Comparative analyses of mitogenomes in the social bees with insights into evolution of long inverted repeats in the Meliponini

The insect mitogenome is typically a compact circular molecule with highly conserved gene contents.Nonetheless,mitogenome structural variations have been reported in specific taxa,and gene rearrangements,usually the tRNAs,occur in different lineages.Because synapomorphies of mitogenome organizations can provide information for phylogenetic inferences,comparative analyses of mitogenomes have been given increasing attention.However,most studies use a very few species to represent the whole genus,tribe,family,or even order,overlooking potential variations at lower taxonomic levels,which might lead to some incorrect inferences.To provide new insights into mitogenome organizations and their implications for phylogenetic inference,this study conducted comparative analyses for mitogenomes of three social bee tribes(Meliponini,Bombini,and Apini)based on the phylogenetic framework with denser taxonomic sampling at the species and population levels.Comparative analyses revealed that mitogenomes of Apini and Bombini are the typical type,while those of Meliponini show diverse variations in mitogenome sizes and organizations.Large inverted repeats(IRs)cause significant gene rearrangements of protein coding genes(PCGs)and rRNAs in Indo-Malay/Australian stingless bee species.Molecular evolution analyses showed that the lineage with IRs have lower dN/dS ratios for PCGs than lineages without IRs,indicating potential effects of IRs on the evolution of mitochondrial genes.The finding of IRs and different patterns of gene rearrangements suggested that Meliponini is a hotspot in mitogenome evolution.Unlike conserved PCGs and rRNAs whose rearrangements were found only in the mentioned lineages within Meliponini,tRNA rearrangements are common across all three tribes of social bees,and are significant even at the species level,indicating that comprehensive sampling is needed to fully understand the patterns of tRNA rearrangements,and their implications for phylogenetic inference.

Isolation and Identification of a Specific cDNA Mapping to the Bam HI-I2 and -LFragments within the Inverted Repeats ofUnique Long Re-gion (IRL) in the Genom e ofMarek′s Disease Herpesvirus (MDV) Oncogenic Strain Beijing-1

Objective\ To understand the transcription of BamHI L DNA fragment from genome of strong virulent GA strain of Marek′s disease herpesvirus (MDV) in lymphoblastoid tumor tissue induced by oncogenic strain Beijing 1 (a specific local strain in China) of MDV. Methods\ Two oligonucleotide primers were synthesized according to the reported sequence of \%meq\% gene an ideal oncogenic candidate and our previously determined sequence of BamHI L fragment of Marek′s disease herpesvirus (MDV), respectively. Reverse transcriptase PCR(RT PCR) assay was performed by using these primers and the mRNA as a template which was isolated from visceral lymphoblastoid tumors obtained from chickens artificially infected with strain Beijing 1 of oncogenic MDV. Southern blot molecular hybridization was further carried out to detect the product of RT PCR with digoxigenin labeled nucleotide probe from BamHI I2 and L fragment in the gene library of MDV strain GA, respectively. Results\ Two probes could simultaneously hybridize this cDNA amplified by RT PCR with a length of about 730 bp. Conclusion\ It is suggested that \%meq\% transcription could extend from the right hand end of BamHI I2 to the adjacent BamHI L, and the BamHI L region was likely to be transcribed in MDV induced lymphoblastoid tumors.

Lirex： A Package for Identification of LongInverted Repeats in Genomes

Long inverted repeats(LIRs) are evolutionarily and functionally important structures in genomes because of their involvement in RNA interference, DNA recombination, and gene duplication. Identification of LIRs is highly complicated when mismatches and indels between the repeats are permitted. Long inverted repeat explorer(Lirex) was developed and introduced in this report. Written in Java, Lirex provides a user-friendly interface and allows users to specify LIR searching criteria, such as length of the region, as well as pattern and size of the repeats. Recombinogenic LIRs can be selected on the basis of mismatch rate and internal spacer size from identified LIRs. Lirex, as a cross-platform tool to identify LIRs in a genome, may assist in designing following experiments to explore the function of LIRs. Our tool can identify more LIRs than other LIR searching tools. Lirex is publicly available at http://124.16.219.129/Lirex.

Phylogenetic placement of Cynomorium in Rosales inferred from sequences of the inverted repeat region of the chloroplast genome

Cynomorium is a herbaceous holoparasite that has been placed in Santalales, Saxifragales, Myrtales, or Sapindales. The inverted repeat （IR） region of the chloroplast genome region is slow evolving and, unlike mitochondrial genes, the chloroplast genome experiences few horizontal gene transfers between the host and parasite. Thus, in the present study, we used sequences of the IR region to test the phylogenetic placements of Cynomorium. Phylogenetic analyses of the chloroplast IR sequences generated largely congruent ordinal relationships with those from previous studies of angiosperm phylogeny based on single or multiple genes. Santalales was closely related to Caryophyllales and asterids. Saxifragales formed a clade where Peridiscus was sister to the remainder of the order, whereas Paeonia was sister to the woody clade of Saxifragales. Cynomorium is not closely related to Santalales, Saxifragales, Myrtales, or Sapindales; instead, it is included in Rosales and sister to Rosaceae. The various placements of the holoparasite on the basis of different regions of the mitochondrial genome may indicate the heterogeneous nature of the genome in the parasite. However, it is unlikely that the placement of Cynomorium in Rosales is the result of chloroplast gene transfer because Cynomorium does not parasitize on rosaceous plants and there is no chloroplast gene transfer between Cynomorium and Nitraria, a confirmed host of Cynomorium and a member of Sapindales.

Structural Analysis of rbcL Gene from an Endangered Plant，（Acanthopanax brachypus）

A 3.2 kb EcoRI DNA fragment containing the complete chloroplast rbcL gene from Acanthopanax brachypus has been cloned. In addition to the 1428 bp coding region encoding 475 amino acids of the gene, 278 bp upstream and 218 bp downstream were also sequenced. Possible ctpl and ctp2, equivalent to prokaryotic-35 and -10 elements, were found as TTGCGC and TACAAT respectively. The 5' untranslated leader region is 194 bp and the putative ribosome binding site in this region is GGAGG,located immediately upstream of the start codon. The 3' unrtanslated region contains two inverted repeat sequences, which in the mRNA might form stem-loop structures. The homology of rbcL amino acid sequence between A. brachypus and tobacco, spinach, pea, alfalfa, maize, rice, pine,Marchantia, Chlamydomonas and Anacystis are, respectively 93.05%,94.11%, 94.53%,89.68%, 92.21%, 2.21%, 92.63%, 87.58% and 80.84%.The promoter regions and part of the 5', 3' non-coding regions of rbcL from various plants were compared.

Relationship between Mutation of IR in the mtr System of Neisseria Gonorrhoeae and Multiple Antibiotic Resistance

To study the relationship between mutation of the inverted repeat sequence （IR） in the multiple transferable resistant system （mtr） of Neisseria gonorrhoeae （NG） and its multiple antibiotic resistance, minimal inhibitory concentrations （MICs） for the clinically isolated strains were tested by agar-dilution-method. The mtr system＇s IR gene of NG was sequenced after amplification by polymerase chain reaction （PCR）. Either two susceptive or five penicillin-resistant strains had no base mutation in IR gene, while all of the 13 strains with multiple-antibiotic-resistance had a singlebase deletion （A/T）. The result suggests that a single-base deletion of the thirteen-base IR sequence in mtr system of NG might result in multiple antibiotic resistance but is not associated with single antibiotic resistance.

Characterization of WAP2 gene in Aegilops tauschii and comparison with homoeologous loci in wheat

The Q/q gene, also known as WAP2, is an important gene for wheat domestication and is a member of the AP2 （APETALA2） class of transcription factors. In the present study, we first isolated the WRAP2 allele （where the superscript ＂t＂ refers to the speciese source, in this case ＂tauschii＂） on chromosome 5D from Aegilops tauschii Coss., the D-genome donor species of common wheat. We found that WRAP2 and the AP2 gene from Arabidopsis share a central core of the AP2 polypeptide, a highly basic 10-amino acid domain, and an AASSGF box, although there are many differences in the 37-amino acid serine-rich acidic domain and the remaining regions. In addition, WRAP2 was highly homologous to the homoeologous loci on 5A and 5B of wheat at both the nucleotide and amino acid level. However, there were some variations that are probably related to gene function. In the first AP2 domain, the amino acids VYL on the 5D and 5A loci were replaced with LLR on 5B. In the 37-amino acid serine-rich acidic domain, WRAP2 on 5D had an extra amino acid insertion. There was also a variation at the 329 amino acid position, which is thought to be related to the appearance of free-threshing wheat. At this position, the amino acid is isoleucine on 5A for the Q allele and valine for the q allele, whereas the amino acid is leucine on 5D and 5B. Furthermore, a Stowaway miniature terminal inverted repeat element （MITE） insertion was present in the ninth intron of WAP2 on 5B of all common wheats and partial tetraploid Triticum turgidum wheats. These results provide new clues for studies into the evolutionary biology of WAP2 and the origin of common wheat.

Enhancing rice grain production by manipulating the naturally evolved cis-regulatory element-containing inverted repeat sequence of OsREM20

Grain number per panicle(GNP)is an important agronomic trait that contributes to rice grain yield.Despite its importance in rice breeding,the molecular mechanism underlying GNP regulation remains largely unknown.In this study,we identified a previously unrecognized regulatory gene that controls GNP in rice,Oryza sativa REPRODUCTIVE MERISTEM 20(OsREM20),which encodes a B3 domain transcription factor.Through genetic analysis and transgenic validation we found that genetic variation in the CArG box-containing inverted repeat(IR)sequence of the OsREM20 promoter alters its expression level and contributes to GNP variation among rice varieties.Furthermore,we revealed that the IR sequence regulates OsREM20 expression by affecting the direct binding of OsMADS34 to the CArG box within the IR sequence.Interestingly,the divergent pOsREM20IR and pOsREM20ΔIR alleles were found to originate from different Oryza rufipogon accessions,and were independently inherited into the japonica and indica subspecies,respectively,during domestication.Importantly,we demonstrated that IR sequence variations in the OsREM20 promoter can be utilized for germplasm improvement through either genome editing or traditional breeding.Taken together,our study characterizes novel genetic variations responsible for GNP diversity in rice,reveals the underlying molecular mechanism in the regulation of agronomically important gene expression,and provides a promising strategy for improving rice production by manipulating the cis-regulatory element-containing IR sequence.

Complete chloroplast genome sequence of Magnolia grandiflora and comparative analysis with related species

Magnolia grandiflora is an important medicinal, ornamental and horticultural plant species. The chloroplast （cp） genome of M. grandiflora was sequenced using a 454 sequencing platform and the genome structure was compared with other related species. The complete cp genome ofM. grandiflora was 159623 bp in length and contained a pair of inverted repeats （IR） of 26563 bp separated by large and small single copy （LSC, SSC） regions of 87757 and 18740 bp, respectively. A total of 129 genes were successfully annotated, 18 of which included introns. The identity, number and GC content of M. grandiflora cp genes were similar to those of other Magnoliaceae species genomes. Analysis revealed 218 simple sequence repeat （SSR） loci, most composed of A or T, contributing to a bias in base composition. The types and abundances of repeat units in Magnoliaceae species were relatively conserved and these loci will be useful for developing M. grandiflora cp genome vectors. In addition, results indicated that the cp genome size in Magnoliaceae species and the position of the IR border were closely related to the length of the ycfl gene. Phylogenetic analyses based on 66 shared genes from 30 species using maximum parsimony （MP） and max- imum likelihood （ML） methods provided strong support for the phylogenetic position of Magnolia. The availability of the complete cp genome sequence of M. grandiflora provides valuable information for breeding of desirable varieties, cp genetic engineering, developing useful molecular markers and phylogenetic analyses in Magnoliaceae.