CN113846176A

CN113846176A - Multi-labeled primer for identifying variety of torreya grandis, cephalospora grandis and cephalotaxus grandis

Info

Publication number: CN113846176A
Application number: CN202110846243.7A
Authority: CN
Inventors: 张苏炯; 陈红星; 李海波; 沈建军; 叶碧欢; 宋其岩; 陈友吾; 胡传久
Original assignee: Natural Resources And Planning Bureau Of Pan'an County; Pan'an Traditional Chinese Medicine Industry Development Promotion Center; Zhejiang Academy of Forestry
Current assignee: Natural Resources And Planning Bureau Of Pan'an County; Pan'an Traditional Chinese Medicine Industry Development Promotion Center; Zhejiang Academy of Forestry
Priority date: 2021-07-26
Filing date: 2021-07-26
Publication date: 2021-12-28

Abstract

The invention relates to a molecular characteristic multiple fluorescence SSR (simple sequence repeat) labeled primer and a method for rapidly identifying new varieties of torreya grandis, namely Yushan torreya grandis, Syringa oblata and Panti torreya grandis. The molecular characteristic multiple fluorescence SSR marker primer has the following nucleotide sequence: primer U7: upstream primer Tg _ U7F: 5 '-FAM-CGCCTCTAACCATGTCCACT-3' downstream primer Tg _ U7R: 5'-AAATTCCAGGGAAACAAAAGAA-3', respectively; primer U734: upstream primer Tg _ U734F: 5 '-HEX-CTATGAGCGGTGGAACCCTA-3' downstream primer Tg _ U734R: 5'-GTGGGAGAAATGCCACAACT-3' are provided. The molecular characteristic multiple fluorescence SSR marker primer can utilize coniferous DNA to rapidly and early identify the Chinese torreya new species Yushan Chinese torreya, big clove and Panti torreya grandis simultaneously, the method is simple, rapid and accurate, and is a molecular means which cannot be replaced by the traditional method for identifying the Chinese torreya species according to the morphological characteristics of fruits.

Description

Multi-labeled primer for identifying variety of torreya grandis, cephalospora grandis and cephalotaxus grandis

(I) technical field

The invention relates to a molecular characteristic multiple fluorescence SSR (simple sequence repeat) labeled primer and a method for rapidly identifying variety Yushan fish torreya, cloves and Panti torreya grandis.

(II) background of the invention

Torreya grandis belongs to Taxaceae (Taxaceae) Torreya genus (Torreya) and is a good variety of Torreya grandis species (Torreya grandis) which is cultivated by grafting after artificial breeding of good variation and is also the only variety cultivated in the current Torreya grandis on a manual scale. Chinese torreya is a unique precious economic tree species in China and has multiple purposes of eating, medicinal use, material use, greening and the like.

Torreya grandis has been cultivated for over 1300 years to date, and is mainly distributed in 10 provinces of Zhejiang, Jiangsu, Anhui, Fujian, Jiangxi, Hunan, Hubei, Sichuan, Yunnan, Guizhou and the like in the south of Yangtze river basin. The seedling grafting propagation of Chinese torreya generates rich variation types, and through years of excellent plant breeding and mass propagation tests, forestry breeding workers in Zhejiang province breed a plurality of Chinese torreya varieties. In 2011, the improved variety of the cephalotaxus (t. grandis 'Xifei') is approved by the forest variety approval committee of the national forestry agency. In recent years, new varieties of 'pearl torreya', 'changchun torreya', 'elephant torreya', 'east white bead', 'crisp kernel torreya', 'Zhuyan torreya', 'butyl torreya', 'Torreya No. 1', 'Torreya No. 2', 'Torreya No. 3', 'big leaf seed torreya', 'Pan an long torreya', 'Yushan torreya' and 'Qian torreya' were examined (acknowledged) in Zhejiang province. Along with the continuous breeding of new varieties of torreya and the development of torreya industry, the identification and intellectual property protection of the new varieties also urgently need to follow up by corresponding molecular technical means.

The identification of Chinese torreya varieties is always based on the traditional morphological classification, namely, the Chinese torreya varieties are classified into round-seed types (large, medium and small round torreya, etc.) and long-seed types (cephalotaxus sinensis, sesamotorreya grandis, Chinese torreya, solanaceae torreya, cephalotaxus fortunei, cephalotaxus turczii, etc.) according to the characters of Chinese torreya fruits or seeds in full bearing period. The identification method needs to identify seeds after fruits mature in one year, and early identification cannot be carried out. Since 2000, some PCR-based molecular marker technologies such as RAPD (random amplified polymorphic DNA), ISSR (simple sequence repeat amplified polymorphism) and SRAP (related sequence amplified polymorphism) have been used in succession for torreya variety classification, genetic variation and population genetic diversity analysis.

However, the molecular markers all adopt universal primers, and the PCR amplification map is not only complex and poor in repeatability, but also low in specificity, and needs a lot of screening work, so that the molecular markers are not suitable for variety identification. SSR (simple repeat sequence; or Microsatelite, Microsatellite) is a codominant marker, can distinguish homozygote and heterozygote, can detect multiple alleles, and has the advantages of rich polymorphism, simple operation, reliable result, good repeatability and the like. In recent years, SSR markers are reported to be developed based on torreya grandis transcriptome sequencing, but the usability of the SSR markers is based on traditional polyacrylamide gel electrophoresis, the artificial interpretation error of a band is large, and the difference of alleles on SSR loci on a plurality of bases is difficult to accurately distinguish; moreover, polymorphism screening work is not carried out on the SSR markers, so that whether the SSR markers can be used for accurately and quickly identifying Chinese torreya varieties is unknown.

The development of fluorescent SSR markers is a capillary electrophoresis detection method based on a DNA sequencing platform, wherein fluorescent groups (such as FAM, HEX, TAMRA and the like) with different colors are used for marking the tail end of one primer in a pair of SSR primers, and a fluorescence detector is used for detecting products, so that the size of an amplification product is automatically identified, the defects of polyacrylamide gel electrophoresis detection by the traditional silver staining method are overcome, and the method has the technical advantages of rapidness, high efficiency, accuracy, sensitivity and the like. The fluorescent multiple SSR labeling technology further combines the advantages of the multiple PCR (multiplex PCR) and the fluorescent SSR labeling technology, a plurality of pairs of SSR primer combinations can be added into a PCR reaction system, and a plurality of genotypes can be detected simultaneously, so that the defects of time and labor waste and high cost in large-batch and multi-batch detection are overcome.

Therefore, the multiple fluorescent SSR marking technology is utilized to develop the characteristic SSR fingerprints (genotypes) of new varieties or excellent and special varieties of the Chinese torreya, reveal the genotype difference among the varieties and have important significance for the identification of the varieties and the protection of intellectual property rights. However, so far, there is no report of utilizing multiple fluorescent SSR marking technology to develop characteristic SSR fingerprints to identify Chinese torreya varieties at home and abroad.

Disclosure of the invention

The invention aims to provide a molecular characteristic multiple fluorescence SSR marker primer and a method for rapidly identifying variety Yushan grand torreya, large clove and Pan grand torreya.

The technical scheme adopted by the invention is as follows:

2 pairs of molecular characteristic multiple fluorescence SSR primers for identifying variety Torreya grandis, Toxicodendron giganteum and Torreya grandis have the following nucleotide sequences:

primer U7:

upstream primer Tg _ U7F: 5 '-FAM-CGCCTCTAACCATGTCCACT-3'

Downstream primer Tg _ U7R: 5'-AAATTCCAGGGAAACAAAAGAA-3'

Primer U734:

upstream primer Tg _ U734F: 5 '-HEX-CTATGAGCGGTGGAACCCTA-3'

Downstream primer Tg _ U734R: 5'-GTGGGAGAAATGCCACAACT-3' are provided.

The primer pair combination is obtained by carrying out polymorphism screening test on a large number of self-developed SSR primers among Chinese torreya varieties to be tested based on a fluorescent SSR marking technology. The multiple fluorescent SSR primers are used for detecting 9 Chinese torreya varieties, 3 different SSR characteristic fingerprints (genotypes) can be stably obtained for 3 Chinese torreya new varieties (Yushan Chinese torreya, big clove and Pangtai torreya), and other Chinese torreya varieties do not have the 3 characteristics, so that the primer group can be used for rapidly identifying the Chinese torreya varieties Yushan Chinese torreya, big clove and Pangtai torreya. It should be noted that the molecular-characteristic multiple-fluorescence SSR primers are only limited to identification of Chinese torreya varieties (whether the Chinese torreya is Yushan Chinese torreya, cloves and Panti torreya), namely, samples to be detected are only limited to Chinese torreya varieties.

The invention also relates to a method for identifying 3 new varieties of Chinese torreya, which comprises the following steps: extracting genome DNA of needle leaves of a Chinese torreya variety to be detected as a template, performing multiplex PCR amplification by using molecular characteristic multiplex fluorescent SSR primer pairs Tg _ U7F/Tg _ U7R and Tg _ U734F/Tg _ U734R as amplification primers, and performing capillary electrophoresis detection on an SSR amplification product, wherein if genotypes displayed on a fluorescence capillary electrophoresis peak picture are 289/295 and 192/222, the Chinese torreya variety to be detected is Yushan Chinese torreya; if the genotypes displayed on the fluorescence capillary electrophoresis peak images are 277/289/295 and 192/216, the Chinese torreya variety to be detected is the large clove; if the genotypes displayed on the fluorescence capillary electrophoresis peak images are 277/295 and 192/216, the Chinese torreya variety to be detected is Pan Chinese torreya, otherwise, the Chinese torreya variety to be detected is not Pan Chinese torreya;

the molecular characteristic multiple fluorescence SSR primer has the following nucleotide sequence:

primer U7:

upstream primer Tg _ U7F: 5 '-FAM-CGCCTCTAACCATGTCCACT-3'

Downstream primer Tg _ U7R: 5'-AAATTCCAGGGAAACAAAAGAA-3'

Primer U734:

upstream primer Tg _ U734F: 5 '-HEX-CTATGAGCGGTGGAACCCTA-3'

Downstream primer Tg _ U734R: 5'-GTGGGAGAAATGCCACAACT-3' are provided.

The method is obtained by optimizing an amplification system of the multiplex fluorescence SSR primer, and comprises the design of 2 pairs of primer sequences marked by U7 and U734, the optimization of the optimal annealing temperature, the proportion of DNA template and primer concentration, the stability test of the system and the like.

Preferably, the multiplex PCR amplification reaction conditions are as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 57.8 ℃ for 15s, and extension at 72 ℃ for 15s for 35 cycles; finally, the temperature is leveled off at 72 ℃ for 2min, and the termination temperature is 4 ℃.

Preferably, the fluorescence capillary electrophoresis detection method is as follows: diluting PCR amplification product, denaturing at 96 deg.C for 5min, quickly freezing at-20 deg.C for 2min, adding ABI 3730XL Genetic Analyzer (ABI, CA, USA), and internal standard GeneScan^TMCapillary electrophoresis detection was performed simultaneously with 500LIZ Size Standard (ABI), and Data Collection 3.0 software (ABI) was used to collect Data.

Specifically, the method comprises the following steps:

(1) taking young and tender coniferous leaves of the Chinese torreya variety to be detected, adding liquid nitrogen and grinding, and extracting genome DNA of the Chinese torreya;

(2) performing multiplex PCR amplification by taking the genomic DNA extracted in the step (1) as a template and the molecular characteristic SSR primer as an amplification primer:

the composition of each 25. mu.L of the PCR reaction system was as follows:

reaction components	Volume (μ L)
		2×T5 Super PCR Mix(PAGE)	12.5
Tg_U7F(10μM)	0.45
		Tg_U7R(10μM)	0.5
Tg_U734F(10μM)	0.5
		Tg_U734R(10μM)	0.55
Template DNA (30 ng/. mu.L)	3.0
		ddH₂O	7.5

The multiplex PCR reaction conditions were as follows:

pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 57.8 ℃ for 15s, and extension at 72 ℃ for 15s for 35 cycles; finally, filling for 2min at 72 ℃, wherein the termination temperature is 4 ℃;

(3) preparing an internal standard: 10ml of Hi-Di and 80 mu of LGeneScan are taken^TM-500LIZ Size Standard, centrifuged, aliquoted 10 μ L per well into a 96 well Standard plate, centrifuged; diluting and centrifuging the PCR amplification product; adding 0.5 mu L/well of the diluted product into a well-distributed 96-well plate, uniformly mixing, centrifuging, placing into a PCR instrument, performing denaturation at 96 ℃ for 5min, then rapidly freezing at-20 ℃ for 2min, and centrifuging to obtain a denatured PCR product; synchronously placing the denatured PCR product and an internal standard into an ABI 3730XL Genetic Analyzer for capillary electrophoresis detection, and collecting Data by using Data Collection 3.0 software;

(4) and (3) data analysis: the raw Data collected by the Data Collection 3.0 software was analyzed by GeneMapper 4.1 software (ABI), and the software system would compare the internal standard GeneScan in the same lane with the position of the target peak^TMComparing with 500LIZ Size Standard, directly reading the accurate peak value (bp number) of the target SSR fragment, recording the allelic variation data of homozygous locus as X/X, and recording the allelic variation data of heterozygous locus as X/Y or X/Y/Z/(polyploid);

(5) and (5) judging a result: if the genotypes displayed on the fluorescence capillary electrophoresis peak images are 289/295 and 192/222, the to-be-detected Chinese torreya variety is Yushan Chinese torreya; if the genotypes displayed on the fluorescence capillary electrophoresis peak images are 277/289/295 and 192/216, the Chinese torreya variety to be detected is the large clove; if the genotypes displayed on the fluorescence capillary electrophoresis peak images are 277/295 and 192/216, the Chinese torreya variety to be detected is Pan Chinese torreya, otherwise, the Chinese torreya variety to be detected is not Pan Chinese torreya.

The invention has the following beneficial effects: the molecular characteristic multiple fluorescence SSR primer can utilize coniferous DNA to rapidly and early identify the Chinese torreya new species, namely, Yushan Chinese torreya, Syringa oblata and Panti torreya grandis at the same time, and the method is simple, rapid and accurate, and is a molecular means which cannot be replaced by the traditional method, namely identifying the Chinese torreya species according to the morphological characteristics of fruits.

(IV) description of the drawings

FIG. 1 shows the result of performing fluorescence capillary electrophoresis detection on the DNA of 9 varieties of Chinese torreya after multiple PCR amplification; a is Torreya grandis (Yushan Torreya grandis) (from Torpan county lake town dry village), B is Torreya grandis (from Torpan county lake town lake mouth village), C is Torpan Torreya grandis (from Torpan county large disc Zhengchang village), D is Torreya grandis (from Torpan county Shang lake town lake mouth village), E is Ganggui (from Torpan county large disc Zhengchang village), F is Torreya grandis (from Pan county lake Zhenghuangyan village), G is Torreya grandis (from Pan county Shang lake dry village), H is Torreya grandis (from Pan county lake green dry village), and I is Torreya grandis (from Pan county lake town Shandong village).

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1:

(1) extracting the genome DNA of the Chinese torreya variety:

taking 0.03g of young leaves preserved by silica gel of 9 Chinese torreya varieties to be detected, adding liquid nitrogen to thoroughly grind, and extracting the genome DNA by using a novel rapid plant genome DNA extraction box (DP3111, Beijing Baitaike) to extract and obtain a crude extract of the genome DNA of the Chinese torreya varieties.

Crude DNA extracts were checked for integrity, purity and concentration by 1.5% agarose gel electrophoresis and DNA/RNA UV spectrophotometer (Nanodrop Technologies, USA). OD₂₆₀/OD₂₈₀>1.8 DNA samples were used for subsequent PCR amplification. The DNA extract was stored at-20 ℃ in a refrigerator for further use.

(2) The sequences of molecular characteristic SSR primers and primer pairs are as follows:

synthesized by Hangzhou Youkang Biotechnology Co.

(3) Multiplex PCR amplification, 25. mu.L PCR reaction system consisted of:

The amplification reaction was performed on a Life ECO type amplification apparatus (Bioer, Hangzhou Bori technology). Amplification conditions: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 57.8 ℃ for 15s, and extension at 72 ℃ for 15s for 35 cycles; finally, the temperature is leveled off at 72 ℃ for 2min, and the termination temperature is 4 ℃.

(4) Preparing an internal standard: 10ml of Hi-Di and 80 mu L of GeneScan are taken^TM-500LIZ Size Standard, centrifuged, aliquoted 10. mu.L per well into 96 well plates, and centrifuged.

(5) And (3) detecting by fluorescence capillary electrophoresis: and (3) taking 5 mu L of PCR amplification product in the step (3), diluting by 100 times, adding 0.5 mu L of PCR amplification product into the well-distributed internal standard plate, uniformly mixing, centrifuging, placing into a PCR instrument, performing denaturation at 96 ℃ for 5min, then rapidly freezing at-20 ℃ for 2min, and centrifuging. Mixing the denatured PCR product with an internal standard GeneScan^TMThe-500 LIZ Size Standard was synchronously placed in ABI 3730XL Genetic Analyzer for capillary electrophoresis detection, and Data Collection 3.0 software was used to collect Data.

(6) Genotype analysis and variety identification: the raw Data collected by the Data Collection 3.0 software was analyzed by GeneMapper 4.1 software. The software system will compare the position of the target peak with the internal standard GeneScan in the same lane^TMAnd (3) comparing the SSR fragments with-500 LIZ Size Standard, and directly reading the accurate peak value (bp) of the target SSR fragment. The allelic variation data of the homozygous loci are recorded as X/X, the allelic variation data of the heterozygous loci are recorded as X/Y or X/Y/Z/(polyploidy), and different Chinese torreya varieties can be identified according to the difference comparison of the allelic variation data. The genotype data of the above 9 varieties of torreya grandis are shown in table 1.

Table 1: genotypes of nine Chinese torreya varieties

Among the 9 varieties of torreya grandis shown in table 1, the genotypes of yushan torreya grandis, syzygium grandis and grandma torreya were 289/295 and 192/222, 277/289/295 and 192/216, and 277/295 and 192/216, respectively, apparently different from those of the other 6 varieties of 277/295 and 192/222, and the genotypes among these 3 new varieties were also different from each other. This shows that U7 and U734 are SSR markers with high polymorphism among torreya grandis varieties, and Tg _ U7 and Tg _ U734 can be used as molecular characteristic SSR primers to identify 3 torreya grandis new varieties at one time, and are powerful tools for variety identification and intellectual property protection.

Sequence listing

<110> development promotion center of traditional Chinese medicine industry in Pan-an county

Natural resources and planning bureau in Pan-an county

Zhejiang Academy of Forestry

<120> multiple-labeled primers for identifying variety of torreya grandis, grandma caryophyllata and grandma torreya grandis

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Unknown (Unknown)

<400> 1

cgcctctaac catgtccact 20

<210> 2

<211> 22

<212> DNA

<213> Unknown (Unknown)

<400> 2

aaattccagg gaaacaaaag aa 22

<210> 3

<211> 20

<212> DNA

<213> Unknown (Unknown)

<400> 3

ctatgagcgg tggaacccta 20

<210> 4

<211> 20

<212> DNA

<213> Unknown (Unknown)

<400> 4

gtgggagaaa tgccacaact 20

Claims

1. The molecular characteristic multiple fluorescence SSR labeled primers for identifying the variety of torreya grandis, grandma caryophyllata and grandma torreya grandis have the following nucleotide sequences:

primer U7:

upstream primer Tg _ U7F: 5 '-FAM-CGCCTCTAACCATGTCCACT-3'

Downstream primer Tg _ U7R: 5'-AAATTCCAGGGAAACAAAAGAA-3', respectively;

primer U734:

upstream primer Tg _ U734F: 5 '-HEX-CTATGAGCGGTGGAACCCTA-3'

Downstream primer Tg _ U734R: 5'-GTGGGAGAAATGCCACAACT-3' are provided.

2. A method for rapidly identifying Yushan fish torreya, lilac and Panti torreya of new varieties of torreya grandis, comprising: extracting genome DNA of needle leaves of the Chinese torreya variety to be detected as a template, taking a molecular characteristic multiple fluorescence SSR primer as an amplification primer, performing multiple PCR amplification, and performing capillary electrophoresis detection on an amplification product, wherein if the genotypes displayed on a fluorescence capillary electrophoresis peak diagram are 289/295 and 192/222, the Chinese torreya variety to be detected is the Yushan torreya grandis; if the genotypes displayed on the fluorescence capillary electrophoresis peak images are 277/289/295 and 192/216, the Chinese torreya variety to be detected is the large clove; if the genotypes displayed on the fluorescence capillary electrophoresis peak images are 277/295 and 192/216, the Chinese torreya variety to be detected is Pan Chinese torreya, otherwise, the Chinese torreya variety to be detected is not Pan Chinese torreya;

primer U7:

upstream primer Tg _ U7F: 5 '-FAM-CGCCTCTAACCATGTCCACT-3'

Downstream primer Tg _ U7R: 5'-AAATTCCAGGGAAACAAAAGAA-3', respectively;

primer U734:

upstream primer Tg _ U734F: 5 '-HEX-CTATGAGCGGTGGAACCCTA-3'

Downstream primer Tg _ U734R: 5'-GTGGGAGAAATGCCACAACT-3' are provided.

3. The method of claim 2, wherein the multiplex PCR amplification reaction conditions are as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 57.8 ℃ for 15s, and extension at 72 ℃ for 15s for 35 cycles; finally, the temperature is leveled off at 72 ℃ for 2min, and the termination temperature is 4 ℃.

4. The method of claim 2, wherein the fluorescence capillary electrophoresis detection method comprises: diluting PCR amplification product, denaturing at 96 deg.C for 5min, quickly freezing at-20 deg.C for 2min, placing into ABI 3730XL Genetic Analyzer, and mixing with internal standard GeneScan^TMThe capillary electrophoresis detection was performed simultaneously with-500 LIZ Size Standard, and Data Collection 3.0 software was used to collect Data.

5. The method of claim 2, characterized in that the method is as follows:

(2) performing multiplex PCR amplification by taking the genomic DNA extracted in the step (1) as a template and the molecular characteristic multiplex SSR primer as an amplification primer:

the composition of each 25. mu.L of the PCR reaction system was as follows:

the multiplex PCR reaction conditions were as follows:

(3) preparing an internal standard: 10ml of Hi-Di and 80 mu of LGeneScan are taken^TM-500LIZ Size Standard, centrifuged, aliquoted 10 μ L per well into a 96 well Standard plate, centrifuged; diluting and centrifuging the PCR amplification product; adding 0.5 mu L/well of the diluted product into a well-distributed 96-well plate, uniformly mixing, centrifuging, placing into a PCR instrument, performing denaturation at 96 ℃ for 5min, then rapidly freezing at-20 ℃ for 2min, and centrifuging to obtain a denatured PCR product; the denatured PCR product was placed in ABI 37 in synchronization with the internal standardPerforming capillary electrophoresis detection on 30XL Genetic Analyzer, and collecting Data by using Data Collection 3.0 software;

(4) and (3) data analysis: the raw Data collected by the Data Collection 3.0 software was analyzed by the GeneMapper 4.1 software, and the software system would compare the internal standard GeneScan in the same lane with the target peak position^TM-500LIZ Size Standard for comparison, directly reading the accurate peak value of the target SSR fragment, recording the allelic variation data of homozygous locus as X/X, and recording the allelic variation data of heterozygous locus as X/Y or X/Y/Z;