CN111304356B - Molecular marker primer combination for rapidly identifying sex traits of Chinese torreya in high throughput manner and application thereof - Google Patents
Molecular marker primer combination for rapidly identifying sex traits of Chinese torreya in high throughput manner and application thereof Download PDFInfo
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Abstract
The invention discloses a group of molecular marker primers for rapidly identifying sex traits of Chinese torreya and application thereof, which are applied to early molecular marker-assisted selection of Chinese torreya breeding. The invention provides a group of molecular marker primers JY1754 capable of identifying the sex characters of Chinese torreya, wherein the PCR amplification product is only 97bp, and the primers are particularly suitable for high-throughput detection platforms such as a fluorescent quantitative PCR instrument and can be used for quickly identifying the sex characters of Chinese torreya germplasm resources. The method for assisting in screening the germplasm resources of the Chinese torreya of different genders is provided based on the developed molecular marker primer combination, has important guiding significance on the phenotype prediction of the sex characters of the Chinese torreya, and has the advantages of simple operation, high throughput, universality of multiple detection and analysis platforms, accurate result and the like.
Description
Technical Field
The invention relates to a group of molecular marker primer combinations for rapidly identifying the sex traits of Chinese torreya in a high-throughput manner, provides a method for assisting in identifying or screening the germplasm resource traits of Chinese torreya of different sexes, and belongs to the field of molecular biology.
Background
Torreya grandis (Torreya grandis fort. ex Lindl. cv. Merrillii Hu) is a tertiary wiggling plant, mainly is a male and female alien plant, occasionally shows a same plant type of male and female, is a unique rare plant in China, is a precious dried fruit integrating edible, medicinal, material and oil extraction, is mainly distributed in Zhejiang, Jiangxi, Jiangsu, Anhui, Fujian, Hunan and other provinces, and the cultivation area of the Torreya grandis (Torreya grandis fort. ex Lindl. cv. Merrillii Hu) is enlarged year by year. The Chinese torreya tree grows slowly in the early stage, the sex is difficult to distinguish, the seedling generally enters the flowering and fructification period within 15-20 years, and the seedling propagation and selling phenomena are frequently caused by the fact that the male plant scion is adopted to pretend to be the female plant variety scion; in addition, the natural pollination effect of most of Chinese torreya produced in the traditional production is limited by the conditions such as environmental climate and the like, so that the yield is low and unstable, in recent years, male plant resources are widely collected and pollen is extracted in the main production area of Chinese torreya, artificial supplementary pollination measures are popularized and implemented, the defect of natural pollination is overcome, and good effect is achieved. Moreover, scientific and technical personnel can realize full natural pollination by digging and screening excellent torreya grandis hermaphrodite type matched planting so as to reduce the cost of artificial supplementary pollination and solve the problems of short flowering phase, low pollination rate and the like of the conventional male plant. Therefore, a method for identifying the early sex of the Chinese torreya should be established as soon as possible for screening male and female scions of the Chinese torreya and exploring and screening more excellent germplasm resources of male plants of the Chinese torreya tree, and a foundation is laid for quality improvement and efficiency improvement of the production link of the Chinese torreya industry.
The development of modern biotechnology provides an effective auxiliary method for early identification of plant sex, namely DNA molecular markers, such as SSR, AFLP, SNP and the like. Although the invention has been reported from the aspects of RAPD, AFLP markers and the like, the operation steps of the invention relate to the processes of enzyme digestion, connection, gel electrophoresis and the like, the operation steps are very complicated, the technical threshold and the detection cost are high, the practicability is poor, and the detection accuracy is not detailed. In addition, the invention patent with the publication number of CN201810144040.1 discloses a specific molecular marker TGMI001 for identifying the sex of torreya grandis at the seedling stage, wherein, a torreya grandis male specific molecular marker TGMI001 is provided, which can be used for identifying the sex of torreya grandis males and females, but the invention does not mention whether the same type of torreya grandis male and female plants can be identified; in addition, the PCR product fragment of the molecular marker TGMI001 is long (461bp), so that the molecular marker TGMI001 is not suitable for high-throughput detection technology platforms such as fluorescent quantitative PCR instrument genotyping and the like (generally, the amplification effect is best when the length of the product fragment is less than 400bp and the length of the product fragment is about 100 bp); moreover, the molecular marker TGMI001 needs relatively long PCR amplification time and subsequent detection of product size through traditional gel electrophoresis, the sensitivity is lower than that of a fluorescent quantitative PCR instrument, the operation technical link is relatively complicated, and time and labor are wasted.
Disclosure of Invention
The invention aims to provide a group of molecular marker primer combinations for rapidly identifying the sex traits of Chinese torreya in a high-throughput manner and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that: a group of molecular marker primers for identifying female and male (including hermaphrodite) sex traits of Chinese torreya, wherein the primer sequence comprises:
JY 1754: f: 5 '-linker sequence-TACCCGACGAC-3',
R：5’-AGGAAACCCCAACTAAAATA-3’。
preferably, the linker sequence is an M13 linker. The purpose of the adaptor sequence is to prevent mismatching, improve the specificity and fragment length of the amplified product, and can be paired with the Tail primer to form a fluorescent product. Since M13 linker is used more often in the art, but other sequences can be used without affecting the achievement of the object of the present invention, and therefore, it is within the scope of the present invention that the linker sequence has other base structures.
Preferably, the linker sequence is tgtaaaacgacggccatt. In addition to this M13 linker sequence, other universal M13 sequences may be used in the present invention.
Preferably, the kit further comprises a Tail primer with a fluorescent group added at the 5' end: 5'-TGTAAAACGACGGCCAGT-3' are provided.
Preferably, the fluorophore is FAM, HEX, NED or PET.
The invention also discloses application of the molecular marker primer in rapid identification of the sex character of Chinese torreya.
The invention also discloses a method for rapidly identifying the sex character of Chinese torreya with high throughput, which comprises the following steps:
(1) primer synthesis: synthesizing a primer JY1754 according to claim 1;
(2) extraction of DNA: extracting genome DNA in the torreya grandis tender leaves to be detected;
(3) and (3) PCR amplification: based on the torreya grandis genome DNA, PCR amplification is carried out by using a primer JY 1754;
(4) detection and analysis of amplification products: and (3) carrying out genotype detection and analysis by using an ABI Q6 Flex real-time fluorescent quantitative PCR system, and further determining the sex character of the Chinese torreya.
Further, the detailed steps are as follows:
(1) primer synthesis: the related biotechnology company is entrusted to synthesize the primer JY 1754;
(2) extraction of DNA: extracting the Genomic DNA in the tender leaves of the Chinese torreya to be detected by using a magnetic bead method Plant Genomic DNA Extraction Kit (Mag-MK Plant Genomic DNA Extraction Kit, Shanghai Biotechnology engineering Co., Ltd.), and performing the specific operation method according to the instruction; finally, 1-2 mul of the extracted genome DNA is taken to be detected on 1.0% agarose gel, and the DNA stock solution is diluted into working solution with the concentration of 50 ng/mul for subsequent use;
(3) fluorescence quantificationThe PCR amplification reaction system is as follows: a PCR reaction system was constructed using Green-2-Go 2 XqPCR-Low-ROX premix (Green-2-Go 2 XqPCR-Low-ROX Mastermix, Shanghai Biotech Co., Ltd.), 2.0. mu.L of genomic DNA template, 5. mu.L of 2 XGreen-2-Go Mastermix, 0.5. mu.L of forward primer (10. mu.M), 0.5. mu.L of reverse primer (10. mu.M), ddH2O is complemented to 10 mu L; the amplification PCR procedure was as follows: at 95 ℃ for 3min, at 95 ℃ for 3s, at 60 ℃ for 50s, for 17 cycles; after the PCR reaction is finished, the characteristics of male plants and hermaphroditic plants of the samples can be directly known based on the amplification condition of the PCR products in the automatic analysis result of the ABI Q6 Flex real-time fluorescent quantitative PCR system platform and Allelic diagnosis Plot; wherein, if PCR amplification products appear (two sample DNAs with determined sex as male or hermaphrodite type are added in advance as internal reference) and are gathered with the internal reference sample into a group, the PCR amplification products can be regarded as the male or hermaphrodite type, and if no PCR amplification products appear and are not gathered with the internal reference sample into a group, the PCR amplification products can be regarded as the female sex type.
In addition, when the typing platform is detected by fluorescence capillary electrophoresis, a Tail primer (5'-TGTAAAACGACGGCCAGT-3') to which a fluorophore (FAM, HEX, NED, PET, or the like) is added is synthesized in advance, and the PCR amplification reaction system is adjusted as follows: a PCR reaction system was constructed using a Dye-free 2 XTaq PCR Mix premix (2 XTaq PCR Master Mix, without Dye, Shanghai Biotech Co., Ltd.), 2.0. mu.L of genomic DNA template, 5. mu.L of 2 XTaq PCR Master Mix, 1.0. mu.L of Tail primer (10. mu.M), 0.5. mu.L of forward primer (10. mu.M), 1.0. mu.L of reverse primer (10. mu.M), ddH2O is complemented to 10 mu L; the amplification PCR procedure was as above; after the PCR reaction is finished, products added with different fluorescent groups can be mutually combined, and then STR typing detection is carried out on the basis of a fluorescent capillary electrophoresis platform ABI3730 XL; wherein, if a PCR amplification product with the length of 97bp appears and the size of the PCR amplification product is consistent with that of an internal reference sample fragment (two sample DNAs with determined sex as male or hermaphroditic type are added in advance as internal references), the PCR amplification product can be regarded as the male (hermaphroditic) type, and if no PCR amplification product with the size appears, the PCR amplification product can be regarded as the female sex type. It is noted that, since the probability of the natural occurrence of hermaphrodite torreya grandis is extremely small, if the result of the measurement is obtainedThe sex type of male plants (hermaphrodite) is considered to be mainly male sex shape.
According to the invention, a molecular marker primer combination with high amplification efficiency is designed and developed according to the type specific sites of male Chinese torreya and hermaphrodite strains; the molecular marker primer combination and the related PCR amplification system are repeatedly optimized, background signal interferences such as primer dimer, nonspecific amplification miscellaneous band and the like are avoided, the total length of an amplification product fragment is only 97bp, the number of required PCR amplification cycles is small, the consumed time is short, the method is particularly suitable for high-throughput typing detection platforms such as a fluorescence quantitative PCR instrument, the like, the whole-process closed-tube operation is realized from PCR amplification to the acquisition of final typing result data, gel electrophoresis detection is not needed, no pollution is caused, and the targets of rapidness, high throughput and high sensitivity are achieved; in addition, the molecular marker primer combination of the invention is added with a joint sequence in advance, and only the Tail primer added with a plurality of fluorescent groups needs to be synthesized to combine a monochromatic multiple detection system for the typing analysis of the fluorescence capillary electrophoresis, thereby reducing the detection cost. Furthermore, if the detection hardware conditions are limited, the primer combination of the present invention can also be used in common PCR instruments and conventional gel electrophoresis detection systems. In conclusion, the primer combination belongs to a rapid, high-throughput and multi-detection-platform universal molecular marker primer combination.
Compared with the prior art, the invention has the beneficial effects that:
(1) the molecular marker primer JY1754 amplification product fragment is only 97bp, and is particularly suitable for high-throughput typing detection platforms such as a fluorescent quantitative PCR instrument, the number of PCR amplification cycles is only 17, the time consumption is short, the whole-process closed-tube operation is realized from the PCR amplification to the acquisition of the final typing result data, the gel electrophoresis detection is not needed, the pollution is avoided, and the characteristics of rapidness, high flux, high sensitivity and the like are realized;
(2) the molecular marker primer JY1754 is suitable for a fluorescence capillary electrophoresis typing detection technology analysis platform, a Tail primer sequence is designed to be added to the 5' end of a forward primer, and single-color multiple PCR product combination can be subjected to capillary electrophoresis detection only by additionally synthesizing 1-4 Tail Tail primers added with fluorescence marks, so that the detection test cost is reduced, the gene typing accuracy and the detection efficiency are improved, and the detection effect is reliable;
(3) the molecular marker primer JY1754 has the advantages of good PCR amplification effect, stable and clear product band shape, good repeatability and easy resolution, and can also be used for agarose electrophoresis detection and analysis; therefore, if the typing detection hardware equipment is limited, and a high-throughput detection platform such as a fluorescence quantitative PCR instrument and the like is not provided, a common PCR instrument and a traditional conventional agarose gel electrophoresis detection system can be used, so that the test cost is further reduced, and the method belongs to multi-platform general molecular markers;
the invention is described in more detail below with reference to the following examples and the accompanying drawings. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
Drawings
FIG. 1 shows the typing effect of JY1754 primer of Torreya grandis germplasm resource DNA sample on the fluorescent quantitative PCR instrument platform (group A, male plant (hermaphrodite) group; group B, female plant group);
FIG. 2 shows the effect of fluorescence capillary electrophoresis typing on the PCR amplification product of JY1754 primer for DNA sample of male torreya (hermaphrodite);
FIG. 3 shows agarose electrophoresis effect of PCR amplification product of JY1754 primer of DNA sample of male torreya (hermaphrodite).
Detailed Description
Example 1
A group of molecular marker primer combinations for rapidly identifying the sex character of Chinese torreya with high flux and application thereof comprise the following detailed steps:
(1) primer synthesis: according to the sequence information in the table 1, the related biotechnology company is entrusted to synthesize a primer JY 1754;
table 1: related primer sequences
(2) Extracting DNA of sample leaves of a Chinese torreya population to be detected (table 2):
extracting the Genomic DNA in the tender leaves of the Chinese torreya to be detected by using a magnetic bead method Plant Genomic DNA extraction Kit (Mag-MK Plant Genomic DNA extraction Kit, Shanghai Biotechnology engineering Co., Ltd.), and performing the specific steps according to the instruction; after extraction, 2 mul of DNA stock solution is respectively taken and detected on 1.0 percent agarose gel; finally, diluting the DNA stock solution into working solution with the concentration of 40 ng/microliter for subsequent use;
(3) the fluorescent quantitative PCR amplification reaction system is as follows: a PCR reaction system was constructed using Green-2-Go 2 XqPCR-Low-ROX premix (Green-2-Go 2 XqPCR-Low-ROX Mastermix, Shanghai Biotech Co., Ltd.), 2.0. mu.L of genomic DNA template, 5. mu.L of 2 XGreen-2-Go Mastermix, 0.5. mu.L of forward primer (10. mu.M), 0.5. mu.L of reverse primer (10. mu.M), ddH2O is complemented to 10 mu L; the amplification PCR procedure was as follows: at 95 ℃ for 3min, at 95 ℃ for 3s, at 60 ℃ for 50s, for 17 cycles; after the PCR reaction is finished, the sex character of the sample can be directly known based on the amplification condition of the PCR product in the automatic analysis result of the ABI Q6 Flex real-time fluorescent quantitative PCR system platform (96 holes) and Allelic diagnosis Plot; wherein, if PCR amplification products appear (two sample DNAs with determined sex as male plant (hermaphrodite) type are added in advance as internal reference) and are gathered with the internal reference sample into a group, the PCR amplification products can be regarded as the male plant (hermaphrodite) type, and if no PCR amplification products appear and are not gathered with the internal reference sample into a group, the PCR amplification products can be regarded as the female plant sex type. Finally, the sex character identification results of all the test samples are recorded by using a table, and the specific typing effect is shown in figure 1. In this example, the 96-well heating module of the fluorescence quantitative platform can be replaced as required, such as 384-well and fluid chip, to achieve the requirement of higher throughput typing detection.
TABLE 2 sex determination result information of 78 Torreya grandis germplasm resources used in the examples of the present invention
Note: the presence and absence of amplification products are indicated by "+" and "-" respectively.
Example 2
A group of molecular marker primer combinations for rapidly identifying the sex character of Chinese torreya with high flux and application thereof comprise the following detailed steps:
(1) primer synthesis: according to the sequence information in the table 1, the related biotechnology company is entrusted to synthesize a primer JY 1754;
table 1: related primer sequences
(2) Extracting DNA of sample leaves of a Chinese torreya population to be detected (table 2):
extracting the Genomic DNA in the tender leaves of the Chinese torreya to be detected by using a magnetic bead method Plant Genomic DNA Extraction Kit (Mag-MK Plant Genomic DNA Extraction Kit, Shanghai Biotechnology engineering Co., Ltd.), and performing the specific steps according to the instruction; after extraction, 2 mul of DNA stock solution is respectively taken and detected on 1.0 percent agarose gel; finally, diluting the DNA stock solution into working solution with the concentration of 40 ng/microliter for subsequent use;
(3) the fluorescent quantitative PCR amplification reaction system is as follows:
based on a fluorescence capillary electrophoresis detection typing platform, a PCR reaction system was constructed using a Dye-free 2 XTaq PCR Mix premix (2 XTaq PCR Master Mix, without Dye, Shanghai Biotech Co., Ltd.), 2.0. mu.L of a genomic DNA template, 5. mu.L of 2 XTaq PCR Master Mix, 1.0. mu.L of a Tail primer (10. mu.M), and a forward primer (10. mu.M)0.5. mu.L, reverse primer (10. mu.M) 1.0. mu.L, ddH2O is complemented to 10 mu L; the amplification PCR program is: at 95 ℃ for 3m, 95 ℃ for 3s, 60 ℃ for 50s, 17 cycles; after the PCR reaction is finished, placing the product on a fluorescence capillary electrophoresis platform ABI3730XL for STR typing detection; wherein, if a PCR amplification product with the length of 97bp appears and the size of the PCR amplification product is consistent with that of an internal reference sample fragment (two sample DNAs with determined sex as male or hermaphroditic type are added in advance as internal references), the PCR amplification product can be regarded as the male (hermaphroditic) type (see figure 2), a conventional agarose gel electrophoresis platform can also be used for detection (see figure 3), if no PCR amplification product with the size appears, the PCR amplification product can be regarded as the female sex type, and the final result can be recorded in a table form. The Tail primer in this example can be synthesized into 1-4 kinds of (A), (B), (C) and (C) as required<FAM>/<HEX>/<NED>/<PET>) And performing PCR amplification on the single colors one by one, and finally combining the single-color multiple PCR mixed products to meet the requirement of higher-flux typing detection.
TABLE 2 sex determination result information of 49 torreya grandis germplasm resources used in the examples of the present invention
Note: the presence and absence of amplification products are indicated by "+" and "-" respectively.
Claims (10)
1. A group of molecular marker primers for rapid high-throughput identification of Chinese torreya sex traits is characterized in that the primers comprise JY1754-F and JY1754-R, and the specific sequences are as follows:
JY 1754-F: 5 '-linker sequence-TACCCGACGAC-3';
JY1754-R：5’- AGGAAACCCCAACTAAAATA-3’。
2. the molecular marker primer of claim 1, wherein the linker sequence is M13 linker.
3. The molecular marker primer of claim 2, wherein the linker sequence is tgtaaaacgacggccatt.
4. The molecular marker primer according to claim 3, further comprising a Tail primer having a fluorescent group added to the 5' end: 5'-TGTAAAACGACGGCCAGT-3' are provided.
5. The molecularly imprinted primer according to claim 4, wherein the fluorescent group is FAM, HEX, NED or PET.
6. Use of the molecular marker primer of any one of claims 1-5 for rapid identification of the sex trait of torreya grandis.
7. A method for rapidly identifying the sex character of Chinese torreya with high throughput comprises the following steps:
(1) primer synthesis: synthesizing primers JY1754-F and JY1754-R of claim 1;
(2) extraction of DNA: extracting genome DNA in a torreya grandis sample to be detected;
(3) and (3) PCR amplification: based on the Torreya grandis genome DNA, primers JY1754-F and JY1754-R are used for PCR amplification, and two sample DNAs with determined sexes of male plants or hermaphrodite plants are added in advance to serve as internal references;
(4) detection and analysis of amplification products: and (3) performing genotype detection and analysis by using a fluorescent quantitative PCR instrument, wherein if a PCR amplification product appears and is aggregated with the internal reference sample into a group, the PCR amplification product can be regarded as a male plant or a hermaphroditic plant type, and if no PCR amplification product appears and is not aggregated with the internal reference sample into a group, the PCR amplification product can be regarded as a female plant sex type, so that the sex character of the torreya grandis is determined.
8. The method of claim 7, wherein: the PCR amplification system in the step (3) is as follows: genomic DNA template 2.0. mu.L, 2 XGreen-2-Go Mastermix 5. mu.L, 10. mu.LJY 1754-F0.5 μ L of M, JY 1754-R0.5 μ L of 10 μ M, ddH2O is complemented to 10 mu L; the amplification PCR procedure was as follows: 95 ℃ for 3min, then 17 cycles of reaction were run, each cycle being 95 ℃ for 3s, 60 ℃ for 50 s.
9. A method for rapidly identifying the sex character of Chinese torreya with high throughput comprises the following steps:
(1) primer synthesis: synthesizing primer JY1754-F, JY1754-R of claim 3 and Tail primer of claim 4;
(2) extraction of DNA: extracting genome DNA in a torreya grandis sample to be detected;
(3) and (3) PCR amplification: based on the torreya grandis genome DNA, carrying out PCR amplification by using a primer JY1754-F, JY1754-R and a Tail primer, and adding two sample DNAs of which the genders are determined to be male plants or hermaphrodite plants in advance as internal references;
(4) detection and analysis of amplification products: and (3) performing detection analysis by using a fluorescence capillary electrophoresis detection typing platform, and further determining the sex character of the Chinese torreya.
10. The method of claim 9, wherein: the PCR amplification system in the step (3) is as follows: genomic DNA template 2.0. mu.L, 2 XTaq PCR Master Mix 5. mu.L, 10. mu.M Tail primer 1.0. mu.L, 10. mu.M JY 1754-F0.5. mu.L, 10. mu.M JY 1754-R1.0. mu.L, ddH2O is complemented to 10 mu L; the amplification PCR procedure was as follows: 95 ℃ for 3min, then 17 cycles of reaction were run, each cycle being 95 ℃ for 3s, 60 ℃ for 50 s.
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