Comparative genome analysis of three euplotid protists provides insights into the evolution of nanochromosomes in unicellular eukaryotic organisms

One of the most diverse clades of ciliated protozoa,the class Spirotrichea,displays a series of unique characters in terms of eukaryotic macronuclear(MAC)genome,including high fragmentation that produces nanochromosomes.However,the genomic diversity and evolution of nanochromosomes and gene families for spirotrich MAC genomes are poorly understood.In this study,we assemble the MAC genome of a representative euplotid(a new model organism in Spirotrichea)species,Euplotes aediculatus.Our results indicate that:(a)the MAC genome includes 35,465 contigs with a total length of 97.3 Mb and a contig N50 of 3.4 kb,and contains 13,145 complete nanochromosomes and 43,194 predicted genes,with the majority of these nanochromosomes containing tiny introns and harboring only one gene;(b)genomic comparisons between E.aediculatus and other reported spirotrichs indicate that average GC content and genome fragmentation levels exhibit interspecifc variation,and chromosome breaking sites(CBSs)might be lost during evolution,resulting in the increase of multi-gene nanochromosome;(c)gene families associated with chitin metabolism and FoxO signaling pathway are expanded in E.aediculatus,suggesting their potential roles in environment adaptation and survival strategies of E.aediculatus;and(d)a programmed ribosomal frameshift(PRF)with a conservative motif 5′-AAATAR-3′tends to occur in longer genes with more exons,and PRF genes play an important role in many cellular regulation processes.

Phylogeny of certain members of Hyrcanus group(Diptera:Culicidae)in China based on mitochondrial genome fragments

Background:Species of the A nopheles hyrcanus group are widely distributed in Palearctic and Oriental regions and some of them are important malaria vectors.The cryptic species of An.hyrcanus group was almost impossible to identify based only on their morphology.The phylogenetic relationship of An.hyrcanus group was also not clear.Methods:Five members of An.hyrcanus group were identified by rDNA ITS2 sequencing as An.yatsushiroensis,An.belenrae,An.kleini,An.lesteri and An.sineroides.The mitochondrial genome fragments were sequenced and annotated using the mitochondrial genome of An.sinensis as reference.Based on the four segments and Joint Data sequences of these species,and other four anopheline species downloaded from GenBank,intraspecific as well as interspecific genetic distances were calculated and the phylogenetic trees were reconstructed by the methods of neighbor joining,maximum parsimony,minimum evolution and maximum likelihood.Findings:Four parts of mitochondrial genomes,which were partial fragments COI+tRNA+COII(F5),ATP6+CO///(F7+F8),A/D7(F19)and IrRNA(F21),were obtained.All fragments were connected as one sequence(referred as Joint Data),which had a total length of 3393 bp.All fragment sequences were highly conservative within species,with the maximum p distance(0.026)calculated by F19 of An.belenroe.The pairwise interspecific p distance calculated by each fragment showed minor or even no difference among An.sinensis,An.kleini and An.belenrae.However,interspecific p distances calculated by the Joint Data sequence ranged from 0.004 (An.belenrae vs An.kleini)to 0.089(An.sineroides vs An.minimus),and the p distances of the six members o f An.hyrcanus group were all less than 0.029.The phylogenetic tree showed two major clades:all subgenus A nopheles species(including six members of An.hyrcanus group,An.otroparvus and An.quadrim aculatus A)and subgenus Cellia(including An.dirus and An.minimus).The An.hyrcanus group was divided into two clusters as{(An.lesteri,An.sineroides)An.yatsushiroensis)and({An.belenrae,An.sinensis)An.kleini)).Conclusions:The An.hyrcanus group in this study could be divided into two clusters,in one of which An.belenrae,An.sinensis and An.kleini were most closely related.More molecular markers would make greater contribution to phylogenetic analysis.

STI PCR: An efficient method for amplification and de novo synthesis of long DNA sequences

Despite continuous improvements,it is difficult to efficiently amplify large sequences from complex templates using current PCR methods.Here,we developed a suppression thermo-interlaced(STI)PCR method for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools.This method uses site-specific primers containing a common 50 tag to generate a stem-loop structure,thereby repressing the amplification of smaller non-specific products through PCR suppression(PS).However,large target products are less affected by PS and show enhanced amplification when the competitive amplification of non-specific products is suppressed.Furthermore,this method uses nested thermointerlaced cycling with varied temperatures to optimize strand extension of long sequences with an uneven GC distribution.The combination of these two factors in STI PCR produces a multiplier effect,markedly increasing specificity and amplification capacity.We also developed a webtool,calGC,for analyzing the GC distribution of target DNA sequences and selecting suitable thermo-cycling programs for STI PCR.Using this method,we stably amplified very long genomic fragments(up to 38 kb)from plants and human and greatly increased the length of de novo DNA synthesis,which has many applications such as cloning,expression,and targeted genomic sequencing.Our method greatly extends PCR capacity and has great potential for use in biological fields.