using software PopGen32 to obtain primer polymorphism, assigning alleles obtained by each pair of primers by using Arabic numerals 0/1, establishing a sapindus and sapindus hirsutus germ plasm 0/1 matrix, calculating genetic distance by using Nysts software, and constructing a phylogenetic tree by using a Clustering module SAHN function.
9. A method for identifying sapindus mukorossi and sapindus mukorossi germplasm using the molecular marker of claim 1, comprising the steps of:
1) extracting DNA of a sapindus mukorossi or sapindus mukorossi young leaf sample to be detected;
2) performing PCR amplification according to the molecular marker by using the DNA extracted in the step 1) as a template;
the PCR adopts a 20 mu L reaction system: comprises 10-50ng template DNA 1-2 μ L, forward and reverse primers 0.2 μ L and 0.2 μ L, respectively, 2 XTaq Plus PCR reaction mixture 10 μ L of Rui Boxing Ke, and appropriate amount of ddH2O; the reaction mixture is MgCl2：3mM；dNTPs：0.4mM；
The PCR reaction program is: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 30s, and extension at 72 ℃ for 10min after 20 cycles; storing at 4 ℃;
3) detecting on a capillary electrophoresis machine;
taking 0.3 mu L of PCR product in the step 2), 0.5 mu L of molecular weight internal standard and 9.5 mu L of deionized formamide, mixing, adding into a PCR plate, denaturing at 95 ℃ for 5min, cooling at 4 ℃, centrifuging, and performing on-machine detection by using 1 Xbuffer Buffer solution; and (3) loading a detection instrument: gene analyzer ABI 3730XL DNA analyzer, capillary ABI 96X 50cm 4331246;
4) after electrophoresis is finished, the original data of the samples are analyzed by GeneMarker V2.2.0 software, and the lengths of the amplified fragments of different samples are obtained. Analyzing data by using software PopGen32 to obtain primer polymorphism, assigning alleles obtained by each pair of primers by using Arabic numerals 0/1, establishing a soapberry and hairball soapberry germplasm 0/1 matrix, calculating genetic distance by using Nysts software, and constructing a phylogenetic tree by using a Clustering module SAHN function;
5) judging the soapberry or the soapberry with the hair lobe according to the result of detecting the germ plasm amplification product.
10. The method of claim 9, wherein in step 2), the molecular marker is one or more of the following primers:
Samuk10G0092500：
an upstream primer: 5'-TTCTTCCGATTGAGCGCCAT-3', respectively;
a downstream primer: 5'-CGAATCCAGTGGCAGTAGCA-3', respectively;
Samuk04G0084900：
an upstream primer: 5'-CTAGCTGTGGGGGCACATAC-3', respectively;
a downstream primer: 5'-GCATATTAGCACCGACCGGA-3', respectively;
Samuk07G0006800：
an upstream primer: 5'-GAAGCCGGATCTAATGGGCA-3', respectively;
a downstream primer: 5'-TCACTCCAACAGCCTTGTCC-3', respectively;
Samuk12G0105900：
an upstream primer: 5'-AGGAGATTCAAGTGGTGGCG-3', respectively;
a downstream primer: 5'-GACGACGTACACTGCTCCAT-3', respectively;
Samuk03G0235800：
an upstream primer: 5'-CAGTCCGATCTCAGCAGCAT-3', respectively;
a downstream primer: 5'-TTGCCCTAGGGTGTTCTTGG-3' are provided.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913810A (en) * | 2018-09-30 | 2018-11-30 | 福建省农业科学院果树研究所 | The molecular labeling and method of Euphoria and its relative genus plant are distinguished in a kind of identification |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913810A (en) * | 2018-09-30 | 2018-11-30 | 福建省农业科学院果树研究所 | The molecular labeling and method of Euphoria and its relative genus plant are distinguished in a kind of identification |
Non-Patent Citations (2)
| Title |
|---|
| CAOWEN SUN: "Genetic structure and biogeographic divergence among Sapindus species: An inter-simple sequence repeat-based study of germplasms in China？", 《INDUSTRIAL CROPS & PRODUCTS》 * |
| 刘宝: "不同地理种源无患子的分子多态性分析", 《浙江农林大学学报》 * |
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