CN113430280B - Molecular marker primer combination for rapidly identifying maturation stage of waxberry fruits and application thereof - Google Patents
Molecular marker primer combination for rapidly identifying maturation stage of waxberry fruits and application thereof Download PDFInfo
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Abstract
The invention discloses a group of molecular marker primer combinations for rapidly identifying the maturation stage characters of waxberry fruits and application thereof, which are expected to be applied to molecular marker-assisted selection of the maturation stage characters of the waxberry fruits. The invention provides a group of molecular marker primer combinations which can rapidly identify the early and late mature periods of waxberry fruits, are BS8.1-25849, are suitable for high-throughput typing detection platforms such as a fluorescent quantitative PCR instrument and the like, have high sensitivity and can be used for the early rapid identification of the mature periods of the waxberry fruits. The method for assisting in screening the excellent waxberry germplasm resources based on the developed molecular marker primer combination has important guiding significance on predicting the waxberry fruit maturity character phenotype, and has the advantages of simplicity in operation, high efficiency, rapidness, low cost and the like.
Description
Technical Field
The invention relates to a group of molecular marker primer combinations for rapidly identifying the mature-period characters of waxberry fruits and application thereof, belonging to the field of molecular biology.
Background
Waxberry (Myrica rubra Sieb. and Zucc.) is a special fruit tree produced in south China, and the fruit is rich in anthocyanin, has high nutritional value and is suitable for fresh eating and processing. According to the characteristics of the maturation stage of the waxberry fruit, the waxberry fruit can be divided into three categories of early, middle and late. However, for the main production of red bayberries in China, Zhejiang province, the current red bayberry cultivars are too single in structure, mainly eat fresh food, have mature periods mainly concentrated from the first ten days to the last ten days in 6 months, have extremely short marketing periods, are concentrated on marketing to cause market saturation and price reduction, are difficult to preserve fruits, have excessive rain in the healthy plum rain season of the mature periods, cause industrial bottlenecks of low commercial fruit rate and low high-quality fruit rate, and are impacted by the red bayberries at other places, so that the industrial development is limited. Therefore, the method takes the market as the guide, improves the variety and optimizes the variety structure, realizes the early maturation period, and has extremely important significance for improving the economic benefit of the current waxberry production.
In addition, the waxberry has a long childhood period (more than 8 years after the seedling propagation and field planting), the seedling is in a seedling stage, the fruit maturation stage is unknown at the early and late stages, and no other characteristics can clearly indicate the fruit maturation stage types in the late development stage and the adult stage, so that the directional breeding process of the waxberry is delayed. In view of this, the method of combining molecular biology technical theory and breeding practice is adopted to realize the directional transformation of the single character of the waxberry, and the method is an effective way for directional breeding of the waxberry in the future. The development of modern biotechnology provides an effective method, namely DNA molecular marker, for the early prediction and identification of the fruit maturation stage characters of the waxberries, and the early identification of the fruit maturation stage characters of the waxberry breeding material is completely possible in a short time by utilizing the specific DNA molecular marker related to the fruit maturation stage characters, so that the breeding process can be greatly accelerated. Therefore, establishing an effective molecular marker combination and a detection method thereof, and realizing high-throughput typing detection-assisted screening become one of the problems to be solved by researchers in the field.
The waxberry fruit mature period character specific site is obtained by DNA re-sequencing results of main cultivars and excellent plant populations (more than 100 parts) of the current local waxberry and developing Genome wide association analysis (GWAS), and the identification accuracy is more reliable through the group verification of large-sample waxberry varieties.
Disclosure of Invention
The invention aims to provide a group of molecular marker primer combinations for rapidly identifying the mature-period traits of waxberry fruits.
In order to achieve the purpose, the invention adopts the technical scheme that: a group of molecular marker primer combinations for rapidly identifying the mature-period traits of waxberry fruits comprises BS8.1-25849-F and BS8.1-25849-R, and the specific sequences are as follows:
BS8.1-25849-F：5’-GCCACCTACACCTACACCGTTAAGGGCAAAAACTA-3’，
BS8.1-25849-R：5’-GAGTCCAATATGAATGCAAGTTAACCAAG-3’。
the invention also discloses application of the molecular marker primer combination in rapid identification of the mature-period characters of the waxberry fruits.
The method comprises the following steps:
(1) primer synthesis: synthesizing the primer BS8.1-25849 of claim 1;
(2) extraction of DNA: extracting genome DNA of waxberry leaves;
(3) and (3) PCR amplification: performing PCR amplification by using the primers BS8.1-25849-F and BS8.1-25849-R synthesized in the step (1) based on the genome DNA of the waxberry leaves, and adding at least one sample DNA with determined waxberry fruit maturation stage characters in advance as an internal reference;
(4) detection and analysis of amplification products: and (3) carrying out genotype detection analysis on the PCR products, and if the PCR amplification products appear and are gathered with the internal reference sample into a group, determining the PCR amplification products as the same fruit maturity character type, and if no PCR amplification products appear, determining the PCR amplification products as other types.
The detailed steps are as follows:
(1) primer synthesis: the biotechnology company was entrusted with the synthesis of primers BS8.1-25849-F and BS 8.1-25849-R;
(2) extraction of DNA:
A. grinding about 0.2g of young leaves of the waxberries into powder by using liquid nitrogen, and putting the powder into a 2mL centrifuge tube;
B. extracting the sample genome DNA by using an Ezup Column type Plant genome DNA extraction Kit (EZ-10Spin Column Plant Genomic DNA Purification Kit, Shanghai Biotech engineering Co., Ltd.), and performing specific operation steps according to the instruction;
C. detecting 1-2 mul on 1.0% agarose gel, and diluting the DNA stock solution into working solution with the concentration of 20 ng/mul for later use;
(3) the PCR amplification reaction system is as follows: sample genomic DNA template 1.0. mu.L (20 ng/. mu.L), 2 XSG Fast qPCR Master Mix (Low Rox, Shanghai Biotech engineering Co., Ltd.) 5. mu.L, forward primer (10. mu.M) 0.5. mu.L, reverse primerPrimer pair (10. mu.M) 0.5. mu.L, ddH2Make up to 10 μ L of O.
(4) And (3) detecting and analyzing PCR amplification products: PCR amplification is a two-step procedure, i.e., 95 ℃ for 3min, (95 ℃ for 3s, 60 ℃ for 30s)28 cycles; after the PCR reaction is finished, the maturity characters of the sample fruits can be known directly by the automatic analysis result (Allelic hybridization Plot) of the ABI Q6 Flex real-time fluorescent quantitative PCR system platform Genotyping functional module without other processes; wherein, if PCR amplification products appear (1 bayberry germplasm sample determined as 'early-maturing' is added as an internal reference) and the bayberry germplasm samples and the internal reference samples are gathered into a group, the bayberry germplasm samples and the internal reference samples can be regarded as the same fruit maturation stage type; in addition, the PCR products are considered to be of other types if they do not occur.
Compared with the prior art, the invention has the beneficial effects that:
the fragment of the amplified product of the molecular marker primer combination BS8.1-25849 is only 84bp, and the method is particularly suitable for high-throughput typing detection platforms such as a fluorescent quantitative PCR instrument, and the PCR amplification in the application process is short in time consumption and only 28 in cycle number; only 1.5 hours are needed from the extraction of the DNA of the waxberry leaf sample to the final identification result; the whole process from PCR amplification to the acquisition of the final typing result data is closed tube operation, subsequent gel electrophoresis detection is not needed, and zero pollution is realized; meanwhile, the amount of the sample genome DNA template is only 20ng, and a typing detection result can be obtained, so that the method has the characteristics of high speed, high flux, high sensitivity and high accuracy.
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FIG. 1 shows the typing effect of the BS8.1-25849 primer of the DNA sample of the waxberry germplasm resource on a platform of a fluorescent quantitative PCR instrument. ("X" indicates that no PCR amplification signal was detected, and the remaining symbols indicate that a PCR amplification signal was detected)
Detailed Description
The present invention will be described more specifically with reference to the following examples and 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.
Example 1
A group of molecular marker primer combinations for identifying the mature-period characters of waxberry fruits and application thereof comprise the following detailed steps:
(1) primer synthesis: according to the sequence information in Table 1, primers BS8.1-25849 were synthesized by Competition Biotechnology engineering (Shanghai) Ltd;
table 1: sequence information of the BS8.1-25849 primer
(2) Extracting DNA of leaves of a waxberry germplasm resource sample to be detected:
A. grinding about 0.2g of young leaves of the waxberries into powder by using liquid nitrogen, and putting the powder into a 2mL centrifuge tube;
B. extracting the sample genome DNA by using an Ezup Column type Plant genome DNA extraction Kit (EZ-10Spin Column Plant Genomic DNA Purification Kit, Shanghai Biotech engineering Co., Ltd.), and performing specific operation steps according to the instruction;
C. detecting 1-2 mul on 1.0% agarose gel, and diluting the DNA stock solution into working solution with the concentration of 20 ng/mul for later use;
(3) the PCR amplification reaction system is as follows: sample genomic DNA template 1.0. mu.L (20 ng/. mu.L), 2 XSG Fast qPCR Master Mix (Low Rox, Shanghai Biotech engineering Co., Ltd.) 5. mu.L, forward primer (10. mu.M) 0.5. mu.L, reverse primer (10. mu.M) 0.5. mu.L, ddH2Make up to 10 μ L of O.
(4) And (3) detecting and analyzing PCR amplification products: PCR amplification is a two-step procedure, i.e., 95 ℃ for 3min, (95 ℃ for 3s, 60 ℃ for 30s)28 cycles; after the PCR reaction is finished, the maturity characters of the sample fruits can be known directly by the automatic analysis result (Allelic hybridization Plot) of the ABI Q6 Flex real-time fluorescent quantitative PCR system platform Genotyping functional module without other processes; wherein, if PCR amplification products appear (1 waxberry germplasm sample DNA which is determined that the fruit maturity character is a type of 'precocity' is added in advance as an internal reference) and are gathered with the internal reference sample into a group, the waxberry germplasm sample DNA can be regarded as the same type and is positive, and the waxberry germplasm sample DNA is marked by a plus sign to preliminarily speculate as a precocity variety; if no PCR amplification product appears or other genotypes are all considered as other types and are negative, the mark is marked with a mark, and the variety is preliminarily presumed to be a medium-maturing or late-maturing variety. Finally, the table is used for recording the fruit maturation stage character identification results (table 2) of all waxberry germplasm samples, the actual fruit maturation stage characters (the waxberry according to the NY/T2761-2015 plant new variety specificity, consistency and stability test guideline) are determined through subsequent observation in the fruiting period, 10 waxberry germplasm fruit maturation stage character identification errors exist, therefore, the final waxberry fruit maturation stage character identification accuracy is about 89.58%, and the specific typing effect is shown in figure 1. Meanwhile, the primer can be prepared into a kit or a biological preparation for the early auxiliary identification of the mature-period character of the waxberry fruit.
In the example, the 96-hole heating module of the fluorescence quantitative platform can be replaced by 384 holes, a fluid chip and the like as required so as to meet the requirement of higher-flux typing detection.
TABLE 2 identification of fruit maturity traits of 96 waxberry germplasm resources used in the examples of the present invention
Note: the typing result graph is shown as "+" for the samples gathered from the internal reference of the red bayberry of "early ripening", and the rest samples are shown as "-".
Claims (4)
1. A group of molecular marker primer combinations for rapidly identifying the mature-period traits of waxberry fruits is characterized in that the primer combinations comprise BS8.1-25849-F and BS8.1-25849-R, and the specific sequences are as follows:
BS8.1-25849-F：5’-GCCACCTACACCTACACCGTTAAGGGCAAAAACTA-3’，
BS8.1-25849-R：5’-GAGTCCAATATGAATGCAAGTTAACCAAG-3’。
2. the application of the molecular marker primer combination of claim 1 in rapidly identifying whether the waxberry fruit mature period trait is an early maturing type.
3. A method for rapidly identifying the mature-period character of a waxberry fruit comprises the following steps:
(1) primer synthesis: synthesizing the primer combination of claim 1, BS8.1-25849-F and BS 8.1-25849-R;
(2) extraction of DNA: extracting genome DNA in a waxberry sample to be detected;
(3) and (3) PCR amplification: based on the waxberry genomic DNA, carrying out PCR amplification by using primers BS8.1-25849-F and BS8.1-25849-R, and adding at least one sample genomic DNA for determining that the maturation stage character of the waxberry fruit is an early-maturing variety as an internal reference in advance;
(4) detection and analysis of amplification products: and (3) carrying out genotype detection and analysis by using a fluorescent quantitative PCR instrument, wherein if a PCR amplification product appears and is gathered with the internal reference sample into a group, the fruit is considered to be of a type that the fruit mature period character is premature, and if no PCR amplification product appears, the fruit is considered to be of another type.
4. The method of claim 3, wherein: the PCR amplification system in the step (3) is as follows: 1.0. mu.L of sample genomic DNA template at 20 ng/. mu.L, 2 XSG Fast qPCR Master Mix 5. mu.L, 0.5. mu.L of 10. mu.M BS8.1-25849-F primer, 0.5. mu.L of 10. mu.M BS8.1-25849-R primer, ddH2O is complemented to 10 mu L; the amplification PCR procedure was as follows: 95 ℃ for 3min, then 28 cycles of reaction were run, each cycle being 95 ℃ for 3s, 60 ℃ for 30 s.
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