The SRAP Analysis And ISSR Analysis Of The Different Splash Types Of Harmonia Axyridis From Maoershan Area

The ladybird beetle,Harmonia axyridis Pallas,is mainly distributed in Asia.It is a highly polymorphic Coccinellidae with a wide geographic,thus lots of taxonomies for Harmonica exist.The research did the SRAP and ISSR markers to 60 phenotypes of H.axyridis Pallas in the base of extract their DNA.Then evaluated the genetic diversity and relationships of 60 H. axyridis Pallas varieties according to SRAP and ISSR markers by amplified with 15 SRAP primers and 15 ISSR primers.The main results showed as follows:(1) Genomie DNA was extracted from H.axyridis Pallas by the method of modified CTAB.Although at the first suspension time we didn't get genomic DNA,at the second suspension time we obtained high quality and purity genomic DNA which can meet the request of PCR reaction.(2) The optimal SRAP-PCR and ISSR-PCR reaction systems for a variety of H.axyridis Pallas are instituted in the study.We optimized five main factors influencing the amplification of SRAP-PCR,and got the suitable reaction system of 20μL:1×buffer,0.625mM of Mg²⁺, 0.15mM of dNTPs,0.25uM of each primer,50～100ng of DNA template,1.5unit of Taq DNA polymerase.A thermal cycle was implemented with the following program:the first five cycles were run at 94℃(1 min),35℃(1 min),and 72℃(1 min) for denaturing,annealing,and extension,respectively.Then,the annealing temperature was raised to 50.3℃for another 35 cycles.After the completion of the 40 cycles,the reaction mixture was incubated for 7 min at 72℃.We optimized five main factors influencing the amplification of ISSR-PCR,and got the suitable reaction system of 20μL:1×buffer,1.25mM of Mg²⁺,0.2mM of dNTPs,0.15uM of each primer,100～150ng of DNA template,1.0unit of Taq DNA polymerase.A thermal cycle was implemented with the following program:94℃(1 min),53.3℃(1 min),and 72℃(1 min) for denaturing,annealing,and extension,respectively for another 35 cycles.After the completion of the 35 cycles,the reaction mixture was incubated for 7 min at 72℃.(3) 326DNA bands were amplified with 15 SRAP primers,of which 214(65.64%) were polymorphic.The similitude soefficient are age form 0.4488 to 0.7008.And 215DNA bands were amplified with 15 ISSR primers,of which 201(93.49%) were polymorphic.The similitude soefficient are age form 0.4494to 0.6966.(4) Polymorphism point ratio,Allele efficiency,Nei exponent,Shannon exponent show that the two groups of the vaginopennous color have accorded genetic diversity basically.The experiment exhibited that the yellow fudus and black spot had higher genetic diversity than others.This result matchs case the value of the mathematics statistics. (5) Form three UPGMA cluster based on SRAP,ISSR and their aggregation,we will know that,both of two markers can division different varieties and exhibit,for example,the yellow fudus and black spot:ab.123456789-duodevigintisignata(1-2) Yuan.and ab.123456789-duodevigintisignata(7-8) Yuan.,ab.1/2123456789-undevigintisignata(6-7) Yuan.and ab.1/2123456789-undevigintisignata(7-8) Yuan.,ab.1/2123456789-undevigintisignata (1-2)(miHi) Yuan.and ab.1/2123456789-undevigintisignata(1-2,4-5) Yuan., ab.12345678-sedecimsignata(Mls.) Yuan.and ab.12345679-sedecimsignata(miHi.) Yuan., ab.34-quattuorsignata(Hem.) Yuan.and ab.36-quattuorsignata(Hem.) Yuan.,ab.1235-octosignata (Hem.)Yuan,and ab.1346-octosignata Yuan.;the black fudus and yellow spot: var.axyridis Pallas and ab.conspicua Fald,ab.circumscripta Hem.and ab.lunata Hem..These results are consistent with the morphological classification.(6) The result shows that there have some different between two markers,they exist best notability relativity with each other by relativity analyzed.When compare to their aggregation result,SRAP is close up to it than ISSR.So SRAP is doughty reliability to the study of varieties genetic diversity and relationships.SRAP marker could be reliably used in genetic diversity analysis of H,axyridis Pallas,all results of this study will be an effective parameter in taxology and pests-control..