Variation Patterns Of RRNA Genes In Pinus (Pinaceae) And Their Evolutionary Implications For Genome Structure, Gene Evolution, And Speciation Inferences

Ribosomal RNA genes (rDNA),a group of tandem repetitive sequence,are well-studied in angiosperms. Usually,1 - 4 pairs of 18S-5.8S-26S rDNA sites and 1-2 pairs of 5S rDNA sites are found in diploid genome of angiosperms. As a special type of multigene family,rDNA undergo concerted evolution by homogenizing forces through processes such as gene conversion,unequal crossing-over. For a long time we assumed strong concerted evolution in rDNAs of various animal and plant species and direct PCR-sequence approach has been widely used in molecular systematic research. However,the variation patterns of rDNA in gymnosperms are little known because of the limited sequence data. In Finns,18S-5.8S-26S rDNA localization were reported for five species,and 5S rDNA sequences were obtained for only three species. Till today there is no report on molecular evolution of 5S rDNA in Pinus. Thus,the rDNA variation pattern and organization in gymnosperm genomes are the primary focus of the present project. The evolutionary implication of our results for genome structure,gene evolution,and speciation inferences are discussed.In this study,fluorescence in situ hybridization (FISH) was used to localize rDNAs on chromosomes in seven Pinus species in order to reveal the rDNA organization. 5S rDNA cloning and sequencing were carried out to analyze the 5S rDNA variation pattern. Genomic in situ hybridization (GISH) was performed for better understanding of genomic relationship among closely relative pines. Among the species selected,P. densata is a putative hybrid between P. tabulaeformis and P. yunnanensis. The analyses of these three types of data in the hybrid and its parents could provide new information on genomic composition and evolutionary mechanisms of the hybrid. The main results are the followings:1. rDNA FISHChromosomal localization of 18S and 5S rDNA was carried out for two soft pines (Subgenus Strobus) and five hard pines (Subgenus Pinus) using FISH. The number of major 18S rDNA sites is generally much more than that in angiosperms and varies markedly among pines. There are seven pairs of 18S sites in Pinus tabulaeformis,five in P. desata,eight in P. yunnanensis,10 in P. massoniana,six in P. latteri,three in P. bungeana,and ten for P. armandi. Furthermore,weak signals are found on the centromeres of many chromosomes. Unlike 18S,pines have 1-2 pairs of 5S rDNA sites,except for P. armandi that has four pairs. Each pine could be discriminated by its rDNA FISH karyotype,although most of these pines cannot be distinguished by traditional karyotype analysis.The two types of rDNAs has different linkage pattern. In diploxylon pines,18S and 5S rDNA usually locate on the same or opposite arm of the same chromosome. 5S is more adjacent to the centromere if both rDNAs are on the same arm. In haploxylon pines,18S and 5S locate either on the same arm of the same chromosome or on different chromosomes. In the former situation,5S is near the telomere. This pattern shows the clear divergence of the two subgenera. At the same time,thedifferentiation of rDNA sites on chromosomes among pines correlates well to their phylogenic positions in Pimis reconstructed with other molecular data. P. densata resembles its parents by combining patterns characteristic of each parent as well as shows new features resulting possibly from recombination and genome reorganization.2 Heterogeneity and evolution of 5S rDNAPattern of intragenomic and interspecific variation of 5S rDNA hi five pines (including subgenus Strobus and Pinus) were studied through cloning and sequencing multiple 5S rDNA copies The length of 5S rDNA unit is 658-728 bp hi diploxylon pines while 499-521 bp hi the haploxylon P. bungeana. The conversed 120 bp gene region contains an intragenic control box related to the gene tanscription. The loops,identified in the secondary structure,are generally more conserved than stems. However,marked high transition/transversion was found on loop E. This could be caused by the presence of pseudogenes. In the spacer region,elements rel...