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.

     

Novel miniature inverted-repeat transposable elements derived from novel <Emphasis Type="Italic">CACTA</Emphasis> transposons were discovered in the genome of the ant <Emphasis Type="Italic">Camponotus floridanus</Emphasis>

Fourteen novel miniature inverted-repeat transposable element (MITE) families are found in the Florida carpenter ant genome, Camponotus floridanus. They constitute approximately 0.63 % of the entire genome. Analysis of their insertion time showed that most members of these MITEs were inserted into their host genome in less than 8 million years ago. In addition, the association between MITEs and the noncoding regions of genes in C. floridanus is random. Interestingly, an autonomous partner (named CfTEC) responsible for the amplification of these MITEs was also found in C. floridanus. Meanwhile, we present evidence, based on searches of publicly available databases, that this autonomous element was widespread in animals. Moreover, structure and phylogenetic analyses supported that TECs might represent a novel cade of transposons intermediate between the classic CACTA transposon and TRCs. Finally, their transposition mechanism and impact on host genome evolution were also discussed.     

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.     

Beta-amylase gene variability in introgressive wheat lines

Variability of the beta-amylase gene in bread wheat, artificial amphidiploids, and derived introgression wheat lines was analyzed. Variation in homeologous beta-amylase sequences caused by the presence of MITE (Miniature Inverted-Repeat Transposable Element) and its footprint has been identified in bread wheat. The previously unknown location of MITE in Triticum urartu and T. aestivum L. beta-amylase gene has been found. These species have a MITE sequence in the third intron of beta-amylase, as opposed to Aegilops comosa and a number of other Triticeae species, which have it in the fourth intron. These two MITEs from Ae. comosa and T. aestivum were shown to have low identity scores. Miosa, an artificial amphidiploid, which has the M genome from Ae. comosa was shown to lose the MITE sequences. This loss might be caused by genomic shock due to allopolyploidization.     

Isolation and Expression Analysis of <Emphasis Type="Italic">PeDWF1</Emphasis> in <Emphasis Type="Italic">Phyllostachys edulis</Emphasis>

Brassinosteroids (BRs) are steroidal hormones that play crucial roles in various processes of plant growth and development. DWF1 encodes a delta(24)-sterol reductase that participates in one of the early stage in the brassinosteroids’ biosynthetic pathway: the conversion of 24-methylenecholesterol to campesterol. Here we report the isolation and expression of one DWF1 homologous gene, PeDWF1, in moso bamboo (Phyllostachys edulis (Carrière) J. Houz.). Sequence analysis revealed that the open reading frame of PeDWF1 was 1686-bp encoding a protein composed of 561 amino acid residues with a calculated molecular weight of 65.1 kD and a theoretic isoelectric point of 8.32. Phylogenetic analysis indicated that PeDWF1 was very close to the cell elongation protein Dwarf1 in rice (Oryza sativa). Furthermore, transient expression of a PeDWF1::GFP fusion protein showed that PeDWF1 was an integral membrane protein most probably associated with the endoplasmic reticulum similar to Dwarf1. Tissue specific expression analysis showed that PeDWF1 was constitutively expressed in moso bamboo with the highest level in shoots and the lowest level in mature leaves. In the early growing stage of shoots, the expression level of PeDWF1 had a rising trend with the increasing height of shoots. These results indicated that PeDWF1 might be involved in the regulation of shoot development by participating in BRs biosynthesis. Moreover, PeDWF1 was heterologously expressed in Escherichia coli and the recombinant protein was about 65 kD, which facilitated further study on the gene function of PeDWF1 in bamboo.     

A MITE Insertion in the Promoter Region of Anthocyanidin Synthase from <Emphasis Type="Italic">Morus alba</Emphasis> L.

Anthocyanins play important role in plant protection and were closely involved with the plant evolution. Anthocyanidin synthase (ANS) is a late key enzyme in the flavonoid pathway which can catalyze leucoanthocyanidins to anthocyanidins. By our study, we found a miniature inverted-repeat transposable element (MITE) inserting in the promoter of ANS gene of mulberry. We used strawberry to evaluate the activities of ANS promoters from Morus alba and Morus notabilis with the method of Agrobacterium-mediated transient expression. The expression patterns of different promoters were also analyzed in transgenic lines of Arabidopsis thaliana and in this study, GUS was used as reporter gene. The 564-bp MITE insertion was strongly required for the activities of ANS promoter and it may reprogram the expression profiles of ANS gene in mulberry. Our results suggested that the MITE insertion was probably involved in either domestication or natural selection.     

Functional analysis of <Emphasis Type="Italic">PI-</Emphasis>like gene in relation to flower development from bamboo (<Emphasis Type="Italic">Bambusa oldhamii</Emphasis>)

Bamboo flowering owns many unique characteristics and remains a mystery. To investigate the molecular mechanisms underlying flower development in bamboo, a petal-identity gene was identified as a PISTILLATA homologue named BoPI from Bambusa oldhamii (bamboo family). Expression analysis showed that BoPI was highly expressed in flower organs and gradually increased during flower development stage, suggesting that BoPI played an important role in flower development. Ectopic expression of BoPI in Arabidopsis caused conversion of sepals to petals. 35S::BoPI fully rescued the defective petal formation in the pi-1 mutant. BoPI could interact with BoAP3 protein in vitro. These results suggested that BoPI regulated flower development of bamboo in a similar way with PI. Besides flower organs, BoPI was also expressed in leaf and branch, which revealed that BoPI may involve in leaf and branch development. Similar to other MIKC-type gene, BoPI contained the C-terminal sequence but its function was controversial. Ectopic expression of the C-terminal deletion construct (BoPI- ΔC) in Arabidopsis converted sepals to petals; BoPI- ΔC interacted with BoAP3 on yeast two-hybrid assay, just like the full-length construct. The result implied that the C-terminal sequence may not be absolutely required for organ identity function in the context of BoPI.     

Two active bamboo <Emphasis Type="Italic">mariner</Emphasis>-like transposable elements (<Emphasis Type="Italic">Ppmar1</Emphasis> and <Emphasis Type="Italic">Ppmar2</Emphasis>) identified as the transposon-based genetic tools for mutagenesis

Bamboo is one of the most important non-timber forest species in the world, but their molecular breeding lags far behind in contrast to other economic plants. Regarding the difficulties of hybridization and gene modification, the transposon-based insertional mutagenesis might be an alternative, feasible way for molecular breeding of bamboo. A systematic search for potential active transposons identified two full-length mariner-like elements (MLEs) (Ppmar1 and Ppmar2) from moso bamboo in the previous study. Both MLEs contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase. Two transposases contain intact DNA-binding motifs and a DD39D catalytic domain which indicates that Ppmar1 and Ppmar2 are likely active. Here, we deployed a heterologous transposition system of Arabidopsis thaliana to study the transposition activity of Ppmar1 and Ppmar2. The results show that both MLEs could transpose in A. thaliana. Excisions of Ppmar1 and Ppmar2 are usually unperfect as they leave 1–4 bp in excision sites. The reinsertions of both Ppmar1 and Ppmar2 occur at TA dinucleotides and prefer to insert into the TA-rich regions. The insertion sites are dispersed and non-linked. Two active bamboo transposons identified here not only could be applied to construction of the bamboo mutant libraries but also would provide another choice for other plant transposon-based gene tagging.     

Genome-wide identification and characterization of aquaporin gene family in moso bamboo (<Emphasis Type="Italic">Phyllostachys edulis</Emphasis>)

Aquaporins (AQPs) are known to play a major role in maintaining water and hydraulic conductivity balance in the plant system. Numerous studies have showed AQPs execute multi-function throughout plant growth and development, including water transport, nitrogen, carbon, and micronutrient acquisition etc. However, little information on AQPs is known in bamboo. In this study, we present the first genome-wide identification and characterization of AQP genes in moso bamboo (Phyllostachys edulis) using bioinformatics. In total, 26 AQP genes were identified by homologous analysis, which were divided into four groups (PIPs, TIPs, NIPs, and SIPs) based on the phylogenetic analysis. All the genes were located on 26 different scaffolds respectively on basis of the gene mapped to bamboo genome. Evolutionary analysis indicated that Ph. edulis was more close to Oryza sativa than Zea mays in the genetic relationship. Besides, qRT-PCR was used to analyze gene expression profiles, which revealed that AQP genes were expressed constitutively in all the detected tissues, and were all responsive to the environmental cues such as drought, water, and NaCl stresses. This data suggested that AQPs may play fundamental roles in maintaining normal growth and development of bamboo, which would contribute to better understanding for the complex regulation mechanism involved in the fast-growing process of bamboo. Furthermore, the result could provide valuable information for further research on bamboo functional genomics.     

Identification of <Emphasis Type="Italic">r</Emphasis> mutations conferring white flowers in the Japanese morning glory (<Emphasis Type="Italic">Ipomoea nil</Emphasis>)

The wild-type Japanese morning glory [Ipomoea nil (L.) Roth.] exhibits blue flowers with red stems, and spontaneous r mutants display white flowers with green stems. We have identified two r mutations, r1-1 and r1-2, that are caused by insertions of Tpn1-related DNA transposable elements, Tpn3 (5.6 kb) and Tpn6 (4.7 kb), respectively, into a unique intron of the CHS-D gene, which is responsible for flower and stem pigmentation. Both Tpn3 and Tpn6, which belong to the En/Spm or CACTA superfamily, are nonautonomous elements lacking transposase genes but containing unrelated cellular DNA segments including exons and introns. Interestingly, r1-2 contains an additional 4-bp insertion at the Tpn3 integration site in r1-1, presumably a footprint caused by the excision of Tpn3. The results strengthen the previous notion that Tpn1 and its relatives are major spontaneous mutagens for generating various floriculturally important traits in I. nil. Since I. nil has an extensive history of genetic studies, molecular identification of classical spontaneous mutations would also facilitate reinterpretation of the abundant classical genetic data available.     

Molecular characteristics of two new <Emphasis Type="Italic">waxy</Emphasis> mutations in China waxy maize

The waxy gene mutation causes waxy maize grain to have a sticky quality. China has numerous waxy maize landraces and is thought to be the place of origin of waxy maize. The most abundant waxy maize resources in China are located in the Yunnan province and its surrounding areas. We collected 57 waxy maize landraces from Yunnan province and cloned and sequenced the waxy gene from its fourth to eighth exon. Two new waxy gene mutations, named wx-Cin4 and wx-124, were identified. The wx-Cin4 mutation is a 466-bp retrotransposon inserted into exon six. The wx-124 mutation is a 116-bp miniature inverted-repeat transposable element inserted into exon seven. This is the first time a 124-type mutation has been found in a maize waxy gene. The discovery of the two specific waxy mutations from landraces collected in Yunnan province provides new evidence supporting the hypothesis that China is the origin area for waxy maize.     

植物中活性MITEs转座子研究进展

MITEs(Miniature inverted-repeat transposable elements)转座子是一种特殊的转座子,其既有DNA转座子的转座特性——"剪切-粘贴"转座方式,又有RNA转座子的高拷贝特性。目前已被报道的MITEs种类和数量虽然很多,但是关于有转座活性的MITEs的报道却甚少。本文总结了近几年来有关活性MITEs的相关报道,发现具有转座活性的MITEs种类大都分布在Tourist家族,分别是m Ping、m Ging、Ph Tourist1、Tmi1和Ph Tst-3,另外还有Stowaway-like家族的d Tstu1和MITE-39以及Mutator家族的Ah MITE1。文中还分析了这些活性MITEs的结构(TIR和TSD)、拷贝数、进化模式以及转座特性等,为鉴定其他活性MITEs以及MITEs转座和扩增机制的研究奠定了基础。     

Overexpression of the repressor gene PvFRI-L from Phyllostachys violascens delays flowering time in transgenic Arabidopsis thaliana

The gene FRIGIDA (FRI) is floral repressor and plays a key role in the timing of Arabidopsis flowering. To study the function of FRI-like genes in bamboo, we isolated a FRI family gene from bamboo Phyllostachys violascens and named it PvFRI-L. Sequence alignment and phylogenetic analysis show that the PvFRI-L protein belongs to the FRL3 (III) subfamily from monocots and contains a conserved FRIGIDA domain. PvFRI-L was located in the nucleus of onion epidermal cells. PvFRI-L was expressed in all tested organs of flowering and non-flowering bamboo plants with a higher expression in non-flowering than in flowering plants. Overexpression of PvFRI-L in Arabidopsis caused late flowering by downregulating flowering locus T and upregulating flowering locus C. A P-box, the binding site involved in gibberellin response, was found only in the promoter region of PvFRI-L but not in that of FRI. Furthermore, PvFRI-L expression in the leaves of Ph. violascens seedlings was downregulated with gibberellic acid treatment. Taking together, our observation suggests that PvFRI-L may be flowering repressor and its delaying floral timing may be regulated by gibberellic acid in bamboo.