CN113718050B - Molecular characteristic SSR primer and method for identifying torreya polyploid variety - Google Patents
Molecular characteristic SSR primer and method for identifying torreya polyploid variety Download PDFInfo
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Abstract
The invention relates to a molecular characteristic SSR marker primer for rapidly identifying a torreya polyploid variety and a torreya polyploid identification method. The nucleotide sequence of the molecular characteristic fluorescent SSR primer is as follows: primer tg_u33: the upstream primer Tg_U33F:5'-FAM-TAAGAGGATTGACATGGCCC-3' downstream primer Tg_U33R:5'-TATTCTGGAGTTTGGACCCG-3'; the molecular characteristic fluorescent SSR primer can be used for rapidly identifying the torreya polyploid variety by utilizing needle DNA, has the advantages of simple, rapid, accurate and efficient method, good repeatability of detection results, is not influenced by seasons, plant development periods and growth environments, and provides references and bases for rapid identification of the torreya polyploid variety, new variety protection, polyploid breeding and other researches. Compared with the traditional morphological identification method, nuclear type analysis method and flow cytometry method, the method has obvious advantages, and is an advanced molecular means for identifying the torreya polyploid variety.
Description
Field of the art
The invention relates to a molecular characteristic SSR marker primer for rapidly identifying a torreya polyploid variety, and also relates to application of the primer in torreya polyploid identification.
(II) background art
The Torreya grandis belonging to Torreya (Torreya) of Taxaceae, which is an excellent variety cultivated by grafting and propagating after artificial breeding of excellent variation in Torreya grandis species (Torreya grandis), and is the only variety cultivated in the Torreya grandis at present on an artificial scale. Chinese torreya is a special precious economic tree species in China, and has multiple purposes of eating, medicinal use, material use, greening and the like. The Chinese torreya has a cultivation history of 1300 years so far and is mainly distributed in Zhejiang, jiangsu, anhui, fujian, jiangxi, hunan, hubei, sichuan, yunnan, guizhou and other provinces in the Yangtze river basin. Due to the influence of long and complex historical environment and natural hybridization, numerous variations are generated, and the variety of natural torreya tree resource is evolved. In addition, the grafting propagation of the torreya grandis produces rich mutation types. These mutation types are the fundamental guarantee for developing new varieties and fine variety breeding of torreya grandis. Through the excellent strain breeding and mass propagation tests for many years, forestry breeding workers in China breed a plurality of Chinese torreya excellent varieties including 'Chinese torreya', 'pearl torreya', 'Chinese torreya', 'ivory' and 'Dongbaizhu', etc. The development of Chinese torreya resource evaluation, germplasm protection and fine breed breeding has great significance for promoting the sustainable development of incense frames.
Polyploid breeding is an important means of plant breeding. Polyploid plants contain three or more sets of chromosome sets, are very common in nature, and generally have the characteristics of high growth speed, high biological yield, strong stress resistance and the like. Triploid torreya is ubiquitous in large adult trees and seedlings. These triploid plants may be fused from a haploid chromosome set for males and a diploid chromosome set for females (Chen Keyong, 1990; huang Shaofu et al, 1990). Tetraploids and other polyploids of torreya have not been found so far. The plant polyploid identification method mainly includes plant morphology identification method, chromosome count method, i.e. karyotype analysis, and Flow Cytometry (FCM). Morphological identification is simple and extensive, but the accuracy is not high; the chromosome counting method is a classical and reliable identification method, but has high requirements on the material taking part of plants and the period in which cells are located, and has complex operation process, large workload and low speed; although the flow cytometry method can rapidly identify the chromosome ploidy level of plants, the flow cytometry method has some defects that (1) the sample preparation time is not suitable to exceed 20 minutes, otherwise, the DNA peak position is deviated, so that the measurement result is influenced; (2) different internal standards are used for detection, and errors exist in the result; different nuclear fluorescent dyes are used for detection, and the results are also obviously different; (3) the measurement costs are high. Therefore, a simple, rapid and efficient method for identifying the torreya polyploids is urgently needed to be developed, which is beneficial to rapidly screening polyploid germplasm materials of torreya natural populations and provides powerful technical support for breeding excellent polyploids of torreya. In recent years, identification of polyploids by using an SSR molecular marker has been successfully applied to plants such as poplar (Gu Huixia and the like, 2015), jujube tree (Liuwei and the like, 2018), locust (Mao Xiugong and the like, 2018) and cottonwood (Li Weijiang and the like, 2019), but so far, rapid identification of Chinese torreya ploidy level by using a molecular characteristic fluorescent SSR marker has not been reported.
(III) summary of the invention
The invention aims to provide a molecular characteristic SSR marker primer for rapidly identifying a torreya polyploid variety, and also relates to application of the primer in torreya polyploid variety identification.
The technical scheme adopted by the invention is as follows:
the molecular characteristic fluorescent SSR marker primer for identifying the torreya polyploid variety has the nucleotide sequence as follows:
primer tg_u33:
the upstream primer Tg_U33F: FAM-TAAGAGGATTGACATGGCCC-3'
Downstream primer tg_u33r:5'-TATTCTGGAGTTTGGACCCG-3';
the molecular characteristic primer Tg_U33 is obtained by screening a large number of EST-SSR primers which are independently developed among the Chinese torreya polyploid varieties to be detected based on a fluorescence SSR marking technology, has high amplification efficiency, good stability and high heterozygosity, can accurately identify whether the Chinese torreya varieties are polyploid, and can identify whether the Chinese torreya varieties are polyploid only by amplifying more than 3 (including 3) alleles by using the primer. The primer modified by the fluorescent group FAM is used for detecting 9 Chinese torreya varieties (8 Chinese torreya varieties are triploid and 1 Chinese torreya variety is diploid through identification by a flow cytometry method), and the detection rate of the polyploid is up to 100%, so that Tg_U33 is a molecular characteristic primer for rapidly identifying Chinese torreya polyploid varieties.
The invention also relates to a method for identifying the torreya polyploid variety, which comprises the following steps:
extracting genomic DNA of a torreya variety needle leaf to be detected as a template, carrying out PCR amplification by taking a molecular characteristic fluorescent SSR primer pair Tg_U33F and Tg_U33R as amplification primers, and carrying out capillary electrophoresis detection on an amplification product, wherein if the number of allele factors detected on a capillary electrophoresis peak diagram is more than or equal to 3, the plant can be confirmed to be polyploidy.
The nucleotide sequence of the molecular characteristic fluorescent SSR primer is as follows:
primer tg_u33:
the upstream primer Tg_U33F:5'-FAM-TAAGAGGATTGACATGGCCC-3'
Downstream primer tg_u33r:5'-TATTCTGGAGTTTGGACCCG-3';
preferably, the PCR amplification reaction conditions are as follows: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 55℃for 15s, extension at 72℃for 15s for a total of 35 cycles; and finally, filling in at 72 ℃ for 2min, wherein the termination temperature is 4 ℃.
Preferably, the fluorescence capillary electrophoresis detection method comprises the following steps: diluting PCR amplified product, denaturing at 96 deg.C for 5min, rapidly freezing the denatured diluted product at-20deg.C for 2min, and adding ABI3730XL Genetic Analyzer (ABI, CA, USA) and internal standard GeneScan TM -500LIZ Size Standard (ABI) capillary electrophoresis detection was performed simultaneously and Data was collected using Data Collection 3.0 software (ABI).
Specifically, the method comprises the following steps:
(1) Taking tender needles of a torreya variety to be detected, adding nitrogen, grinding, and extracting genomic DNA of the torreya;
(2) Taking the genomic DNA extracted in the step (1) as a template, and taking the molecular characteristic SSR primer as an amplification primer for PCR amplification:
every 20 mu L of PCR reaction system consists of the following components:
the PCR conditions were as follows:
pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 55℃for 15s, extension at 72℃for 15s for a total of 35 cycles; finally, filling the mixture into a flat state for 2min at 72 ℃ and keeping the termination temperature at 4 ℃;
(1) And (3) preparing an internal standard: 10ml Hi-Di and 80 mu LGene scan were taken TM Mixing-500 LIZ SizeStandard, centrifuging, packaging into 96-well internal standard plate with 10 μl per well, and centrifuging; diluting and centrifuging PCR amplification products; adding the diluted product into a 96-well internal standard plate which is distributed according to 0.5 mu L/well, uniformly mixing, centrifuging, placing into a PCR instrument, denaturing for 5min at 96 ℃, then rapidly freezing for 2min at-20 ℃, centrifuging to obtain a denatured PCR product, synchronously placing the denatured PCR product and an internal standard into ABI3730 XLGenetic Analyzer for capillary electrophoresis detection, and collecting Data by using Data Collection 3.0 software;
(2) Data analysis: raw Data collected by Data Collection 3.0 software was analyzed by GeneMapper 4.1 software (ABI), the software system will be based on the position of the target peak and the internal standard GeneScan in the same lane TM -500LIZ Size Standard, directly reading out the accurate peak value (bp number) of the target SSR fragment, wherein the allelic variation data of the homozygous site is recorded as X/X, and the allelic variation data of the heterozygous site is recorded as X/Y or X/Y/Z/;
(3) And (3) judging results: if the allele factors detected on the capillary electrophoresis peak diagram are more than or equal to 3, the torreya variety to be detected is polyploid, otherwise, the torreya variety to be detected is not polyploid.
The beneficial effects of the invention are mainly as follows: the molecular characteristic fluorescent SSR primer can be used for rapidly identifying the torreya polyploid variety by utilizing needle DNA, has the advantages of simple, rapid, accurate and efficient method, good repeatability of detection results, is not influenced by seasons, plant development periods and growth environments, and provides references and bases for rapid identification of the torreya polyploid variety, new variety protection, polyploid breeding and other researches. Compared with the traditional morphological identification method, nuclear type analysis method and flow cytometry method, the method has obvious advantages, and is an advanced molecular means for identifying the torreya polyploid variety.
(IV) description of the drawings
FIG. 1 shows the detection results of the flow cytometry and the fluorescence capillary electrophoresis for the identification of 9 Chinese torreya varieties, wherein A1-I1 are the detection results of the flow cytometry, and A2-I2 are the detection results of the fluorescence capillary electrophoresis; the Chinese medicinal composition is prepared from (A) Yushan fruit Chinese torreya (from Shang Huzhen Huang Linkeng village), B Dafeng Chinese torreya (from deep Chuan village under village and under Xanthan mountain), C Dafeng Chinese torreya (from deep Chuan village under mountain rear mountain), D Dafeng Chinese lilac (from An Wen street Wang Yinkeng village), E Dafeng Chinese torreya (from Shang Hu Zhen plate Chinese torreya village), F Dafeng Chinese lilac (from Dashi Zhenhan village), G Dachang Chinese torreya (from An Wen mountain community dock), H Dachang Chinese torreya (from An Wen street cloud mountain community dock), and I Dafeng Chinese lilac (from Shang Hu forward yellow rock village).
(fifth) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
example 1:
(1) Extraction of genomic DNA of torreya grandis varieties:
taking 0.03g of young leaves preserved by 9 Chinese torreya varieties (identified by a flow cytometry method, wherein 8 Chinese torreya varieties are triploid and 1 Chinese torreya varieties are diploid), adding liquid nitrogen, thoroughly grinding, and extracting genome DNA by using a novel rapid plant genome DNA extraction box (DP 3111, beijing baitaike) so as to extract and obtain a crude genome DNA extract of the Chinese torreya varieties.
The crude DNA extract was checked for integrity, purity and concentration by 1.5% agarose gel electrophoresis and DNA/RNA uv spectrophotometry (Nanodrop Technologies, USA). OD (optical density) 260 /OD 280 >1.8 for subsequent PCR amplification. The DNA extract is stored in a refrigerator at-20 ℃ for standby.
(2) Molecular characteristic SSR primers, the sequences of the primer pairs are as follows:
primer tg_u33:
the upstream primer Tg_U33F:5'-FAM-TAAGAGGATTGACATGGCCC-3'
Downstream primer tg_u33r:5'-TATTCTGGAGTTTGGACCCG-3';
synthesized by Hangzhou Kangshen Biotechnology Co.
(3) The PCR amplification, 20. Mu.L PCR reaction system consisted of:
| reaction components | Volume (mu L) |
| 2×T5 Super PCR Mix(PAGE) | 10.0 |
| Tg_U33F(10μM) | 1.5 |
| Tg_U33R(10μM) | 1.5 |
| 20 ng/. Mu.L template DNA | 3.0 |
| ddH 2 O | 4.0 |
The amplification reaction was performed on a Life ECO type amplification apparatus (Bioer, hangzhou Bosch technology). Amplification conditions: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 55℃for 15s, extension at 72℃for 15s for a total of 35 cycles; and finally, filling in at 72 ℃ for 2min, wherein the termination temperature is 4 ℃.
(4) And (3) preparing an internal standard: 10ml Hi-Di and 80. Mu.L GeneScan were taken TM Mixing-500 and LIZ Size Standard, centrifuging, and packaging into 96-well internal standard plate with 10 μl per well, and centrifuging.
(5) Fluorescent capillary electrophoresis detection: taking 5 mu L of the PCR amplification product in the step (3), diluting 100 times, taking 0.5 mu L/hole, adding into a distributed 96-hole internal standard plate, uniformly mixing, centrifuging, putting into a PCR instrument, denaturing for 5min at 96 ℃, quickly freezing for 2min at-20 ℃, and centrifuging. The denatured PCR products and internal standard GeneScan were used TM -500LIZ Size Standard was simultaneously placed into ABI3730XL Genetic Analyzer for capillary electrophoresis detection and Data were collected using Data Collection 3.0 software.
(6) Genotype analysis and variety identification: raw Data collected by Data Collection 3.0 software was analyzed using GeneMapper 4.1 software. The software system will be based on the location of the target peak and the internal standard GeneScan in the same lane TM -500LIZ Size Standard to directly read out the exact peak (bp) of the SSR fragment of interest. Allelic variation data for homozygous sites are recorded as X/X, allelic variation data for heterozygous sites are recorded as X/Y or X/Y/Z/(polyploid). And judging whether the torreya grandis variety is polyploid according to the number of amplified allelic variation (alleles). Genotype data of the 9 torreya varieties are shown in table 1.
Table 1: genotype of nine Chinese torreya varieties
Of the 9 Torreya grandis varieties shown in Table 1, 8 Torreya grandis varieties numbered from A to H have genotypes of 204/207/225, and 8 Torreya grandis varieties numbered I have genotypes of 207/225. Indicating that the varieties numbered A through H have 3 alleles and are triploid; the variety numbered I has 2 alleles and is diploid, which is fully consistent with the ploidy levels identified by flow cytometry. The method shows that the U33 locus of the Chinese torreya variety is highly heterozygous, tg_U33 can be used as a molecular characteristic SSR primer to identify the ploidy level of the Chinese torreya variety, and the method is an advanced molecular means for identifying the Chinese torreya polyploid variety.
Sequence listing
<110> Zhejiang province forestry science institute
Strong natural resource and planning bureau
Strong and safe county traditional Chinese medicine industry development promotion center
<120> molecular characteristic SSR primer and method for identifying torreya polyploid variety
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 2
Claims (4)
1. A method of identifying a torreya polyploid variety, the method comprising: extracting genomic DNA of a torreya variety needle leaf to be detected as a template, taking a molecular characteristic fluorescent SSR primer pair Tg_U33F and Tg_U33R as amplification primers, carrying out PCR amplification, carrying out capillary electrophoresis detection on an amplification product, and if the genotype detected on a capillary electrophoresis peak diagram is 204/207/225, confirming that the plant is triploid, otherwise, confirming that the plant is not triploid;
the nucleotide sequence of the molecular characteristic fluorescent SSR primer is as follows:
primer tg_u33:
the upstream primer Tg_U33F:5'-FAM-TAAGAGGATTGACATGGCCC-3'
Downstream primer tg_u33r:5'-TATTCTGGAGTTTGGACCCG-3'.
2. The method of claim 1, wherein the PCR amplification reaction conditions are as follows: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 55℃for 15s, extension at 72℃for 15s for a total of 35 cycles; and finally, filling in at 72 ℃ for 2min, wherein the termination temperature is 4 ℃.
3. The method of claim 1, wherein the fluorescent capillary electrophoresis detection method is as follows: diluting PCR amplified product, denaturing at 96 deg.C for 5min, rapidly freezing the denatured diluted product at-20deg.C for 2min, adding ABI3730XL Genetic Analyzer, and adding internal standard GeneScan TM -500LIZ Size Standard simultaneous capillary electrophoresis detection and Data Collection with Data Collection 3.0 software.
4. The method according to claim 1, characterized in that the method is as follows:
(1) Taking tender needles of a torreya variety to be detected, adding nitrogen, grinding, and extracting genomic DNA of the torreya;
(2) Taking the genomic DNA extracted in the step (1) as a template, and taking the molecular characteristic SSR primer as an amplification primer for PCR amplification:
the PCR reaction system consisted of, per 20. Mu.L:
2×T5 Super PCR Mix 10.0 μL
10μM Tg_U33F 1.5 μL
10μM Tg_U33R 1.5 μL
20 ng/. Mu.L template DNA 3.0. Mu.L
ddH 2 O 4.0 μL
The PCR conditions were as follows:
pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 55℃for 15s, extension at 72℃for 15s for a total of 35 cycles; finally, filling the mixture into a flat state for 2min at 72 ℃ and keeping the termination temperature at 4 ℃;
（3）10ml Hi-Di and 80 mu LGene scan were taken TM Mixing-500 and LIZ Size Standard, centrifuging, and sub-packaging 10 mu L of the mixture in 96-well internal standard plates, and centrifuging; diluting and centrifuging PCR amplification products; adding the diluted product into a 96-well internal standard plate which is distributed according to 0.5 mu L/well, uniformly mixing, centrifuging, placing into a PCR instrument, denaturing for 5min at 96 ℃, then rapidly freezing for 2min at-20 ℃, centrifuging to obtain a denatured PCR product, synchronously placing the denatured PCR product and an internal standard into ABI3730XL Genetic Analyzer for capillary electrophoresis detection, and collecting Data by using Data Collection 3.0 software;
(4) Data analysis: raw Data collected by Data Collection 3.0 software was analyzed by GeneMapper 4.1 software, the software system will be based on the position of the target peak and the internal standard GeneScan in the same lane TM -500LIZ Size Standard, directly reading out the accurate peak value of the target SSR fragment, wherein the allelic variation data of the homozygous site is recorded as X/X, and the allelic variation data of the heterozygous site is recorded as X/Y or X/Y/Z;
(5) And (3) judging results: if the genotype detected on the capillary electrophoresis peak diagram is 204/207/225, the torreya variety to be detected is triploid, otherwise, the torreya variety to be detected is not triploid.
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