CN115896333B - Method for identifying Jinyu No. 1 tea tree strain by utilizing SSR fingerprint - Google Patents
Method for identifying Jinyu No. 1 tea tree strain by utilizing SSR fingerprint Download PDFInfo
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Abstract
The invention provides a method for identifying Jinyu No.1 tea tree strains by utilizing SSR fingerprint. The invention uses 44 good tea tree varieties from 12 provinces as sample materials, designs 76 SSR primers in the whole genome of tea tree to carry out polymorphism screening, and the identification process mainly comprises total DNA extraction of tea tree, primer design, PCR amplification and allele polymorphism statistics, finally establishes 5 pairs of primers as core primers for identification of Jinyu No.1 varieties, and can effectively distinguish the Jinyu No.1 novel varieties from other 43 tea tree varieties, thereby being beneficial to protection and popularization of the Jinyu No.1 novel varieties and providing a quick and accurate method for identification of true and false of Jinyu No.1 tea varieties.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a method for identifying Jinyu No. 1 tea tree strains by utilizing SSR fingerprint.
Background
The Jinyu No. 1 is a single plant which is rich in sprout and strong in resistance and is named as 'Jinyu No. 1', and is named as 'Jinyu No. 1' after a new sprout is tender and grows well in the head of a new sprout which is found in a group tea garden by a local tea grower in Jinzhai county of Liu an Anhui province. And then, cutting and propagating by using short spikes, and successively establishing a maternal garden for cultivation management. Through observation and cultivation for decades, the strain is found to have high yield, high resistance, early germination and She Feizhuang buds, and is very suitable for manufacturing Liuan melon slices. The strain has the characteristics of early germination, high fresh leaf yield, strong stress resistance, good quality of the prepared green tea and the like, has higher popularization and application economic benefit, and is suitable for cultivation in high-quality green tea areas.
The Jinyu No. 1 belongs to shrub type, early-growing variety, semi-open tree pose, dense branch density, dense bud leaf density, leaf length of 7.3cm, leaf width of 4.0cm, middle leaf class, leaf shape medium ellipse, she Shenping, leaf vein of 7 pairs, hard leaf, luster, green in leaf color, light green in tender bud color, less fuzz, 73.33g of one bud three-leaf hundred bud, oblique growth on leaf blade, tip tapering, she Yuanping, leaf micro-bulge, she Chirui, dense and medium.
The yield per mu of the Jinyu No.1 is 448.5kg according to the standard of one bud and two leaves, and the yield per mu of the spring tea is 1-2 times of that of other varieties, and the Jinyu No.1 has higher popularization value. The dry tea has high chemical quality, contains free amino acid 5.70%, tea polyphenols 23.20%, catechin 12.85%, caffeine 2.33%, theanine 2.45%, and phenol-ammonia ratio 4.07, and is suitable for preparing green tea. Making six-an melon slices, wherein the appearance of the melon slices is sheet-shaped, and the melon slices are deep yellow green and relatively uniform; the soup is green, yellow and slightly blue, and has sediment; the flavor is fresh and rich, the faint scent is pure, and the flavor is relatively flat; tender and soft leaves, green and yellow, and even and bright. The Liuan melon slices manufactured by using Jinyu No.1 as a raw material are popular in the market, and have high germination time, high quality and higher price than similar products in the market, so that the Liuan melon slices have popularization value.
In order to better protect and popularize new varieties of Jinyu No. 1 tea trees and effectively distinguish the authenticity of fresh leaves and dried tea, a quick and simple method is needed to be established to accurately identify the new variety of Jinyu No. 1 tea trees.
Disclosure of Invention
The invention aims to provide a method for identifying Jinyu No. 1 tea tree strains by utilizing SSR fingerprint.
The invention uses 44 good tea tree varieties from 12 provinces as sample materials, designs 76 SSR primers in the whole genome of tea tree to carry out polymorphism screening, and the identification process mainly comprises total DNA extraction of tea tree, primer design, PCR amplification and allele polymorphism statistics, finally establishes 5 pairs of primers as core primers for identification of Jinyu No. 1 varieties, and can effectively distinguish the Jinyu No. 1 novel varieties from other 43 tea tree varieties, thereby being beneficial to protection and popularization of the Jinyu No. 1 novel varieties and providing a quick and accurate method for identification of true and false of Jinyu No. 1 tea varieties.
In order to achieve the aim of the invention, in a first aspect, the invention provides a primer combination for amplifying SSR markers of Jinyu No.1 tea tree line, which comprises 5 SSR markers, namely CS-SSR 37, CS-SSR 40, CS-SSR 45, CS-SSR 51 and CS-SSR 54; the primers used for amplifying the primers are respectively shown as SEQ ID NO. 1-2, SEQ ID NO. 3-4, SEQ ID NO. 5-6, SEQ ID NO. 7-8 and SEQ ID NO. 9-10.
In a second aspect, the invention provides a detection reagent or kit comprising said SSR primer combination.
In a third aspect, the invention provides application of the SSR primer combination in the identification and breeding of Jinyu No. 1 tea tree lines.
In a fourth aspect, the invention provides application of the SSR primer combination in molecular marker assisted breeding of Jinyu No. 1 tea tree lines.
In a fifth aspect, the invention provides application of the SSR primer combination in construction of SSR finger print of Jinyu No.1 tea tree strain.
In a sixth aspect, the invention provides a method for identifying Jinyu No.1 tea tree lines by utilizing SSR fingerprint, comprising the following steps:
1) Extracting genome DNA of a tea tree sample to be detected;
2) Taking the extracted DNA as a template, and carrying out PCR reaction by utilizing the SSR primer combination;
3) The PCR amplification products were analyzed.
Preferably, the PCR amplification system is: 40 ng/. Mu.L of DNA template 1. Mu.L, 10. Mu.M of each of the upstream and downstream primers 0.5. Mu.L, 2 XEs Taq MasterMix 5. Mu.L, ddH 2 O3. Mu.L.
Preferably, the PCR amplification procedure is: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 57℃for 30s, elongation at 72℃for 30s,30 cycles; finally, the extension is carried out for 5min at 72 ℃.
Preferably, the amplification product is detected by capillary electrophoresis.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the primer provided by the invention is based on tea tree genome analysis, and has the characteristics of high polymorphism and large quantity compared with EST-SSR. And 5 pairs of core primers are finally determined to be used for identifying the novel Jinyu No.1 line through a large number of primer screening.
The SSR fingerprint technology is adopted, and the technology is an application genetic germplasm analysis method developed along with the development of molecular biology in recent years, has the characteristics of good stability, simplicity in operation and high accuracy, and provides an accurate, rapid and simple method for identifying the Jinyu No. 1 excellent strain.
The materials selected by the invention are not limited by seasons, environments and test time, and DNA extraction can be carried out on any organ in any period of growth of the Jinyu No. 1 strain, or the dried tea stored for a period of time is prepared, so that the identification result is not affected.
The invention selects Fragment Analyzer TM full-automatic capillary electrophoresis system for primer screening, which has the characteristics of high flux, safety, convenience, high sensitivity and the like, is favorable for quickly and accurately establishing SSR fingerprint, and shortens the period of identifying excellent strain of Jinyu No. 1.
Drawings
FIG. 1 is a diagram showing agarose gel electrophoresis of DNA extracted in a preferred embodiment of the present invention.
FIG. 2 is a diagram showing PCR amplification capillary electrophoresis of 44 tea varieties with 5 pairs of core primers according to the preferred embodiment of the present invention.
FIG. 3 is a three-time repeated capillary electrophoresis gel of 5 pairs of core primer pairs Jinyu No. 1 fresh leaves and dried tea in a preferred embodiment of the present invention.
Detailed Description
The invention provides a method for identifying novel strain of Jinyu No.1 tea tree by utilizing SSR fingerprint technology, which can identify novel variety resources of Jinyu No.1 tea tree more accurately from the molecular level of plant genes.
The invention utilizes SSR fingerprint technology to identify Jinyu No. 1, wherein 5 specific polymorphic microsatellite SSR sites in the genome of the tea tree are involved, and repeat units of different sites consist of two bases and three bases, which are microsatellite types with rich and high polymorphism in the genome of the tea tree:
(1) The SSR sequence of the primer pair 1 corresponding to the specific site is as follows: ATATATATATATATATAT (marker CS-SSR 37)
The conserved sequences flanking marker CS-SSR 37 are as follows:
Left conserved sequence:
The right conserved sequence:
The sequence of the primer pair 1 designed according to the SSR primer design principle is (SEQ ID NO: 1-2):
An upstream primer: 5'-ACATTCGACGAACTCTACAC-3' (Tm: 56.4 ℃ C.)
A downstream primer: 5'-TGAGTTGAGGGCTAAGGTA-3' (Tm: 55 ℃ C.)
(2) The primer pair 2 corresponds to a specific site SSR sequence: ATATATATATATATATAT (Mark CS-SSR 40)
The conserved sequences on both sides of the marker CS-SSR 40 are as follows:
Left conserved sequence:
The right conserved sequence:
The sequence of the primer pair 2 designed according to the SSR primer design principle is (SEQ ID NO: 3-4):
An upstream primer: 5'-TGACGGGTGGTGATTGAA-3' (Tm: 54.3 ℃ C.)
A downstream primer: 5'-AGAAAAGAATCCGCACGAAA-3' (Tm: 54.3 ℃ C.)
(3) The primer pair 3 corresponds to a specific site SSR sequence: TCTCTCTCTCTCTCTCTCTCTCTC (Mark CS-SSR 45)
The conserved sequences flanking marker CS-SSR 45 are as follows:
Left conserved sequence:
The right conserved sequence:
the sequence of the primer pair 3 designed according to the SSR primer design principle is (SEQ ID NO: 5-6):
An upstream primer: 5'-TAGATTTCAAAGGCAGAGCA-3' (Tm: 54.3 ℃ C.)
A downstream primer: 5'-CAGTGACGCAGAAGATAAGA-3' (Tm: 56.4 ℃ C.)
(4) The primer pair 4 corresponds to a specific site SSR sequence: CGCCGCCGCCGCCGCCGCCGCCGCCGC (Mark CS-SSR 51)
The conserved sequences flanking marker CS-SSR 51 are as follows:
Left conserved sequence:
The right conserved sequence:
The sequence of the primer pair 4 designed according to the SSR primer design principle is (SEQ ID NO: 7-8):
an upstream primer: 5'-TCCTCCATCTTCTTGCTCCT-3' (Tm: 58.4 ℃ C.)
A downstream primer: 5'-TCCTCAGTCCCAACATACCA-3' (Tm: 58.4 ℃ C.)
(5) The SSR sequence of the primer pair 5 corresponding to the specific site is as follows: GAGAGAGAGAGAGAGAGAGAGAGAGAGA (Mark CS-SSR 54)
The conserved sequences flanking marker CS-SSR 54 are as follows:
Left conserved sequence:
The right conserved sequence:
The sequence of the primer pair 5 designed according to the SSR primer design principle is (SEQ ID NO: 9-10):
an upstream primer: 5'-GTTTGGCACTATCATCTCC-3' (Tm: 55 ℃ C.)
A downstream primer: 5'-GTGAATTCCTACCTTAGGTC-3' (Tm: 56.4 ℃ C.)
The primer screening steps are as follows: extracting total DNA of the selected sample, selecting SSR sites and designing and screening primers according to the whole genome sequence of tea trees, performing PCR amplification and agarose gel electrophoresis primary screening, re-screening by a Fragment Analyzer TM full-automatic capillary electrophoresis system, and screening core primers according to electrophoresis results.
Wherein, the total DNA of tea tree is extracted by adopting an improved CTAB method to extract dry tea sold in the market and fresh leaves picked in the original place. The whole genome sequence of the tea tree is subjected to SSR site selection and primer design screening, and specifically, in 10 ten thousands of sequences containing SSR sites, two bases and three bases are microsatellite types which are rich in the genome of the tea tree and have higher polymorphism. Therefore, according to the principles, 500 sequences containing SSR sites are selected, 76 pairs of primers are successfully designed, wherein the principle of SSR primer screening is as follows:
1) The length of the primer is 18-22bp, and the target fragment is about 250 bp;
2) GC content is 40% -60%, and three or four continuous bases are avoided in the primer sequence;
3) The annealing temperature is 52-60 ℃, preferably about 57 ℃, and the difference between the Tm values of the upstream primer and the downstream primer is not more than 4 ℃;
4) The 3' end of the primer avoids A or more than 3 continuous bases, and primer dimer and hairpin structure are avoided as much as possible.
The primer screening method is as follows: the PCR amplification and capillary electrophoresis primary screening is carried out by sucking 2.5 mu L of PCR products, carrying out primary screening by a Fragment Analyzer TM full-automatic capillary electrophoresis system, selecting PCR products with high amplification rate, bright bands and good peaks by comparing the PCR products with target fragments, carrying out secondary screening by a Fragment Analyzer TM full-automatic capillary electrophoresis system, and accurately reflecting the difference between allelic loci and screening out primers with high polymorphism.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
EXAMPLE 1 extraction of total DNA from tea leaves
1. The selected tea tree varieties are shown in Table 1.
TABLE 1 tea tree variety name Table
2. The method adopts a CTAB method to extract total DNA by taking the selected tea tree variety as a material, and comprises the following specific operation processes:
(1) 0.1g tea leaves were weighed into a mortar pre-cooled with liquid nitrogen, and quickly ground with liquid nitrogen until the sample material was powdered and transferred to a 2mL centrifuge tube. 700. Mu.L of CTAB extract (preheated to 65 ℃ C.) and 10. Mu.L of beta-mercaptoethanol were added to the centrifuge tube. After the sample is moved in, shaking up, water bath is carried out for 15min at 65 ℃, and shaking up and down for 6-8 times every 5 min.
(2) 600. Mu.L of the nucleic acid extract was added thereto, and the mixture was centrifuged at 12000rpm for 10 minutes. 500. Mu.L of the supernatant was placed in a new 1.5mL centrifuge tube, 500. Mu.L of isopropyl alcohol was added, the mixture was shaken up and down 6-8 times, centrifuged at 12000rpm for 5min, and the supernatant was discarded.
(3) Adding 500 μl of 70% ethanol, blowing and mixing the bottom precipitate, centrifuging at 12000rpm for 5min, and discarding the supernatant. Repeating the step (3) once.
(4) The centrifuge tube cover is opened, the remaining ethanol is dried in a fume hood, 100 mu L of sterilized water is added for uniform mixing for standby, the nucleic acid content is measured, and 0.8% agarose electrophoresis is used.
The agarose gel electrophoresis of the extracted DNA is shown in FIG. 1.
Example 2 selection of SSR sites and primer Primary screening
In the obtained 10 ten thousand sequences containing SSR sites, the multi-base repeat is a microsatellite type which is relatively rich in tea tree genome and has higher polymorphism, so 500 sequences containing SSR sites are selected, 76 pairs of primers (synthesized by general biology (Anhui) Co., ltd.) are successfully designed, wherein the principle of SSR primer screening is as follows:
1) The length of the primer is 18-22bp, and the target fragment is about 250 bp;
2) GC content is 40% -60%, and three or four continuous bases are avoided in the primer sequence;
3) The annealing temperature is 52-60 ℃, preferably about 57 ℃, and the difference between the Tm values of the upstream primer and the downstream primer is not more than 4 ℃;
4) The 3' end of the primer avoids A or more than 3 continuous bases, and primer dimer and hairpin structure are avoided as much as possible.
EXAMPLE 3 full automatic capillary electrophoresis Primary screening for PCR amplification and Fragment Analyzer TM
The total reaction system was 10. Mu.L, in which 40 ng/. Mu.L of DNA solution was 1. Mu.L, 10. Mu.M upstream and downstream primers were each 0.5. Mu.L, 2 XEs Taq Master mix (Dye) was 5. Mu.L, ddH 2 O3. Mu.L (reagent was from Kangshengmbh), and after centrifugation, 20. Mu.L of mineral oil was added to seal against evaporation during PCR amplification.
The PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 57℃for 30s, elongation at 72℃for 30s,30 cycles; finally, the mixture is extended at 72 ℃ for 5min and stored at 4 ℃. And (3) carrying out full-automatic capillary electrophoresis on the PCR product by Fragment Analyzer TM, selecting a single product with high amplification rate and clear band, and carrying out primary screening of the primer by comparing the single product with a target fragment.
Example 4Fragment Analyzer TM full-automatic capillary electrophoresis System double Screen
The specific operation flow is as follows:
1. preparation of the reagent:
1) And (3) glue preparation: 45mL of dsDNA 905 Separation Gel (Separation Gel) was added with 2.5. Mu.L of dye (INTER CALATING DYE) and thoroughly mixed.
2) 5X 930dsDNA buffer solution: the 5 Xcapillary conditioning solution (InterBuffer) was diluted 5-fold.
3) 5 X capillary wash solution (CAPILLARY CONDITIONING SOLUTION): 5 Xcapillary wash solution (CAPILLARY CONDITIONING SOLUTION) was diluted 5-fold and 1mL of capillary wash solution (CAPILLARY CONDITIONING SOLUTION) was added to each 96-well plate to avoid air bubbles.
4) Marker: mu.L of 1-500bp Markers each was added to a 96-well plate, blocked with 20. Mu.L of Mineral oil (Mineral oil) per well, and centrifuged.
5) Sample preparation: 22. Mu.L of Dilution buffer (Dilution buffer) and 2.5. Mu.L of LPCR product were added to each well of a 96-well plate, and 24. Mu.L of 1-500bp DNA molecular weight standard (RANGE DNA LADDER) was added to the last well, and the wells were centrifuged to avoid air bubbles.
2. The operation steps are as follows: and placing the prepared reagent in a designated position of the instrument, and clicking an operation program of the instrument.
3. Data recording and result analysis: the capillary electrophoresis results were shown using PROSize TM 2.0.0 software, the highest peak band was selected from each band, and specific values were recorded. The following requirements are set on the requirement standard of the primer re-screening: i) The main band is clear, no redundant miscellaneous bands exist, and the peak shape is good; ii) high polymorphism values, multiple alleles; iii) The size of the band difference between two similar allelic sites is larger than 4bp; iv) performing three-time repeated amplification on the primer conforming to the first three points, selecting the primer with good repeatability and stability, and finally selecting 5 pairs of primers as core primers for identifying Yu 1 (figure 2). Primer pairs specific alleles amplified for 44 varieties are shown in table 2:
Table 25 primer pairs 44 tea variety capillary electrophoresis strip statistics (bp)
The invention can rapidly and accurately identify Jinyu No. 1 from 44 tea leaves in table 1, and the primer three-time repeated capillary electrophoresis fingerprint is shown in fig. 3, and the lowest resolution of a Fragment Analyzer TM full-automatic capillary electrophoresis system is 2bp, so that the allowed range of the primer to the band amplified by Jinyu No. 1 variety is +/-3 bp.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (4)
1. The primer combination is used for amplifying SSR markers of tea tree strain No. 1 of golden Yu, and is characterized by being used for amplifying 5 SSR markers, namely CS-SSR 37, CS-SSR 40, CS-SSR 45, CS-SSR 51 and CS-SSR 54; the primer combination is SEQ ID NO. 1-10.
2. The use of the primer combination for amplifying the SSR marker of the Jinyu No. 1 tea tree line in the identification and the breeding of the Jinyu No. 1 tea tree line according to claim 1.
3. Use of the primer combination for amplifying the SSR marker of the tea tree line 1 of kuyu 1 in molecular marker assisted breeding of the tea tree line 1 of kuyu 1.
4. The use of the primer combination for amplifying the golden yu No. 1 tea tree line SSR marker of claim 1 in constructing golden yu No. 1 tea tree line SSR finger print.
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| CN202211487207.7A CN115896333B (en) | 2022-11-24 | 2022-11-24 | Method for identifying Jinyu No. 1 tea tree strain by utilizing SSR fingerprint |
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