Genetic Relationship And Evolution Among Citrus And Its Close And Distant Relatives Based On Chloroplast Genome

Citrus is the most cultivated fruit tree in the world and has important commercial value and scientific research value.Since the establishment of Citrus,many scholars have conducted a series of studies on the classification and evolution of Citrus,but a widely supported conclusion has not been obtained.The chloroplast genome is cytoplasmic inheritance.Its structure is stable,and its genetic evolution is conservative.It retain many variations accumulated in evolution.The study of citrus chloroplast genome is beneficial to reveal the genetic relationship and evolution process of Citrus and its close and distant relatives from the cytoplasmic level and provide a reference for the identification and classification and breeding evaluation of citrus germplasm resources.In this study,Citrus and its close and distant relatives were used as experimental materials.The analysis were carried out from two aspects: chloroplast genomic molecular markers and highly variable regions cloning sequencing.Main results were as follows:1.Researches based on molecular markers of chloroplast genome1.1 Development of cpInDel markers and cpSSR markersWith the chloroplast genome of sweet orange as reference,the highly variable regions of the chloroplast genome of 14 samples were assembled,and 4 different In Dels were found by comparative analysis.Aimed at these different In Dels,4 pairs of cpInDels primers were successfully designed and named as HVar-cp INDEL.SSRs sites of the sweet orange chloroplast genome were detected by MISA(Microsatellite identification tool)first,then Seqkit was used to locate the SSRs composed of two or more nucleotide repeats.13 SSRs sites were found with differences among species.Aimed at these different SSRs,13 cpSSRY primers were designed.1.2 Analysis of genetic diversity by cpInDel markers and cpSSR markersThe newly designed markers and published cpSSR markers were used for the analysis of genetic diversity.The newly designed cpInDel and cpSSRY primers can detect polymorphisms in 100% of the experimental materials,and the published cpSSR markers are only 66.7%.Compared with the cpInDel,cpSSR markers produced more polymorphic bands in this study.A total of 21 polymorphic bands were amplified by cpInDel primers.An average of 5 polymorphic bands can be detected per marker.The amplified fragments ranged from 178 bp to 491 bp.The average polymorphism information content(PIC)was0.47,with a range of 0.24 to 0.69.A total of 112 polymorphic bands were amplified by cpSSR markers,with an average of 6 polymorphic bands per marker.The amplified fragments ranged from 160 bp to 349 bp,the polymorphism information content(PIC)average was 0.55,and the variation range was 0.04 to 0.80.The electrophoresis bands of cpInDel and cpSSR markers were analyzed,and it was found that the samples could be effectively distinguished by different bands.Results of the study showed that the cpInDel markers give more accurate distinguishing effect than the cpSSR markers at species and/or above taxa level.The cluster analysis of cpInDel and cpSSR markers obtained similar clustering results.Minor differences were found in the relationship between the lower-rank taxa and the classification of a few genotypes.Mandarin,pummelo,citron,kumquat and trifoliate orange were clustered into C.reticulata,C.grandis,C.medica,Fortunella and P.trifoliate by both markers.The cytoplasmic origin of P.trifoliate hybrids were found to be different from P.trifoliate and that of lemon,liminia and lime were different from C.medica.Wangcang zhoupigan,a representative variety of C.speciosa,has a different cytoplasmic origin from other mandarin varieties.Ziyang xiangcheng and Korean xiangcheng may have different cytoplasmic origin though they were both named as C.junos.cpSSR markers classify mandarin,sweet orange and sour orange,ichang papeda and honghe papeda into three clusters,representing C.reticulate,C.sinensis and Papeda,while cpInDel markers show that they are closely related,and classify them into one clusters.Indian wild mandarin were clustered into C.medica by cpInDel,and were clustered into a single cluster by cpSSR.2.Researches based on the cloned sequences of the hypervariable regions of the chloroplast genomeThirty-two samples representing each genus or showing differences in electropherogram and eight samples representing Citrus distant relatives were selected for the cloning and sequencing analysis of the three hypervariable chloroplast genomes regions.Consensus sequence of each sample was considered as the representative sequence of the sample and a total of 120 representative sequences were obtained and used in the following analysis.MEGA cluster analysis showed that all samples were divided into 18 groups with a genetic distance between 0.0014 to 0.0015.8 samples representing Citrus distant relatives,4 samples representing Citrus close relatives,and the three basic species of the genus Citrus were clustered into corresponding clusters.Hezhou guposhan yegan,Indian wild mandarin and Honghe papeda were clustered into individual cluster respectively.The sequence alignment analysis showed that the hypervariable region 2 have less sequence variation types,generally SNPs or short In Dels unique in a few genotypes.In hypervariable region 1 and 3,not only SNPs,but also long In Dels are found to present in all samples and exhibited regular variation among taxa.There are two regularly mutation sites in hypervariable region 1,and their variation characteristics are consistent with the evolutionary level of the species in traditional research.Therefore,based on the sequence variation of hypervariable region 1,and supplemented by hypervariable regions 2 and 3,the study on the evolution of Citrus and its close and distant relatives was carried out.In hypervariable region 1,major variations were observed at the middle and 3’ end in the mutation site 1.The variation types at the3’ end are TTAG,TTAC,TGAC,and GGAC.TTAG motif are found in Zanthoxylum and Toddalia only,which are considered to be more primitive species in Rutacea,so it is inferred that the TTAG-containing sequence is more primitive than other sequence types.The variation types at the middle can be classified as Poly A type and Poly A intervening by other base types.It is difficult to find evolution clues from the variations,but the variations could be used in genotype identification.All samples can be divided into two clusters according to the presence or absence of AGAA motif at variation site 2.The samples without AGAA motif,including more Citrus close and distant relatives,such as Zanthoxylum,Toddalia,Eremocitrus,Fortunella,P.trifoliate,Clausena excavata and Murraya koenigii,were considered to be more primitive by previous studies.Therefore,it is considered that the absence of AGAA motif may be more primitive.The samples containing AGAA motif can be classified into three groups further.The samples excluding ATATTCTCT in front of AGAA or the corresponding reverse complementary motif AGAGAATAT were classified into one group,including Clausena Burm,C.aurantium and C.limon.The samples containing TATTCTCTA in front of AGAA were classified into one group,including Murraya exotica,C.ichangensis,and C.reticulata.The samples containing GAGAATATA in front of AGAA were classified into one group,including C.mangshanensis,C.grandis and C.sinensis.3.Comparison analysis of molecular markers and cloning of hypervariable regions of chloroplast genomeThe cloning results of the chloroplast genome hypervariable regions showed that the fragments with length differences in electrophoresis have sequence differences,and there were also differences in the fragments without length differences,especially SNP.Through length differences of the cloning sequences and the constitution of In Del and SNP,samples can be distinguished.Similar clustering results were obtained with cloning sequences and molecular markers,but there were differences in the classification of Indian wild orange,Yichang orange 2586,Mangshan yegan and Hezhou guposhan yegan.The cluster analysis of cloning sequence can effectively distinguish species and the lowerrank taxa.In conclusion,17 cp Indel and cpSSR markers are developed efficiently and used in analysis of 48 genotypes.Cloning and sequencing of the hypervariable region in 40 genotypes was carried out to test and verify the molecular marker results and further elucidate the relationship among Citrus and its close and distant relatives.Nearly consistent results are obtained in cluster analysis with data from cp Indel,cpSSR and cloning and sequencing.The cluster results are similar to the traditional taxonomy based on morphology.But obvious different cytoplasmic sources were found within the traditional taxonomic groups.According to some characteristic motifs,the evolution of Citrus was probed.The developed markers and obtained sequences were of value for citrus germplasm identification and evaluation.