Genetic Diversity Of Crataegus Spp. Revealed By Chloroplast DNA

Hawthorn(Crataegus spp.) is one of important germplasm resources of fruit trees. Exactly discriminating the genus and variety of the hawthorn is the foundation that the hawthorn resource to get preservation,research and utilization.For a long time,the identification of the hawthorn variety mainly depends on morphological characteristics which have more problems.The molecular markers,RAPD,ISSR and SSR,are being applied to the hawthorns gradually which mainly aiming at nuclear genome.Using thirty-nine genotypes belong to eight Crataegus species,this article analyse the polymorphism of the cpDNA of Crataegus spp.by cpDNA PCR-RFLP and cpSSR,and make a simple discussion to the category and evolution.The main results were as follows:(1) Among ten universal primer pairs of chloroplast genome,five primer pairs(cp01, cp03,cp04,cp05,cpl0) were screened for cpDNA PCR-RFLP analysis.The result showed that five primer pairs generated special bands from Crataegus with different fragment size. The primer cp05 generated the shortest bands,about 300～400 bp;the primer cp04 generated the longest bands,about 2000～3000 bp;and the remaining three primer pairs(cp01,cp03, cp10) generated similar size bands,the amplified fragments ranged from 1000 to 2000 bp. The other five primer pairs have no amplified products.(2) Above mentioned five universal primer pairs of chloroplast genome were used to amplify cpDNA non-coding regions in eight Crataegus species including thirty-nine genotypes.The PCR products were digested by seven restriction enzymes and the fragments digested by two restriction enzymes(HinfⅠand TaqⅠ) were highly variable in Crataegus species.The results showed that interspecies of the genus Crataegus had higher cpDNA variations,but no variable bands were detected in different genotypes of C.pinnatifida.(3) UPGMA cluster analysis of the eight Crataegus species based on cpDNA PCR-RFLP marker resulted in three clusters.ClusterⅠincluded C.songarica C.(Sect.Orientales), C.pinnatifida Bge.and C.pinnatifida var.major(Sect.Pinnatifidae).ClusterⅡcontained five wild species,they are four species of Sect.Sanguineae(C.kansuensis Wils.,C.dahurica Koehne,C.sanguinea Pall.,C.maximouiczii Schneid.) and C.brettschneideri Schneid.of Sect. Pinnatifidae.ClusterⅢonly had C.altaica(Loud.)Lange.It was distinct with others and the distance coefficient was 0.91.(4) Among ten universal chloroplast simple sequence repeat(cpSSR) primers,six primers(ccmp2,ccmp3,ccmp4,ccmp6,ccmp7,ccmp10) were screened for cpSSR analysis. And the specific products of three primers showed cpSSR polymorphisms.(5) UPGMA cluster analysis of thirty-nine hawthorns based on cpSSR marker resulted in three clusters.ClusterⅠincluded C.songarica C.(Sect.Orientales),twenty-nine genotypes of C.pinnatifida Bge.and C.pinnatifida var.major(Sect.Pinnatifidae).ClusterⅡcontained six wild species,they are five species of Sect.Sanguineae(C.kansuensis Wils.,C.dahurica Koehne,C.altaica(Loud.)Lange,C.sanguine Pall.,C.maximowiczii Schneid.) and C. brettschneideri of Sect.Pinnatifidae(include three varieties).ClusterⅢonly had C. pinnatifida 'Xinbinruanzi'.(6) Two molecular markers,cpDNA PCR-RFLP and cpSSR,could detect interspecific genetic diversity of the genus Crataegus.Compared with PCR-RFLP,cpSSR has some advantages in detecting intraspecific genetic diversity of the Crataegus.The classification results based on cpDNA PCR-RFLP marker are not completely in accordance with that analyzed by cpSSR marker,and the classification results based on cpDNA polymorphisms have big difference with traditional morphological classification of Crataegus,this revealed very complicated origination and evolution of the genus Crataegus.