Genome size and sequence composition of moso bamboo: A comparative study

Moso bamboo (Phyllostachys pubescens) is one of the world’s most important bamboo species. It has the largest area of all planted bamboo—over two-thirds of the total bamboo forest area—and the highest economic value in China. Moso bamboo is a tetraploid (4x=48) and a special member of the grasses family. Although several genomes have been sequenced or are being sequenced in the grasses family, we know little about the genome of the bambusoids (bamboos). In this study, the moso bamboo genome size was estimated to be about 2034 Mb by flow cytometry (FCM), using maize (cv. B73) and rice (cv. Nipponbare) as internal references. The rice genome has been sequenced and the maize genome is being sequenced. We found that the size of the moso bamboo genome was similar to that of maize but significantly larger than that of rice. To determine whether the bamboo genome had a high proportion of repeat elements, similar to that of the maize genome, approximately 1000 genome survey sequences (GSS) were generated. Sequence analysis showed that the proportion of repeat elements was 23.3% for the bamboo genome, which is significantly lower than that of the maize genome (65.7%). The bamboo repeat elements were mainly Gypsy/DIRS1 and Ty1/Copia LTR retrotransposons (14.7%), with a few DNA transposons. However, more genomic sequences are needed to confirm the above results due to several factors, such as the limitation of our GSS data. This study is the first to investigate sequence composition of the bamboo genome. Our results are valuable for future genome research of moso and other bamboos.     

Genome-wide characterization of the biggest grass, bamboo, based on 10,608 putative full-length cDNA sequences

Background

With the availability of rice and sorghum genome sequences and ongoing efforts to sequence genomes of other cereal and energy crops, the grass family (Poaceae) has become a model system for comparative genomics and for better understanding gene and genome evolution that underlies phenotypic and ecological divergence of plants. While the genomic resources have accumulated rapidly for almost all major lineages of grasses, bamboo remains the only large subfamily of Poaceae with little genomic information available in databases, which seriously hampers our ability to take a full advantage of the wealth of grass genomic data for effective comparative studies.

Results

Here we report the cloning and sequencing of 10,608 putative full length cDNAs (FL-cDNAs) primarily from Moso bamboo, Phyllostachys heterocycla cv. pubescens, a large woody bamboo with the highest ecological and economic values of all bamboos. This represents the third largest FL-cDNA collection to date of all plant species, and provides the first insight into the gene and genome structures of bamboos. We developed a Moso bamboo genomic resource database that so far contained the sequences of 10,608 putative FL-cDNAs and nearly 38,000 expressed sequence tags (ESTs) generated in this study.

Conclusion

Analysis of FL-cDNA sequences show that bamboo diverged from its close relatives such as rice, wheat, and barley through an adaptive radiation. A comparative analysis of the lignin biosynthesis pathway between bamboo and rice suggested that genes encoding caffeoyl-CoA O-methyltransferase may serve as targets for genetic manipulation of lignin content to reduce pollutants generated from bamboo pulping.     

毛竹基因组大小测定

毛竹(Phyllostachys edulis)属禾本科(Poaceae)竹亚科(Bambusoideae)刚竹属(Phyllostachys), 是我国分布和栽培面积最大的经济竹种, 有着广泛的开发前景。本实验以水稻(Oryza sativa)为内标, 用流式细胞仪对水稻和竹子样品的PI发射荧光强度进行测定, 通过比较水稻与毛竹样品峰值的倍数关系, 计算出毛竹的基因组大小。对24组样品进行重复测试, 测得毛竹基因组大小为 2 075.025±13.08 Mb, 即2 C DNA含量为4.24 pg(以1 pg DNA = 0.978×109 bp计算)。毛竹基因组大小测定为毛竹基因组文库的建立及其基因组学研究奠定了重要基础。     

Genome-wide characterization and evolution analysis of long terminal repeat retroelements in moso bamboo (<Emphasis Type="Italic">Phyllostachys edulis</Emphasis>)

The availability of nearly complete moso bamboo genome sequences permits the detailed discovery and cross-species comparison of transposable elements (TEs) between Bambusoideae and other Poaceae species at the whole genome level. Long terminal repeat retroelements (LTR-retroelements) are the single largest components of most plant genomes and can substantially impact the genome in various ways. Through a combination of structure- and homology-based approaches, we initially investigated 982 LTR-retroelement families comprising 2,004,644 LTR-retroelement sequences, which accounted for more than 40% of the moso bamboo genome. Further analysis revealed that the ratio of solo LTRs to intact elements (S/I) in moso bamboo is significantly low (approximately 0.28:1), indicating that bamboo LTR-retroelements might have undergone relatively low frequencies of unequal recombination and illegitimate recombination. Phylogenetic analysis revealed four Ty1-copia and five Ty3-gypsy evolutionary lineages that were present before the divergence of eudicot and monocot species, but the scales and timeframes within which they proliferated significantly varied across families and lineages. Insertion time estimates showed that LTR-retroelements were amplified for approximately 0~3 million years and had longer periods of activity than those of rice and Arabidopsis. These findings suggest that the expansion of LTR-retroelements might be responsible for host large genome size during moso bamboo evolution.     

毛竹基因组大小测定

毛竹（Phyllostachys edulis）属禾本科（Poaceae）竹亚科（Bambusoideae）刚竹属（Phyllostachys）,是我国分布和栽培面积最大的经济竹种,有着广泛的开发前景。本实验以水稻（Oryza sativa）为内标,用流式细胞仪对水稻和竹子样品的PI发射荧光强度进行测定,通过比较水稻与毛竹样品峰值的倍数关系,计算出毛竹的基因组大小。对24组样品进行重复测试,测得毛竹基因组大小为2075.025±13.08Mb,即2C DNA含量为4.24pg（以1pg DNA=0.978×10^9bp计算）。毛竹基因组大小测定为毛竹基因组文库的建立及其基因组学研究奠定了重要基础。     

An optimal proportion of mixing broad‐leaved forest for enhancing the effective productivity of moso bamboo

Moso bamboos (Phyllostachys edulis) are important forestry plants in southern China, with substantial roles to play in regional economic and ecological systems. Mixing broad‐leaved forests and moso bamboos is a common management practice in China, and it is fundamental to elucidate the interactions between broad‐leaved trees and moso bamboos for ensuring the sustainable provision of ecosystem services. We examine how the proportion of broad‐leaved forest in a mixed managed zone, topology, and soil profile affects the effective productivity of moso bamboos (i.e., those with significant economic value), using linear regression and generalized additive models. Bamboo's diameter at breast height follows a Weibull distribution. The importance of these variables to bamboo productivity is, respectively, slope (25.9%), the proportion of broad‐leaved forest (24.8%), elevation (23.3%), gravel content by volume (16.6%), slope location (8.3%), and soil layer thickness (1.2%). Highest productivity is found on the 25° slope, with a 600‐m elevation, and 30% broad‐leaved forest. As such, broad‐leaved forest in the upper slope can have a strong influence on the effective productivity of moso bamboo, ranking only after slope and before elevation. These factors can be considered in future management practice.     

Genome-wide characterization and evolution analysis of miniature inverted-repeat transposable elements (MITEs) in moso bamboo (<Emphasis Type="Italic">Phyllostachys heterocycla</Emphasis>)

Main conclusion

Moso bamboo MITEs were genome-wide identified first time, and data shows that MITEs contribute to the genomic diversity and differentiation of bamboo. Miniature inverted-repeat transposable elements (MITEs) are widespread in animals and plants. There are a large number of transposable elements in moso bamboo (Phyllostachys heterocycla var. pubescens) genome, but the genome-wide information of moso bamboo MITEs is not known yet. Here we identified 362 MITE families with a total of 489,592 MITE-related sequences, accounting for 4.74 % of the moso bamboo genome. The 362 MITE families are clustered into six known and one unknown super-families. Our analysis indicated that moso bamboo MITEs preferred to reside in or near the genes that might be involved in regulation of host gene expression. Of the seven super-families, three might undergo major expansion event twice, respectively, during 8–11 million years ago (mya) ago and 22–28 mya ago; two might experience a long expansion period from 6 to 13 mya. Almost 1/3 small RNAs might be derived from the MITE sequences. Some MITE families generate small RNAs mainly from the terminals, while others predominantly from the central region. Given the high copy number of MITEs, many siRNAs and miRNAs derived from MITE sequences and the preferential insertion of MITE into gene regions, MITEs may contribute to the genomic diversity and differentiation of bamboo.

     

Detailed Analysis of a Contiguous 22-Mb Region of the Maize Genome

Most of our understanding of plant genome structure and evolution has come from the careful annotation of small (e.g., 100 kb) sequenced genomic regions or from automated annotation of complete genome sequences. Here, we sequenced and carefully annotated a contiguous 22 Mb region of maize chromosome 4 using an improved pseudomolecule for annotation. The sequence segment was comprehensively ordered, oriented, and confirmed using the maize optical map. Nearly 84% of the sequence is composed of transposable elements (TEs) that are mostly nested within each other, of which most families are low-copy. We identified 544 gene models using multiple levels of evidence, as well as five miRNA genes. Gene fragments, many captured by TEs, are prevalent within this region. Elimination of gene redundancy from a tetraploid maize ancestor that originated a few million years ago is responsible in this region for most disruptions of synteny with sorghum and rice. Consistent with other sub-genomic analyses in maize, small RNA mapping showed that many small RNAs match TEs and that most TEs match small RNAs. These results, performed on ∼1% of the maize genome, demonstrate the feasibility of refining the B73 RefGen_v1 genome assembly by incorporating optical map, high-resolution genetic map, and comparative genomic data sets. Such improvements, along with those of gene and repeat annotation, will serve to promote future functional genomic and phylogenomic research in maize and other grasses.     

Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice

Summary The entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice (Oryza sativa cv. Nipponbare) was identified using clone banks that cover the chloroplast and mitochondrial genomes. The mitochondrial fragments that were homologous to chloroplast DNA were mapped and sequenced. The nucleotide sequences around the termini of integrated chloroplast sequences in the rice mtDNA revealed no common sequences or structures that might enhance the transfer of DNA. Sixteen chloroplast sequences, ranging from 32 bases to 6.8 kb in length, were found to be dispersed throughout the rice mitochondrial genome. The total length of these sequences is equal to approximately 6% (22 kb) of the rice mitochondrial genome and to 19% of the chloroplast genome. The transfer of segments of chloroplast DNA seems to have occurred at different times, both before and after the divergence of rice and maize. The mitochondrial genome appears to have been rearranged after the transfer of chloroplast sequences as a result of recombination at these sequences. The rice mitochondrial DNA contains nine intact tRNA genes and three tRNA pseudogenes derived from the chloroplast genome.     

Aluminum Tolerance in Moso Bamboo (<Emphasis Type="Italic">Phyllostachys pubescens</Emphasis>)

Moso bamboo (Phyllostachys pubescens) is widely distributed in the acid soil region of Southern China, where great potential of aluminum (Al) toxicity exists. To evaluate the Al tolerance of Moso bamboo, seed germination and root elongation were compared with two rice cultivars, and physical and physiological damages were examined under various levels of Al stress. Results showed that Moso bamboo seed germination was inhibited when Al concentration increased to 500 μM, and the median lethal concentration was 2,000 μM. Comparatively, the rice seed germination was not inhibited even at a concentration of 2,000 μM Al. Aluminum accumulated mainly in the cell wall of root apices, and entered into protoplasts as treating time prolonged and/or Al concentration increased, which resulted in apoptosis. The bamboo root epidermis degraded significantly in the presence of 2,000 μM Al. In conclusion, Moso bamboo is moderately weak in Al tolerance.     

Universal primers for the amplification of chloroplast microsatellites in grasses (Poaceae)

Attempts to design truly universal primers to amplify chloroplast microsatellites have met with limited success due to nonconservation of repeat loci across widely divergent taxa. We have used the complete chloroplast genome sequences of rice, maize and wheat to design five pairs of primers that amplify homologous mononucleotide repeats across the Poaceae (grasses). Sequencing confirmed conservation of repeat motifs across subfamilies and a preliminary study in Anthoxanthum odoratum revealed polymorphism at two loci with a haplotype diversity value of 0.495. These primers provide a valuable tool to study cytoplasmic diversity in this extensively studied and economically important range of taxa.     

The nuclear genome of Brachypodium distachyon: analysis of BAC end sequences

Due in part to its small genome (~350 Mb), Brachypodium distachyon is emerging as a model system for temperate grasses, including important crops like wheat and barley. We present the analysis of 10.9% of the Brachypodium genome based on 64,696 bacterial artificial chromosome (BAC) end sequences (BES). Analysis of repeat DNA content in BES revealed that approximately 11.0% of the genome consists of known repetitive DNA. The vast majority of the Brachypodium repetitive elements are LTR retrotransposons. While Bare-1 retrotransposons are common to wheat and barley, Brachypodium repetitive element sequence-1 (BRES-1), closely related to Bare-1, is also abundant in Brachypodium. Moreover, unique Brachypodium repetitive element sequences identified constitute approximately 7.4% of its genome. Simple sequence repeats from BES were analyzed, and flanking primer sequences for SSR detection potentially useful for genetic mapping are available at . Sequence analyses of BES indicated that approximately 21.2% of the Brachypodium genome represents coding sequence. Furthermore, Brachypodium BES have more significant matches to ESTs from wheat than rice or maize, although these species have similar sizes of EST collections. A phylogenetic analysis based on 335 sequences shared among seven grass species further revealed a closer relationship between Brachypodium and Triticeae than Brachypodium and rice or maize. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. N. Huo and G.R. Lazo contributed equally to this work.     

The chloroplast generps 4 as a tool for the study ofPoaceae phylogeny

Phylogenetic analyses of 28Poaceae species based on the chloroplastrps 4 gene are presented using parsimony and distance methods. Two monocots from other families were used as outgroups. The chloroplast generps 4 was amplified, cloned, and sequenced for each species. The inferred phylogenetic trees were compared to recent classifications and are shown to fit their general features. There is a dichotomy in our tree between the pooid group and the other grasses. This is in contradiction with other molecular phylogenies, where the bamboos appear first within the family. This result led us to discuss some hypotheses about the relationships of the bambusoids with the other groups of grasses, and also about the relative position of rice and bamboo, which are found close to each other in our trees.     

毛竹GRF转录因子家族的全基因组鉴定与分析

利用生物信息学方法,于毛竹(Phyllostachys edulis (Carr.) Lehaie)全基因组中鉴定获得18个GRF转录因子,并对其理化特性、保守结构域、系统发育关系、mi R396靶位点以及基因表达模式进行了分析。结果表明,18个Pe GRF蛋白长度为170～551 aa,分子量为18.5～58.8 k D;这些Pe GRF蛋白均具有QLQ和WRC结构域,部分Pe GRF含有FFD和TQL保守结构域。对毛竹、拟南芥(Arabidopsis thaliana (L.) Heynh)和水稻(Oryza sativa L.)的系统进化分析结果显示,毛竹18个GRF可分为3个亚类,且单子叶植物毛竹和水稻的GRF转录因子亲缘关系更近。mi R396靶位点预测分析结果发现,在13个Pe GRF基因序列的编码区存在毛竹mi R396结合位点; Pe GRF基因表达模式分析结果显示,Pe GRF主要在毛竹的竹笋中表达。     

Spatial heterogeneity and carbon contribution of aboveground biomass of moso bamboo by using geostatistical theory

Moso bamboo extensively distributes in southeast and south Asia, and plays an important role in global carbon budget. However, its spatial distribution and heterogeneity are poorly understood. This research uses geostatistics theory to examine the spatial heterogeneity of aboveground biomass (AGB) of moso bamboo, and uses a point kriging interpolation method to estimate and map its spatial distribution. Results showed that (1) spatial heterogeneity and spatial pattern of moso bamboo’s AGB can be revealed by an exponential semivariance model. The analysis of the model structure indicating that the AGB spatial heterogeneity is mainly composed of spatial autocorrelation components, and spatial autocorrelation range is from 360 to 41,220 m; (2) kriging standard deviation map showing the level of the model errors indicates that the AGB spatial distribution by point kriging interpolation method is reliable; (3) the average AGB of moso bamboo in Anji County is 44.228 Mg hm⁻², and carbon density is 20.297 Mg C hm⁻². The total AGB of moso bamboo accounts for 16.97% of the total forest-stand biomass in Zhejiang province. The total carbon storage of moso bamboo in China is 68.3993 Tg C, accounting for 1.6286% of the total forest carbon storage. This implies the important contribution of moso bamboo in regional or national carbon budget.     

Insights into the bamboo genome: syntenic relationships to rice and sorghum

Bamboo occupies an important phylogenetic node in the grass family and plays a significant role in the forest industry.We produced 1.2 Mb of tetraploid moso bamboo(Phyllostachys pubescens E.Mazel ex H.de Leh.)sequences from 13 bacterial artificial chromosome(BAC)clones,and these are the largest genomic sequences available so far from the subfamily Bambusoideae.The content of repetitive elements(36.2%)in bamboo is similar to that in rice.Both rice and sorghum exhibit high genomic synteny with bamboo,which suggests that rice and sorghum may be useful as models for decoding Bambusoideae genomes.