CN104328119B - The related microsatellite molecular marker of megalobrama amblycephala growth traits and application - Google Patents
The related microsatellite molecular marker of megalobrama amblycephala growth traits and application Download PDFInfo
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- CN104328119B CN104328119B CN201410654344.4A CN201410654344A CN104328119B CN 104328119 B CN104328119 B CN 104328119B CN 201410654344 A CN201410654344 A CN 201410654344A CN 104328119 B CN104328119 B CN 104328119B
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Abstract
The invention discloses the related microsatellite molecular marker of megalobrama amblycephala growth traits and application, the present invention is using point group's partition method, the association analysis of molecular labeling and character is realized, two molecular labelings significantly correlated with growth traits are finally filtered out, Mam116 primer is F:5 ' CTATTTACAGTTTCATGCTTTCCTC 3 ', R:5’‑ATCCCGTCCGCCGCTTACT‑3’；Mam166 primer is F:5 ' GGTACTGTTTGTGCTGGGC 3 ', R:5’‑CTGCTCACTCAACTTATTGTAGGTC‑3’.The present invention carries out the Multiple range test of different genotype character to the two marks, to determine inferior position and advantage allele, individual of the selection containing positive effect genotype is used as seed selection parent in the related breeding of megalobrama amblycephala character, avoid the individual containing negative effect genotype, experimental work amount can be reduced, accelerate breeding efficiency.
Description
Technical field
The present invention relates to fish molecular marking supplementary breeding technology, and in particular to related to megalobrama amblycephala growth traits micro- to defend
Star molecular labeling and application.
Background technology
Megalobrama amblycephala (Megalobrama amblycephala Yih, 1955), also known as blunt snout bream, are under the jurisdiction of Osteichthyes,
Cypriniformes, Cyprinidae, triangular bream category, its NATURAL DISTRIBUTION is limited only in a few lake of the middle and lower reach of Yangtze River.Because of its delicious flavour, feeding habits
Extensively, grow and soon, easily fish for and extensively liked by consumer and culturist.Due to generally existing inbreeding in the production of megalobrama amblycephala fingerling
Phenomenon cause germplasm degenerate the conventional seed of aggravation growth speed it is slower.Although China selected grew that speed compared with
Fast megalobrama amblycephala Pujiang 1, but seed can not still meet the market demand.It need further to strengthen megalobrama amblycephala germ plasm resource prison
Survey, assess and protect, select and grow fast, the megalobrama amblycephala improved seeds (Wang Weimin, 2009) of good stress resistance.
The growth traits of fish is quantitative character, is multiple gene controls, these genes occupy certain on chromosome
Region, referred to as quantitative trait locus (Quantitative Trait Locus).The QTL of one quantitative character be not it is a lot,
It there is major gene resistance (Li Ning, 1997).These QTL one or two major gene resistance is with regard to that can reflect a quantitative trait phenotypes
More than the 10%-50% of variation.Therefore we can control the gene or mark of these characters by finding, to the warp of fish
Character of helping carries out genetic improvement.Microsatellite DNA, also known as simple tandem repetitive sequence (simple sequence repeat,
SSR), it is, by the nucleotide sequence that 2~6 bases are the multiple tandem sequence repeats of elementary cell, to be distributed widely in Eukaryotic base
Because of (Hamad a et al., 1982) in group, due to it, to have codominant inheritance, easy to detect, polymorphism information content high etc. excellent
Point, is widely used in the analysis of aquatic livestock population genetic variations, the structure of genetic linkage mapses, QTL positioning and mark auxiliary
(Sun Xiaowen, 2004 in terms of selection and use；Liao et al.,2009).Zhang Yifeng etc. (2008) is using from genetic linkage map
Spectrum and the 47 pairs of micro-satellite primers delivered carry out body weight, body length and body to Bai Shi carps and C carpiovarwuyuanensis cold-resistant strain from F2 generations
Height has carried out correlation analysis, as a result finds 7 sites related to growth traits, and find the related genotype of 3 kinds of characters.
Liu Xiande etc. (2013) screens 2 and microsatellite marker that large yellow croaker growth traits is closely related and its corresponding protogene
Type.
Conventional seed selection is selected according to the phenotype rather than genotype of character, lacks pre- player to objective trait
Section, breeding cycle length, efficiency are low.The development of modern molecular biology technique makes according to the gene or molecular labeling related to character
Progress is selected to possibility.Have not yet to see the report of megalobrama amblycephala growth traits related molecular marker.
The content of the invention
Object of the present invention is to provide the related microsatellite molecular marker Mam116 of megalobrama amblycephala growth traits and
Mam166, Mam116 primer are F:CTATTTACAGTTTCATGCTTTCCTC, R:ATCCCGTCCGCCGCTTACT；Mam166
Primer be F:GGTACTGTTTGTGCTGGGC, R:CTGCTCACTCAACTTATTGTAGGTC；The molecular labeling is megalobrama amblycephala
The QTL positioning and molecular mark work of growth traits lay the foundation.
It is another object of the present invention to provide the microsatellite molecular marker related to megalobrama amblycephala growth traits in group
Application in head triangular bream marker assisted selection, i.e., the advantage allele based on molecular labeling screens the excellent individual of growth traits.
The present invention is achieved by the following technical solutions：
The microsatellite molecular marker related to megalobrama amblycephala growth traits, is obtained by following steps screening：
In June, (1) 2009, maternal to 42 tails from Liangzi Lake, Yuni Lake, Poyang Lake, 50 tail male parents carry out artificial breeding
Grow, breeding filial generation is cultivated in same pond.The megalobrama amblycephala F1 generation that in March, 2011 salvages in pond at random is numbered,
Measure body length, body height and body weight.According to weight data, the great individual in 0.4kg of selective body is designated as big group of individuals (Top) and body
The individual less than 0.28kg is designated as small group of individuals (Below), each 96 tail again；
(2) isozyme of the megalobrama amblycephala F1 generation experimental subjects of clip step (1), isozyme is extracted using ammonium acetate method
Genomic DNA as template, enter performing PCR amplification to microsatellite locus from 17 pairs of micro-satellite primers；
(3) megalobrama amblycephala F1 generation individual passes through 8% (W/V) non denatured polyacrylamide in the PCR primer of 17 microsatellite locus
Amine gel electrophoresis and the polymorphism of cma staining method detection site, it is each using PopGene (Version 3.2) software statistics
The number of alleles (number of alleles, Na) and observation heterozygosity (Observed of microsatellite locus
heterozygosity,Ho).Using the GLM general linear models in SAS softwares to megalobrama amblycephala growth traits and microsatellite locus
Relevance carry out Least square analysis, production traits indicator difference conspicuousness between same marker genetype is tested simultaneously
Carry out Multiple range test；
(4) correlation analysis is carried out with 17 pairs of microsatellite markers to the growth traits of megalobrama amblycephala by general linear model,
As a result find in 17 microsatellite locus, site Mam116, Mam166 show significantly correlated with body length, body weight, body height
(P<0.05)；
(5) genotype to 2 sites carries out Multiple range test analysis, determines Pros and Cons allele, final to determine
Mam116 preponderant genotype is 172/176,154/176,176/176 and 176/182；Mam166 preponderant genotype is 129/
129th, 123/123,123/129 and 123/135.
Application of the related microsatellite molecular marker of megalobrama amblycephala growth traits in megalobrama amblycephala marker assisted selection, was applied
Cheng Wei：The genotype of individual is analyzed, by whether the preponderant genotype containing Mam116 sites and Mam166 sites is individual to judge
Whether body is the excellent individual of growth traits, so as to select to be parent.
Compared with prior art, the present invention has advantages below：
(1) present invention is using point group's partition method, from batch breeding, using body weight as standard in the megalobrama amblycephala raised with the pool, choosing
Select individual individual each 96 tail with body weight less than 0.28kg of the body weight more than 0.4kg to be analyzed, so as to realize molecular labeling
With the association analysis of character.
(2) present invention increases the sample size of big group of individuals and small group of individuals, to can more quickly and accurately obtain
Obtain the mark related to character.
(3) present invention carries out different genotype character to 2 marks that there is pole significant difference with body length, body weight, body height
Multiple range test, experimental work amount is reduced to a certain extent, while saving certain human and material resources, financial resources.
(4) present invention carries out the Multiple range test of different genotype character to 2 marks, to determine inferior position and advantage equipotential base
Cause, finally with marker assisted selection, seed selection parent can be used as by individual of the selection containing positive effect genotype in practice process as far as possible
This, avoids the individual containing negative effect genotype.
Embodiment
Technical scheme of the present invention, is the ordinary skill in the art if not otherwise specified.
Embodiment 1：
The screening of microsatellite marker related to megalobrama amblycephala growth traits：
(1) material source
In June, 2009, maternal to 42 tails from Liangzi Lake, Yuni Lake, Poyang Lake, 50 tail male parents carry out artificial propagation,
Breeding filial generation is cultivated in same pond.Salvage the megalobrama amblycephala F1 generation in pond in March, 2011 at random, numbering measures body
Long, body height and weight data, clip fin ray is stored in standby in 100% alcohol.According to weight data, selective body it is great in
0.4kg individual, is designated as big group of individuals (Top), and body weight is less than 0.28kg individual each 96 tail, is designated as small group of individuals (Below),
Microsatellite genotyping is carried out as experimental subjects；Simultaneously using each group of 60 tails individual as with megalobrama amblycephala growth traits phase
The big microcommunity of microsatellite locus preliminary screening is closed, other 36 individuals will be each organized as associated with megalobrama amblycephala growth traits
The big microcommunity of microsatellite locus checking.
(2) micro-satellite primers
17 pairs of micro-satellite primers are that shown in table 1, it is limited that primer gives birth to the service of work biotechnology by Shanghai in the present invention
Company synthesizes.
17 pairs of micro-satellite primers information in the present invention of table 1
T：Annealing temperature；Na：Number of alleles；Ho：Observe heterozygosity.
(3) extraction of megalobrama amblycephala STb gene
The DNA of megalobrama amblycephala isozyme is extracted using ammonium acetate method, the DNA of extraction is by 1% agarose gel electrophoresis and Nan
The ultramicrospectrophotometers of oDrop 2000 detect its integrality and concentration, and -20 DEG C save backup.
(4) amplification of microsatellite DNA
Each PCR reaction systems cumulative volume is 20 μ l, (contains Mg including 10 × buffer2+) 2.0 μ l, dNTPs (10m
Mol) 0.4 μ l, upstream and downstream primer (10 μm of ol) each 0.5 μ l, Taq enzyme 1U, DNA profiling about 100ng.PCR reaction conditions are：94℃
Pre-degeneration 5min；94 DEG C of denaturation 30s, primer renaturation temperature 30s, 72 DEG C of extension 45s, 35 circulations；72 DEG C of extension 10min.Expand
Volume increase thing detects its polymorphism by 8% (W/V) native polyacrylamide gel electrophoresis and cma staining.
(5) data statistics and analysis
With number of alleles (the number of of each microsatellite locus of PopGene (Version 3.2) software statistics
Alleles, Na) and observation heterozygosity (Observed heterozygosity, Ho).Utilize the general lines of GLM in SAS softwares
Property model Least square analysis is carried out to megalobrama amblycephala growth traits and the relevance of microsatellite locus, to same marker genetype it
Between production traits indicator difference conspicuousness test and carry out Multiple range test.Observed value is less than the genotype of 4 times, because lacking point
Analysis value, do not take statistics analysis herein.
(6) preliminary screening of microsatellite locus related to megalobrama amblycephala growth traits
1) polymorphism of microsatellite locus
17 pairs of microsatellites used in this experiment can expand stable clearly band.204 etc. are detected altogether in 17 sites
Position gene, wherein site Mam144, Mam12, Mam46 polymorphism highest, number of alleles is more than 14, and heterozygosity is big
In 0.8.Site Mam147 polymorphism is relatively low, and the number of alleles in two groups is respectively 5.The allele of big group of individuals
Number is between 5~17, and average number of alleles is 9.5, and heterozygosity is located between 0.5658~0.8928, and average heterozygosity is
0.7627.The number of alleles of small group of individuals is between 4~18, and average number of alleles is 10.2, and heterozygosity is located at 0.4489
Between~0.8851, average heterozygosity is 0.7150.The genetic diversity of two groups is not significantly different.
2) megalobrama amblycephala microsatellite marker and the correlation analysis of growth traits
Correlation analysis is carried out with 17 pairs of microsatellite markers to the growth traits of megalobrama amblycephala using general linear model, as a result
It was found that in 17 microsatellite locus, site Mam166 and Mam116 show extremely significantly correlated (P with body length, body weight, body height
<0.01)。
Embodiment 2：
The positives and negatives of site Mam166 and Mam116 genotype：
Genotype to 2 sites carries out Multiple range test, is inferred to the inferior position equipotential base on each microsatellite markers
Cause, so as to tentatively judge that above genotype is played or positive or negative effect to character.
The genotype in site of the present invention reads site PCR primer polyacrylamide with Quantity One softwares and coagulated
Shown stripe size (bp) is defined after gel electrophoresis and cma staining.Such as site Mam116,121/121 genotype refers to logical
Cross the band that primer only amplifies 121bp；154/176 genotype refers to amplifies 154bp and 176 liang of rule by primer
Band.
Genotype progress Multiple range test analysis to 2 sites is found, in the Mam116 of site, 11 kinds of genes are obtained altogether
Type, wherein being not significantly different (P in body weight between 121/121,121/129,121/133 genotype>0.05) it is, but significantly low
In other genotype (P<0.05)；176/182 except with 154/176,176/176,172/176 without (P outside the significance difference opposite sex>
0.05), it is significantly higher than other genotype (P<0.05)；123/123,154/176,154/172,154/182,176/176,172/
176,172/180 between any two without significant difference (P>0.05).In terms of body length and body height, 154/172,154/182,172/
(P is not significantly different between 180,176/176,172/176,154/176,123/123,176/182>0.05), its phenotypic number
It is significantly higher than 121/121,121/129,121/133 genotype individuals (P<0.05)；It is significantly higher than in body long side 121/133
121/129(P<0.05), and 121/121 in terms of body height, (P is not significantly different between 121/129,121/133 genotype>
0.05)。
In the Mam166 of site, 8 kinds of genotype are obtained altogether, in terms of body weight, body length and body height, 123/123,123/129,
(P is not significantly different between 123/135,129/129 genotype>0.05), but it is significantly higher than 261/269,261/279,269/
269,269/279 genotype (P<0.05)；In body weight and body senior middle school, 261/269,261/279,269/269,269/279 gene
There was no significant difference between type (P>0.05)；In body long side, 269/279 genotype is substantially less than other genotype (P<
0.05).The Multiple range test of the individual phenotype of two site different genotypes is as follows：
The Multiple range test of the individual phenotype of the site Mam116 different genotypes of table 2
The Multiple range test of the individual phenotype of the site Mam166 different genotypes of table 3
Note：Same letter represents difference not significantly (P in same row in table 2 and table 3>0.05), different letters represent difference
Significantly (P<0.05).
The preponderant genotype for determining Mam116 is 172/176,154/176,176/176 and 176/182；Mam166 advantage
Genotype is 129/129,123/123,123/129 and 123/135.
Embodiment 3：
Application of the microsatellite molecular marker related to megalobrama amblycephala growth traits in megalobrama amblycephala marker assisted selection, application
Process is as follows：
1) 3 maximum positive-effect genotype of average, the 3 of average minimum are chosen respectively from the analysis result in two sites
Individual negative effect genotype alternatively standard (deficiency do not take), the results are shown in Table 4.
The positive-effect and negative effect genotype in 4 two sites of table
2) carry out genotyping in 72 F1 generations (embodiment 1) individual of checking colony, choose wherein containing just or
The individual of negative effect genotype carries out the Multiple range test and significance difference analysis of growth traits, the results are shown in Table 5.
The ontoanalysis of the positive or negative effector type of the checking of table 5 colony
Note：Same letter represents difference not significantly (P in same growth traits>0.05), different letters represent significant difference
(P<0.05).N is the F1 generation number of individuals detected.
3) can intuitively it see from result, in two marks of Mam116 and Mam166, as long as including protogene
The growth traits value of type individual will be significantly higher than the growth traits value with inferior position genotype individuals.Therefore Mam116 can be utilized
Or individual of the Mam16 marks selection containing positive effect genotype is as seed selection parent, and the individual containing negative effect genotype is avoided, from
And the workload of breeding is reduced, improve breeding efficiency.
SEQUENCE LISTING
<110>Hua Zhong Agriculture University Hubei hundred holds aquatic products breeding Co., Ltd
<120>The related microsatellite molecular marker of megalobrama amblycephala growth traits and application
<130>The related microsatellite molecular marker of megalobrama amblycephala growth traits and application
<160> 4
<170> PatentIn version 3.1
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
ctatttacag tttcatgctt tcctc 25
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
atcccgtccg ccgcttact 19
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence
<400> 3
ggtactgttt gtgctgggc 19
<210> 4
<211> 25
<212> DNA
<213>Artificial sequence
<400> 4
ctgctcactc aacttattgt aggtc 25
Claims (1)
1. megalobrama amblycephala growth traits related microsatellite molecular marker Mam116 or Mam166 amplimer in megalobrama amblycephala to giving birth to
Application in terms of the related breeding of long character, the growth traits is that body length, body weight or body are high；
The application process is：The genotype of individual is analyzed, by whether the advantage containing Mam116 sites and Mam166 sites
Whether genotype is come to judge individual be the excellent individual of growth traits, so as to select to be parent；Wherein Mam116 advantage base
Because type is 172/176,154/176,176/176 and 176/182；Mam166 preponderant genotype be 129/129,123/123,
123/129 and 123/135；Numeral in the genotype refers to that Quantity One softwares read site PCR primer by poly-
Shown stripe size after acrylamide gel electrophoresis and cma staining, unit is bp, and when two numbers in genotype
When word is identical, only one of which amplified band is represented；
Wherein, microsatellite molecular marker Mam116 amplimer is：
F:5’-CTATTTACAGTTTCATGCTTTCCTC-3’
R:5’-ATCCCGTCCGCCGCTTACT-3’；
Microsatellite molecular marker Mam166 amplimer is：
F:5’-GGTACTGTTTGTGCTGGGC-3’
R:5’-CTGCTCACTCAACTTATTGTAGGTC-3’。
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| CN107653323B (en) * | 2016-07-23 | 2021-05-04 | 华中农业大学 | Megalobrama amblycephala transferrin gene SNP molecular marker and application thereof |
| CN106554996B (en) * | 2016-08-31 | 2020-01-24 | 华中农业大学 | Megalobrama amblycephala transferrin receptor gene SNP molecular marker and application thereof |
| CN107190053B (en) * | 2017-03-13 | 2021-01-08 | 北京林业大学 | Cypress microsatellite molecular marker combination, primer screening method and application thereof |
| CN110791511B (en) * | 2019-11-21 | 2023-02-28 | 上海海洋大学 | Hypoxia-resistant megalobrama amblycephala growth character gene and positioning method and application thereof |
| CN113528677B (en) * | 2021-08-11 | 2022-02-18 | 华中农业大学 | Leaf-specific notopterygium plateau loach microsatellite molecular marker, and primer and application thereof |
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| CN102653785B (en) * | 2011-03-03 | 2013-10-23 | 华中农业大学 | Identifying method of megalobrama amblycephala family by microsatellite |
| CN102487860B (en) * | 2011-11-24 | 2013-06-05 | 华中农业大学 | Method for screening megalobrama amblycephala group combinations with hybrid vigor |
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