Identification of Yunhe pears in Zhejiang province based on LFY2int2 and SSR analyses

Abstract: 【Objective】Yunhe pears are a group of Chinese sand pears(Pyrus pyrifolia) native to Lishui,Zhejiang province,China and can be divided into two types,Laoxueli and Zhenxiangli,which are important and unique germplasm resources for their large fruits and/or lateness in ripening compared with the current sand pear cultivars.Some fine selections of Yunhe pears were collected and planted under unified conditions as candidates for selection and breeding.However,some of these selections showed a high level of similarity on morphological characteristics and phenological phases,suggesting the possibility of synonyms.So far,little has been known about the genetic background of Yunhe pears,especially Zhenxiangli.To provide a basis for the breeding of Yunhe pear cultivars,their genetic background and relationships among these fine selections needs to be estimated.【Methods】Eleven fine selections of Laoxueli and one Zhenxiangli accession were collected from Yunhe and Jingning in Lishui,grafted onto 'Cuiguan' and grown under uniform conditions.One semi-wild accession,' YHHL',and three other Laoxueli accessions('XHXL','BPXL1' and 'BPXL2') were collected from local orchards.Young leaves of these 16 accessions were collected and the total genomic DNA was extracted by using CTAB methods for the following PCRs.The second intron of the LEAFY2 gene,LFY2int2,was amplified by using primers of LFY2-F and LFY2- R.The PCR products were directly sequenced,and more than three TA clones were sequenced when there were intra-individual divergent copies.The LFY2int2 sequences of Chinese sand pears and Meili native to Zhejiang province and P.betuleaforlia obtained in our previous study were aligned with sequences obtained in this study using Clustal X.Phylogenetic trees based on Maximum Parsimony methods were constructed using MEGA6 with sequence of Malus domestica(GenBank accession number DQ535886) as an outgroup.12 SSR markers explored in previous studies were selected.NH026 a,CH02bl0,CH01bl2 and CH01f02 were genomic SSRs,and each forward primer was fluorescently-labeled by Fam(blue) or Hex(green).The other 8 markers were explored from pear transcriptomes and the primer pairs were provided by the explorer,each forward primer was added by a tail(M13 universal sequence) to the 5' end,and the additional primer M13 was fluorescently-labeled by Fam or Hex.The PCR and sequencing of the SSRs were carried out according to the referenced studies.The number of alleles,expected heterozygosity(H_e),observed heterozygosity(H_o) and polymorphism information content(PIC)were calculated by using MStool.The sequencing results of the marker length(bp value) were transformed into a 0/1 value by DataTrans,then a dendrogram using UPGMA(unweighted pair-group method with arithmetic average) cluster analysis based on DICE identity was constructed by NTSYSYpc 2.02.【Results】Direct sequencing of LFY2int2 PCR products showed that 'YHHL' had one overlapped site,while'Zhenxiangli' had six,so TA cloning was carried out for 'Zhenxiangli' The other 14 accessions shared the same results with multiple overlapped sites and one indel at site 480 bp,and two accessions,' Laoxueli1' and 'Laoxueli 3',were randomly selected for TA cloning.As a result,four distinct LFY2int2 copies(a,b1,b2,c) were observed among the 16 accessions.Alignment of these four copies produced 11 substitutions and one indel,and copy b1 and b2 had only one substitution difference.'YHHL' had two copies of b1 and b2,'Zhenxiangli' had c and b2,and the other accessions had copies a and b1.The phylogenetic tree based on the LFY2int2 sequences showed that copy c of 'Zhenxiangli1' formed a separating clade,while bl and b2 were in clade Ⅰ with Chinese sand pears such as 'Yiwulizi' and 'Puguali' and the copy a from Laoxueli was in clade Ⅱ with two copies from Meili,which belong to the paralog of the LFY2int2-Del2 copy types.Among the 12 SSR markers,only TXY77 failed in amplification of five accessions.Each primer pair produced 2 to 6 alleles,and the H_ewas from 0.062 5 to 0.688 3,the H_o was from 0.062 5 to 1,and the PIC was from 0.058 7 to 0.707 3.Among the markers,8 showed a low level of polymorphisms,which could only discriminate 'Zhenxiangli' and/or 'YHHL' from the other 16 accessions.The dendrogram of the 16 Yunhe pear accessions based on UPGMA showed that 'Zhenxiangli' and 'YHHL' were separated from the other accessions at the coefficient of 0.45 and 0.7,respectively,while the rest accessions were clustered into two groups.Group 1 consists of six Laoxueli accessions including four fine individuals,XHXL and BPXL in group 2,there were eight accessions,among which Laoxueli4 and'Bopixueli2' were identical,while 'Laoxueli3' and 'Laoxueli10' were identical.【Conclusion】Phylogenetic analyses based on LFY2int2 sequences suggested that Laoxueli and the unknown 'YHHL' belonged to the Chinese sand pear,while 'Zhenxiangli' which had the special c copy types,did not.The dendrogram based on SSRs showed that ' Zhenxiangli' and ' YHHL' were not genetically closely related to Laoxueli accessions,and two sections of the putative synonyms were identified.According to their morphological characteristics,'Zhenxiangli' was possibly a hybrid between the Chinese sand pear and P.communis,which is worth further study by investigating the local cultivars of P.communis.