Extensive gene rearrangements in the chloroplast DNAs of Chlamydomonas species featuring multiple dispersed repeats

We have constructed a physical and gene map for the chloroplast DNA (cpDNA) of the unicellular green alga Chlamydomonas gelatinosa, a close relative of Chlamydomonas reinhardtii. At 285 kb, the C. gelatinosa cpDNA is 89 kb larger than its C. reinhardtii counterpart. The alterations in the order of 77 genes on the cpDNAs of these green algae are attributable to nine inversions and one event of expansion/contraction of the inverted repeat. These rearrangements are much more extensive than those previously reported between the cpDNAs of the closely related Chlamydomonas moewusii and Chlamydomonas pitschmannii. Because the divergence level of the C. gelatinosa and C. reinhardtii chloroplast-encoded large subunit rRNA gene sequences is equivalent to that of the corresponding C. moewusii and C. pitschmannii sequences, our results may suggest that, in the same period of time, there have been more numerous rearrangements in the lineage comprising C. gelatinosa and C. reinhardtii than in the lineage comprising C. moewusii and C. pitschmannii. Alternatively, given that substitution rates in chloroplast genes are not necessarily uniform across lineages, the extensive rearrangements between the C. gelatinosa and C. reinhardtii cpDNAs may reflect a longer divergence period for this pair of Chlamydomonas species compared to that for the C. moewusii/C. pitschmannii pair. We have also found that, like its C. reinhardtii homologue but unlike its C. moewusii and C. pitschmannii counterparts, the C. gelatinosa cpDNA features a large number of dispersed repeated sequences that are readily detectable by Southern blot hybridization with homologous fragment probes. Assuming that the two pairs of closely related Chlamydomonas species diverged at about the same time, these data suggest that the susceptibility of Chlamydomonas cpDNAs to rearrangements is correlated with the abundance of repeated sequences. Preliminary characterization of a 345-bp C. gelatinosa cpDNA region containing a repeated sequence by both DNA sequencing and Southern blot analysis has revealed no sequence homology between this region and the cpDNAs of C. reinhardtii and other Chlamydomonas species.      

The complete structure of the cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) chloroplast genome: Its composition and comparative analysis

The complete nucleotide sequence of the cucumber (C. sativus L. var. Borszczagowski) chloroplast genome has been determined. The genome is composed of 155,293 bp containing a pair of inverted repeats of 25,191 bp, which are separated by two single-copy regions, a small 18,222-bp one and a large 86,688-bp one. The chloroplast genome of cucumber contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species), and 37 tRNA genes (30 tRNA species), with 18 of them located in the inverted repeat region. Of these genes, 16 contain one intron, and two genes and one ycf contain 2 introns. Twenty-one small inversions that form stem-loop structures, ranging from 18 to 49 bp, have been identified. Eight of them show similarity to those of other species, while eight seem to be cucumber specific. Detailed comparisons of ycf2 and ycf15, and the overall structure to other chloroplast genomes were performed.     

Deep genomic analysis of Coelastrella saipanensis (Scenedesmaceae,Chlorophyta): comparative chloroplast genomics of Scenedesmaceae

Many species belonging to the coccoid green algae genus Coelastrella are considered potential candidates for the large-scale production of natural pigments and biofuels. However, little is known about the structural, functional and molecular aspects of the chloroplast genomes (cpDNAs) of this genus. In the present study, the complete sequence of the cpDNA of strain FACHB-2138, which was further identified as Coelastrella saipanensis Hanagata based on morphological and molecular analyses, was elucidated. The 196 140 bp cpDNA sequence that was assembled as a circular map was found to possess the typical quadripartite structure. The two identical copies of 11 897 bp inverted repeat (IR) sequences were separated from one another by single copy regions. The large single copy region (LSC) was 104 949 bp, whereas the small single copy region (SSC) was 67 397 bp. The cpDNA encoded a total of 96 unique genes, which included 67 protein-coding genes, three rRNA genes and 26 tRNA genes. A total of 19 group I introns were annotated in this genome. Comparative analyses with three species from the family Scenedesmaceae showed C. saipanensis had a slightly expanded genome, higher GC content and less skewed distribution of its genes between the two DNA strands than that of the other three species. The cpDNA data deduced from the present study helps to expand our present understanding of plant systematics and phylogenetic reconstruction, and identify the possible biotechnological applications of the species belonging to the studied taxa.     

The complete chloroplast genome of the green algae <Emphasis Type="Italic">Hariotina reticulata</Emphasis> (Scenedesmaceae,Sphaeropleales, Chlorophyta)

In this study, the chloroplast genome of Hariotina reticulata was fully sequenced and compared to other Sphaeropleales chloroplast genomes. It is 210,757 bp larger than most Sphaeropleales cpDNAs. It presents a traditional chloroplast structure, and contains 103 genes, including 68 protein-coding genes, six rRNA genes and 29 tRNA genes. The coding region constitutes of 43% of the whole cpDNA. Eighteen introns are found in 11 genes and six introns are unique for Hariotina. 11 open reading frames are identified among these introns. The synteny between Hariotina and Acutodesmus cpDNAs is in general identical, while within Sphaeropleales order, high variability in cpDNA architecture is indicated by general high DCJ distances. Ankyra judayi exhibits the greatest dissimilarity in gene synteny to the others and share some unique gene clusters with Treubaria triappendiculata. The phylogenomic analyses show that A. judayi is clustered with Treubariaceae species and sister to Chlorophyceae incertae sedis and other Sphaeropleales species. The monophyly of Sphaeropleales is rejected.     

An improved chloroplast DNA extraction procedure for whole plastid genome sequencing

Background

Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs.

Methodology/Principal Findings

We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40–50% cpDNA purity is achieved with our method.

Conclusion

Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.     

Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis

Plants have varying abilities to tolerate chilling (low but not freezing temperatures), and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance.     

The Chloroplast Genome Sequence of Date Palm (Phoenix dactylifera L. cv. ‘Aseel’)

Date palm (Phoenix dactylifera L.) is an economically important and widely cultivated palm of the family Arecaceae. We sequenced the complete date palm chloroplast genome (cpDNA) from Pakistani cv. ??Aseel??, using a combination of Sanger-based and next-generation sequencing technologies. Being very similar to a sequence from a Saudi Arabian date palm cultivar ??Khalas?? published recently, the size of the genome was 158,458?bp with a pair of inverted repeat (IR) regions of 27,276?bp that were separated by a large single-copy (LSC) region of 86,195?bp and a small single-copy (SSC) region of 17,711?bp. Genome annotation demonstrated a total of 138 genes, of which 89 were protein coding, 39 were tRNA, and eight were rRNA genes. Comparison of cpDNA sequences of cultivars ??Aseel?? and ??Khalas?? showed following intervarietal variations in the LSC region; (a) two SNPs in intergenic spacers and one SNP in the rpoc1 gene, (b) polymorphism in two mono-nucleotide simple sequence repeats (SSR), and (c) a 4-bp indel in the accD-psaI intergenic spacer. The SSC region has a polymorphic site in the mono-nucleotide SSR located at position 120,710. We also compared cv. ??Aseel?? cpDNA sequence with partial P. dactylifera cpDNA sequence entries deposited in Genbank and identified a number of potentially useful polymorphisms in this species. Analysis of date palm cpDNA sequences revealed a close relationship with Typha latifolia. Occurrence of small numbers of forward and inverted repeats in date palm cpDNA indicated conserved genome arrangement.     

Investigation of chloroplast DNA heteroplasmy in Medicago sativa L. using cultured tissue

Summary Medicago sativa L. cv Regen S is heteroplasmic for chloroplast DNA (cpDNA). Previous analyses of regenerated plants have shown a predominance of one of the cpDNAs which we have designated type A (the other we have designated type B). Studies of the replication of the two cpDNAs in tissue culture were carried out using leaflet expiants with defined cpDNA types and a distinguishing probe. The explants obtained showed a bias toward type A cpDNA during tissue culture. The data suggest that chloroplasts with different DNAs in a common nuclear background can multiply at different rates.     

鲁桑叶绿体基因组序列及特征分析

鲁桑(Morus multicaulis)是亚洲地区栽培的重要经济作物。以鲁桑品种日本胡橙为实验材料, 利用高通量测序技术对鲁桑叶绿体基因组进行测序, 获得NCBI登录号(KU355297), 并研究鲁桑的叶绿体基因组结构。结合前人对蒙桑(M. mongolica)、印度桑(M. indica)和川桑(M. notabilis)的研究结果, 对鲁桑的系统进化关系进行了探讨。研究结果表明: 鲁桑叶绿体基因组是一个典型的四部分结构, 全长159 154 bp, 共注释130个基因, 包含85个蛋白质编码基因(18个基因在反向重复区重复)、37个转运RNA (tRNA)基因和8个核糖体RNA (rRNA)基因。生物信息学分析表明, 在鲁桑中共搜索到82个SSR位点, 单核苷酸、二核苷酸、三核苷酸、四核苷酸和五核苷酸重复基序个数分别为63、7、2、9和1个, 并没有发现六核苷酸; 其中单核苷酸重复在鲁桑的叶绿体基因组SSR中占76.8%。采用MEGA 6.0软件, 通过最大似然法和近邻结合法对包括4个桑属物种在内的15个物种的叶绿体基因组序列进行聚类分析, 2种方法得到的聚类结果均为鲁桑和蒙桑聚在一起。研究结果对叶绿体基因组工程研究及桑属种间的分子标记开发和优良品种培育具有一定的参考价值。     

Complete sequence and organization of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome

The nucleotide sequence of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome was completed (DQ119058). The circular double-stranded DNA, consisting of 155,527 bp, contained a pair of inverted repeat regions (IRa and IRb) of 25,187 bp each, which were separated by small and large single copy regions of 86,879 and 18,274 bp, respectively. The presence and relative positions of 113 genes (76 peptide-encoding genes, 30 tRNA genes, four rRNA genes, and three conserved open reading frames) were identified. The major portion (55.76%) of the C. sativus chloroplast genome consisted of gene-coding regions (49.13% protein coding and 6.63% RNA regions; 27.81% LSC, 9.46% SSC and 18.49% IR regions), while intergenic spacers (including 20 introns) made up 44.24%. The overall G-C content of C. sativus chloroplast genome was 36.95%. Sixteen genes contained one intron, while two genes had two introns. The expansion/contraction manner of IR at IRb/LSC and IR/SSC border in Cucumis was similar to that of Lotus and Arabidopsis, and the manner at IRa/LSC was similar to Lotus and Nicotiana. In total, 56 simple sequence repeats (more than 10 bases) were identified in the C. sativus chloroplast genome.     

The chloroplast genomes of Bryopsis plumosa and Tydemania expeditiones (Bryopsidales,Chlorophyta): compact genomes and genes of bacterial origin

Background

Species of Bryopsidales form ecologically important components of seaweed communities worldwide. These siphonous macroalgae are composed of a single giant tubular cell containing millions of nuclei and chloroplasts, and harbor diverse bacterial communities. Little is known about the diversity of chloroplast genomes (cpDNAs) in this group, and about the possible consequences of intracellular bacteria on genome composition of the host. We present the complete cpDNAs of Bryopsis plumosa and Tydemania expeditiones, as well as a re-annotated cpDNA of B. hypnoides, which was shown to contain a higher number of genes than originally published. Chloroplast genomic data were also used to evaluate phylogenetic hypotheses in the Chlorophyta, such as monophyly of the Ulvophyceae (the class in which the order Bryopsidales is currently classified).

Results

Both DNAs are circular and lack a large inverted repeat. The cpDNA of B. plumosa is 106,859 bp long and contains 115 unique genes. A 13 kb region was identified with several freestanding open reading frames (ORFs) of putative bacterial origin, including a large ORF (>8 kb) closely related to bacterial rhs-family genes. The cpDNA of T. expeditiones is 105,200 bp long and contains 125 unique genes. As in B. plumosa, several regions were identified with ORFs of possible bacterial origin, including genes involved in mobile functions (transposases, integrases, phage/plasmid DNA primases), and ORFs showing close similarity with bacterial DNA methyltransferases. The cpDNA of B. hypnoides differs from that of B. plumosa mainly in the presence of long intergenic spacers, and a large tRNA region. Chloroplast phylogenomic analyses were largely inconclusive with respect to monophyly of the Ulvophyceae, and the relationship of the Bryopsidales within the Chlorophyta.

Conclusions

The cpDNAs of B. plumosa and T. expeditiones are amongst the smallest and most gene dense chloroplast genomes in the core Chlorophyta. The presence of bacterial genes, including genes typically found in mobile elements, suggest that these have been acquired through horizontal gene transfer, which may have been facilitated by the occurrence of obligate intracellular bacteria in these siphonous algae.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1418-3) contains supplementary material, which is available to authorized users.     

The possible role of BAX and BI-1 genes in chilling-induced cell death in cucumber fruit

The aim of the present study was to investigate the possible role of BAX and BI-1 genes in chilling injury of cucumber fruit. BAX and BI-1 gene expressions were assayed under 2 ± 1 °C. Meanwhile, cell death, cellular integrity, specific chromatin fragmentation and nucleus morphology in cucumber (Cucumis sativus L. cv. Zhexiu-1) fruits were determined. Results indicated that BAX and BI-1 genes were activated by low temperature and the expression level of the BAX was much higher than BI-1. At the same time, electrolyte leakage and cell death were increased coupled with nuclear envelope disassembly and DNA fragmentation during the occurrence of chilling injury. In addition, characteristic features of programmed cell death were induced as well as the initiation of chilling injury. The interaction of BAX and BI-1 might predetermine the cell life or death in response to cold stimulus.     

Small, repetitive DNAs contribute significantly to the expanded mitochondrial genome of cucumber

Closely related cucurbit species possess eightfold differences in the sizes of their mitochondrial genomes. We cloned mitochondrial DNA (mtDNA) fragments showing strong hybridization signals to cucumber mtDNA and little or no signal to watermelon mtDNA. The cucumber mtDNA clones carried short (30-53 bp), repetitive DNA motifs that were often degenerate, overlapping, and showed no homology to any sequences currently in the databases. On the basis of dot-blot hybridizations, seven repetitive DNA motifs accounted for >13% (194 kb) of the cucumber mitochondrial genome, equaling >50% of the size of the Arabidopsis mitochondrial genome. Sequence analysis of 136 kb of cucumber mtDNA revealed only 11.2% with significant homology to previously characterized mitochondrial sequences, 2.4% to chloroplast DNA, and 15% to the seven repetitive DNA motifs. The remaining 71.4% of the sequence was unique to the cucumber mitochondrial genome. There was <4% sequence colinearity surrounding the watermelon and cucumber atp9 coding regions, and the much smaller watermelon mitochondrial genome possessed no significant amounts of cucumber repetitive DNAs. Our results demonstrate that the expanded cucumber mitochondrial genome is in part due to extensive duplication of short repetitive sequences, possibly by recombination and/or replication slippage.     

秦巴山区漆树nrDNAITS和cpDNA序列分子进化特点的分析

应用PCR产物直接测序法分析漆树种群nrDNA （核糖体DNA） ITS序列和cpDNA序列（matK、rbcL、psbA-trnH）的碱基差异,初步研究两套植物基因组的变异速率。结果表明：nrDNA ITS序列共有518 bp,有变异位点15处,变异位点百分率为2.9%,（G+C）含量为61.8%。cpDNA序列合并后长度1 907 bp,有变异位点20处,变异位点百分率为1.05%,（G+C）含量为36.1%。通过对ITS序列核糖型（Ribotype）和叶绿体序列单倍型（Haploype）进行分析发现,秦巴山区漆树区域性分布明显,不同区域拥有自己独特的单倍型,漆树居群历史近期没有扩张。nrDNA ITS序列较叶绿体序列进化较快,变异速率较快。nrDNA ITS序列及叶绿体matK、psbA-trnH适合漆树的亲缘地理学研究。     

Recombination of Chlamydomonas chloroplast DNA occurs more frequently in the large inverted repeat sequence than in the single-copy regions

Summary It is well documented that chloroplast DNA (cpDNA) recombination occurs at a relatively high frequency during sexual reproduction of unicellular green algae from the Chlamydomonas genus. Like the cpDNAs of most land plants, those of Chlamydomonas species are divided into two single-copy regions by a large inverted repeat sequence, part of which encodes the chloroplast rRNA genes. In the present study, we scored the inheritance of polymorphic loci spanning the entire chloroplast genome in hybrids recovered from reciprocal interspecific and F₁ crosses between Chlamydomonas eugametes and C. moewusii, and from these data, estimated the density of recombination junctions within each region of recombinant cpDNAs. Our results indicate that recombination junctions occur at highly variable frequencies across the three main domains of the chloroplast genome. The large inverted repeat sequence was found to exhibit at least a five-fold higher density of recombination junctions compared to one of the singlecopy regions, whereas junctions in the latter region were five-fold more abundant relative to those in the other single-copy region. This marked difference in the densities of recombination junctions implies that the extent of genetic linkage between two given chloroplast loci will depend not only on their physical distance, but also on their locations within the genome.     

藻类植物叶绿体基因组研究进展

藻类植物的cpDNA结构复杂,普遍缺失反向重复序列IR,且存在IR的藻类植物种类的cpDNA也有IR变短退化迹象.藻类植物的cpDNA包含的基因一般比高等植物要多,编码能力更强.藻类植物cpDNA全序列的测定方法主要是Fosmid文库构建,配合使用Long-PCR技术.该文对国内外有关藻类植物叶绿体基因组结构、叶绿体编码基因、叶绿体基因组在藻类系统发育中的应用以及藻类植物叶绿体基因组的提取和序列测定方法等进行综述,为藻类植物的系统发育和叶绿体起源以及功能基因组学的研究提供理论依据.     

Gene rearrangements in Chlamydomonas chloroplast DNAs are accounted for by inversions and by the expansion/contraction of the inverted repeat

To gain insight into the mutational events responsible for the extensive variation of chloroplast DNA (cpDNA) within the green algal genus Chlamydomonas, we have investigated the chloroplast gene organization of Chlamydomonas pitschmannii, a close relative of the interfertile species C. eugametos and C. moewusii whose cpDNAs have been well characterized. At 187 kb, the circular cpDNA of C. pitschmannii is the smallest Chlamydomonas cpDNA yet reported; it is 56 and 105 kb smaller than those of its C. eugametos and C. moewusii counterparts, respectively. Despite this substantial size difference, the arrangement of 77 genes on the C. pitschmannii cpDNA displays only three noticeable differences from the organization of the corresponding genes on the collinear C. eugametos and C. moewusii cpDNAs. These changes in gene order are accounted for by the expansion/contraction of the inverted repeat and one or two inversions in a single-copy region. In land plant cpDNAs, these kinds of events are also responsible for gene rearrangements. The large size difference between the C. pitschmannii and C. eugametos/C. moewusii cpDNAs is mainly attributed to multiple events of deletions/additions as opposed to the usually observed expansion/contraction of the inverted repeat in land plant cpDNAs. We also found that the mitochondrial genome of C. pitschmannii is a circular DNA molecule of 16.5 kb which is 5.5 and 7.5 kb smaller than its C. moewusii and C. eugametos counterparts, respectively.     

Involvement of polyamines in the chilling tolerance of cucumber cultivars

The possible involvement of polyamines (PAs) in the chilling tolerance of cucumber (Cucumis sativus L. cv Jinchun No. 3 and cv Suyo) was investigated. Plants with the first expanded leaves were exposed to 3 degrees C or 15 degrees C in the dark for 24 h (chilling), and then transferred to 28 degrees C/22 degrees C under a 12-h photoperiod for another 24 h (rewarming). Chilling-tolerant cv Jinchun No. 3 showed a marked increase of free spermidine (Spd) in leaves, once during chilling and again during rewarming. Putrescine increased significantly during rewarming, but the increase of spermine was slight. Any of these PAs did not increase in chilling-sensitive cv Suyo during either period. PA-biosynthetic enzyme activities appear to mediate these differences between cultivars. Pretreatment of Spd to cv Suyo prevented chill-induced increases in the contents of hydrogen peroxide in leaves and activities of NADPH oxidases and NADPH-dependent superoxide generation in microsomes and alleviated chilling injury. Pretreatment of methylglyoxal-bis-(guanylhydrazone), a PA biosynthesis inhibitor, to chilled cv Jinchun No. 3 prevented Spd increase and enhanced microsomal NADPH oxidase activity and chilling injury. The results suggest that Spd plays important roles in chilling tolerance of cucumber, probably through prevention of chill-induced activation of NADPH oxidases in microsomes.