Species Identification Using SSR,ITS And Chloroplast Genome Comparative Analysis On Peach

China,where peach originated,has the richest peach species resource.With more and more peach species integrated into peach breeding project and many cultivars introduced into China,an efficient and accurate peach species identification method is necessary to be explored.At the same time,the genome information is crucial for peach genetic engineering to keep up with the discoveries of new genes.This study was presented as three parts: firstly,based on the representative accessions in National Peach Germplasm Repository(NPGR),a set of simple sequence repeat(SSR)panel was screened for peach species identification,and the reliability of the SSR panel were demonstrated by the following unknown accessions species identification and interspecific hybrids genotype analysis.Secondly,the ITS sequence structure features and interspecific diversity were analyzed by colon sequencing,the potential of ITS as a DNA barcoding for peach is analyzed;thirdly,six complete nucleotide sequences of the chloroplast genome were sequenced on six peach representative species accessions,the structure features and diversity were analyzed.The details are as follows:1.Peach species identification and hybrid genotyping using SSR markers(1)Basing on 20 representative accessions in NPGR,78 SSRs covered 8 linkages were screened,18 SSR markers were finally selected for five peach species discrimination.(2)A basic genotype database was established based on five peach species and 18 selected SSR loci through fluorescent-labeled SSR markers PCR and capillary electrophoresis analysis.(3)Eight unknown peach accessions were successfully identified,and three interspecific hybrids genotypes of ‘P.persica × P.davidiana’,‘P.persica × P.kansuensis’ and ‘P.persica × P.ferganensis’ were also analyzed reliably,which demonstrated the SSR panel and species genotype database were reliable.2.Peach ITS sequence feature exploration and species identification(1)The mainly features of peach were acquired by clone sequencing on 20 representative accessions.The ITS1 and ITS2 sequnce sizes are 228-232 bp and 278-280 bp respectively,the length ratio of ITS1 and ITS2 sequence is 0.82,the GC content of ITS sequence is 63.9%-65.2%.(2)The varieties of ITS sequence were represented by five peach species sequence alignment.There are 20 informative sites in ITS sequence altogether,11 and 9 sites located in ITS1 and ITS2 sequence respectively.The 20 information sites were showed as base transversion,the repeat times variation of mononucleotide unit,one or more bases insertion,the position variation caused by other base insertion,and the complex variation of base transition and base transversion.The base transversion shared the most,which amountted to 65% of all the variation sites.(3)Part of the 20 ITS informative sites are characterized by peach species.There are obvious specific information sites in P.mira,P.kansuensis,P.ferganensis and P.persica,and no complete informative sites in P.davidiana,which show the transitive character from P.kansuensis to P.persica.The feasibility of the interspecific informative sites were tested by the other representative accessions in NPGR,the result accorded with their real identity,which demonstrated the potential value of ITS sequence as the DNA barcoding for peach species identification.3.Peach chloroplast genome sequencing and genome structure comparative analysis(1)The complete nucleotide sequences of the chloroplast genomes for 6 representative peach species were obtained through assembly of Illumina paired-end reads produced by total DNA sequencing.The total genome size ranges from 157 628 bp to 158 129 bp,with an IR of 26 380-26 413 bp,LSC of 85 764-86 276 bp,and SSC of 19 025-19 122 bp.The GC content of 6 species is 36.7% consistently.The genome encodes 112 different genes,including 78 unique protein-coding genes,30 tRNA genes and 4 ribosomal RNA genes.(2)SSR loci were variety from 111 to 119 in six peach chloroplast genomes.Among of them,2-3 loci are mononucleotide SSR,3 loci are dinucleotide SSR,3-4 loci are tetranucleotide SSR,and one locus is pentanucleotide SSR.Of the mononucleotide SSR loci,(A)n and(T)n were polymorphic microsatellites in the chloroplast genome of peach.Comparative analysis indicated the mononucleotide SSR of(C)n is absent in P.davadiana and P.davadiana var.Potaninii.,and(TAAA)n is absent in P.persica and P.ferganensis.A total of 21 long repeats of 30-40 bases with a sequence identity 100% in 6 peach plastid genomes repeated from two to four times,of which 13 are forward and 8 are inverted repeats.(3)Phylogenetic tree were constructed by complete chloroplast genome sequence and plant universal DNA barcoding sequence of matK+rbcL respectively,all of which accorded with the morphology classification.Availability of complete chloroplast genome sequence is crucial to understanding phylogenetic relationships among 6 peach species and will facilitate peach plastid genetic engineering.