Identification and characterization of jute LTR retrotransposons

Abstract

Long Terminal Repeat (LTR) retrotransposons constitute a significant part of eukaryotic genomes and play an important role in genome evolution especially in plants. Jute is an important fiber crop with a large genome of 1250 Mbps. This genome is still mostly unexplored. In this study we aimed at identifying and characterizing the LTR retrotransposons of jute with a view to understanding the jute genome better. In this study, the Reverse Transcriptase domain of Ty1-copia and Ty3-gypsy LTR retrotransposons of jute were amplified by degenerate primers and their expressions were examined by reverse transcription PCR. Copy numbers of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy elements were determined by dot blot analysis. Sequence analysis revealed higher heterogeneity among Ty1-copia retrotransposons than Ty3-gypsy and clustered each of them in three groups. Copy number of RT genes in Ty1-copia was found to be higher than that of Ty3-gypsy elements from dot blot hybridization. Cumulatively Ty1-copia and Ty3-gypsy may constitute around 19% of the jute genome where two groups of Ty1-copia were found to be transcriptionally active. Since the LTR retrotransposons constitute a large portion of jute genome, these findings imply the importance of these elements in the evolution of jute genome.

Acknowledgments

The authors are grateful to Bangladesh Jute Research Institute (BJRI) for supplying jute seeds and leaves. The authors are also thankful to Mr. Abu Ashfaqur Sajib for his valuable suggestions in preparing the manuscript.

Figures and Tables

Figure 1 PCR amplified products of RT domains of Ty1-copia and Ty3-gypsy retrotransposons of jute genome. Lane 1: Ty3-gypsy PCR products, Lane 2: Ty1-copia PCR products, Lane 3: Control PCR without template, Lane 4: 1 Kb⁺ ladder. Genomic DNA was isolated from mature leaves of jute variety O-9897 and after PCR amplification, the PCR amplified products were run in 2% agarose gel.

Figure 2 Multiple Sequence Alignments (MSA) of predicted amino acid sequences of the reverse transcriptase domains of Ty1-copia retrotransposons of jute and other species (Table 1). (a) is the Group I, (b) is the Group II and (c) is group III according to the phylogenetic tree.

Figure 3 Multiple Sequence Alignments (MSA) of predicted amino acid sequences of the reverse transcriptase domains of Ty3-gypsy retrotransposons of jute and other species (Table 2). (a) is the Group I, (b) is the Group II and (c) is Group III according to the phylogenetic tree.

Figure 4 RT-PCR products of jute Ty1-copia and Ty3-gypsy retrotransposons. Lane 1: PCR from isolated RNA only as a control, Lane 2: RT-PCR of Ty1-copia, Lane 3: RT-PCR of Ty3-gypsy. RNA was isolated from the mature leaves of jute variety O-9897.

Figure 5 (A) Phylogenetic tree of amino acid sequences of RT domain of Ty1-copia retrotransposons of jute and other species. The phylogenetic tree was constructed using NJ method and displayed using MEGA4. The bootstrap consensus tree was inferred from 1,000 replicates showing bootstrap values higher than 50%. Among the three groups based on this phylogenetic tree, Group I and Group III contain jute genomic Ty1-copia sequences and transcribed Ty1-copia sequences respectively. The transcribed sequences are denoted by ‘JTECrt’ whereas the genomic sequences are denoted by ‘JTEC’. (B) Phylogenetic tree of amino acid sequences of RT domain of Ty3-gypsy retrotransposons of jute and other species. The phylogenetic tree was constructed using NJ method and displayed using MEGA4. The bootstrap consensus tree was inferred from 1,000 replicates showing bootstrap values higher than 50%. According to this phylogenetic tree, Group I, Group II and Group III are like athila, CRM and reina type Ty3-gypsy retroelements.

Figure 6 Determination of the copy number of Ty1-copia and Ty3-gypsy sequences in jute genome where (a) is for Ty1-copia and (b) is for Ty3-gypsy. For Ty1-copia 10 µg, 8 µg and 5 µg of genomic DNA corresponding to 100 ng, 50 ng, 40 ng and 25 ng of PCR products were immobilized on membrane followed by probing with non-radioactive labeled PCR probe. For Ty3-gypsy 20 µg and 10 µg of genomic DNA corresponding to 200 ng, 100 ng and 50 ng of PCR products were immobilized on membrane followed by probing with non-radioactive labeled PCR probe.

Table 1 List of Ty1-copia retrotransposon extracted from GyDB which were used for phylogenetic analysis

Table 2 List of Ty3 retroelement sequences extracted from GyDB used for phylogenetic analysis

Table 3 Synonymous divergence (dS), nonsynonymous divergenece (dN) and dN:dS ratios of Ty-1 copia and Ty-3 gypsy elements of jute

Table 4 Ty1-copia Z values (above) and significance P-values (below) for the codon based Z-test for positive selection