CN108642187A - Inbred male Macrobrachium rosenbergii analysis of genetic diversity method - Google Patents
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Abstract
The invention discloses inbred male Macrobrachium rosenbergii analysis of genetic diversity methods, include the following steps：Step 1, the inbred male Macrobrachium rosenbergii sample for acquiring various trait, and extract DNA；Step 2, selection microsatellite marker combination, and design Specific PCR primers group and expanded；Step 3, evaluation PCR product number, observe allelic gene typing and size；Number of alleles A, the allele of each male shrimp of step 4, record replicate number G, observation heterozygosity Ho, it is expected heterozygosity He, the precision runout value P based on Hardy's Weinberg equilibrium law, and analyze microsatellite locus difference；Gene flow Nm is calculated according to population gene frequency parameter；Gene spacing is measured with to gene Nei Y-factor method Ys.The present invention is based on microsatellite analysis methods, provide inbred male Macrobrachium rosenbergii analysis of genetic diversity method, are very suitable for studying different close relative's hero shrimp group genetic structures, are the high-quality male parent of Macrobrachium rosenbergii.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a genetic diversity analysis method for inbreeding male macrobrachium rosenbergii.
Background
Macrobrachium rosenbergii (De Man 1879) is a large freshwater shrimp, and gradually becomes an important aquaculture species in China, southeast Asia and even the world due to the characteristics of wide water quality change bearing range, easy cultivation, tender and smooth meat quality, high commercial value and the like. Currently, the increasing yield of macrobrachium rosenbergii still cannot meet the increasing consumption and market demands, so that the factors for limiting the yield improvement of the macrobrachium rosenbergii need to be further broken through to greatly improve the yield. Since male shrimps grow faster than females, have large mature heads and high culture yield [1], the method mainly solves the limiting factors causing sex and individual difference in seedling culture and breeding.
People research the genetic diversity of the macrobrachium rosenbergii, such as the analysis of the genetic diversity of different populations. The different types of male shrimps are produced for the following reasons: (1) inherent factors of individuals such as genetic differences and age of metamorphosis; (2) environmental factors such as limited space and resources cause competition; (3) and the social factors inside the population such as the level of the population status, the territory and the like. In recent years, the modern molecular marker technology of microsatellites is widely used for biological breeding and genetic research because of solving the problem of subtle genetic difference which cannot be found by allelic enzyme technology, and has the advantages of high polymorphism, co-dominant inheritance, high repeatability, easiness in detection and the like. At present, the macrobrachium rosenbergii is subjected to autonomous or artificial long-term inbreeding to cause germplasm degradation, and identification and differentiation are urgently required. In addition, the male morphological inheritance of the macrobrachium rosenbergii is uncontrollable, so that the genetic difference accuracy evaluation of the male parent is insufficient, and the fine breeding is seriously influenced, so that the individual growth genetic diversity of the inbred mating morphological male shrimps is found by utilizing the microsatellite marking technology, which is favorable for solving the practical problem.
At present, scholars at home and abroad use a microsatellite molecular marker technology to research the genetic diversity of macrobrachium rosenbergii, but mainly research the genetic diversity of different populations, and obtain less heritage information between the same sex obtained by natural or artificial inbreeding. The genetic diversity of wild macrobrachium rosenbergii species is researched by using 6 microsatellite loci, and the average allelic factor of each locus is highly diversified, however, the genetic diversity of the species is only obtained by the allelic factor of each microsatellite marker locus is limited.
Disclosure of Invention
In order to solve the defects of the technology, the invention provides a genetic diversity analysis method for inbreeding male macrobrachium rosenbergii.
In order to solve the technical problems, the invention adopts the technical scheme that:
the genetic diversity analysis method for the inbred male macrobrachium rosenbergii comprises the following steps:
step 1, collecting inbred male macrobrachium rosenbergii samples with different properties, and extracting DNA;
step 2, selecting a microsatellite marker combination, and designing a specific PCR primer group for amplification;
step 3, evaluating the number of PCR products, and observing the allelic gene type and size;
step 4, recording the allelic base factor A and the allelic gene copy number G of each male shrimp, observing the heterozygosity Ho and the expected heterozygosity He, and analyzing the microsatellite locus difference based on the exact deviation value P of the Harvard-Weinberg equilibrium law; calculating the gene flow Nm according to the population allele frequency parameter; determining the gene spacing by using a gene Nei coefficient method;
wherein, the microsatellite marker combination in the step 2 comprises the following microsatellite markers or complementary sequences of nucleotide sequences thereof: a microsatellite marker 1, a microsatellite marker 2, a microsatellite marker 3, a microsatellite marker 4, a microsatellite marker 5, a microsatellite marker 6 and a microsatellite marker 7; wherein,
the nucleotide sequence of the microsatellite marker 1 is shown as SEQ ID NO. 1;
the nucleotide sequence of the microsatellite marker 2 is shown as SEQ ID NO. 2;
the nucleotide sequence of the microsatellite marker 3 is shown as SEQ ID NO. 3;
the nucleotide sequence of the microsatellite marker 4 is shown as SEQ ID NO. 4;
the nucleotide sequence of the microsatellite marker 5 is shown as SEQ ID NO. 5;
the nucleotide sequence of the microsatellite marker 6 is shown as SEQ ID NO. 6;
the nucleotide sequence of the microsatellite marker 7 is shown as SEQ ID NO. 7.
Further, the specific PCR primer set in step 2 comprises:
the nucleotide sequence of the specific primer pair of the microsatellite marker 1 is shown as SEQ ID NO.8 and SEQ ID NO. 9;
the nucleotide sequence of the specific primer pair of the microsatellite marker 2 is shown as SEQ ID NO.10 and SEQ ID NO. 11;
the nucleotide sequence of the specific primer pair of the microsatellite marker 3 is shown as SEQ ID NO.12 and SEQ ID NO. 13;
the nucleotide sequence of the specific primer pair of the microsatellite marker 4 is shown as SEQ ID NO.14 and SEQ ID NO. 15;
the nucleotide sequence of the specific primer pair of the microsatellite marker 5 is shown as SEQ ID NO.16 and SEQ ID NO. 17;
the nucleotide sequence of the specific primer pair of the microsatellite marker 6 is shown as SEQ ID NO.18 and SEQ ID NO. 19;
the nucleotide sequence of the specific primer pair of the microsatellite marker 7 is shown as SEQ ID NO.20 and SEQ ID NO. 21.
Further, the traits of the inbred male macrobrachium rosenbergii sample include blue brachial type, orange brachial type and small idiotype.
Further, the degree of genetic differentiation is analyzed using F statistics, such as the population allele frequency parameter FIS、FITAnd FSTIn which F isISAverage inbred coefficient, F, for local populationITAverage inbred coefficient for the entire population and FSTCalculating the Nm of gene flow according to the Wright method for the average inbreeding coefficient among related local populations：Nm＝FST0.25(1-FST)/FST.FIS。
The invention provides a genetic diversity analysis method for inbreeding male macrobrachium rosenbergii based on a microsatellite analysis method, and the result shows that all microsatellite loci show height difference, the microsatellite marker combination is easy to identify and highly polymorphic, is very suitable for researching different inbreeding male shrimp group genetic structures, provides basic genetic information for breeding of high-quality male parents of the macrobrachium rosenbergii, has certain significance for regulating and controlling genetic development of the male macrobrachium rosenbergii, provides genetic information for breeding of the male parents, and provides a basis for improving the fry quality.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
[ example 1]
1.1 sample Collection
200 adult male macrobrachium rosenbergii bred by the Guangxi population inbreeding is provided by the national fine breed center of macrobrachium rosenbergii (Guangxi Nanning), bred in a fine breed center laboratory culture pond (5 multiplied by 1.8m) for 4 months, and classified into three different morphologic types of male shrimps, namely blue brachium longarm type (BCM), orange brachium longarm type (OCM) and small individual type (SM) based on color, behavior, growth characteristics and zigzag chela. 150 pieces of 50 pieces of 3 morphological types are collected, and the body weights of BCM, OCM and SM are 14.48 +/-1.05 g, 10.04 +/-1.15 g and 5.86 +/-0.66 g respectively.
1.2DNA extraction
Extracting the genomic DNA of abdominal muscle tissues of the macrobrachium rosenbergii according to the steps of a Wizard genomic DNA purification kit (purchased from Promega corporation, USA), measuring the light absorption values (OD) of an extracting solution at 260nm and 280nm by using an ultraviolet spectrophotometer, measuring the DNA concentration and purity according to the ratio of OD260nm/OD280nm, and keeping the solution at the temperature of-20 ℃ after fixing the volume by using double-distilled liquid to 1: 40.
1.3PCR amplification
Preparation of PCR reaction solution, total 50. mu.L: 10.0. mu.L of 5 XPCR buffer, 5.0. mu.L of MgCl2(2.5mM), 1.0. mu.L dNTP (0.2mM), 2.0. mu.L of upstream and downstream primers (0.4. mu.M), 24.75. mu.L of sterile ddH2O20.25 mu L of Taq polymerase, 2.0 mu L of LDNA template and sterile deionized water.
The DAN microsatellite primer design is shown in Table 1.
TABLE 1 Macrobrachium rosenbergii microsatellite locus characterization
F: forward direction, R: reverse direction
The PCR reaction program is: pre-denaturation at 95 ℃ for 3min, 40 cycles of denaturation at 95 ℃ for 30s, reaction at the actual annealing temperature (48-60 ℃) of each pair of primers for 30s, extension at 72 ℃ for 30s, final extension at 72 ℃ for 40s, and heat preservation at 4 ℃.
After the PCR amplification product is detected by 2.5% agarose gel electrophoresis, red gel is stained with red, and ultraviolet color development is carried out. Then, the number of PCR products was evaluated by polyacrylamide electrophoresis, the reaction solution was added to a gel system containing 1 XTBE buffer, run for 8min under 75V, and then observed under a gel imaging system (AlphaInotech, USA), and allele typing and size were observed with a 20bp extended DNA ladder (Linza, USA) as a reference standard visual method.
1.4 data analysis
Recording the allele factor (A), allele copy number (G), observed heterozygosity (Ho), expected heterozygosity (He), exact deviation based on Hardy-Winberg equilibrium law for each male shrimpValues (P) and microsatellite locus differences were analyzed using ARLEQUIN 3.11. The degree of genetic differentiation is statistically analyzed using F, e.g. population allele frequency parameters (F)IS，FIT，FST) Gene flow (Nm) was calculated according to Wright method, formula: nm ═ FST0.25(1-FST)/FST.FIS. And determining the gene spacing by using a gene pair Nei coefficient method, and constructing a dendrogram by using POPGEN1.32 software based on a non-weighted pairing arithmetic mean method.
[ example 2 ] results and analysis
2.1 genetic diversity of the population
As can be seen from Table 2, the genetic information of the loci is polymorphic except for position 5 of SM, which is removed in all statistical analyses. The male shrimp populations of the 3 morphotypes showed high genetic differences, with allelic factors of 2.885, 3.762 and 2.627 for BCM, OCM, SM, respectively, and observed heterozygosity of 0.5561, 0.474 and 0.475, respectively. The exact deviation value (P) of the haben-wenberg equilibrium law was evaluated using the Markov chain method and subsequently corrected by Bonferroni multiple hypothesis experiments. The results show that most locus sites are significantly offset.
TABLE 2 genetic diversity of three morphotype male Macrobrachium rosenbergii satellite loci
G: gene copy number, a: allelic factor, Ho: observation of heterozygosity, He: desired heterozygosity, P: the Hardy-Weinberg equilibrium law is biased, - - -, indicates no polymorphism, a-indicates very significant, and a + indicates no significance.
2.2 sample genetic differentiation and relationships
As can be seen from table 3, the FST of the samples is 0.2877 on average, indicating that the differentiation of the samples shows significant difference. The mean value Nm of the sample gene stream is 0.7452, the Nm of the site 7 is 1.1074, the Nm of the site 5 is 1.10740.2044. F of all samplesITAnd FISThe values show significant differences (P)<0.05). As can be seen from Table 4, the maximum gene distance was 1.0369 for BCM and SM, and the minimum gene distance was 0.7497 for BCM and OCM.
TABLE 3 three groups of Macrobrachium rosenbergii microsatellite locus F statistical values
Nm: gene flow value
TABLE 4 Gene spacing based on Macrobrachium rosenbergii microsatellite locus Nei data
Discussion of 3
The microsatellite molecular marker technology is widely applied to the field of aquaculture as a most efficient neutral molecular marker tool. At present, scholars at home and abroad use a microsatellite molecular marker technology to research the genetic diversity of macrobrachium rosenbergii, but mainly research the genetic diversity of different populations, and obtain less heritage information between the same sex obtained by natural or artificial inbreeding. In the research, 3 closely related male shrimp groups are researched by taking easily-recognized and highly polymorphic macrobrachium rosenbergii microsatellite loci as a standard. The results show that all microsatellite loci display high diversity; in addition, BCM, OCM, SM contained 24, 28, 22 different alleles, respectively, indicating that the alleles in 3 groups of male shrimps had higher diversity and abundance. The genetic diversity of wild macrobrachium rosenbergii species is researched by using 6 microsatellite loci, and the average allelic factor of each locus is highly diversified, however, the genetic diversity of the species is only obtained by the allelic factor of each microsatellite marker locus is limited.
The degree of gene heterozygosity, also known as gene diversity, is the optimal parameter for indicating gene differences. As can be seen from the experimental results, the observed heterozygosity (Ho) mean was lower than the expected heterozygosity (He) mean in all the sampling groups, indicating a decrease in heterozygosity in male macrobrachium rosenbergii. The observed heterozygosity averages in the 3 samples ranged from 0.474 to 0.5561.
FISIndicating the degree of bias in the random pairing of genes, a positive value indicating significant deficit in heterozygosity, and a negative value indicating the presence of excess heterozygosity. In this study FISA value of-0.0189 indicates that the heterozygote was in excess and the excess was small. F at all sites except site 1 and site 2ISThe values are negative, indicating that these sites have an excess of heterozygotes, which may be due to null alleles, genotyping errors, inbreeding, or the Wahlund effect.
Chi fang (X)2) And likelihood (G)2) For testing significant deviations in genetic loci (P)<0.05) population of the Hardy-Winberg equilibrium law. In this study, all gene loci, except for the 3 gene loci of OCM, deviate from Harvard-Winberg equilibrium, probably due to the presence of null alleles. In addition, the intergenic distance of the 3 groups of samples ranged from 0.7497 to 1.0369, indicating that the 8 microsatellite markers have very high diversity, indicating that the influence of the environment on the genetic variation among the groups of samples is in a homodomain distribution. The maximum values of BCM and SM indicate the maximum distance between the BCM and the OCM genes, and the small distance between the BCM and the OCM genes indicates that the BCM and the OCM genes are genetically similar or homologous.
In recent years, researchers have studied the genetic diversity of macrobrachium rosenbergii breeding species and wild species in different countries or regions. Based on the same population inbreeding, 3 male shrimps with different morphologies are selected to develop genetic diversity research, and specific site genes causing 3 morphologic differences are unknown, wherein the differences can be caused by genetic factors, food sources, living spaces and other environmental factors, self internal population factors and the like. The significant differences between male macrobrachium rosenbergii are not only expressed in the morphology that accounts for their genetic potential, but also in the hepatic index and biochemical characteristics. The study results showed that after 4 months of culture, the average weight of BCM was 23.34 + -1.26 g, the average weight of OCM was 18.39 + -1.37 g, and the average weight of SM was 9.06 + -0.53 g, which accounted for 21%, 62.5%, 16.5% of the study sample population, respectively. Thus, they differ in morphology, anatomy and physiology.
The microsatellite molecular marker is more applied to the research of the genetic diversity of the macrobrachium rosenbergii population, the genetic information of inbred male shrimps is not reported, and the genetic diversity performance of the male macrobrachium rosenbergii is researched to provide genetic information for the male parent breeding and improve the seedling quality because the genetic form of the male macrobrachium rosenbergii is uncontrollable. As can be seen from the above discussion, the 3 types of male macrobrachium rosenbergii through the inbreeding have genetic difference, and the genetic difference has morphology and physiology, so the research result can provide basic genetic information for the breeding of the high-quality male parent of the macrobrachium rosenbergii and has certain significance for controlling the genetic development of the male macrobrachium rosenbergii.
The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above examples, and those skilled in the art may make variations, modifications, additions or substitutions within the technical scope of the present invention.
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<213> Artificial Sequence (Artificial Sequence)
<400>12
atttgcggaa ggatgtgttc 20
<210>13
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
tatgcttccg gctcgtatg 19
<210>14
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
tatcagcagc agcagagaag 20
<210>15
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
gtctgtttgt gcaggtggag 20
<210>16
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
ggcattccgt tgttgttg 18
<210>17
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
cgctcttgtt gtttcttctc 20
<210>18
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
atttcatccg gttttgttcc 20
<210>19
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
agctcggatc cactagtaac g 21
<210>20
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
ttgttatccg tccacaattc c 21
<210>21
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
attaccgcct ttgagtgagc 20
Claims (4)
1. The genetic diversity analysis method for the inbred male macrobrachium rosenbergii is characterized by comprising the following steps:
step 1, collecting inbred male macrobrachium rosenbergii samples with different properties, and extracting DNA;
step 2, selecting a microsatellite marker combination, and designing a specific PCR primer group for amplification;
step 3, evaluating the number of PCR products, and observing the allelic gene type and size;
step 4, recording the allelic base factor A and the allelic gene copy number G of each male shrimp, observing the heterozygosity Ho and the expected heterozygosity He, and analyzing the microsatellite locus difference based on the exact deviation value P of the Harvard-Weinberg equilibrium law; calculating the gene flow Nm according to the population allele frequency parameter; determining the gene spacing by using a gene Nei coefficient method;
wherein, the microsatellite marker combination in the step 2 comprises the following microsatellite markers or complementary sequences of nucleotide sequences thereof: a microsatellite marker 1, a microsatellite marker 2, a microsatellite marker 3, a microsatellite marker 4, a microsatellite marker 5, a microsatellite marker 6 and a microsatellite marker 7; wherein,
the nucleotide sequence of the microsatellite marker 1 is shown as SEQ ID NO. 1;
the nucleotide sequence of the microsatellite marker 2 is shown as SEQ ID NO. 2;
the nucleotide sequence of the microsatellite marker 3 is shown as SEQ ID NO. 3;
the nucleotide sequence of the microsatellite marker 4 is shown as SEQ ID NO. 4;
the nucleotide sequence of the microsatellite marker 5 is shown as SEQ ID NO. 5;
the nucleotide sequence of the microsatellite marker 6 is shown as SEQ ID NO. 6;
the nucleotide sequence of the microsatellite marker 7 is shown as SEQ ID NO. 7.
2. The method of claim 1, wherein: the specific PCR primer group in the step 2 comprises:
the nucleotide sequence of the specific primer pair of the microsatellite marker 1 is shown as SEQ ID NO.8 and SEQ ID NO. 9;
the nucleotide sequence of the specific primer pair of the microsatellite marker 2 is shown as SEQ ID NO.10 and SEQ ID NO. 11;
the nucleotide sequence of the specific primer pair of the microsatellite marker 3 is shown as SEQ ID NO.12 and SEQ ID NO. 13;
the nucleotide sequence of the specific primer pair of the microsatellite marker 4 is shown as SEQ ID NO.14 and SEQ ID NO. 15;
the nucleotide sequence of the specific primer pair of the microsatellite marker 5 is shown as SEQ ID NO.16 and SEQ ID NO. 17;
the nucleotide sequence of the specific primer pair of the microsatellite marker 6 is shown as SEQ ID NO.18 and SEQ ID NO. 19;
the nucleotide sequence of the specific primer pair of the microsatellite marker 7 is shown as SEQ ID NO.20 and SEQ ID NO. 21.
3. The method of claim 1, wherein: the traits of the inbred male macrobrachium rosenbergii sample comprise blue long arm type, orange long arm type and small individual type.
4. The method of claim 1, wherein: the degree of genetic differentiation is statistically analysed by F, e.g. population allele frequency parameter FIS、FITAnd FSTIn which F isISAverage inbred coefficient, F, for local populationITAverage inbred coefficient for the entire population and FSTCalculating the gene flow Nm according to the Wright method for the average inbreeding coefficient among related local populations, wherein the calculation formula is as follows: nm ═ FST0.25(1-FST)/FST.FIS。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110016510A (en) * | 2019-04-22 | 2019-07-16 | 宁波大学 | A kind of molecular labeling for Macrobrachium rosenbergii hereditary and selection |
| CN111118174A (en) * | 2020-01-10 | 2020-05-08 | 浙江省农业科学院 | Macrobrachium rosenbergii sex identification method based on PCR and sequencing technology |
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| CN106434974A (en) * | 2016-11-22 | 2017-02-22 | 河北大学 | Microsatellite primer for macrobrachium nipponensis diversity analysis and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110016510A (en) * | 2019-04-22 | 2019-07-16 | 宁波大学 | A kind of molecular labeling for Macrobrachium rosenbergii hereditary and selection |
| CN110016510B (en) * | 2019-04-22 | 2022-06-07 | 宁波大学 | Molecular marker for genetic breeding of macrobrachium rosenbergii |
| CN111118174A (en) * | 2020-01-10 | 2020-05-08 | 浙江省农业科学院 | Macrobrachium rosenbergii sex identification method based on PCR and sequencing technology |
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