Phylogeny and evolution of Cervidae based on complete mitochondrial genomes

Mitochondrial DNA sequences can be used to estimate phylogenetic relationships among animal taxa and for molecular phylogenetic evolution analysis. With the development of sequencing technology, more and more mitochondrial sequences have been made available in public databases, including whole mitochondrial DNA sequences. These data have been used for phylogenetic analysis of animal species, and for studies of evolutionary processes. We made phylogenetic analyses of 19 species of Cervidae, with Bos taurus as the outgroup. We used neighbor joining, maximum likelihood, maximum parsimony, and Bayesian inference methods on whole mitochondrial genome sequences. The consensus phylogenetic trees supported monophyly of the family Cervidae; it was divided into two subfamilies, Plesiometacarpalia and Telemetacarpalia, and four tribes, Cervinae, Muntiacinae, Hydropotinae, and Odocoileinae. The divergence times in these families were estimated by phylogenetic analysis using the Bayesian method with a relaxed molecular clock method; the results were consistent with those of previous studies. We concluded that the evolutionary structure of the family Cervidae can be reconstructed by phylogenetic analysis based on whole mitochondrial genomes; this method could be used broadly in phylogenetic evolutionary analysis of animal taxa.     

Phylogenetic analysis of diprotodontian marsupials based on complete mitochondrial genomes

Australidelphia is the cohort, originally named by Szalay, of all Australian marsupials and the South American Dromiciops. A lot of mitochondria and nuclear genome studies support the hypothesis of a monophyly of Australidelphia, but some familial relationships in Australidelphia are still unclear. In particular, the familial relationships among the order Diprotodontia (koala, wombat, kangaroos and possums) are ambiguous. These Diprotodontian families are largely grouped into two suborders, Vombatiformes, which contains Phascolarctidae (koala) and Vombatidae (wombat), and Phalangerida, which contains Macropodidae, Potoroidae, Phalangeridae, Petauridae, Pseudocheiridae, Acrobatidae, Tarsipedidae and Burramyidae. Morphological evidence and some molecular analyses strongly support monophyly of the two families in Vombatiformes. The monophyly of Phalangerida as well as the phylogenetic relationships of families in Phalangerida remains uncertain, however, despite searches for morphological synapomorphy and mitochondrial DNA sequence analyses. Moreover, phylogenetic relationships among possum families (Phalangeridae, Petauridae, Pseudocheiridae, Acrobatidae, Tarsipedidae and Burramyidae) as well as a sister group of Macropodoidea (Macropodidae and Potoroidae) remain unclear. To evaluate familial relationships among Dromiciops and Australian marsupials as well as the familial relationships in Diprotodontia, we determined the complete mitochondrial sequence of six Diprotodontian species. We used Maximum Likelihood analyses with concatenated amino acid and codon sequences of 12 mitochondrial protein genomes. Our analysis of mitochondria amino acid sequence supports monophyly of Australian marsupials+Dromiciops and monophyly of Phalangerida. The close relatedness between Macropodidae and Phalangeridae is also weakly supported by our analysis.     

Comparing heat stress effects on male-fertile and male-sterile tomatoes

To separate the effects of heat stress on male and female reproductive tissues, male-sterile (MSs) and male-fertile tomatoes (MFs) were placed in growth chambers at 12 h day/12 h night temperatures of 28/22, 30/24 or 32/26 °C from flower appearance to seed maturation (daily mean temperatures of 25, 27 or 29 °C). Pollen from MFs was applied individually to MS flowers. As MFs were self-pollinated, heat stress was experienced by both male and female tissues. At growth temperatures of 29 °C fruit number, fruit weight per plant, and seed number per fruit were only 10%, 6·4% and 16·4%, respectively, compared with those at 25 °C. Heat stress also adversely affected fruitset in MSs, especially when experienced by donor pollen. No fruit at all developed on MSs receiving pollen produced at 29 °C, even when ovule development, pollen germination and subsequent embryo development all took place at 25 °C. Effects on fruitset in MSs were reduced if donor pollen had not experienced heat stress. MSs grown at 29 °C but receiving pollen developing at 25 °C produced 73% as much fruit (both on number and weight basis), had 40% as high fruitset and produced 87% of the seed per fruit as MSs grown at 25 °C. This use of male-sterile and male-fertile lines of tomato provides new evidence that impairment of pollen and anther development by elevated temperature will be an important contributing factor to decreased fruit set in tomato, and possibly other crops, with global warming.     

Sequencing complete mitochondrial and plastid genomes

Organelle genomics has become an increasingly important research field, with applications in molecular modeling, phylogeny, taxonomy, population genetics and biodiversity. Typically, research projects involve the determination and comparative analysis of complete mitochondrial and plastid genome sequences, either from closely related species or from a taxonomically broad range of organisms. Here, we describe two alternative organelle genome sequencing protocols. The "random genome sequencing" protocol is suited for the large majority of organelle genomes irrespective of their size. It involves DNA fragmentation by shearing (nebulization) and blunt-end cloning of the resulting fragments into pUC or BlueScript-type vectors. This protocol excels in randomness of clone libraries as well as in time and cost-effectiveness. The "long-PCR-based genome sequencing" protocol is specifically adapted for DNAs of low purity and quantity, and is particularly effective for small organelle genomes. Library construction by either protocol can be completed within 1 week.     

Phylogenetic analyses and improved resolution of the family Bovidae based on complete mitochondrial genomes

Efforts have been made to investigate the phylogeny of the family Bovidae; however, the relationships within this group still remain controversial. To further our understanding of the relationships, we sequenced the mitochondrial genome of the Himalayan goral, Naemorhedus goral, an IUCN Redlist near threatened conservation dependent species. Then we conducted molecular phylogenetic relationships of the Bovidae based on Bayesian and Maximum Likelihood methods. The results indicate that the basal divergence within the Bovidae is between the Bovinae and a strongly supported clade of the remaining Bovidae species. The two Neotragus species (the suni and pygmy antelope) clustered with the impala, Aepyceros melampus (Aepycerotinae), and together they formed the most basal of the non-Bovinae. All the genera of the Antilopinae clustered together except Neotragus, which suggested that the Antilopinae was a paraphyletic subfamily. The present study confirmed a close relationship between the genera Capricornis and Naemorhedus while supporting their designation as separate genera and suggested that the Capricornis-Naemorhedus-Ovibos clade (serows, gorals, and the muskox) should be placed in the Caprinae. Bison, Bos, and Tragelaphus (bison & cattle and kudus and nyalas) were paraphyletic. The very close relationship between Bison and Bos suggested that Bos and Bison should be integrated into a single Bos genus. Saiga and Pantholops (the Chiru or Tibetan Antelope), unique genera which have sometimes been lumped together, were placed in different groups: Saiga within the Antilopinae and Pantholops at the base of the Caprinae. Our results also supported a new taxonomy which places the three species of Hemitragus into three monospecific genera: the genus Hemitragus is restricted to the Himalayan tahr, and two new genera are created: Arabitragus for the Arabian tahr and Nilgiritragus for the Nilgiri tahr.     

Comparisons among two fertile and three male-sterile mitochondrial genomes of maize

We have sequenced five distinct mitochondrial genomes in maize: two fertile cytotypes (NA and the previously reported NB) and three cytoplasmic-male-sterile cytotypes (CMS-C, CMS-S, and CMS-T). Their genome sizes range from 535,825 bp in CMS-T to 739,719 bp in CMS-C. Large duplications (0.5-120 kb) account for most of the size increases. Plastid DNA accounts for 2.3-4.6% of each mitochondrial genome. The genomes share a minimum set of 51 genes for 33 conserved proteins, three ribosomal RNAs, and 15 transfer RNAs. Numbers of duplicate genes and plastid-derived tRNAs vary among cytotypes. A high level of sequence conservation exists both within and outside of genes (1.65-7.04 substitutions/10 kb in pairwise comparisons). However, sequence losses and gains are common: integrated plastid and plasmid sequences, as well as noncoding "native" mitochondrial sequences, can be lost with no phenotypic consequence. The organization of the different maize mitochondrial genomes varies dramatically; even between the two fertile cytotypes, there are 16 rearrangements. Comparing the finished shotgun sequences of multiple mitochondrial genomes from the same species suggests which genes and open reading frames are potentially functional, including which chimeric ORFs are candidate genes for cytoplasmic male sterility. This method identified the known CMS-associated ORFs in CMS-S and CMS-T, but not in CMS-C.     

Construction of backcrossedGelidium male-sterile and male-fertile lines and their growth comparison

The male sterility gene from a male-sterile, green,Gelidium vagum line was introduced to a wild-type line through repeated backcrossing and selection for five generations. The plants from the recurrent parent, the male-sterile green, the backcross-5 fertile and the backcross-5 male-sterile lines were compared for their growth performance. The backcross-5, red, male-sterile plants grew at a significantly higher rate than the plants from other lines, suggesting that male-sterile gametophytes would likely be better candidates for aquaculture than normal wild-type plants of this species. This study also provides evidence that the growth rate ofG. vagum decreases as a consequence of reproduction.     

Progress in the complete mitochondrial genomes of the Acari

蜱螨亚纲包括蜱类和螨类,是节肢动物中物种多样性最高的类群之一.本文综述了当前已测序的28种蜱螨线粒体基因组的研究成果.概括起来,蜱螨线粒体基因组具有以下特点:(1)大小变异显著,其中柑橘全爪螨Panonychus citri线粒体基因组在目前已测节肢动物中最小(13077 bp);(2)一般碱基组成偏向A和T,但6种蜱螨具有相反的GC-偏斜(正值);(3)基因组的碱基组成及A+T富集区的位置、长度和拷贝数等变异显著,其中4种叶螨的A+T含量最高,其A+T富集区在目前已测节肢动物中最短(44 -57 bp);(4)基因高度重排,特别是真螨总目的种类,但重排与高分类阶元无相关性;(5)真螨总目部分螨类的tRNA基因极度缩短,不能形成经典的三叶草二级结构.作者建议要进一步测定更多蜱螨的线粒体基因组,验证蜱螨非典型tRNA基因的生物学功能性,分析蜱螨线粒体基因组的分子进化机制,开展蜱螨线粒体转录组研究等.     

Analysis of three leafminers' complete mitochondrial genomes

Liriomyza trifolii (Burgess), Liriomyza huidobrensis (Blanchard), and Liriomyza bryoniae (Kaltenbach), are three closely related and economically important leafminer pests in the world. This study examined the complete mitochondrial genomes of L. trifolii, L. huidobrensis and L. bryoniae, which were 16141 bp, 16236 bp and 16183 bp in length, respectively. All of them displayed 37 typical animal mitochondrial genes and an A + T-rich region. The genomes were highly compact with only 60–68 bp of non-coding intergenic spacer. However, considerable differences in the A + T-rich region were detected among the three species. Results of this study also showed the two ribosomal RNA genes of the three species had very limited variable sites and thus should not provide much information in the study of population genetics of these species. Data generated from three leafminers' complete mitochondrial genomes should provide valuable information in studying phylogeny of Diptera, and developing genetic markers for species identification in leafminers.     

The rice mitochondrial genomes and their variations

Based on highly redundant and high-quality sequences, we assembled rice (Oryza sativa) mitochondrial genomes for two cultivars, 93-11 (an indica variety) and PA64S (an indica-like variety with maternal origin of japonica), which are paternal and maternal strains of an elite superhybrid rice Liang-You-Pei-Jiu (LYP-9), respectively. Following up with a previous analysis on rice chloroplast genomes, we divided mitochondrial sequence variations into two basic categories, intravarietal and intersubspecific. Intravarietal polymorphisms are variations within mitochondrial genomes of an individual variety. Intersubspecific polymorphisms are variations between subspecies among their major genotypes. In this study, we identified 96 single nucleotide polymorphisms (SNPs), 25 indels, and three segmental sequence variations as intersubspecific polymorphisms. A signature sequence fragment unique to indica varieties was confirmed experimentally and found in two wild rice samples, but absent in japonica varieties. The intersubspecific polymorphism rate for mitochondrial genomes is 0.02% for SNPs and 0.006% for indels, nearly 2.5 and 3 times lower than that of their chloroplast counterparts and 21 and 38 times lower than corresponding rates of the rice nuclear genome, respectively. The intravarietal polymorphism rates among analyzed mitochondrial genomes, such as 93-11 and PA64S, are 1.26% and 1.38% for SNPs and 1.13% and 1.09% for indels, respectively. Based on the total number of SNPs between the two mitochondrial genomes, we estimate that the divergence of indica and japonica mitochondrial genomes occurred approximately 45,000 to 250,000 years ago.     

蜱螨线粒体基因组研究进展

蜱螨亚纲包括蜱类和螨类, 是节肢动物中物种多样性最高的类群之一。本文综述了当前已测序的28种蜱螨线粒体基因组的研究成果。概括起来, 蜱螨线粒体基因组具有以下特点： （1）大小变异显著, 其中柑橘全爪螨Panonychus citri线粒体基因组在目前已测节肢动物中最小（13 077 bp）; （2）一般碱基组成偏向A和T, 但6种蜱螨具有相反的GC-偏斜（正值）; （3）基因组的碱基组成及A+T富集区的位置、 长度和拷贝数等变异显著, 其中4种叶螨的A+T含量最高, 其A+T富集区在目前已测节肢动物中最短（44~57 bp）; （4）基因高度重排, 特别是真螨总目的种类, 但重排与高分类阶元无相关性; （5）真螨总目部分螨类的tRNA基因极度缩短, 不能形成经典的三叶草二级结构。作者建议要进一步测定更多蜱螨的线粒体基因组, 验证蜱螨非典型tRNA基因的生物学功能性, 分析蜱螨线粒体基因组的分子进化机制, 开展蜱螨线粒体转录组研究等。     

Evolutionary sequence analysis of complete eukaryote genomes

Background  

Gene duplication and gene loss during the evolution of eukaryotes have hindered attempts to estimate phylogenies and divergence times of species. Although current methods that identify clusters of orthologous genes in complete genomes have helped to investigate gene function and gene content, they have not been optimized for evolutionary sequence analyses requiring strict orthology and complete gene matrices. Here we adopt a relatively simple and fast genome comparison approach designed to assemble orthologs for evolutionary analysis. Our approach identifies single-copy genes representing only species divergences (panorthologs) in order to minimize potential errors caused by gene duplication. We apply this approach to complete sets of proteins from published eukaryote genomes specifically for phylogeny and time estimation.     

The complete nucleotide sequence and multipartite organization of the tobacco mitochondrial genome: comparative analysis of mitochondrial genomes in higher plants

Tobacco is a valuable model system for investigating the origin of mitochondrial DNA (mtDNA) in amphidiploid plants and studying the genetic interaction between mitochondria and chloroplasts in the various functions of the plant cell. As a first step, we have determined the complete mtDNA sequence of Nicotiana tabacum. The mtDNA of N. tabacum can be assumed to be a master circle (MC) of 430,597 bp. Sequence comparison of a large number of clones revealed that there are four classes of boundaries derived from homologous recombination, which leads to a multipartite organization with two MCs and six subgenomic circles. The mtDNA of N. tabacum contains 36 protein-coding genes, three ribosomal RNA genes and 21 tRNA genes. Among the first class, we identified the genes rps1 and rps14, which had previously been thought to be absent in tobacco mtDNA on the basis of Southern analysis. Tobacco mtDNA was compared with those of Arabidopsis thaliana, Beta vulgaris, Oryza sativa and Brassica napus. Since repeated sequences show no homology to each other among the five angiosperms, it can be supposed that these were independently acquired by each species during the evolution of angiosperms. The gene order and the sequences of intergenic spacers in mtDNA also differ widely among the five angiosperms, indicating multiple reorganizations of genome structure during the evolution of higher plants. Among the conserved genes, the same potential conserved nonanucleotide-motif-type promoter could only be postulated for rrn18-rrn5 in four of the dicotyledonous plants, suggesting that a coding sequence does not necessarily move with the promoter upon reorganization of the mitochondrial genome.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. Hagemann