| China is one of the originating and distributing centers of Diospyros Linn.,where abundant valuable genetic resources exist.Retrotransposon are ubiquitous in eukaryotic organisms,constituting a major portion of the nuclear genome(often more than half of the total DNA) in plant,and play a major role in plant genome size,structure,gene function as well gene and genome evolution.Retrotransposon-based molecular markers are valuable tools to reveal the behavior of retrotransposons in their host genome. Therefore,the development and utilization of retrotransposon-based molecular markers in Diospyros Linn.will be of considerable interest for improving technical tools and extending obtainable genetic information concerning Diospyros Linn.germplasm resources.In the present study,we carried out a series of research in this aspect,and the main results were as follows:1.31 RNaseH-LTR(Long terminal repeat) sequences of Ty1-copia like retrotransposon were isolated from the genome of Diospyros kaki Thunb.'Luotiantianshi' using suppression PCR method.Sequence multiple alignment and the phylogenetic analysis indicated that at least 10 subgroups were amplified.High sequence heterogeneity which were due to nucleotide replacement,insertion or deletion and amino acid replacement,stop codon and frameshift mutations in translated putative amino acid sequences were observed among the different subgroups.But some clones showed high similarity within the same subgroup,of which behavior like quasispecies-like population. Potential regulatory motifs from transcription,such as 'CAAT box' and 'TATA box' and some conserved cis-acting regulator elements were discovered within LTR regions in the great number of sequences using MatInspector and PLACE software.The sequences have been deposited in the NCBI GenBank database under accession numbers of EU068698-EU068728.2.26 retrotransposon primers were designed based on 15 out of 31 RNaseH-LTR sequences,which included 8 LTR forward primers,10 LTR reverse primers and 8 PPT (Polypurine tract) primers.To enhance the specificity and reliability of PCR amplification, long length and high annealing temperature(Tm) value of designed primers were thoroughly considered in primer disigh.In this study,the length of retrotransposon primer was 23bp on average;Tm value was set beyond 55℃universally with an average of 60.2℃.Of which 24 primers were successfully amplified by IRAP,giving rise to reproducible and reliable bands.3.The optimal IRAP(Inter-retrotransposon amplified polymorphism) and REMAP (Retrotransposon-microsatellite amplified polymorphism) retrotransposon-based molecular marker systems were successfully established in Diospyros Linn.on the basis of optimizing the concentration of several components in PCR reaction,the value of Tm and the PCR amplification program,which were:in 20μL reaction system,Buffer 1×, template DNA 50 ng,Mg2+ 2.0 mmol/mL,dNTPs 0.25 mmol/L,primer 0.40μmol/L Taq DNA polymerase 1.0U and mineral oil 20μL.The optimal amplification program was:1 cycle at 95℃,5min;1 cycle at 95℃,1min;56℃1min;ramp+0.6℃/s to 72℃;44 cycles of 72℃,2min.1 cycle at 72℃,72℃8min.4.SSAP(Sequence-specific amplified polymorphism) retrotransposon molecular marker was also developed successfully and several key elements influencing its performance were analyzed.Moreover,the features of different retrotransposon applied for the study of genetic relationship in 28 Diospyros kaki Thunb.were compared.The results showed that polymorphisms detected in the insertion pattern of all the retrotransposons revealed that each can be used for SSAP.All elements including different retrotransposons,the kind of adapter primers and the base composition and the order of the selective nucleotides in selective amplification had influence on the performance of SSAP analysis.Different retrotransposon had an unique behavior in accessions tested,which may likely reflect distinct evolutionary histories and activities of particular retrotransposon.Taken together,our results would offer a technique approach for obtaining more resolved and detailed genetic phylogenetic information,estimating genetic diversity and distinguishing germplasm in Diospyros kaki Thunb.by retrotransposon-based SSAP molecular marker.5.ISTR(Inverse sequence-tagged repeat) retrotransposon molecular marker system based on universal primers originally derived from coconut was established in Diospyros Linn.,and was further used for the detection of genetic variability and genetic relationship analysis in 32 Diospyros Spp..IRAP was also used for the same set of genotypes analysis, and the performance of these two methods was compared.The results showed that both IRAP and ISTR could be applied for the genetic analysis in Diospyros Linn..IRAP could distinguish all the genotypes examined including bud sports.ISTR also presented good discrimination capability,but it was not capable for differentiate 'Matsumoto-wase' from its original variety 'Fuyuu'.The cluster pattern between IRAP and ISTR shared much similarities,however,some slight differences was showed.IRAP possessed the higher values of all the parameters for evaluating the efficiency of marker system including loci per assay unit,average number of polymorphic bands per assay unit,Effective Multiplex Ratio,Diversity Index and Marker Index than ISTR whether at the inter-specific level or at the intra-specific level.So,collection data suggested that Diospyros-specific IRAP retrotransposon marker techniques are superior to the universal primer-based ISTR retrotransposon marker in our study.6.Feasibility of IRAP for genetic relatedness analysis particularly for that of inter-species was investigated.Subsequently,comparative studies concerning the level of polymorphism and cluster results between IRAP and DAMD(Directed amplification of minisatellite-region DNA) molecular techniques were conducted.The results showed that IRAP could be capable for the genetic relatedness analysis between different species of Diospyros Linn.DAMD,although used for the ftrst time in the genus of Diospyros,also indicated good amplification,and it could distinguish bud sports examined.Species tested almost could be grouped well according to their different collection locality using two methods,however,there were incongruent concerning the genetic relatedness of species collected from tropic and subtropic regions with that from temperate regions in China. IRAP and ISTR produced almost comparable values of the polymorphic level,average amplified loci per assay unit and diversity index,whereas the former revealed the higher comprehensive performance than the later due to its higher multiplex effective ratio.In all, collective results suggested IRAP was an ideal marker system in the future for the detection of genetic polymorphism both at the inter-species level and at the intra-species level.Moreover,our results indicated that DAMD was also a potential powerful marker tool for the future genetic analysis in Diospyros Linn.7.Capability of IRAP for tiny differences detection within a cultivar was examined on the 11 'Mopanshi' genotypes which was selected and reported by Zhangfang town, Fangshan district of Beijing city,the most centralized regions of 'Mopanshi'.These genotypes had unique properties compared to the standard 'Mopanshi' cultivar.The results showed that 4 out of total 19 amplifiable primers,PPT3,PPT6,SAF5 and SAR10, was polymorphic primers between different 'Mopanshi' mutations.By using dichotomy classification,11 'Mopanshi' mutations could be completed distinguished by these 4 primers.In UPGMA dendrogram,'Mopanshi' mutations grouped tightly each other and with standard cultivar 'BZMP',and the branch pattern was significantly distinct from that between species,as well as those of different cultivars within the same species.The measurement of similarity coefficients revealed that the similarity coefficient between 'mopanshi' mutations was from 0.90('MP2' and 'MP4','MP2' and 'MP8','MP2' and 'MP9') to 0.98('MP7' and 'MP8'),and the mean was 0.94.Moreover,Between 'Mopanshi' mutations and the standard cultivar 'BZMP',the mean similarity coefficient was 0.91,which was over that of between species and that of between different cultivars, but approximately similar to that of between bud sports.Therefore,in conclusion,the present study ascertained that the variation of 11 'Mopanshi' mutations from Fandshan district of Beijing was genetic variability,but not alternation due to environment, development stage and nutrition status etc.;11 'Mopanshi' might be the bud sports arose from standard 'Mopanshi' cultivar in view of the great similarity between 11 genotypes, as well as between these mutations and the standard 'Mopanshi' cultivar;IRAP molecular technique could distinguish the tiny mutations,which implied that this method was an powerful genetic tool for the future cultivar identification,particularly for genotypes of great genetic similarity,such as bud sports.8.IRAP,REMAP and SSAP molecular markers were exploited for the germplasm identification.The results showed that these primers can be used both within and between species;each of 16 primers out of 26 produced numerous amplified loci and abundant polymorphism bands by IRAP(Inter-Retrotransposon Amplified Polymorphism) in 28 genotypes of Diospyros spp..Moreover,successful amplifications were also obtained when these primers were further used for REMAP and SSAP analysis.Each types of molecular marker produced unique fingerprint in 28 genotypes tested,and among of all used primers or primer combinations 2 IRAP primers(PPT3 and SAR10),4 SSAP primer combinations(SAR10/EA06,SAR9/MC03,SAR9/MC11 and SAR1/MC11) could be used individually for all germplasm differentiation.Furthermore,15 accessions that can been divided into 5 groups according to their similarity of morphologic characteristics and known relationships of bud sports were selected,and the genetic similarity within groups were computed to measure discrimination power of each molecular marker system, the result indicated that IRAP is the most sensitive and efficient one among 3 molecular techniques in germplasm identification.9.IRAP,REMAP,SSAP and AFLP were compared in terms of their informativeness and efficiency in a study of genetic relationships among 28 genotypes in the genus Diospyros.The results showed that IRAP represented a higher level of polymorphism and a greater information content,as assessed by the diversity index(DI), than the rest of molecular markers.The lower level of polymorphism for AFLP were obtained than IRAP and SSAP,which,nevertheless was the most efficient marker system due to its capacity to reveal the highest number of bands per reaction and because of the high values achieved for a considerable number of indexes.The value of marker index (MI) is lower for SSAP than that of the AFLP,but the former had a higher level of polymorphism than the latter.The correlation coefficients of similarity were statistically significant for all four marker systems used,but the relatively lower correlation with other markers was observed for REMAP.For all markers a high similarity in dendrogram topologies was obtained,which generally divided 28 genotypes examined into 3 groups according to different species and their different geographic origin.However,some differences in the positioning of some genotypes at the main groups were revealed due to different marker systems.In summary,we recommend,in the genus Diospyros,IRAP for the assessment of genetic diversity and germplasm discrimination,particular for theidentification of genetically very similar germplasm;and SSAP and AFLP were preferred for the study of phylogenetic,as well as the creation of a genetic linkage map.10.The transferability of retrotransposon primers derived from 'Luotian-tianshi' persimmon(Diospyros kaki Thunb.) in Diospyros spp.and other 13 kinds of fruit crops was investigated by way of IRAP retrotransposon molecular marker.Our results as follows:Retrotransposon primers from 'Luotian-tianshi' could be used well not only in inter-species and intra-species in Diospyros,but also in the other 13 kinds of fruit crops analyzed,which may be attribute to the horizontal and vertical transfer characters of retrotransposon.14 primers representated 53.85%primers tested could be amplified in 14 kinds of fruit crops simultaneously.Primers showed different amplified performance in different fruit crops,and the outstanding performance of transferability was revealed in four fruit crops including Diospros spp.,grape,Citrus and peach.Furthermore,based on IRAP molecular data sets from the effective primers amplification corresponding to the different fruit crops,genetic similarity coefficients were calculated and UPGMA cluster dendrograms were constructed.The results indicated that these effective primers could further offer a good potential tool for germplasm differentiation,parentage identification, classification and phylogenetic study,as well as genetic diversity assessment.The conclusion that retrotransposon primers could be of transferability among different plant species was further confirmed by examining the transferability of retrotransposon primers derived from published primers in Rosaceae,Gramineae and Solanaceae in 13 kinds of fruit crops.Our results were incongruent with previous researchs that thought retrotransposon primers was of species-specific and species-specific primers was one of a perquisite for the development of molecular markers.This is the first successful report of transferability of retrotransposon primers derived from 'Luotian-tianshi' of Diospyros to other fruit crops,and to our knowledge,it is also one of the limited number of related report concerning the transferability of retrotransposon primers available at present.It provided not only the proof of transferability of retrotransposon primers in different plant species,but also the precious resources of primers for other fruit crops,which is benefit for saving cost of retrotransposon primer development and for popularizing retrotransposon molecular markers.11.More than 963 cultivars named as Japanese persimmon exist in China,but hitherto most of them are PCA type.No PVNA and PVA types have been reported.In the present study,from the aspect of morphology,cytology,biochemistry and molecular biology,we investigated '90-1-10' which was introduced from Luotian county of Hubei province where the first Chinese native PCNA Japanese persimmon 'Luotian-tianshi' was found.The results were as follows:'90-1-10' had the similar tree type and fruit morphologic traits with other Chinese native persimmon germplasm,suggesting that it should be considered as a Chinese native persimmon,which was further confirmed by molecular marker analysis.The results of the tannin cell size test showed that '90-1-10' should be belong to non-astringent type persimmon.The results of the observation of brown in fruit flesh and soluble tannin content test indicated that it should be a pollination variant non-astringent(PVNA) type persimmon.In addition,'90-1-10' exhibited remarkable differences with 'Luotian-tianshi' and the other variations from 'Luotian-tianshi' in fruit morphology,the pattern of astringency-loss and genetic composition,implying that it should be a new germplasm genetically distinct from 'Luotian-tianshi'.In summary,collective results demonstrated that '90-1-10' should be a Chinese native PVNA type persimmon.This is the first report on existence of a PVNA type persimmon in China hitherto.This germplasm would be an important source for the phylogenetic study on Chinese native PCNA type persimmon and would have a potential role for breeding in the future. |
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