The complete chloroplast genome sequence of the Chinese endemic species Sorbus setschwanensis (Rosaceae) and its phylogenetic analysis

Sorbus setschwanensis Koehne is a pinnate-leaved Sorbus s.str. species endemic to China with narrow distribution and intriguing phylogeny that needs wider attention. In this paper, the complete chloroplast (cp) genome of S. setschwanensis is reported, and its phylogenetic position is analyzed. The complete cp genome of S. setschwanensis is 160 064 bp in size with 36.50% GC content. It has a typical quadripartite structure including a pair of inverted repeat regions (IRs) of 26 378 bp that separates a large single copy (LSC) region of 86 013 bp and a small single copy (SSC) region of 19 295 bp. The cp genome encodes 108 genes, comprising 76 protein-coding genes, 28 tRNA genes and 4 rRNA genes. Additionally, 52 simple sequence repeats (SSRs) and 43 dispersed repeats were identified. Comparison of the whole cp genome with those of other Sorbus species showed an overall high degree of sequence similarity, but there are six highly variable regions (trnR-atpA, petN-psbM, ndhC-trnV, trnE-trnT, trnT-trnL and rpl32-trnL) located in intergenic spacers that may be useful as molecular markers in future population genetic and phylogenetic studies in the genus. Phylogenetic analyses based on 108 coding genes from 25 species in Rosaceae revealed that S. setschwanensis is nested within Sorbus sect. Sorbus together with other pinnately leaved species, but does not form a sister lineage to S. rufopilosa belonging to the same series Multijugae. Thus, the systematic position of S. setschwanensis and relationships with other species in the genus needs to be further studied.     

大粒香水稻叶绿体基因组特征分析

大粒香作为贵州重要的优质稻资源,推广种植面积大,并且在乡村振兴过程中为社会带来了较高的经济效益。然而,目前对大粒香基因组学的理论研究报道较少。为揭示大粒香水稻叶绿体基因组(cpDNA)特征及系统发育关系,该研究对大粒香叶绿体进行测序,并分析其基因组特征。结果表明:(1)大粒香水稻cpDNA全长134 563 bp,包括1个大单拷贝区(LSC,80 864 bp)、1个小单拷贝区(SSC,12 347 bp)和2个反向重复序列区(IR_s,20 676 bp)。(2)注释到129个基因,可分为蛋白编码、tRNA和rRNA三类基因,数量分别为85个、36个和8个。(3)密码子偏好分析显示,大粒香cpDNA密码子偏好A碱基或U(T)碱基,亮氨酸密码子使用了1 944次,使用次数最多; 半胱氨酸的密码子仅使用198次,使用次数最少。(4)检测到129个SSR,其中有95个单核苷酸重复且大部分SSR序列由A/T碱基组成。(5)系统发育分析结果显示,大粒香与热带粳稻亲缘关系最近,聚为一类。该研究结果揭示了大粒香叶绿体基因组信息,并确定了大粒香系统发育所属分支。     

Changes in habitat specificity of species at their climatic range boundaries

Species are thought to have more restricted niches towards their range boundaries, although this has rarely been quantified systematically. We analysed transect data for 41 butterfly species along climatic gradients within Britain and show that 71% of species have broader niches at sites with milder winters. Shifts in habitat associations are considerable across most species' ranges; averaged across all 41 species, we estimate that if 26% of individuals were associated with the favoured habitat on the species' warmest transect, then 70% of individuals would be confined to this habitat on the species' coldest transect. Species with more southerly distributions in Britain showed the greatest changes in their habitat associations. We conclude that geographic variation in realized niche breadth is common and relatively large, especially near range boundaries, and should be taken into account in conserving species under changing climates.     

Complete nucleotide sequence of the sugarcane (Saccharum officinarum) chloroplast genome: a comparative analysis of four monocot chloroplast genomes.

The complete nucleotide sequence of the chloroplast genome of sugarcane (Saccharum officinarum) has been determined. It is a circular double-stranded DNA molecule, 141,182 bp in size, and is composed of a large single copy of 83,048 bp, a small single copy of 12,544 bp, and a pair of inverted repeat regions of 22,795 bp each. A comparative analysis among monocots showed that the sugarcane chloroplast genome was very similar to maize but not to rice or wheat. Between sugarcane and maize at the rps16-trnQ (UUG) region, however, a length polymorphism was identified. With regard to insertions/deletions equal to or longer than 5 bp, a total of 53 insertion and 31 deletion events were identified in the sugarcane chloroplast genome. Of the 84 loci identified, a pair of direct repeat sequences was located side by side in a tandem fashion in 47 loci (56.0%). A recombination event during plant evolution is discussed at two sites between the sugarcane and tobacco chloroplast genomes.     

Molecular phylogeny of Dipterocarpaceae in Indonesia based on chloroplast DNA

In order to construct a molecular phylogeny of Indonesian Dipterocarpoideae (Dipterocarpaceae), PCR-RFLP of the chloroplast regions rbcL, petB, psbA, psaA, and trnL-F was performed with seven restriction enzymes in 129 samples including 58 species from nine genera. In the strict consensus tree with Monotes kerstingii as outgroup Indonesian Dipterocarpaceae were divided into two major clades. One clade (bootstrap value=71) consisted of Upuna, Cotylelobium, Anisoptera, Vatica, Dipterocarpus (tribe Dipterocarpeae, bootstrap value=83) and Dryobalanops (tribe Shoreae, bootstrap value=99) in a basal position. The second clade consisted of Hopea, Parashorea, and Shorea (tribe Shoreae) with 95% bootstrap support. Tribe Dipterocarpeae is monophyletic, tribe Shoreae is polyphyletic since Dryobalanops is sister to tribe Dipterocarpeae. In the neighbour-joining tree the sister group position of Dryobalanops to tribe Dipterocarpeae is not supported by the bootstrap analysis. Alternatively, we used Upuna borneensis as outgroup. The effect of outgroup selection on tree topology, taxonomic classification and the interpretation of character evolution is discussed.     

Complete chloroplast genomes of Liliaceae (s.l.) species: comparative genomic and phylogenetic analyses

We first report the complete chloroplast (cp) genome of Fritillaria taipaiensis and determine its characteristics, sequence divergence and phylogenetic relationships by comparing it with complete cp genomes of Liliaceae s.l. (including e.g. Nartheciaceae, Amaryllidaceae and Asparagaceae) species obtained from NCBI Genbank. We show that the ycf1, ycf15 and infA genes have become pseudogenes or are lost in some of the seventeen Liliaceae species, and that dispersed repeats are prevailing among the four types of repeats (dispersed, palindromic, complement and tandem repeats). The number of simple sequence repeats ranged from 53 to 84 in the seventeen species, with mononucleotide repeats being the most abundant, followed by dinucleotides. A total of nine genes with positive selection sites were identified (atpB, atpE, ndhF, ndhH, petB, rpl2, rpl20, rpl22 and ycf2). Furthermore, we examined 19 mutational hotspot regions, including three coding regions (rps16, infA and rpl22) and sixteen non-coding regions. A phylogenetic analysis of the complete cp genomes and protein-coding sequences showed that Fritillaria is most closely related to Lilium. Moreover, Asparagus and Polygonatum, Hosta and Yucca are closely related to the Liliaceae. These results will contribute to further study of evolutionary patterns and phylogenetic relationships in Liliaceae s.l.     

Major lineages within Apiaceae subfamily Apioideae: a comparison of chloroplast restriction site and DNA sequence data

Traditional sources of taxonomic characters in the large and taxonomically complex subfamily Apioideae (Apiaceae) have been confounding and no classification system of the subfamily has been widely accepted. A restriction site analysis of the chloroplast genome from 78 representatives of Apioideae and related groups provided a data matrix of 990 variable characters (750 of which were potentially parsimony-informative). A comparison of these data to that of three recent DNA sequencing studies of Apioideae (based on ITS, rpoCl intron, and matK sequences) shows that the restriction site analysis provides 2.6–3.6 times more variable characters for a comparable group of taxa. Moreover, levels of divergence appear to be well suited to studies at the subfamilial and tribal levels of Apiaceae. Cladistic and phenetic analyses of the restriction site data yielded trees that are visually congruent to those derived from the other recent molecular studies. On the basis of these comparisons, six lineages and one paraphyletic grade are provisionally recognized as informal groups. These groups can serve as the starting point for future, more intensive studies of the subfamily.     

Complete structure of the chloroplast genome of a legume, Lotus japonicus.

The nucleotide sequence of the entire chloroplast genome (150,519 bp) of a legume, Lotus japonicus, has been determined. The circular double-stranded DNA contains a pair of inverted repeats of 25,156 bp which are separated by a small and a large single copy region of 18,271 bp and 81,936 bp, respectively. A total of 84 predicted protein-coding genes including 7 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids species were assigned on the genome based on similarity to genes previously identified in other chloroplasts. All the predicted genes were conserved among dicot plants except that rpl22, a gene encoding chloroplast ribosomal protein CL22, was missing in L. japonicus. Inversion of a 51-kb segment spanning rbcL to rpsl6 (positions 5161-56,176) in the large single copy region was observed in the chloroplast genome of L. japonicus. The sequence data and gene information are available on our World Wide Web database at http://www.kazusa.or.jp/en/plant/database.html.     

Enhanced dynamic range in a genetically encoded Ca sensor

Genetically encoded fluorescence resonance energy transfer (FRET) indicators are powerful tools for real-time detection of second messenger molecules and activation of signal proteins. However, these fluorescent protein-based sensors typically display marginal FRET efficiency. To improve their FRET efficiency for optical imaging and screening, we developed a number of fluorescent protein mutants based on cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). To improve FRET ratios, which were initially within a narrow dynamic range, we used DNA shuffling to develop a new FRET pair called 3xCFP/Venus. The optimized 3xCFP/Venus pair exhibited higher FRET ratios than CyPet/YPet, which has one of the greatest dynamic ranges of protein-based FRET pairs. We converted this FRET pair to a Ca²⁺ FRET indicators using circular permutation Venus (cpVenus) linked with 3xCFP to form 3xCFP/cpVenus, which displayed an ∼11-fold change in dynamic range in response to Ca²⁺ binding. The enhanced dynamic range for Ca²⁺ concentration detection using 3xCFP/cpVenus was confirmed in PC12 cells using previously established indicators (TN-XXL, ECFP/cpCitrine). To our knowledge, this FRET pair displays the largest dynamic range so far among genetically-encoded sensors, and can be used for sensitive FRET detection.     

Seasonal changes in taxon richness and abundance of mobile invertebrates inhabiting holdfast of annual kelp Ecklonia radicosa (Phaeophyceae,Lessoniaceae) at the central Pacific coast of Japan

To investigate seasonal changes in the taxon richness and abundance of mobile invertebrates inhabiting holdfasts of the warm temperate annual kelp Ecklonia radicosa, five holdfasts were collected monthly at the central Pacific coast of Japan from April to November 2014. During the study period, there was little variation in holdfast height and diameter, which ranged from 5.9 to 8.5 cm and from 7.1 to 10.8 cm, respectively. In total 7087 animals were collected from 40 holdfasts (177.2 individuals inds./holdfast, on average). The number of mobile invertebrates gradually increased from May (15 ± 9.9 inds./holdfast) to August (346 ± 152.5 inds./holdfast), with over 300 inds./holdfast until October before rapidly decreasing in November (110 ± 85.6 inds./holdfast). Similarly, taxon richness increased gradually from April (4.3 ± 1.0 taxa/holdfast) to August (11.0 ± 3.7 taxa/holdfast), and decreased in November (8.6 ± 2.3 taxa/holdfast). Interestingly, hundreds of mobile invertebrates inhabited holdfasts of kelp plants that had shed their blade in October and November. Taxon composition from August to October and the number of invertebrates from July to November were comparable to data previously reported for perennial kelps. In this study, the importance of annual Ecklonian species as biogenic habitats was demonstrated for the first time.     

Differentiation of Populus species using chloroplast single nucleotide polymorphism (SNP) markers--essential for comprehensible and reliable poplar breeding

Within the genus Populus several species belonging to different sections are cross-compatible. Hence, high numbers of interspecies hybrids occur naturally and, additionally, have been artificially produced in huge breeding programmes during the last 100 years. Therefore, determination of a single poplar species, used for the production of 'multi-species hybrids' is often difficult, and represents a great challenge for the use of molecular markers in species identification. Within this study, over 20 chloroplast regions, both intergenic spacers and coding regions, have been tested for their ability to differentiate different poplar species using 23 already published barcoding primer combinations and 17 newly designed primer combinations. About half of the published barcoding primers yielded amplification products, whereas the new primers designed on the basis of the total sequenced cpDNA genome of Populus trichocarpa Torr. & Gray yielded much higher amplification success. Intergenic spacers were found to be more variable than coding regions within the genus Populus. The highest discrimination power of Populus species was found in the combination of two intergenic spacers (trnG-psbK, psbK-psbl) and the coding region rpoC. In barcoding projects, the coding regions matK and rbcL are often recommended, but within the genus Populus they only show moderate variability and are not efficient in species discrimination.     

A new set of mono‐ and dinucleotide chloroplast microsatellites in Fagaceae

A new set of 14 chloroplast microsatellites, represented by mono‐ and dinucleotide repeats, was optimized in the three main species of the Fagaceae (Castanea sativa, Fagus sylvatica and Quercus petraea). The intraspecific variation was tested in some natural populations. The polymorphic microsatellites displayed two or three variants. Conservation of the primer pairs was checked on an additional set of six species of the Fagaceae and on Fraxinus excelsior. All the primer pairs produced a fragment of the expected size in the Fagaceae species while no amplification was obtained with 36% of the primers in F. excelsior.     

New chloroplast microsatellite markers suitable for assessing genetic diversity of Lolium perenne and other related grass species

Background and Aims

Lolium perenne (perennial ryegrass) is the most important forage grass species of temperate regions. We have previously released the chloroplast genome sequence of L. perenne ‘Cashel’. Here nine chloroplast microsatellite markers are published, which were designed based on knowledge about genetically variable regions within the L. perenne chloroplast genome. These markers were successfully used for characterizing the genetic diversity in Lolium and different grass species.

Methods

Chloroplast genomes of 14 Poaceae taxa were screened for mononucleotide microsatellite repeat regions and primers designed for their amplification from nine loci. The potential of these markers to assess genetic diversity was evaluated on a set of 16 Irish and 15 European L. perenne ecotypes, nine L. perenne cultivars, other Lolium taxa and other grass species.

Key Results

All analysed Poaceae chloroplast genomes contained more than 200 mononucleotide repeats (chloroplast simple sequence repeats, cpSSRs) of at least 7 bp in length, concentrated mainly in the large single copy region of the genome. Nucleotide composition varied considerably among subfamilies (with Pooideae biased towards poly A repeats). The nine new markers distinguish L. perenne from all non-Lolium taxa. TeaCpSSR28 was able to distinguish between all Lolium species and Lolium multiflorum due to an elongation of an A₈ mononucleotide repeat in L. multiflorum. TeaCpSSR31 detected a considerable degree of microsatellite length variation and single nucleotide polymorphism. TeaCpSSR27 revealed variation within some L. perenne accessions due to a 44-bp indel and was hence readily detected by simple agarose gel electrophoresis. Smaller insertion/deletion events or single nucleotide polymorphisms detected by these new markers could be visualized by polyacrylamide gel electrophoresis or DNA sequencing, respectively.

Conclusions

The new markers are a valuable tool for plant breeding companies, seed testing agencies and the wider scientific community due to their ability to monitor genetic diversity within breeding pools, to trace maternal inheritance and to distinguish closely related species.     

The complete chloroplast genomes of three Hamamelidaceae species: Comparative and phylogenetic analyses

Hamamelidaceae is an important group that represents the origin and early evolution of angiosperms. Its plants have many uses, such as timber, medical, spice, and ornamental uses. In this study, the complete chloroplast genomes of Loropetalum chinense (R. Br.) Oliver, Corylopsis glandulifera Hemsl., and Corylopsis velutina Hand.‐Mazz. were sequenced using the Illumina NovaSeq 6000 platform. The sizes of the three chloroplast genomes were 159,402 bp (C. glandulifera), 159,414 bp (C. velutina), and 159,444 bp (L. chinense), respectively. These chloroplast genomes contained typical quadripartite structures with a pair of inverted repeat (IR) regions (26,283, 26,283, and 26,257 bp), a large single‐copy (LSC) region (88,134, 88,146, and 88,160 bp), and a small single‐copy (SSC) region (18,702, 18,702, and 18,770 bp). The chloroplast genomes encoded 132–133 genes, including 85–87 protein‐coding genes, 37–38 tRNA genes, and 8 rRNA genes. The coding regions were composed of 26,797, 26,574, and 26,415 codons, respectively, most of which ended in A/U. A total of 37–43 long repeats and 175–178 simple sequence repeats (SSRs) were identified, and the SSRs contained a higher number of A + T than G + C bases. The genome comparison showed that the IR regions were more conserved than the LSC or SSC regions, while the noncoding regions contained higher variability than the gene coding regions. Phylogenetic analyses revealed that species in the same genus tended to cluster together. Chunia Hung T. Chang, Mytilaria Lecomte, and Disanthus Maxim. may have diverged early and Corylopsis Siebold & Zucc. was closely related to Loropetalum R. Br. This study provides valuable information for further species identification, evolution, and phylogenetic studies of Hamamelidaceae plants.     

虎杖叶绿体基因组结构与变异分析

虎杖(Reynoutria japonica Houtt.)为蓼科(Polygonaceae)蓼族(Polygoneae)虎杖属(Reynoutria Houtt.)植物,是一种传统的中草药,具有利湿退黄、清热解毒、散瘀止痛、止咳化痰的功效。本研究采取高通量测序技术获得5个虎杖品种的叶绿体全基因组序列,并与NCBI已公布的蓼族何首乌(Fallopia multiflora)和金线草(Antenoron filiforme)等植物的叶绿体全基因组序列进行了基因组学和系统发育分析。通过基因组学分析发现,5种虎杖的叶绿体基因组大小有163 376 bp和163 371 bp两种情况,并呈现出典型的环状四分体结构,85 784 bp的一条较长的单拷贝区(large single-copy region,LSC),18 616 bp的一条较短的单拷贝区(small single-copy region,SSC),还有两条长度一致的反向重复区,分别为IRa区和IRb区相间隔分布。通过注释得到161个基因,其中蛋白编码基因106个,rRNA编码基因10个,tRNA编码基因45个。总GC含量为36.7%,其中LSC、SSC和IR区域的GC含量分别为34.8%、30.7%和42.7%。对比不同虎杖品种叶绿体的全基因组分析发现,trnk-UUU、rpoC1、petD、rpl16、ndhA、rpl12基因的编码区发生了变异。系统发育分析显示,虎杖5个品种聚为一枝,处在已知11种蓼族材料最原始的位置,与何首乌构成姐妹群。