Genetic Diversity Analysis Of Yane Goose By ISSR Molecular Markers

Yane goose originated from Liu'An region of Anhui Province,but is now distributed in Lang-xi county which belongs to Xuancheng City of Anhui Province.Yane goose, which is listed the key protected geese by Angricultural Ministry,is rare middle-typed grey goose at present.Inter-simple sequence repeat(ISSR) marker technic,first set up by Zietkiewicz(1994), is amplified and polymorphic molecule marker.It is based on PCR technic,and design primer by SSR widely existed in Eukaryotic gene.It doesn't need sequencing in advance, so it could carry out PCR amplification for inter-simple sequence repeat genomic fragment with reverse arrangement and little interval.Then,the polymorphic map is gained from PCR amplification product through electrophoresis.This experiment has a lower cost, more specificity and stability,and it needs a small amount of DNA.Its primer design is easier than that of SSR,without knowing the base sequence of two ends.Furthermor,the polymorphism is high.The experiment took the Yane goose as the research materials,which were sampled from the original goose farm of Langxi in Anhui Province,and did the research on the pedigree and genectic diversity of goose community by using the technology of ISSR.The results of this research will offer relevant data in the breed characteristics of Yane goose, and apply to the goose-protecting practical work in some extent.The results were as follows:(1) This experiment was to determine the reaction condition of ISSR-PCR.Using the genomie DNA extracted from Yane geese,the orthogonal design was used to optimize ISSR-PCR amplification system in five levels of five factors(Taq DNA polymerase,Mg²⁺, DNA template,dNTPs and primer),respectively.Based on the preliminary experiment,the density within factors of ISSR-PCR was further optimized.The results showed that a better ISSR amplification was obtained with the reaction system containing 0.20μmol/L primer, 0.28mmol/L dNTPs,40ng templet DNA of Yane goose,1.5mmol/L Mg²⁺,1.0U Taq DNA polymerase in the total volume of 25uL.The temperature used for PCR was 94℃for 5min,followed by 35 cycle of 94℃for 1 min,55℃for 1 min,and 72℃for 1 min,and terminated with a 10 min DNA extension step at 72℃.The ISSR-PCR amplification products were stored at 4℃.(2) After the optimal reaction condition was determined,43 ISSR primer was sifted out to amplify 94 samples,and 216 locations were found.Each location had a amount of pedigree cord of 2～7,averagely 5.Each pedigree cord had an amount of molecule of 150～2000bp.Polymorphic location produced by the 43 primer added up to 145,67.13%in proportion to the overall amount,and the PPB is between 16.67～100%,which accounted for 65.50%in average.(3)According to ISSR figure,the Similarity index and Genetic distance were calculated between every two bodies random,and a Yan Goose genetic distance matrix composed of 94 samples was constructed.According to this matrix,a Yane Goose population pedigree figure was constructed by analyzing the relationship among the 94 samples with Unweighted pair group mothed with airthcmean(UPGMA).(4)It's the first time to take the techonology of ISSR to analyse and research the genetic mutation of Yane goose.The ISSR-PCR amplification prodcuts in Yane geese were highly steady,and were tested to have more polymorphic locations than other molecule marker methods.In the whole experiment,there was low frequency to appear the unsteady state of amplication reaction caused by casual factors,which fully approved that it was feasible to analyse genetic diversity of Yane goose by ISSR.At the same time,it's a convenient and low cost analytic technic.The experimental results will supply the direct data in the further research for germplasm characteristics and subsidiary breeding of Yane goose.