Genetic Analysis And A Preliminary Study On The Precisely Identification System Of Eucalyptus Germplasm

Eucalyptus germplasm materials in China mainly come from Australia where is the source area of eucalyptus.As the world’s fastest-growing tree species and has higher economic benefits,Eucalyptus has become the most representative tree species of fast-growing plantation and strategic tree species in southern China.The SSR marker was conducted in order to offer a powerful and efficient testing means for studies of population genetic diversity and germplasm identification of eucalyptus.Meanwhile,it can effectively promote the selection of improved varieties and the utilization of germplasm resources of eucalyptus.In this research,eucalyptus SSR molecular markers were developed by using the Eucalyptus genomic sequences and EST sequences of eucalyptus from the Genbank database.This will 159 eucalyptus germplasm materials as the main research object,to analysis the genetic diversity of eucalyptus germplasm resources,and to explore the genetic relationship among the eucalyptus species.Through selecting 4 SSRs which have the same exactly conservative sequences among different individuals in the species to premalinary construct an accurate identification system of eucalyptus interspecific germplasm resources.The main results are showed as follows:1.In this study,28,691 genome sequences and 16,566 expressed sequence tags(ESTs)of eucalyptus were derived from the Genbank database.A total of 2,292 SSR loci were sought out from 1,785 effective sequences,accounted for 12.64% of the total sequences which contain SSRs.By analysis of SSR loci information,it was found that SSR motif length was negatively correlated with the abundance of SSRs.In the EST sequences of eucalyptus,triplet repeat motifs were the most abundant,and dinucleotide repeats motifs had the highest frequencies.Subsequently,395 pairs of primers were synthesized based on all of the SSRs,and preliminary screened 340 pairs SSR primers by the method of optimizing SSR-PCR reaction system.Those primers can provide insights for evaluating genetic diversity of Eucalyptus and analyses of eucalyptus phylogenetics by SSR markers.2.In the research,6 eucalyptus DNA samples were used as the control group,a genomic DNA pooling as the treatment group to analyze the applicability of SSR primers screening by genomic DNA pooling.By re-sequencing analysis of the PCR amplification products,the control group and the treatment group have no significant difference.By applicating the genomic DNA pooling technology in re-sequencing the SSR ampliconic sequences of eucalyptus after sequences alignment and comparative analysis on SSRs,the results were revealed that the 283 pairs of SSR primers had a very good application in genetic diversity analysis and phyligenetic studies.Furthermore,39 species of eucalyptus were used for PCR reaction by 283 pair of SSR primers,and the electrophoresis results were satisfactory with clear strap,stable and good repetition in 268 pairs of primers,and those SSR primers have a relatively strong versatility in subgenus Symphyomyrtus.3.In the study,110 pairs of SSR primers which have steady and specificity amplification were be selected to PCR amplification,A total of 200 alleles were obtained from the analysis of PCR results among all 159 accessions by 110 pairs of SSR primers with an average of 1.818 alleles per pairs of primers ranging from 1 to 7.The number of homozygous allele was 162(81%),and the number of heterzoygous alleles was 38(19%).The results of genetic diversity showed that the mean value of effective number of alleles(Ne)was 1.172,and the average Shannon’s information index(I)was 0.181.The average observed heterozygosity(Ho)was 0.068,and the average value of polymorphism information content(PIC)was 0.182 in 47 polymorphic SSR loci.The highest genetic diversity level were found in the three loci eSSR-GR018,eSSR-GR083,and eSSR-GR109 by comprehensive comparison.And the three SSR loci would play an important roles in genetic diversity and germplasms identification among eucalyptus germplasm resources.The principal coordinate analysis showed that the classification in 110 SSR loci was basically agree with morphological of 42 eucalyptus species,and it proved that there had a significant genetic distance between Corymbia and Eucalyptus.The results obtained from UPGMA analysis indicated that the relationship between Eucalyptus cloeziana and E.pauciflora was relatively closer genetic distant,and they could be more likely to produce hybrids.The research provided a theoretical foundation and the basis for seed selection for eucalyptus germpalsm resources.4.The Blast results on Genbank database of ampliconic sequences by 20 SSR primers pairs which had same sizes of ampliconic sequences in each individuals among intraspecies of eucalyptus,indicated that different SSR loci were corresponding to different functional protein.By comparing the characteristics of sequences of eucalyptus interspecies and different individuals on 20 SSR loci indicated that the variation of sequences between eucalyptus interspecies were mainly due to the difference of the numbers of repetition and base mutation on flanking sequences;the variation of sequences between individuals within species were mainly due to the difference of the numbers of repetition.Genetic relationship analysis of 103 accessions of eucalyptus germplasm materials showed that several possible eucalyptus combinations of cross breeding were be suggested,it provided a theoretical foundation for selecting seeds of eucalyptus cross breeding.5.According to the features analysis of ampliconic sequences in 4 SSR loci found that apart from the variations of SSR motifs and SSR repeat numbers,base mutation was quite abundant in flanking regions(FRs)of the alleles sequenced.This conclusion is verified that the variability mechanism of SSR alleles in length is complicate.The precisely identification of barcodes among interspecific of eucalyptus germplasm was constructed with the combination of SSR repeat numbers and the specific bases in FRs on 4 SSR loci.12 species of eucalyptus were precisely indentificated in this identification system.The results turned out that the study of the construction of precisely identification system for eucalyptus germplasm had been gained preliminary achievement.The analysis of the barcodes which shared multiple species of eucalyptus indicated that those eucalyptus species have relatively closer genetic relationships,and this conclusion provided the biological basis for eucalyptus cross-breeding works.