Complete Chloroplast Genome of Sedum sarmentosum and Chloroplast Genome Evolution in Saxifragales

Comparative chloroplast genome analyses are mostly carried out at lower taxonomic levels, such as the family and genus levels. At higher taxonomic levels, chloroplast genomes are generally used to reconstruct phylogenies. However, little attention has been paid to chloroplast genome evolution within orders. Here, we present the chloroplast genome of Sedum sarmentosum and take advantage of several available (or elucidated) chloroplast genomes to examine the evolution of chloroplast genomes in Saxifragales. The chloroplast genome of S. sarmentosum is 150,448 bp long and includes 82,212 bp of a large single-copy (LSC) region, 16.670 bp of a small single-copy (SSC) region, and a pair of 25,783 bp sequences of inverted repeats (IRs).The genome contains 131 unique genes, 18 of which are duplicated within the IRs. Based on a comparative analysis of chloroplast genomes from four representative Saxifragales families, we observed two gene losses and two pseudogenes in Paeonia obovata, and the loss of an intron was detected in the rps16 gene of Penthorum chinense. Comparisons among the 72 common protein-coding genes confirmed that the chloroplast genomes of S. sarmentosum and Paeonia obovata exhibit accelerated sequence evolution. Furthermore, a strong correlation was observed between the rates of genome evolution and genome size. The detected genome size variations are predominantly caused by the length of intergenic spacers, rather than losses of genes and introns, gene pseudogenization or IR expansion or contraction. The genome sizes of these species are negatively correlated with nucleotide substitution rates. Species with shorter duration of the life cycle tend to exhibit shorter chloroplast genomes than those with longer life cycles.     

Complete Chloroplast Genome of the Wollemi Pine (Wollemia nobilis): Structure and Evolution

The Wollemi pine (Wollemia nobilis) is a rare Southern conifer with striking morphological similarity to fossil pines. A small population of W. nobilis was discovered in 1994 in a remote canyon system in the Wollemi National Park (near Sydney, Australia). This population contains fewer than 100 individuals and is critically endangered. Previous genetic studies of the Wollemi pine have investigated its evolutionary relationship with other pines in the family Araucariaceae, and have suggested that the Wollemi pine genome contains little or no variation. However, these studies were performed prior to the widespread use of genome sequencing, and their conclusions were based on a limited fraction of the Wollemi pine genome. In this study, we address this problem by determining the entire sequence of the W. nobilis chloroplast genome. A detailed analysis of the structure of the genome is presented, and the evolution of the genome is inferred by comparison with the chloroplast sequences of other members of the Araucariaceae and the related family Podocarpaceae. Pairwise alignments of whole genome sequences, and the presence of unique pseudogenes, gene duplications and insertions in W. nobilis and Araucariaceae, indicate that the W. nobilis chloroplast genome is most similar to that of its sister taxon Agathis. However, the W. nobilis genome contains an unusually high number of repetitive sequences, and these could be used in future studies to investigate and conserve any remnant genetic diversity in the Wollemi pine.     

Insights from the Complete Chloroplast Genome into the Evolution of Sesamum indicum L

Sesame (Sesamum indicum L.) is one of the oldest oilseed crops. In order to investigate the evolutionary characters according to the Sesame Genome Project, apart from sequencing its nuclear genome, we sequenced the complete chloroplast genome of S. indicum cv. Yuzhi 11 (white seeded) using Illumina and 454 sequencing. Comparisons of chloroplast genomes between S. indicum and the 18 other higher plants were then analyzed. The chloroplast genome of cv. Yuzhi 11 contains 153,338 bp and a total of 114 unique genes ({"type":"entrez-nucleotide","attrs":{"text":"KC569603","term_id":"496538580","term_text":"KC569603"}}KC569603). The number of chloroplast genes in sesame is the same as that in Nicotiana tabacum, Vitis vinifera and Platanus occidentalis. The variation in the length of the large single-copy (LSC) regions and inverted repeats (IR) in sesame compared to 18 other higher plant species was the main contributor to size variation in the cp genome in these species. The 77 functional chloroplast genes, except for ycf1 and ycf2, were highly conserved. The deletion of the cp ycf1 gene sequence in cp genomes may be due either to its transfer to the nuclear genome, as has occurred in sesame, or direct deletion, as has occurred in Panax ginseng and Cucumis sativus. The sesame ycf2 gene is only 5,721 bp in length and has lost about 1,179 bp. Nucleotides 1–585 of ycf2 when queried in BLAST had hits in the sesame draft genome. Five repeats (R10, R12, R13, R14 and R17) were unique to the sesame chloroplast genome. We also found that IR contraction/expansion in the cp genome alters its rate of evolution. Chloroplast genes and repeats display the signature of convergent evolution in sesame and other species. These findings provide a foundation for further investigation of cp genome evolution in Sesamum and other higher plants.     

The Complete Plastid Genome Sequence of Madagascar Periwinkle Catharanthus roseus (L.) G. Don: Plastid Genome Evolution,Molecular Marker Identification,and Phylogenetic Implications in Asterids

The Madagascar periwinkle ( Catharanthus roseus in the family Apocynaceae) is an important medicinal plant and is the source of several widely marketed chemotherapeutic drugs. It is also commonly grown for its ornamental values and, due to ease of infection and distinctiveness of symptoms, is often used as the host for studies on phytoplasmas, an important group of uncultivated plant pathogens. To gain insights into the characteristics of apocynaceous plastid genomes (plastomes), we used a reference-assisted approach to assemble the complete plastome of C . roseus , which could be applied to other C . roseus -related studies. The C . roseus plastome is the second completely sequenced plastome in the asterid order Gentianales. We performed comparative analyses with two other representative sequences in the same order, including the complete plastome of Coffea arabica (from the basal Gentianales family Rubiaceae) and the nearly complete plastome of Asclepias syriaca (Apocynaceae). The results demonstrated considerable variations in gene content and plastome organization within Apocynaceae, including the presence/absence of three essential genes (i.e., accD, clpP, and ycf1) and large size changes in non-coding regions (e.g., rps2-rpoC2 and IRb-ndhF). To find plastome markers of potential utility for Catharanthus breeding and phylogenetic analyses, we identified 41 C . roseus -specific simple sequence repeats. Furthermore, five intergenic regions with high divergence between C . roseus and three other euasterids I taxa were identified as candidate markers. To resolve the euasterids I interordinal relationships, 82 plastome genes were used for phylogenetic inference. With the addition of representatives from Apocynaceae and sampling of most other asterid orders, a sister relationship between Gentianales and Solanales is supported.     

Complete Plastid Genome of the Brown Alga Costaria costata (Laminariales,Phaeophyceae)

Costaria costata is a commercially and industrially important brown alga. In this study, we used next-generation sequencing to determine the complete plastid genome of C. costata. The genome consists of a 129,947 bp circular DNA molecule with an A+T content of 69.13% encoding a standard set of six ribosomal RNA genes, 27 transfer RNA genes, and 137 protein-coding genes with two conserved open reading frames (ORFs). The overall genome structure of C. costata is nearly the same as those of Saccharina japonica and Undaria pinnatifida. The plastid genomes of these three algal species retain a strong conservation of the GTG start codon while infrequently using TGA as a stop codon. In this regard, they differ substantially from the plastid genomes of Ectocarpus siliculosus and Fucus vesiculosus. Analysis of the nucleic acid substitution rates of the Laminariales plastid genes revealed that the petF gene has the highest substitution rate and the petN gene contains no substitution over its complete length. The variation in plastid genes between C. costata and S. japonica is lower than that between C. costata and U. pinnatifida as well as that between U. pinnatifida and S. japonica. Phylogenetic analyses demonstrated that C. costata and U. pinnatifida have a closer genetic relationship. We also identified two gene length mutations caused by the insertion or deletion of repeated sequences, which suggest a mechanism of gene length mutation that may be one of the key explanations for the genetic variation in plastid genomes.     

The Tarenaya hassleriana Genome Provides Insight into Reproductive Trait and Genome Evolution of Crucifers

The Brassicaceae, including Arabidopsis thaliana and Brassica crops, is unmatched among plants in its wealth of genomic and functional molecular data and has long served as a model for understanding gene, genome, and trait evolution. However, genome information from a phylogenetic outgroup that is essential for inferring directionality of evolutionary change has been lacking. We therefore sequenced the genome of the spider flower (Tarenaya hassleriana) from the Brassicaceae sister family, the Cleomaceae. By comparative analysis of the two lineages, we show that genome evolution following ancient polyploidy and gene duplication events affect reproductively important traits. We found an ancient genome triplication in Tarenaya (Th-α) that is independent of the Brassicaceae-specific duplication (At-α) and nested Brassica (Br-α) triplication. To showcase the potential of sister lineage genome analysis, we investigated the state of floral developmental genes and show Brassica retains twice as many floral MADS (for MINICHROMOSOME MAINTENANCE1, AGAMOUS, DEFICIENS and SERUM RESPONSE FACTOR) genes as Tarenaya that likely contribute to morphological diversity in Brassica. We also performed synteny analysis of gene families that confer self-incompatibility in Brassicaceae and found that the critical SERINE RECEPTOR KINASE receptor gene is derived from a lineage-specific tandem duplication. The T. hassleriana genome will facilitate future research toward elucidating the evolutionary history of Brassicaceae genomes.     

Comparative Genome Analysis and Phylogenetic Relationship of Order Liliales Insight from the Complete Plastid Genome Sequences of Two Lilies (Lilium longiflorum and Alstroemeria aurea)

Monocots are one of the most diverse, successful and economically important clades of angiosperms. We attempt to analyse the complete plastid genome sequences of two lilies and their lengths were 152,793bp in Lilium longiflorum (Liliaceae) and 155,510bp in Alstroemeria aurea (Alstroemeriaceae). Phylogenetic analyses were performed for 28 taxa including major lineages of monocots using the sequences of 79 plastid genes for clarifying the phylogenetic relationship of the order Liliales. The sister relationship of Liliales and Asparagales-commelinids was improved with high resolution. Comparative analyses of inter-familial and inter-specific sequence variation were also carried out among three families of Liliaceae, Smilacaceae, and Alstroemeriaceae, and between two Lilium species of L . longflorum and L . superbum . Gene content and order were conserved in the order Liliales except infA loss in Smilax and Alstroemeria . IR boundaries were similar in IRa, however, IRb showed different extension patterns as JLB of Smilax and JSB in Alstroemeria . Ka/Ks ratio was high in matK among the pair-wise comparison of three families and the most variable genes were psaJ, ycf1, rpl32, rpl22, matK, and ccsA among the three families and rps15, rpoA, matK, and ndhF between Lilium.     

The Complete Plastid Genome Sequence of the Haptophyte Emiliania huxleyi: a Comparison to Other Plastid Genomes

The complete nucleotide sequence of the plastid genome of thehaptophyte Emiliania huxleyi has been determined. E. huxleyiis the most abundant coccolithophorid and has a key role inthe carbon cycle. It is also implicated in the production ofdimethylsulphide (DMS), which is involved in cloud nucleationand may affect the global climate. Here, we report the plastidgenome sequence of this ecologically and economically importantspecies and compare its gene content and arrangement to otherknown plastid genomes. The genome is circular and consists of105,309 bp with an inverted repeat of 4,841 bp. In terms ofboth genome size and gene content E. huxleyi cpDNA is substantiallysmaller than any other from the red plastid lineage. The geneticinformation is densely packed, with 86.8% of the genome specifying110 identified protein-coding genes, 9 open reading frames,28 different tRNAs, and 3 rRNAs. A detailed comparison to otherplastid genomes, based on gene content, gene function, and genecluster analysis is discussed. These analyses suggest a closerelationship of the E. huxleyi cpDNA to the chlorophyll c-containingplastids from heterokonts and cryptophytes, and they supportthe origin of the chromophyte plastids from the red algal lineage.     

Complete Chloroplast Genome of Tanaecium tetragonolobum: The First Bignoniaceae Plastome

Bignoniaceae is a Pantropical plant family that is especially abundant in the Neotropics. Members of the Bignoniaceae are diverse in many ecosystems and represent key components of the Tropical flora. Despite the ecological importance of the Bignoniaceae and all the efforts to reconstruct the phylogeny of this group, whole chloroplast genome information has not yet been reported for any members of the family. Here, we report the complete chloroplast genome sequence of Tanaecium tetragonolobum (Jacq.) L.G. Lohmann, which was reconstructed using de novo and referenced-based assembly of single-end reads generated by shotgun sequencing of total genomic DNA in an Illumina platform. The gene order and organization of the chloroplast genome of T. tetragonolobum exhibits the general structure of flowering plants, and is similar to other Lamiales chloroplast genomes. The chloroplast genome of T. tetragonolobum is a circular molecule of 153,776 base pairs (bp) with a quadripartite structure containing two single copy regions, a large single copy region (LSC, 84,612 bp) and a small single copy region (SSC, 17,586 bp) separated by inverted repeat regions (IRs, 25,789 bp). In addition, the chloroplast genome of T. tetragonolobum has 38.3% GC content and includes 121 genes, of which 86 are protein-coding, 31 are transfer RNA, and four are ribosomal RNA. The chloroplast genome of T. tetragonolobum presents a total of 47 tandem repeats and 347 simple sequence repeats (SSRs) with mononucleotides being the most common and di-, tri-, tetra-, and hexanucleotides occurring with less frequency. The results obtained here were compared to other chloroplast genomes of Lamiales available to date, providing new insight into the evolution of chloroplast genomes within Lamiales. Overall, the evolutionary rates of genes in Lamiales are lineage-, locus-, and region-specific, indicating that the evolutionary pattern of nucleotide substitution in chloroplast genomes of flowering plants is complex. The discovery of tandem repeats within T. tetragonolobum and the presence of divergent regions between chloroplast genomes of Lamiales provides the basis for the development of markers at various taxonomic levels. The newly developed markers have the potential to greatly improve the resolution of molecular phylogenies.     

Implications of the Plastid Genome Sequence of Typha (Typhaceae, Poales) for Understanding Genome Evolution in Poaceae

Plastid genomes of the grasses (Poaceae) are unusual in their organization and rates of sequence evolution. There has been a recent surge in the availability of grass plastid genome sequences, but a comprehensive comparative analysis of genome evolution has not been performed that includes any related families in the Poales. We report on the plastid genome of Typha latifolia, the first non-grass Poales sequenced to date, and we present comparisons of genome organization and sequence evolution within Poales. Our results confirm that grass plastid genomes exhibit acceleration in both genomic rearrangements and nucleotide substitutions. Poaceae have multiple structural rearrangements, including three inversions, three genes losses (accD, ycf1, ycf2), intron losses in two genes (clpP, rpoC1), and expansion of the inverted repeat (IR) into both large and small single-copy regions. These rearrangements are restricted to the Poaceae, and IR expansion into the small single-copy region correlates with the phylogeny of the family. Comparisons of 73 protein-coding genes for 47 angiosperms including nine Poaceae genera confirm that the branch leading to Poaceae has significantly accelerated rates of change relative to other monocots and angiosperms. Furthermore, rates of sequence evolution within grasses are lower, indicating a deceleration during diversification of the family. Overall there is a strong correlation between accelerated rates of genomic rearrangements and nucleotide substitutions in Poaceae, a phenomenon that has been noted recently throughout angiosperms. The cause of the correlation is unknown, but faulty DNA repair has been suggested in other systems including bacterial and animal mitochondrial genomes.     

Synthesis of 2-(4-Hydroxyphenyl)naphthalene-1,8-dicarboxylic Anhydride,a Phytoalexin Isolated from Unripe Banana (Musa acuminata)

2-(4-Hydroxyphenyl)naphthalene-1,8-dicarboxylic anhydride, a component of the phytoalexin that has been isolated from the peel of unripe banana (Musa acuminata), was synthesized from 3-bromoacenaphthene.     

The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis

Actinidia chinensis is an important economic plant belonging to the basal lineage of the asterids. Availability of a complete Actinidia chloroplast genome sequence is crucial to understanding phylogenetic relationships among major lineages of angiosperms and facilitates kiwifruit genetic improvement. We report here the complete nucleotide sequences of the chloroplast genomes for Actinidia chinensis and A. chinensis var deliciosa obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. The total genome size ranges from 155,446 to 157,557 bp, with an inverted repeat (IR) of 24,013 to 24,391 bp, a large single copy region (LSC) of 87,984 to 88,337 bp and a small single copy region (SSC) of 20,332 to 20,336 bp. The genome encodes 113 different genes, including 79 unique protein-coding genes, 30 tRNA genes and 4 ribosomal RNA genes, with 16 duplicated in the inverted repeats, and a tRNA gene (trnfM-CAU) duplicated once in the LSC region. Comparisons of IR boundaries among four asterid species showed that IR/LSC borders were extended into the 5’ portion of the psbA gene and IR contraction occurred in Actinidia. The clap gene has been lost from the chloroplast genome in Actinidia, and may have been transferred to the nucleus during chloroplast evolution. Twenty-seven polymorphic simple sequence repeat (SSR) loci were identified in the Actinidia chloroplast genome. Maximum parsimony analyses of a 72-gene, 16 taxa angiosperm dataset strongly support the placement of Actinidiaceae in Ericales within the basal asterids.     

沙冬青属植物叶绿体基因组对比和系统发育分析

该研究以沙冬青和矮沙冬青叶绿体基因组为研究对象,比较分析其基因组结构和系统发育关系。结果显示: (1)沙冬青和矮沙冬青的叶绿体基因组具有典型的四段式结构,全长为153 935 bp和154 140 bp,其中大单拷贝区(LSC)分别为83 891 bp和84 126 bp,小单拷贝区(SSC)分别为18 022 bp和18 014 bp,以及长度各自为26 011 bp和26 000 bp的成对反向重复区(IRs);沙冬青和矮沙冬青均注释130个基因,包括85个蛋白编码基因(PCGs),37个转运RNA(tRNA)以及8个核糖体RNA(rRNA)。(2)沙冬青和矮沙冬青的叶绿体基因组中分别检测出26和15个回文重复序列,39和50个串联重复序列,23和34个散在重复序列。同时都鉴定出96个SSRs位点,包括74和73个单核苷酸重复,5和6个二核苷酸重复,以及各有17个复合SSR位点;边界分析显示两者IR区相似,但仍有一定差异。(3)通过近邻结合法(NJ)对沙冬青和矮沙冬青在内的17种蝶形花亚科以及2种云实亚科植物的叶绿体基因组构建的系统发育树显示,沙冬青和矮沙冬青以较高的支持率聚为一个独立分枝。该研究结果为沙冬青属的种间鉴别、SSR分子标记开发、保育工作、种群动态以及进一步研究坡塔里族的演化过程奠定了理论基础。     

喜马红景天叶绿体基因组特征及其系统发育分析

以青藏高原特有药用植物——喜马红景天（Rhodiola himalensis）为试验材料,利用高通量测序技术对喜马红景天进行叶绿体基因组测序、组装和注释,获得完整的叶绿体基因组。结果显示：喜马红景天叶绿体基因组全长为151 074 bp,GC含量为37.8%,具有1个长单拷贝区、1个短单拷贝区和1对反向重复区的典型四分体结构,其序列长度分别为82 309、17 017、25 874 bp;叶绿体基因组共编码130个基因,其中编码蛋白的基因86个、编码tRNA的基因37个、编码rRNA的基因7个;叶绿体基因组共检测出25 513个密码子,其中编码亮氨酸（Leu）的密码子占比最大;喜马红景天IRa和IRb区的rps19和ycf1基因缺失,长单拷贝区的trnH基因收缩;喜马红景天与圣地红景天（R. sacra）亲缘关系最近;短单拷贝区域的单核苷酸多态性（SNP）变异频率最高。本研究报道了喜马红景天的叶绿体基因组,并对其进行了组装、注释和序列分析,为今后开展喜马红景天的遗传多样性研究和合理开发利用提供理论依据。     

Mycorrhizal dependency of banana (Musa acuminata,AAA group) cultivar

Seven banana cultivars (Musa acuminata, AAA group) were inoculated with two species of vesicular arbuscular mycorrhizal (VAM) fungi (Glomus mosseae and Glomus macrocarpum) in a greenhouse experiment. Inoculated plants had generally greater shoot dry weight and shoot phosphorus concentrations compared to the noninoculated plants. A great variation in dependency on mycorrhizal colonization was observed among the banana cultivars. Cv. Williams showed the highest relative mycorrhizal dependency (RMD) and cv. Poyo the lowest. For all the cultivars studied, inoculation with G. macrocarpum resulted in the highest RMD values. Both root dry weight and root hair length or density of the noninoculated plants were inverserly correlated with the RMD values of cultivars.     

The Complete Plastid Genome of Lagerstroemia fauriei and Loss of rpl2 Intron from Lagerstroemia (Lythraceae)

Lagerstroemia (crape myrtle) is an important plant genus used in ornamental horticulture in temperate regions worldwide. As such, numerous hybrids have been developed. However, DNA sequence resources and genome information for Lagerstroemia are limited, hindering evolutionary inferences regarding interspecific relationships. We report the complete plastid genome of Lagerstroemia fauriei. To our knowledge, this is the first reported whole plastid genome within Lythraceae. This genome is 152,440 bp in length with 38% GC content and consists of two single-copy regions separated by a pair of 25,793 bp inverted repeats. The large single copy and the small single copy regions span 83,921 bp and 16,933 bp, respectively. The genome contains 129 genes, including 17 located in each inverted repeat. Phylogenetic analysis of genera sampled from Geraniaceae, Myrtaceae, and Onagraceae corroborated the sister relationship between Lythraceae and Onagraceae. The plastid genomes of L. fauriei and several other Lythraceae species lack the rpl2 intron, which indicating an early loss of this intron within the Lythraceae lineage. The plastid genome of L. fauriei provides a much needed genetic resource for further phylogenetic research in Lagerstroemia and Lythraceae. Highly variable markers were identified for application in phylogenetic, barcoding and conservation genetic applications.     

Comparative Complete Chloroplast Genome Analyses and Contribution to the Understanding of Chloroplast Phylogeny and Adaptive Evolution in Subgenus Anguinum

Russian Journal of Genetics - The subgenus Anguinum (G. Don. ex W.D.J. Koch) N. Friesen disjunctly distributes in the high mountains from south-western Europe to eastern Asia and in northeastern...     

Complete Plastid Genome Sequencing of Four Tilia Species (Malvaceae): A Comparative Analysis and Phylogenetic Implications

Tilia is an ecologically and economically important genus in the family Malvaceae. However, there is no complete plastid genome of Tilia sequenced to date, and the taxonomy of Tilia is difficult owing to frequent hybridization and polyploidization. A well-supported interspecific relationships of this genus is not available due to limited informative sites from the commonly used molecular markers. We report here the complete plastid genome sequences of four Tilia species determined by the Illumina technology. The Tilia plastid genome is 162,653 bp to 162,796 bp in length, encoding 113 unique genes and a total number of 130 genes. The gene order and organization of the Tilia plastid genome exhibits the general structure of angiosperms and is very similar to other published plastid genomes of Malvaceae. As other long-lived tree genera, the sequence divergence among the four Tilia plastid genomes is very low. And we analyzed the nucleotide substitution patterns and the evolution of insertions and deletions in the Tilia plastid genomes. Finally, we build a phylogeny of the four sampled Tilia species with high supports using plastid phylogenomics, suggesting that it is an efficient way to resolve the phylogenetic relationships of this genus.     

贡蕉胚性细胞悬浮系的建立和植株再生

鲜食蕉品种的高度不育性和多倍性制约了用传统育种方法培育生产实践中所需的新品种 ,建立稳定的胚性细胞悬浮系是香蕉生物技术育种的前提。以目前国内尚未建立该体系的鲜食蕉品种贡蕉 (AA)未成熟雄花序的第 1～ 15位花梳为外植体 ,对胚性细胞悬浮系的建立和植株再生体系进行了优化。结果表明 ,5～ 6个月的培养后可获得分生小球体和浅黄色、松散易碎的胚性愈伤组织。 9μmol/L 2,4 D对外植体愈伤组织的诱导效果最好 ,诱导率为 40.96 % ,胚性愈伤组织诱导率可达7.45 % ,其中5.79%的胚性愈伤组织来源于第 6～12号位置的花梳。胚性愈伤组织悬浮培养后 ,通过 3个月的筛选和继代培养 ,可得到均质的胚性细胞悬浮系。该培养体系合适继代周期为 15d ,继代时合适的起始接种量为每 30mL培养基加 2mLPCVECS。培养 6个月的胚性细胞在体细胞胚诱导培养基中培养15d后可见到白色半透明体细胞胚的发生 ,体细胞胚诱导率为 2 80× 10³个 mLPCV。成熟体细胞胚的萌发率为 17 2 8% ,其中发育成正常的再生植株的百分率为 14 16 %。