| Microsatellite is frequece little sequence on DNA,it have some feature of much amount,wide disposition,high polymorphism,codominant inheritance,and so on.many reaserchers study deeply on The genetic diversity and breeding of sheep by the feature of the maker of microsatellite.In the area of the eastnorth of our country,the main population is northeast (half)wool sheep,Mongolia sheep,china merino,the xiaoweihan sheep and abroad mutton sheep breedings,but the optimize program about the crossing of this sheep is no fina conclusion.this research select several microsatellite sites as gene maker,analysis polymorphism and genetic distance connected with production data ,in order to predict the heterosis between the breeds of sufllok,pull doset sheep and xiaoweihan sheep,northeast wool sheep , preliminary screening the optimize cross combinations.Since 1980's,much multi-lamb main gene were found.we have discover so much mutational genes as BMPR-IB(Bone Morphogenetic Protein Receptor IB),BMP15(Bone Morphogenetic Protein 15),GDF9(Growth differentitation factor 9).it is found that the xiaoweihan sheep and hu sheep have the same mutation with the Booroola sheep, which has a mutation on the 746 basic radical (A746G).The main population sheep in northeast of our country is northeast wool sheep,china merino,Mongolia sheep and so on ,none had study about the question if there are mutational multi-lamb main gene in this breeds. This research built a reasonable molecular biology detecting method ,and test the multi-lamb main gene in this breeds, providing basic data for theory study and production practice using this genes.The genetic diversity for xiaoweihan sheep, northeast wool sheep, suffolk , Polled Dorset , German Mutton Merino sheep,dorper sheep was studied using 4 microsatellite DNA markers, The genetic heterorygosity,polymorphism information content(PIC),private alleles frequency,dominate alleles frequency,and Nei Standard Genetic Distance were determined to evaluate the genetic variability within breeds and genetic relationship among breeds, connected with analysis of product recorder , the optimistical cross combination is selcted.1,the optimization PCR system of the four microsatellite site is found by studying, amplification consequence is tested by 6% denaturing polyacrylamide gel electrophoresis and silver staining, then gotten distinguishing and detailed band of this genotypes.2,Point polymorphism of the four microsatellite are abundant , the PIC is between 0.8644 and 0.7242, allelomorphic gene is between 10～5, utility allele between 7.14～4.0; genetic heterorygosity is between 0.8669～0.7515.3,By Nei standard genetic distance , we find that: The maximal Nei Standard Genetic Distance among them was between German mutto merino sheep and northeast wool sheep ,the number is 1.08723, the follows in order were respectively northeast wool sheep and Xiaoweihan sheep. The minimal Nei Standard Genetic Distance among them was between polles dorset sheep and doper sheep ,only achieve 0.21738,the follows in order were respectively northeast wool sheep and Xiaoweihan sheep,achieve 0.34397,the result is reasonable,because it is a fact the dorper is a result of crossing selection between polled dorset and Persia sheep, Mongolia is the origin of northeast wool sheep and Xiaoweihan sheep.4,By neighbor joining method,the dorper sheep and polled dorset sheep are clustered firstly,then clustered by German mutton sheep and APS. Northeast wool sheep and Xiaoweihan sheep who have lately relationship are clustered along.5,analysis of the average increased weight in on hundred days of this four cross combination F1's produce recorder show that the two cross combination of Northeast wool sheep and German Mutton Merio,xiaoweihan sheep and APS have the maximum heterosis ratio , achieved 0.11 and 0.109 .Correlation analysis of genetic distance , parents heterozygosis degree and heterosis ratio and the average day increased weight have been done by the analysis modular of software of SPSS, it show that it is direct correlation between genetic distance and heterosis ratio, P=0.048<0.05,r=0.952;it is no sense of correlation between father and mother's heterozygosis degree,P>0.05;mother's heterozygosis degree have inverse correlation with the average day increased weight.7,In order to know the difference between the several combination on the average day increased weight,we do analysis of variance about it.the result is P>0.05,so the difference between the several combination on the average day increased weight is nonsignificant.8,By cluster analysis, genetic distance, heterosis ratio conclusion and produre recorder,we think that German Mutton Merio♂X Northeast wool sheep♀,APS and dorset and dorper♂X xiaoweihan sheep♀are optimized combination. In order to compare the polymorphism between this six sheep populations,then, we detected SNP of BMPRIB and BMP15 gene by SSCP method after design three pairs of primer to cover all extron base on all sequence of the two gene,and analyzed the result of sequencing of DNA part which have polymorphism on SSCP. FecXI loc on X chromosome, heterotic female sheep can increase one ovulation and 0.6 yean per foetus, homozygotic type female sheep have small thin and flat ridge pattern ovaries,which have no signal about operative follicle so that is apogeny . FecXH linked with X chromosome also,and heterotic female sheep who have FecXH is property ,but the homozygotic type female sheep is infertility.it is found that BMP15 gene have mutable point linked with the phaenotype of FecXI and FecXH after sequence analysis,one have a mutation of T-A on 92 site in DNA ,the other of C-T on 67 site.by the methods of PCR-SSCP and restricted enzyme cutting,we detected the site of FecXI and FecXH on BMP15 gene of the six sheep population,and found that it have no polymorphism.it show that the FecXI and FecXH site on BMP15 gene have nothing to do with yean trait of the six sheep population.FecB gene is localized on the sixth chromosome of sheep, it have additive effect on ovulating, and dominant effect on yean number.one FecB can increase ovulating 1.3～1.6 and yean number 0.9～1.2 per female sheep;two copy can increase ovulating 2.7～3.0 and yean 1.1～1.7.the mutation on BMPRIB 746 loc (A-G) has the same phaenotype with FecB gene. By the method of PCR-SSCP,the experiment find the mutation on the BMPRIB gene of the six sheep population. and we find that BB genetype and B allele frequency of xiaoweihan sheep's are higher significantly than others. The polymorphism on BMPRIB gene 746 loc can be one of MAS for superfoetation trait of sheep.the BB genetype had not been found in the introduced mutton sheep breeds,only a sprinkle of AB genetype found which is no statistics sense.the reason is that the father have little B allele.the northeast wool sheep all have recorder of two lamb in this experiment,but we could not find BB genetype except a small quantity of AB genetype,so we get a conclusion that superfoetation of northeast wool sheep have other mechanism or influenced by environment mostly.During experiment of SSCP,we optimize the concentration and degree of cross linking of polyacrylamide gel,actually selct degree of cross linking 29:1 and concentration of 12%. The result proved that PCR-SSCP is a deliberate,simple and precise method in Polymorphism detection,this method can find mutable point exactly in DNA sequence, it is a useful tool for multi-lamb main gene detection. |
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