Analysis Of Polymorphism And Development Of InDel Molecular Markers In Poncirus Trifoliata

Trifoliate orange(Poncirus trifoliata),a small tree of the Poncirus Raf.in the Rutaceae family,has become one of the most important rootstocks in the subfamily Citrus as a rootstock with high resistance and excellent grafting compatibility.Trifoliate orange germplasm resources are abundant and widely distributed in China,from Zhejiang in the east to Guangxi in the south,Tibet in the west,and Shandong in the north.However,there is a wealth of polymorphism in the biological traits of different trifoliate orange germplasm in different regions,and few people have conducted systematic evaluation of different trifoliate orange germplasm,especially at the molecular level.Therefore,a comprehensive evaluation of the polymorphism of trifoliate orange germplasm resources and the development of molecular markers are of great significance for the selection and tracing of citrus rootstocks.In this study,we collected 54 trifoliate orange germplasm from China and different countries or regions,systematically analyzed their biological characteristics,and systematically compared their resistance traits such as cold resistance and disease resistance,and analyzed the genetic polymorphism of trifoliate orange germplasm based on whole genome resequencing technology,and screened the specific markers that showed polymorphism in different trifoliate orange germplasm to provide a theoretical basis for the selection and breeding of new rootstock varieties in the future.The main results of the study are as follows.(1)Abundance of polymorphisms in biological traits in Trifoliate orange germplasm.Forty grafted and 14 live trifoliate orange germplasm from different regions were collected and preserved,and the morphological observation of the fruits of live trifoliate orange germplasm and the determination of the growth potential of live seedlings showed that the fruits of PT38 and PT43 were larger,had more single seeds and fuller seed development,and their stem thickness was larger than the remaining 12 live trifoliate orange germplasm at 6 weeks after sowing.The morphology of the 40 grafted trifoliate orange germplasm showed that the leaf shape of trifoliate orange germplasm differed greatly,among which the leaf shape of PT31and PT54 was similar to that of Citrus sinensis.(2)Trifoliate orange germplasm differed significantly in cold resistance.Fifty-three isolated leaves of trifoliate orange germplasm were treated with low temperature-5℃,and the relative conductivity and enzyme activities of SOD,POD and CAT were measured after 24 h.The results showed that PT28,PT38 and PT44from high latitude regions(Japan,Henan and Shandong)had relatively higher SOD and POD enzyme activities,indicating that their leaves were less damaged by low temperature and more resistant to cold,while PT42 and PT54 from low latitude region Guangxi had higher relative conductivity,lower SOD,POD and CAT enzyme activities and more damaged by low temperature,indicating that they were less resistant to cold.(3)Trifoliate orange germplasm does not differ significantly in its resistance to citrus ulcer disease.To evaluate the susceptibility of different trifoliate orange germplasm to citrus canker(Xanthomonas citri subsp citri,Xcc),different methods of Xcc inoculation identification were first compared on in vitro and in vivo materials.In vitro inoculation with 10~5～10~7 CFU/m L concentration of injection inoculation was favorable for late In vivo inoculation with 10~3～10~5 CFU/m L was more favorable for the evaluation of Xcc susceptibility.The Xcc susceptibility evaluation of 52 trifoliate orange germplasm revealed that PT3,PT7,PT25,PT35,PT47,PT48,PT51 and PT52showed slightly weaker symptoms at 8 dpi of Xcc inoculation,while PT1,PT2,PT13and PT29 showed extreme susceptibility to Xcc and more severe leaf damage,but there was no significant difference in the relative bacterial content of leaves of each trifoliate orange germplasm.(4)Polymorphism enrichment by InDel and SNP.The total high-quality genomic DNA of 54 extracted trifoliate orange germplasm was subjected to whole-genome resequencing,and an average of 14.67 G of valid data was obtained for each trifoliate orange germplasm after data filtering.Using common trifoliate orange(Citrus trifoliata,GCA＿018350135.1)as the reference genome,the 54 resequenced data were analyzed for comparison.A total of 2112686 InDel site and 12622684 SNP site were obtained,PT29 contained three specific InDel markers,PT5 and PT31 each contained two specific InDel markers,while PT46 and PT54 each contained one specific InDel marker.The remaining 15 InDel markers showed two or more band types in different trifoliate orange germplasm.