CN105624307B - Microsatellite primers and identification method for identifying crassostrea hongkongensis, crassostrea gigas and crassostrea pacificus and hybrid species thereof - Google Patents
Microsatellite primers and identification method for identifying crassostrea hongkongensis, crassostrea gigas and crassostrea pacificus and hybrid species thereof Download PDFInfo
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Abstract
The invention discloses a microsatellite primer and an identification method for identifying crassostrea hongkongensis, crassostrea gigas and pacific crassostrea and hybrid species thereof. The microsatellite primer comprises: 5'-CGACTGGTGGGAGTTTCTGAC-3' is used as a reference material; 5'-GCCGCTTCTATCTCCTTTGC-3' is added. Because the external form of the oysters often changes greatly along with different living environments, the oysters are difficult to identify simply by relying on morphological characteristics; oysters at the larval stage are more difficult to distinguish based on morphological features. The microsatellite primer disclosed by the invention can be used for quickly and accurately identifying individual Crassostrea hongkongensis, Crassostrea gigas and Crassostrea pacifica by adopting the identification method disclosed by the invention, has potential application value in individual identification of hybrid among the Crassostrea hongkongensis, the Crassostrea gigas and the Crassostrea pacifica, and has the advantages of simplicity, visual, accurate and effective result, no influence of environment and development period, low cost and the like.
Description
The technical field is as follows:
the invention belongs to the field of shellfish molecular markers and species identification in the aquatic biotechnology, and particularly relates to a microsatellite primer and an identification method for identifying crassostrea hongkongensis, crassostrea gigas and pacific crassostrea and hybrid species thereof.
Background art:
microsatellite DNA, also known as Short Tandem Repeats (STRs), simple repeat sequences (SSRs), and Simple Sequence Length Polymorphisms (SSLP), refers to nucleotide sequences composed of 1-6 nucleotides in tandem as a unit in the genome. In recent years, microsatellite markers have been widely used for species identification and hybrid analysis due to their advantages such as simplicity and high efficiency.
The Concha Ostreae belongs to the order of mollusca, Bivalvia, Pteriales, and Ostreidae, and is in the group of globularity distribution; the oyster meat is delicious in taste and rich in various amino acids essential to human bodies, is an important marine organism resource, is one of the most important mariculture economic groups in the world, and the total culture yield and the unit area yield of the oyster meat are in the top of all shellfish culture types. Oysters are also the economic shellfishes with the largest yield in China, wherein the crassostrea hongkongensis, crassostrea gigas and pacific oysters with higher economic value are artificially cultured in China. Crassostrea hongkongensis (Crassostrea hongkongensis) likes to live in the place near the river mouth or near the river mouth where fresh water is injected, and is mainly distributed in Zhejiang, Fujian, Guangdong, Guangxi and Hainan, and the annual output is more than 100 million tons. The oyster meat has large meat column, white and tender meat quality and delicious taste, and the market price of the oyster meat far exceeds that of other oyster varieties. Is an oyster group with extremely high economic value and growing in warm and near shore, and is a main economic species for breeding in south China. Crassostera gigas (Crassostreariakensis) is distributed in the south and north coastal areas of China, mainly concentrated in the Guangdong and Guangxi areas, and distributed in the south and north low-salt seas of China. The meat flavor is delicious, the nutrition is rich, the method is an important seawater culture economic shellfish in southern provinces of China, and the history of culture is nearly two hundred years. Pacific oysters (Crassostra gigas) are distributed in the north of the Yangtze river in China, are cultivated in the world, are mainly concentrated in Liaoning, Shandong and other places in China, and have annual output of nearly 80 million tons. The meat has the advantages of high growth speed, strong environmental adaptability and delicious meat taste, and is deeply favored by consumers. The external form of oysters is very changed with different living environments, and is difficult to identify simply by means of morphological features. The oyster soft body has small differences in the anatomical structure, chromosome number and karyotype, and provides little classification evidence. In recent years, molecular biology based on differences between mitochondrial genes such as COI and 16S rDNA and ITS sequences is used for identification of the three genes, but the differences between the gene sequences are small, and the gene sequences are difficult to distinguish by simple methods such as electrophoresis; HRM methods, while simple, require a relatively large instrument support; and the sequencing test has longer period and higher cost. A simple and efficient method for carrying out large-scale species identification on individual Crassostrea hongkongensis, Crassostrea gigas and Crassostrea pacifica is urgently needed.
In addition, with the increasing demand of oyster breeding industry for new and superior species, interspecific hybridization research is continuously carried out among oysters, and researchers successively develop the hybridization combination of hong Kong giant oysters, clear oysters and pacific oysters, and prove that sperm and eggs of part of interspecific hybridized oysters can be combined and normally develop. Genetic identification of the obtained filial generation is necessary because gynogenesis, androgenesis, haploid development and the like often occur in the interspecific hybrid of oysters. There is an urgent need for a simple method for molecular identification of hybrid individuals of crassostrea hongkongensis, crassostrea gigas, and crassostrea pacificus.
The invention content is as follows:
the invention aims to provide a microsatellite primer and an identification method for simply, efficiently and accurately identifying crassostrea hongkongensis, crassostrea gigas and pacific crassostrea pacifica and hybrid species thereof.
The invention screens 1 pair of microsatellite (SSR) primers from 150 pairs of crassostrea hongkongensis microsatellite primers, performs PCR amplification on genome DNA of 90 individuals of crassostrea hongkongensis, crassostrea gigas and crassostrea pacifica, can simply and efficiently perform species identification through an amplification map, and has potential application value for identifying individual hybrids among the crassostrea hongkongensis, the crassostressostressostressostressostressostressostressostressostressostressoides and the crassostressostressostressostressostressostressostresso.
The invention relates to a microsatellite primer for identifying crassostrea hongkongensis, crassostrea gigas, pacific crassostrea and hybrid species thereof, which is characterized in that the microsatellite primer (SSR primer Ch409) is as follows:
5'-CGACTGGTGGGAGTTTCTGAC-3' (shown as SEQ ID NO. 1);
5'-GCCGCTTCTATCTCCTTTGC-3' (shown as SEQ ID NO. 2).
The invention relates to an identification method of crassostrea hongkongensis, crassostrea gigas, pacific crassostrea and hybrids thereof, which is characterized by comprising the following steps:
extracting the genome DNA of the oyster sample, using the genome DNA as a template, performing PCR amplification by using the microsatellite primer, performing gel electrophoresis on a PCR product, and then performing typing judgment;
the specific band range of the crassostrea hongkongensis generated by the SSR primer Ch409 is 308 bp-347 bp, the specific band range of the crassostrea hongkongensis is 362 bp-422 bp, the specific band range of the pacific crassostrea pacifica is 206 bp-242 bp, and allelic gene overlapping regions do not exist among the specific band range, so that individuals of the three crassostressostressostrea hongkongensis can be obviously distinguished. If 1-2 bands appear in the interval of 308 bp-347 bp of the PCR product, the oyster sample is crassostrea hongkongensis, 1 band appears to indicate that the individual is homozygote, and 2 bands appear to indicate that the individual is heterozygote; if 1-2 stripes appear in the interval of 362 bp-422 bp, the oyster sample is a clear oyster, the individual is a homozygote if 1 stripe appears, and the individual is a heterozygote if 2 stripes appear; if 1-2 bands appear in the range of 206 bp-242 bp, the oyster sample is Pacific oyster, 1 band appears to indicate that the individual is homozygote, and 2 bands appear to indicate that the individual is heterozygote; if 1 band respectively appears in the interval of 308 bp-347 bp and the interval of 362 bp-422 bp of the PCR product, namely the individual simultaneously possessing 1 specific band of the parent is a filial generation of the crassostrea hongkongensis and the crassostrea gigas, and any band lacking the crassostrea gigas is a false hybrid; if 1 band respectively appears in the 308 bp-347 bp interval and the 206 bp-242 bp interval of the PCR product, namely an individual simultaneously possessing 1 specific band of a parent is a first filial generation of the hybrid of the crassostrea hongkongensis and the pacific oyster, and any band lacking in the first filial generation is a false hybrid; if 1 band respectively appears in 362 bp-422 bp interval and 206 bp-242 bp interval of PCR product, that is, the individual possessing 1 specific band of parent is the first filial generation of the hybrid of Ming oyster and Pacific oyster, and any one of the bands is false hybrid.
The preferable PCR reaction system for PCR amplification by using the microsatellite primer and taking the genome DNA as a template is as follows: 50ng of DNA template, 10mM of Buffer, 0.2mM of each of microsatellite primer F and microsatellite primer R, 0.2mM of dNTP and Mg2+2.0Mm, 1U Taq DNA polymerase, and 25 μ L sterilized water; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; extension at 72 ℃ for 10 min.
The invention screens out a pair of microsatellite primers which can be commonly used in Crassostrea hongkongensis, Crassostrea gigas and Crassostrea pacifica from 150 pairs of Crassostrea hongkongensis microsatellite primers, can distinguish three types of oysters according to the size of an amplification band, and has clear band type, good repeatability and strong reliability: ch409 and the individual identification of the hybrid among the three also has potential application value.
Because the external form of the oysters often changes greatly along with different living environments, the oysters are difficult to identify simply by relying on morphological characteristics; oysters at the larval stage are more difficult to distinguish based on morphological features. The microsatellite primer disclosed by the invention can be used for quickly and accurately identifying individual Crassostrea hongkongensis, Crassostrea gigas and Crassostrea pacifica by adopting the identification method disclosed by the invention, has potential application value in individual identification of hybrid among the Crassostrea hongkongensis, the Crassostrea gigas and the Crassostrea pacifica, and has the advantages of simplicity, visual, accurate and effective result, no influence of environment and development period, low cost and the like.
Description of the drawings:
FIG. 1 is the comparison graph of the electrophoretic staining of microsatellite primer Ch409 among Pacific oyster, Crassostrea gigas and Crassostrea hongkongensis; 1-9 are Pacific oyster individuals, wherein 2, 3, 4, 5, 6, 7 and 8 are heterozygotes, and the others are homozygotes, 10-19 are clear oyster individuals, wherein 11, 12, 14, 16, 17 and 19 are heterozygotes, and the others are homozygotes, and 20-29 are hong Kong giant oyster individuals, wherein 20, 21, 25, 26, 27 and 29 are heterozygotes, and the others are homozygotes.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
the method comprises the following steps of extracting genome DNA of 90 individuals of Crassostrea hongkongensis, Crassostrea gigas and Crassostrea pacifica by adopting a phenol chloroform extraction method:
the adductor muscle is cut into pieces, the pieces are put into a 1.5mL centrifuge tube after water is absorbed by clean filter paper, 400 mu l of lysate and 10 mu l of proteinase K (10mg/mL) are added, the mixture is evenly mixed on a shaker, and the mixture is digested in water bath at 55 ℃ for 3-5 h until the lysate is clear. The mixture was extracted three times with an equal volume of saturated phenol (200. mu.L), chloroform/isoamyl alcohol (24:1) (200. mu.L). The DNA was precipitated with 1mL of absolute ethanol, washed with 70% ethanol, air-dried at room temperature, and dissolved in 100. mu.L of ultrapure water. DNA concentration and purity were determined by UV spectrophotometer and DNA integrity was checked by 1% agarose gel electrophoresis. Thus, genomic DNAs of Crassostrea hongkongensis, Crassostrea gigas and Crassostrea pacifica were obtained.
Taking the extracted genome DNA of the three oysters as a template to carry out PCR amplification reaction:
the total volume of the PCR reaction system is 25 muL, wherein the components and the final concentration are respectively as follows: 50ng of DNA template, 10mM of Buffer, 0.2mM of each of the primers (microsatellite primer Ch409 sequences F and R), 0.2mM of dNTP, and Mg2+2.0Mm, 1U Taq DNA polymerase, made up to 25. mu.L with sterile water.
Wherein the sequence of the microsatellite primer Ch409 is as follows:
5'-CGACTGGTGGGAGTTTCTGAC-3' (shown as SEQ ID NO. 1);
5'-GCCGCTTCTATCTCCTTTGC-3' (shown as SEQ ID NO. 2).
The reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and repeating the three steps from denaturation to extension for 30 times; extension at 72 ℃ for 10 min.
After the PCR reaction is finished, 0.5 mu L of amplification product is subjected to 8% non-denaturing macroacrylamide gel electrophoresis and 22V voltage-stabilizing electrophoresis for 4 hours, after the electrophoresis is finished, the gel is taken down from a glass plate and is placed in a tray, the tray is washed twice by deionized water, and then 0.1% AgNO is added3Solution (AgNO)31g, 37% Formaldehyde 1.5mL, dH2O1000 mL) for 10 min. Adding deionized water, washing once again, adding color developing solution (2% NaOH, 0.4% formaldehyde, 0.2% NaS)2O3、dH2O1000 mL) until the bands are clear, scanned under a UMAX scanner, and then typed. The specific result is shown in FIG. 1, and it can be seen from FIG. 1 that 1 band appears in the 308 bp-347 bp interval for the homozygote of Crassostrea hongkongensis, and 2 bands appear in the 308 bp-347 bp interval for the heterozygote of Crassostrea hongkongensis; the homozygote with oyster has 1 band in the interval of 362 bp-422 bp, and the heterozygote with oyster has 1 band in the interval of 362 bp-422 bp2 strips; the homozygous oyster Pacific oyster has 1 band in 206 bp-242 bp interval, and the heterozygous oyster Pacific oyster has 2 bands in 206 bp-242 bp interval.
Therefore, if the sample is an oyster sample to be detected, extracting DNA of the sample according to the method, taking the sample as a template, performing PCR amplification by using a microsatellite primer Ch409, performing non-denaturing macroacrylamide gel electrophoresis on a PCR product, then after dyeing, parting, and comparing the result of the electrophoresis with a standard map according to the result of the electrophoresis, wherein if 1-2 bands appear in a 308 bp-347 bp interval, the DNA sample is a hong Kong macrooyster, 1 band appears to indicate that the individual is a homozygote, and 2 bands appear to indicate that the individual is a heterozygote; if 1-2 bands appear in the interval of 362 bp-422 bp, the DNA sample is a clear oyster, 1 band appears to indicate that the individual is a homozygote, and 2 bands appear to indicate that the individual is a heterozygote; if 1-2 bands appear in the interval of 206 bp-242 bp, the DNA sample is Pacific oyster, 1 band appears to indicate that the individual is homozygote, and 2 bands appear to indicate that the individual is heterozygote. The potential application method of the marker in identifying the hybrid between crassostrea hongkongensis, crassostrea gigas and crassostrea pacificus is as follows: only 1 band respectively appears in the interval of 308 bp-347 bp and the interval of 362 bp-422 bp of the sample, namely an individual simultaneously possessing 1 specific band of a parent is a filial generation of true hong Kong giant oyster and clear oyster, and any band lacking the hybrid is a false hybrid; only 1 band respectively appears in the 308 bp-347 bp interval and the 206 bp-242 bp interval of the sample, namely an individual simultaneously possessing 1 specific band of a parent is a first filial generation of the hybridization of the real hong Kong giant oyster and the Pacific oyster, and any band lacking the first filial generation is a false hybrid; only 1 band respectively appears in the 362 bp-422 bp interval and the 206 bp-242 bp interval of the sample, namely an individual simultaneously possessing 1 specific band of a parent is a true first filial generation of the hybrid of the clear oyster and the pacific oyster, and any band lacking the false hybrid is a false hybrid.
Claims (2)
1. An identification method of crassostrea hongkongensis, crassostrea gigas, pacific crassostrea gigas and hybrid species thereof is characterized by comprising the following steps:
extracting the genome DNA of the oyster sample, using the genome DNA as a template, performing PCR amplification by using a microsatellite primer, performing gel electrophoresis on a PCR product, and then performing typing judgment;
if 1-2 bands appear in the interval of 308 bp-347 bp of the PCR product, the oyster sample is crassostrea hongkongensis, 1 band appears to indicate that the individual is homozygote, and 2 bands appear to indicate that the individual is heterozygote; if 1-2 stripes appear in the interval of 362 bp-422 bp, the oyster sample is a clear oyster, the individual is a homozygote if 1 stripe appears, and the individual is a heterozygote if 2 stripes appear; if 1-2 bands appear in the range of 206 bp-242 bp, the oyster sample is Pacific oyster, 1 band appears to indicate that the individual is homozygote, and 2 bands appear to indicate that the individual is heterozygote; if 1 band respectively appears in the interval of 308 bp-347 bp and the interval of 362 bp-422 bp of the PCR product, namely, the individual simultaneously possessing 1 specific band of the parent is a first filial generation of the crassostrea hongkongensis and the crassostrea gigas; if 1 band respectively appears in the 308 bp-347 bp interval and the 206 bp-242 bp interval of the PCR product, namely, the individual simultaneously possessing 1 specific band of the parent is a first filial generation of the crassostrea hongkongensis and the pacific oyster; if 1 band respectively appears in the interval of 362 bp-422 bp and the interval of 206 bp-242 bp of the PCR product, namely, the individual simultaneously possessing 1 specific band of the parent is a first filial generation of the oyster with Ming oyster and Pacific oyster;
the microsatellite primer comprises:
F:5’-CGACTGGTGGGAGTTTCTGAC-3’；
R:5’-GCCGCTTCTATCTCCTTTGC-3’。
2. the method of claim 1, wherein the genomic DNA is used as a template, and the microsatellite primer of claim 1 is used for PCR amplification, and the PCR reaction system is usedComprises the following steps: 50ng of DNA template, 10mM of Buffer, 0.2mM of each of microsatellite primer F and microsatellite primer R, 0.2mM of dNTP and Mg2+2.0Mm, 1U Taq DNA polymerase, and 25 μ L sterilized water; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; extension at 72 ℃ for 10 min.
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| CN108103211B (en) * | 2018-01-31 | 2021-03-05 | 中国科学院南海海洋研究所 | Microsatellite primer and identification method for tridacna elongata and tridacna nova |
| CN108950016B (en) * | 2018-08-08 | 2021-07-16 | 中国科学院南海海洋研究所 | Microsatellite primer, kit and identification method for identifying crassostrea hongkongensis, crassostrea gigas and hybridized first filial generation thereof and application |
| CN110592230B (en) * | 2019-09-03 | 2021-09-21 | 中国科学院南海海洋研究所 | Microsatellite primer, kit and identification method for identifying crassostrea hongkongensis and crassostrea viticola and first filial generation thereof |
| CN110760595B (en) * | 2019-11-06 | 2021-07-27 | 中国科学院南海海洋研究所 | Identification primer and method suitable for tridacna species common in trioyster and south China sea and hybridized species of tridacna species |
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| CN103907529A (en) * | 2013-12-18 | 2014-07-09 | 中国科学院南海海洋研究所 | Breeding method capable of fixing oyster distant hybrid backcross advantages |
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| Phenotypic trait analysis of diploid and triploid hybrids from female Crassostrea hongkongensis × male C. gigas;Yuehuan Zhang等;《Aquaculture》;20140902;第434卷;307-314 * |
| 应用微卫星标记鉴别施氏鲟、达氏鳇及其杂交子代;刘洋等;《水产学杂志》;20150228;第28卷(第1期);18-23 * |
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