Chloroplast Genome Evolution Of Calycanthaceae And Highly Variable Chloroplast Markers Development Of Flowering Plants

Chloroplast is specific in green plants, its genomic information has been widely utilized in comparative genomics and plant phylogenetic studies. For the former we mainly analysized the evolution of chloroplast genome of Calycanthaceae to explore the angiosperm chloroplast genome insertion/deletion, inversion, repeat of short segments and other structural variation patterns. Meanwhile, we studied the sequence differentiation of Calycanthaceae genomes at different classification levels. For the latter, the major dilemmas of the plant phylogenetic studies is information shortage of markers. Therefore, in the second part of the paper, we intended to do some research in the development of chloroplast genome markers.I Studies on comparative of Calycanthaceae chloroplast genomeCalycanthaceae is a small family of Laurales which contains10species,3or4genera and2subfamilies—Calycanthoideae and Idiospermoidcae. The phylogenetic relationships of Calycanthaceae are relatively clear, thus it can be used to explore the sequence differentiation and the direction of structure evolution of the chloroplast genome. In this paper, we sequensed4chloroplast genomes of Calycanthaceae, coupled with chloroplast genome of Calycanthus floridus Linn which has been published already to reveal the variation of the chloroplast genome at subfamily, genus and species levels.The size of Calycanthaceae chloroplast genome is153,250-154,746bp, encoding a total of113genes. There are significant sequence differentiation between Calycanthoideae and Idiospermoideae with a degree of0.0175(π). Although differentiation is earlier in the two species of Calycanthus than that of Chimonanthus, the chloroplast genome sequence differentiation is quite limited. The most frequent structural variation occur in the chloroplast genome is insertion/deletion. The probability of its occurrence decreased significantly as its length increases, together with bias of deletions. Repeats are common micro-structural changes of the chloroplast genome, usually3-5bases. There are a total of8inversion events checked in the chloroplast genome of Calycanthaceae, including two independent events occur in both Calycanthus and Chimonanthus. The cause of such internal mechanisms still need further exploration. The rate of nucleotide substitutions in coding region is strong relatedand to gene function. The evolution rate of photosynthesis-related gene is generally low. The evolution rate of ycfl is the fastest in the chloroplast genome of Calycanthaceae.II Highly variable chloroplast markers development of flowering plantsAt present, plant molecular systematics and DNA barcoding techniques rely heavily on the use of chloroplast gene sequences. Because of the relatively low evolutionary rates of chloroplast genes, there are very few choices suitable for molecular studies on angiosperms at low taxonomic levels, and for DNA barcoding of species.We scanned the entire chloroplast genomes of12genera to search for highly variable regions. The sequence data of9genera were from GenBank and3genera were of our own. We identified nearly5%of the most variable loci from all variable loci in the chloroplast genomes of each genus, and then selected23loci that were present in at least three genera. The23loci included4coding regions,2introns, and17intergenic spacers. Of the23loci, the most variable (in order from highest variability to lowest) were intergenic regions ycfl-a, trnK, rpl32-trnL, and trnH-psbA, followed by trnSUGA-trnGucc, petA-psbJ, rpsl6-trnQ, ndhC-trnV, ycfl-b, ndhF, rpoB-trnC, psbE-petL, and rbcL-accD. Three loci, trnSUGA-trnGUCC, trnT-psbD, and trnW-psaJ, showed very high nucleotide diversity per site (π values) across three genera. Other loci may have strong potential for resolving phylogenetic and species identification problems at the species level. The loci accD-psal, rbcL-accD, rp132-trnL, rps16-trnQ, and ycfl are absent from some genera. To amplify and sequence the highly variable loci identified in this study, we designed primers from their conserved flanking regions. We tested the applicability of the primers to amplify target sequences in eight species representing basal angiosperms, monocots, eudicots, rosids, and asterids, and confirmed that the primers amplified the desired sequences of these species.Chloroplast genome sequences contain regions that are highly variable. Such regions are the first consideration when screening the suitable loci to resolve closely related species or genera in phylogenetic analyses, and for DNA barcoding.