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Molecular Cloning of Cellulose Synthase Gene, SpCesA1 from Developing Xylem of Shorea parvifolia spp. parvifolia
Abstract
This study reported the isolation and in silico characterization of full-length cellulose synthase ( CesA ) cDNA from Shorea parvifolia spp. parvifolia , an important tropical hardwood tree species. Cellulose synthase (CesA) is a member of processive glycosyltransferases that involved in cellulose biosynthesis of plants. The full-length of SpCesA1 cDNA with size 3308 and 3120 bp open reading frames encoding a 1040 amino acid was isolated using RT-PCR and RACE-PCR approaches. The predicted SpCesA1 protein contained N-terminal cysteine rich zinc binding domain, 7 putative transmembrane helices (TMH), 4 U-motifs that contain a signature D, D, D, QxxRW motif, an alternating conserved region (CR-P) and 2 hypervariable regions (HVR). These entire shared domain structures suggest the functional role of SpCesA1 is involved in cellulose biosynthesis in secondary vascular tissues of S. parvifolia spp. parvifolia . Sequence comparison also revealed the high similarity (87%) among SpCesA1 and PtrCesA2 of Populus tremuloides . This further implies the involvement of SpCesA1 in catalyzes the cellulose biosynthesis of secondary cell wall rather than primary cell wall. Thus, identification of new CesA genes from tropical tree genomes is essential for enhancing knowledge of cellulose biosynthesis in trees that has many fundamental and commercial implications.
... Cellulose synthase, a multienzyme complex is the key enzyme in cellulose biosynthesis and the first CesA gene in plants was reported from cotton fibre (Pear et al. 1996). Subsequently, they were isolated and characterized from several plant species including A. thaliana (Richmond and Somerville 2000), Zea mays (Holland et al. 2000), Hordeum vulgare (Burton et al. 2004), Populus tremuloides , Pinus taeda (Nairn et al. 2008), Shorea parvifolia (Lau et al. 2009), Oryza sativa , Betula platyphylla (Liu et al. 2012) and Leucaena leucocephala (Vishwakarma et al. 2012). ...
... The expression of the EtCesA3 transcripts was 87-fold higher in developing secondary xylem tissues, in comparison to the primary tissues like leaf and internodes, which was analogous to the 50-fold abundant expression of EgCesA3 in the secondary xylem tissues of E. grandis (Ranik and Myburg 2006). Correspondingly, the expression of EtCesA1, EtCesA2 and EtCesA3 in developing xylem tissues was similar to the secondary xylem tissue specific predominant expression of PtCesA1, PtCesA2 and PtCesA3 in Pinus taeda (Nairn and Haselkorn 2005), PtrCesA3 in Populus tremuloides , BplCesA8, BplCesA7 and BplCesA4 in Betula platyphylla (Liu et al. 2012), Ll-7CesA and Ll-8CesA in Leucaena leucocephala (Vishwakarma et al. 2012) and SpCesA1 in Shorea parvifolia (Lau et al. 2009), which reveals the expression of specific groups of the CesA genes during the secondary cell wall formation in woody perennials. ...
... In the present study the full-length genes were isolated from the developing xylem tissues of E. tereticornis with size ranging from 2940 bp for EtCesA1, 3114 bp for EtCesA2 and 2406 bp for EtCesA3. The sizes of the full length CDS are comparable to their orthologues from other tree species like Betula platyphylla (Liu et al. 2012), Leuceana (Vishwakarma et al. 2012), Populus (Wu et al. 2000), Eucalyptus grandis (Ranik and Myburg 2006) and Shorea parvifolia (Lau et al. 2009). ...
Cellulose synthases (CesA) represent a group of β-1, 4 glycosyl transferases involved in cellulose biosynthesis. Recent reports in higher plants have revealed that two groups of CesA gene families exist, which are associated with either primary or secondary cell wall deposition. The present study aimed at identifying developing secondary xylem specific cellulose synthase genes from Eucalyptus tereticornis, a species predominantly used in paper and pulp industries in the tropics. The differential expression analysis of the three EtCesA genes using qRT-PCR revealed 49 to 87 fold relative expression in developing secondary xylem tissues. Three full length gene sequences of EtCesA1, EtCesA2 and EtCesA3 were isolated with the size of 2940, 3114 and 3123 bp, respectively. Phytohormone regulation of all three EtCesA genes were studied by exogenous application of gibberellic acid, naphthalene acetic acid, indole acetic acid and 2, 4-epibrassinolide in internode tissues derived from three-month-old rooted cuttings. All three EtCesA transcripts were up-regulated by indole acetic acid and gibberellic acid. This study demonstrates that the increased cellulose deposition in the secondary wood induced by hormones can be attributed to the up-regulation of xylem specific CesAs.
... Cellulose synthase, a multienzyme complex is the key enzyme in cellulose biosynthesis and the first CesA gene in plants was reported from cotton fibre (Pear et al. 1996). Subsequently, they were isolated and characterized from several plant species including A. thaliana (Richmond and Somerville 2000), Zea mays (Holland et al. 2000), Hordeum vulgare (Burton et al. 2004), Populus tremuloides , Pinus taeda (Nairn et al. 2008), Shorea parvifolia (Lau et al. 2009), Oryza sativa , Betula platyphylla (Liu et al. 2012) and Leucaena leucocephala (Vishwakarma et al. 2012). ...
... The expression of the EtCesA3 transcripts was 87-fold higher in developing secondary xylem tissues, in comparison to the primary tissues like leaf and internodes, which was analogous to the 50-fold abundant expression of EgCesA3 in the secondary xylem tissues of E. grandis (Ranik and Myburg 2006). Correspondingly, the expression of EtCesA1, EtCesA2 and EtCesA3 in developing xylem tissues was similar to the secondary xylem tissue specific predominant expression of PtCesA1, PtCesA2 and PtCesA3 in Pinus taeda (Nairn and Haselkorn 2005), PtrCesA3 in Populus tremuloides , BplCesA8, BplCesA7 and BplCesA4 in Betula platyphylla (Liu et al. 2012), Ll-7CesA and Ll-8CesA in Leucaena leucocephala (Vishwakarma et al. 2012) and SpCesA1 in Shorea parvifolia (Lau et al. 2009), which reveals the expression of specific groups of the CesA genes during the secondary cell wall formation in woody perennials. ...
... In the present study the full-length genes were isolated from the developing xylem tissues of E. tereticornis with size ranging from 2940 bp for EtCesA1, 3114 bp for EtCesA2 and 2406 bp for EtCesA3. The sizes of the full length CDS are comparable to their orthologues from other tree species like Betula platyphylla (Liu et al. 2012), Leuceana (Vishwakarma et al. 2012), Populus (Wu et al. 2000), Eucalyptus grandis (Ranik and Myburg 2006) and Shorea parvifolia (Lau et al. 2009). ...
Cellulose synthases (CesA) represent a group of β-1, 4 glycosyl transferases involved in cellulose biosynthesis. Recent reports in higher plants have revealed that two groups of CesA gene families exist, which are associated with either primary or sec-ondary cell wall deposition. The present study aimed at identifying developing secondary xylem specific cellulose synthase genes from Eucalyptus tereticornis, a species predominantly used in paper and pulp industries in the tropics. The differen-tial expression analysis of the three EtCesA genes using qRT-PCR revealed 49 to 87 fold relative expression in developing secondary xylem tissues. Three full length gene sequences of EtCesA1, EtCesA2 and EtCesA3 were isolated with the size of 2940, 3114 and 3123 bp, respectively. Phytohormone regulation of all three EtCesA genes were studied by exogenous appli-cation of gibberellic acid, naphthalene acetic acid, indole acetic acid and 2, 4-epibrassinolide in internode tissues derived from three-month-old rooted cuttings. All three EtCesA transcripts were upregulated by indole acetic acid and gibberellic acid. This study demonstrates that the increased cellulose deposition in the secondary wood induced by hormones can be attributed to the upregulation of xylem specific CesAs.. 2014 Isolation of developing secondary xylem specific cellulose synthase genes and their expression profiles during hormone signalling in Eucalyptus tereticornis. J. Genet. 93, 403–414]
... Cellulose synthase shares certain similar domain structures across species. These include a zinc finger, several transmembrane domains, conserved residues and 2 hypervariable regions (HVR), HVRI and HVRII [2], [3]. ...
... The HVRII region is one of the plant-specific regions with high sequence divergence, along with the conserved region (CR-P) which instead shows high sequence conservations [3]. These plant-specific domains are thought to be involved in functions unique to plant such as the binding of sucrose synthase, interaction with proteins associated with cytoskeleton or other accessory proteins. ...
Neolamarckia cadamba or locally known as Kelampayan, is one of the fast growing plantation tree species that holds great prospect as a renewable bioresources for plywood, pulp and paper, and biofuel industries. Sufficient information on cellulose synthase (CesA) gene, especially the hypervariable region II (HVRII) component involved in wood formation of Kelampayan is imperative for future applications. This region is thought to play a role in interaction with other unique cell-type-specific proteins involved in the biosynthesis of cellulose. The aim of this study was to identify and clone the HVRII regions of cellulose synthase gene from the developing xylem tissues of Kelampayan. The cDNA of cellulose synthase HVRII regions was amplified using reverse transcription-PCR (RT-PCR) approach using degenerate primers. Three clones, namely NcCesA1HVRII (520bp), NcCesA2HVRII (580bp) and NcCesA3HVRII (620bp) were successfully sequenced and characterized. NcCesA1HVRII and NcCesA3HVRII were clustered into two distinct clades implicated with secondary cell wall development whereas NcCesA2HVRII has renamed to NcCslD1HVRII due its high similarity with various plants' CslD-HVRII. This study provides an easier and faster access to NcCesAHVRII sequences to further understand the role of NcCesA/NcCslD protein for future applications such as selecting trees with optimal cellulose content required for specific industries.
... In addition, there are at least six classes of CesA proteins exist in plants. In general, CesA protein consists of a zinc finger, two hypervariable regions (HVR I and II), several transmembrane domains and conserved residues [1], [7], [8], [19]. The hypervariable region II (HVRII) is made up of around 500bp to 600bp and the amino acid sequences between highly conserved motifs of ALYG and VISCG are associated with this HVRII region. ...
... With the gene expression and comparative studies that have been done previously elsewhere, these CesA proteins are confirmed to be associated with the function in the development of primary and secondary cell wall in plant species such as N. cadamba, Zea mays, Gossypium hirsutum, Oryza sativa, Populus tremuloides, S. parvifolia ssp. parvifolia and Eucalyptus [10], [7], [16], [17], [18], [19]. The NJ tree consists of the three clades that are associated with primary (P) cell wall synthesis, secondary (S) cell wall synthesis and CslD (C) protein between plant species. ...
The present study is aimed to isolate and characterize the hypervariable (HVRII) region in cellulose synthase gene from the developing xylem tissues of a tropical timber tree species, Neolamarckia macrophylla. N. macrophylla is locally known as red kelampayan and it has been selected as one of the important reforestation tree species in Malaysia. RT-PCR was carried out by using the degenerate primers and one of the three amplified DNA bands was successfully sequenced and characterized. The sequence was named as NmCesA1HVRII and it was clustered in a distinct clade that is associated with secondary cell wall development. This study has generated a useful genomic resource for a better understanding about the HVRII region of CesA gene in N. macrophylla and its function which is important in future applications, genetic improvement of N. macrophylla. This also facilitates the future selection of trees with optimal cellulose content required for certain specific industries as well as synthesizing of artificial cellulose, hence increasing the economic development and growth in the country.
... As we used partial sequences of TaER-1 and TaER-2 for detecting the SNPs and associating them with the traits, definitely, some genetic associations might have not been discovered. In future, candidate genes with full length sequences consisting of intron, exon, promoter, 5'-end, 3'-end un-translated regions are recommended for the detection of SNPs that can provide locus to the candidate gene (Zhu et al. 2008, Lau et al. 2009). ...
Candidate gene association studies implicate the detection of contributing single nucleotide polymorphism (SNP) for the target traits and have been recommended as a promising technique to anatomize the complex characters in plants. ERECTA gene in plants controls different physiological functions. In this study, we identified SNPs in 1.1 kb partial sequences of TaER-1 and TaER-2 of wheat (Triticum aestivum L.). Thirty-nine SNPs were identified in the coding regions of TaER-1 gene in 33 wheat genotypes, of which 20 SNPs caused non-synonymous mutations while 19 SNPs produced synonymous mutations; while 31 SNPs were located in the coding regions of TaER-2 gene in 26 genotypes, of which 18 SNPs caused non-synonymous mutations and 13 SNPs caused synonymous mutations. In addition, 32 SNPs in TaER-1 and 9 SNPs in TaER-2 were also identified in the non-coding regions. Moreover, the significant genetic associations of SNPs of TaER-1 and TaER-2 genes with carbon isotope discrimination, stomatal conductance, photosynthetic rate, transpiration rate, intrinsic water use efficiency (iWUE), leaf length, leaf width, stomatal density, epidermal cell density, and stomatal index were noted in wheat genotypes. This study confirms the importance of TaER-1 and TaER-2 genes which could improve iWUE of wheat by regulating leaf gas exchange and leaf structural traits. These identified SNPs may play a critical role in molecular breeding by means of marker-assisted selection.
... EgCesA3 has been proven to be highly expressed in xylem tissues that carried out secondary cellulose biosynthesis (Ranik and Myburg, 2006). S. parvifolia CesA1 that is closely related to NcCesA1 also showed high similarity to PtrCesA2 of P. tremuloides that catalysed cellulose biosynthesis in secondary cell walls (Lau et al., 2009). Fig. 9. Neighbour-joining phylogenetic tree generated from multiple alignments of full-length amino acid sequences of NcCesA1 and selected species. ...
This study reported the isolation and in silico characterization of full-length cellulose synthase (CesA) cDNA from Neolamarckia
Association genetics study is a powerful approach to detect the potential genetic variants (i.e., SNPs) underlying the common and complex adaptive traits. Once the quantitative frait nucleotides are identified, such powerful approach provides significant advantages to the forest industry. Hence, attempts were made to discover SNPs from Neolamarckia cadamba partial C4H (3,538 bp) and CAL) (2,354 bp) DNA sequences and further associate those SNPs with basic wood density. Overlapping primers were designed in flanking the partial C4H and CAD DNA from 12 N. cadamba frees. The amplified DNA fragments were sequenced and the basic wood density measurements were determined for each free. The sequence variation analyses revealed that there were 60 and 32 SNPs detected in the paitial C41-I and CAD DNA sequences, respectively. Those SNPs were distributed throughout the exon, infron, 5'-UTR and 3'-UTR regions. The total nucleotide diversities were m 0.00302 andO 0.004 2. The synonymous mutations (m 0.00983; O 0.02 0) were more commonthan nonsynonymous mutations (ic = 0.00045; 6 = 0.00089) for both C41-I and CAD genes. LD declined linearly over short distance at the loci examined. Association genetics study also revealed that 4 and 6 SNPs from C41-I and CAD genes, respectively were in significant associations with basic wood density of N. cadamba (p<0.05). The genetic variation identified by the SNP markers, once validated, will facilitate the selection of N. cadantha parental lines or seedlings with optimal quality through Gene-assisted Selection (GAS) approach.
Candidate-gene-based association study which involves the identification of causative Single Nucleotide Polymorphisms (SNPs) for excellent fraits has been proposed as a promising approach to dissect complex fraits in forest frees. Hence, the goal of this study was to identify the genetic association among SNPs from Cinnamate 4-Hydroxylase (C41]) and Cinnaniyl Alcohol Dehydrogenase (CAD) genes and an array of wood properties namely, specific gravity, wood density, fiber-length, cell wall thickness and microfibril angle from Acacia mangiuni Superbulk frees. Sequence variations within these two genes in 12 A. mangium Superbulk frees were examined and wood properties were measured The data obtained was tested using General Linear Model (GLM) within TASSEL software. Two SNPs were identified in the exon of C4H of which all the SNPs caused nonsynonymous mutations whereas five SNPs were identified in the CAD exons along with one deletion mutation. In addition, two SNPs were also identified in the CAD introns. Variation in these two lignin biosynthesis genes might change the stnictural, functional or biochemical properties of the enzyme being produced, and therefore possibly lead to changes in phenotypic characteristic of the frees. The genetic association study also revealed that SNPs in CAD gene do associate with the wood density, specific gravity and cell wall thickness (p<0.05). However, no significant results were obtained for SNPs in C4H gene with wood properties studied. Thickening of cell wall is affected by the arrangement of biopolymer aggregates which comprise of cellulose, hemicellulose and lignin. Results indicated that SNP in CAD gene might alter the lignin biosynthesis and thus lead to changes in phenotypic characteristics of the frees. Overall, the study has demonstrated that SNP is very useful in association genetic study to identify Quantitative Trait Nucleotide (QE[N) which then leads to Gene-assisted Selection (GAS) in the free breeding programme.
Neolamarckia cadamba (or locally known as kelampayan) is an important fast growing plantation free species that confers various advantages for timber industry as a sfrategy for reducing the logging pressure on natural forests for wood production to an acceptable level. Hence, attempts were made to develop a set of EST-SSR markers for kelampayan frees based on the EST sequences of kelampayan (NcdbEST) and further assessed the polymorphisms and frarisferability of the markers to other species. In this study, 155 (2.34%) out of 6,622 EST sequences which contain 232 SSRs were mined from NcdbEST. Of these, 97 ESTs were assigned with putative functions and gene ontology tenns. Eighteen EST-SSR markers were developed according to the criteria, and further chanicterized and validated by using 50 individuals of kelampayan from two selected mother trees. The markers exhibited a considerable high level of polymorphism in kelampayan trees with an average of 4.17 and 4.11 alleles per locus, and PlC values of 0.465 and 0.537, respectively for mother frees Ti and T2. Parentage assignment analysis snggests a high probability for kelampayan frees to be predominantly outcrossed. The transferability rate was ranging from 16.7-94.4% among the five cross-genera species of kelampayan. The present study is the first report of the development of EST-SSR markers in kelampayan. These markers will be valuable genomic resources that could pave the way for exploiting the genotype data for comparative genome mapping, association genetics, population genetics studies and molecular breeding of kelampayan and other indigenous tropical free species in future.
A detailed study was carried out to discover single nucleotide polymorphisms (SNPs) from Neolamarckia cadamba partial XTH (~1283bp) and CesA (778bp) DNA sequences and further associates those SNPs with basic wood density. Primers were designed in flanking the partial XTH and CesA genes from 15 N. cadamba trees. The amplified DNA fragments were sequenced and the basic wood density measurements were determined for each tree. The sequence variation analyses revealed that 34 SNPs (2.65% occurrence) and 3 SNPs (0.39% occurrence) were found in 15 partial genomic DNA sequences of NcXTH1 and NcCesA1, respectively. All the SNPs were discovered in both exon and intron regions. NcXTH1 examined sites showed higher nucleotide diversities of π = 0.00402 and θw = 8.919 when compared to NcCesA1 (π = 0.00127; θw = 0.9226). The LD decayed slowly with distance of polymorphic sites in a linear pattern with the mean R2 value of 0.000687. Association genetics study showed that 2 SNPs from NcXTH1 genes were significantly associated with basic wood density (p<0.05) of N. cadamba. Once the gene-associated SNP markers in NcXTH1 genes are validated, it could be potentially used as a tool in Gene-Assisted Selection (GAS) of N. cadamba trees. This study has also demonstrated that the candidate-gene based association genetics is a powerful approach to dissect complex adaptive traits for organism lacking a genome sequence or reference genomic resources.
Isolation of high quality RNA fromRosaceae species is particularly difficult. These plants contain considerable amounts of plant polyphenolic compounds and polysaccharides
that copurify with RNA, often rendering it unsuitable for either cDNA synthesis and/or hybridization in northern analyses.
We describe a method for RNA isolation from pear leaves that is modified from that of Manning (1990). The procedure includes
i) an extraction with phenol and PVPP, to remove proteins and polyphenols ii) two purifications by LiCl, with a 2-butoxyethanol
treatment between the LiCl steps. The method results in high quality RNA suitable for RT-PCR and northern blot experiments.
Cellulose, a microfibrillar polysaccharide consisting of bundles of β-1,4-glucan chains, is a major component of plant and
most algal cell walls and is also synthesized by some prokaryotes. Seed plants and bacteria differ in the structures of their
membrane terminal complexes that make cellulose and, in turn, control the dimensions of the microfibrils produced. They also
differ in the domain structures of their CesA gene products (the catalytic subunit of cellulose synthase), which have been localized to terminal complexes and appear to
help maintain terminal complex structure. Terminal complex structures in algae range from rosettes (plant-like) to linear
forms (bacterium-like). Thus, algal CesA genes may reveal domains that control terminal complex assembly and microfibril structure. The CesA genes from the alga Mesotaenium caldariorum, a member of the order Zygnematales, which have rosette terminal complexes, are remarkably similar to seed plant CesAs, with deduced amino acid sequence identities of up to 59%. In addition to the putative transmembrane helices and the D-D-D-QXXRW
motif shared by all known CesA gene products, M. caldariorum and seed plant CesAs share a region conserved among plants, an N-terminal zinc-binding domain, and a variable or class-specific
region. This indicates that the domains that characterize seed plant CesAs arose prior to the evolution of land plants and
may play a role in maintaining the structures of rosette terminal complexes. The CesA genes identified in M. caldariorum are the first reported for any eukaryotic alga and will provide a basis for analyzing the CesA genes of algae with different types of terminal complexes.
Genetic improvement of cell wall polymer synthesis in forest trees is one of the major goals of forest biotechnology that could possibly impact their end product utilization. Identification of genes involved in cell wall polymer biogenesis is essential for achieving this goal. Among various candidate cell wall-related genes, cellulose synthase-like D (CSLD) genes are intriguing due to their hitherto unknown functions in cell wall polymer synthesis but strong structural similarity with cellulose synthases (CesAs) involved in cellulose deposition. Little is known about CSLD genes from trees. In the present article PtrCSLD2, a first CSLD gene from an economically important tree, aspen (Populus tremuloides) is reported. PtrCSLD2 cDNA was isolated from an aspen xylem cDNA library and encodes a protein that shares 90% similarity with Arabidopsis AtCSLD3 protein involved in root hair tip growth. It is possible that xylem fibers that also grow by intrusive tip growth may need expression of PtrCSLD2 for controlling the length of xylem fibers, a wood quality trait of great economical importance. PtrCSLD2 protein has a N-terminal cysteine-rich putative zinc-binding domain; eight transmembrane domains; alternating conserved and hypervariable domains; and a processive glycosyltransferases signature, D, D, D, QXXRW; all similar to aspen CesA proteins. However, PtrCSLD2 shares only 43-48% overall identity with the known aspen CesAs suggesting its distinct functional role in cell wall polymer synthesis perhaps other than cellulose biosynthesis. Based on Southern analysis, the aspen CSLD gene family consists of at least three genes and this gene copy estimate is supported by phylogenetic analysis of available CSLDs from plants. Moreover, gene expression studies using RT-PCR and in situ mRNA hybridization showed that PtrCSLD2 is expressed at a low level in all aspen tissues examined with a slightly higher expression level in secondary cell wall-enriched aspen xylem as compared to primary cell wall enriched tissues. Together, these observations suggest that PtrCSLD2 gene may be involved in the synthesis of matrix polysaccharides that are dominant in secondary cell walls of poplar xylem. Future molecular genetic analyses will clarify the functional significance of CSLD genes in the development of woody trees.
Recent molecular genetic data suggest that cellulose synthase (CesA) genes coding for the enzymes that catalyze cellulose biosynthesis (CESAs) in Arabidopsis and other herbaceous plants belong to a large gene family. Much less is known about CesA genes from forest trees. To isolate new CesA genes from tree species, discriminative but easily obtainable homologous DNA probes are required. Hypervariable regions (HVRII)
of CesA genes represent highly divergent DNA sequences that can be used to examine structural, expressional and functional relationships
among CesA genes. We used a reverse transcriptase-polymerase chain reaction (RT-PCR)-based technique to identify HVRII regions from
eight types of CesA genes and two types of CesA-like D (CslD) genes in quaking aspen (Populus tremuloides Michx.). Comparison of these aspen CESA/CSLD HVRII regions with the predicted proteins from eight full-length CesA/CslD cDNAs available in our laboratory and with searches for aspen CesA/CslD homologs in the recently released Populus trichocarpa Torr. & A. Gray. genome confirmed the utility of this approach in identifying several CesA/CslD gene members from the Populus genome. Phylogenetic analysis of 56 HVRII domains from a variety of plant species suggested that at least six distinct classes
of CESAs exist in plants, supporting a previous proposal for renaming HVRII regions as class-specific regions (CSR). This
method of CSR cloning could be applied to other crop plants and tree species, especially softwoods, for which the whole genome
sequence is unlikely to become available in the near future because of the large size of these genomes.