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The transcriptome of Utricularia vulgaris, a rootless plant with minimalist genome, reveals extreme alternative splicing and only moderate sequence similarity with Utricularia gibba.

Bárta J, Stone JD, Pech J, Sirová D, Adamec L, Campbell MA, Štorchová H - BMC Plant Biol. (2015)

Bottom Line: We also found a strong correspondence in the presence or absence of root-associated genes between the U. vulgaris transcriptome and U. gibba genome, which indicated that the loss of some root-specific genes had occurred before the divergence of the two rootless species.The species-rich genus Utricularia offers a unique opportunity to study adaptations related to the environment and carnivorous habit and also evolutionary processes responsible for considerable genome reduction.Our study is the first comparison of two global sequence data sets in Utricularia.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The species of Utricularia attract attention not only owing to their carnivorous lifestyle, but also due to an elevated substitution rate and a dynamic evolution of genome size leading to its dramatic reduction. To better understand the evolutionary dynamics of genome size and content as well as the great physiological plasticity in this mostly aquatic carnivorous genus, we analyzed the transcriptome of Utricularia vulgaris, a temperate species with well characterized physiology and ecology. We compared its transcriptome, namely gene content and overall transcript profile, with a previously described transcriptome of Utricularia gibba, a congener possessing one of the smallest angiosperm genomes.

Results: We sequenced a normalized cDNA library prepared from total RNA extracted from shoots of U. vulgaris including leaves and traps, cultivated under sterile or outdoor conditions. 454 pyrosequencing resulted in more than 1,400,000 reads which were assembled into 41,407 isotigs in 19,522 isogroups. We observed high transcript variation in several isogroups explained by multiple loci and/or alternative splicing. The comparison of U. vulgaris and U. gibba transcriptomes revealed a similar distribution of GO categories among expressed genes, despite the differences in transcriptome preparation. We also found a strong correspondence in the presence or absence of root-associated genes between the U. vulgaris transcriptome and U. gibba genome, which indicated that the loss of some root-specific genes had occurred before the divergence of the two rootless species.

Conclusions: The species-rich genus Utricularia offers a unique opportunity to study adaptations related to the environment and carnivorous habit and also evolutionary processes responsible for considerable genome reduction. We show that a transcriptome may approximate the genome for gene content or gene duplication estimation. Our study is the first comparison of two global sequence data sets in Utricularia.

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PCR amplification with theLCB-1-P lyasespecific primers. An agarose gel (1.2%) electrophoresis of PCR fragments amplified from the gene encoding LCB-1-P lyase (isogroup 000007) in U. vulgaris with different templates. 1–6: cDNA prepared from RNA extracted from two different plant individuals, 7: genomic DNA. NC: negative control with water instead of DNA. (A) PCR with exon-specific primers UV405_F1 and UV405_R1. (B). PCR with intron-specific primer UV405_F2 and exon-specific primer UV405_R1. Annealing temperature is indicated above the lanes. Standard of molecular weights is shown on the both sides of the gel.
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Fig3: PCR amplification with theLCB-1-P lyasespecific primers. An agarose gel (1.2%) electrophoresis of PCR fragments amplified from the gene encoding LCB-1-P lyase (isogroup 000007) in U. vulgaris with different templates. 1–6: cDNA prepared from RNA extracted from two different plant individuals, 7: genomic DNA. NC: negative control with water instead of DNA. (A) PCR with exon-specific primers UV405_F1 and UV405_R1. (B). PCR with intron-specific primer UV405_F2 and exon-specific primer UV405_R1. Annealing temperature is indicated above the lanes. Standard of molecular weights is shown on the both sides of the gel.

Mentions: We selected two isogroups of U. vulgaris with very high isotig counts for more detailed analysis. After aligning all 277 isotigs of the isogroup 00007, we found that all of them were derived from the same locus, because only one sequence variant (contig) corresponded to each exon of the homologous Arabidopsis gene, At1g27980, coding for sphingoid long-chain base 1-phosphate lyase (LCB-1-P lyase) (Additional file 3). We assigned eight contigs to eight introns based on a comparison with the homologous Arabidopsis gene. The retention of variable numbers of introns was responsible for the observed extreme AS in this isogroup. Only one isotig 00648 contained the correct ORF with genetic information for a functional protein. To confirm AS experimentally, we designed primers targeted to exon 6 or intron 6 (forward) and exon 15 (reverse) and ran PCR (Figure 3). The size of PCR fragment generated from genomic DNA (2.4 kb) with exon-specific primers UV405_F1 and UV405_R1 agreed with the expected size of this genomic region (2,353 bp). The amplification of cDNA produced a strong band (1.3 kb) corresponding to correctly spliced mRNA with no introns (1,377 bp) and several weak upper bands most likely derived from partially spliced mRNA with retained introns. The primers spanning from intron 6 to exon 15 (UV405_F2 and UV405_R1) produced a PCR fragment from genomic DNA as well as one strong band (1.1 kb) and a few weaker ones from cDNA. The strong band amplified from cDNA provided evidence for intron 6 retention, because no amplification with this primer pair could occur if only correctly spliced mRNA were present in the transcript pool.Figure 3


The transcriptome of Utricularia vulgaris, a rootless plant with minimalist genome, reveals extreme alternative splicing and only moderate sequence similarity with Utricularia gibba.

Bárta J, Stone JD, Pech J, Sirová D, Adamec L, Campbell MA, Štorchová H - BMC Plant Biol. (2015)

PCR amplification with theLCB-1-P lyasespecific primers. An agarose gel (1.2%) electrophoresis of PCR fragments amplified from the gene encoding LCB-1-P lyase (isogroup 000007) in U. vulgaris with different templates. 1–6: cDNA prepared from RNA extracted from two different plant individuals, 7: genomic DNA. NC: negative control with water instead of DNA. (A) PCR with exon-specific primers UV405_F1 and UV405_R1. (B). PCR with intron-specific primer UV405_F2 and exon-specific primer UV405_R1. Annealing temperature is indicated above the lanes. Standard of molecular weights is shown on the both sides of the gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4358910&req=5

Fig3: PCR amplification with theLCB-1-P lyasespecific primers. An agarose gel (1.2%) electrophoresis of PCR fragments amplified from the gene encoding LCB-1-P lyase (isogroup 000007) in U. vulgaris with different templates. 1–6: cDNA prepared from RNA extracted from two different plant individuals, 7: genomic DNA. NC: negative control with water instead of DNA. (A) PCR with exon-specific primers UV405_F1 and UV405_R1. (B). PCR with intron-specific primer UV405_F2 and exon-specific primer UV405_R1. Annealing temperature is indicated above the lanes. Standard of molecular weights is shown on the both sides of the gel.
Mentions: We selected two isogroups of U. vulgaris with very high isotig counts for more detailed analysis. After aligning all 277 isotigs of the isogroup 00007, we found that all of them were derived from the same locus, because only one sequence variant (contig) corresponded to each exon of the homologous Arabidopsis gene, At1g27980, coding for sphingoid long-chain base 1-phosphate lyase (LCB-1-P lyase) (Additional file 3). We assigned eight contigs to eight introns based on a comparison with the homologous Arabidopsis gene. The retention of variable numbers of introns was responsible for the observed extreme AS in this isogroup. Only one isotig 00648 contained the correct ORF with genetic information for a functional protein. To confirm AS experimentally, we designed primers targeted to exon 6 or intron 6 (forward) and exon 15 (reverse) and ran PCR (Figure 3). The size of PCR fragment generated from genomic DNA (2.4 kb) with exon-specific primers UV405_F1 and UV405_R1 agreed with the expected size of this genomic region (2,353 bp). The amplification of cDNA produced a strong band (1.3 kb) corresponding to correctly spliced mRNA with no introns (1,377 bp) and several weak upper bands most likely derived from partially spliced mRNA with retained introns. The primers spanning from intron 6 to exon 15 (UV405_F2 and UV405_R1) produced a PCR fragment from genomic DNA as well as one strong band (1.1 kb) and a few weaker ones from cDNA. The strong band amplified from cDNA provided evidence for intron 6 retention, because no amplification with this primer pair could occur if only correctly spliced mRNA were present in the transcript pool.Figure 3

Bottom Line: We also found a strong correspondence in the presence or absence of root-associated genes between the U. vulgaris transcriptome and U. gibba genome, which indicated that the loss of some root-specific genes had occurred before the divergence of the two rootless species.The species-rich genus Utricularia offers a unique opportunity to study adaptations related to the environment and carnivorous habit and also evolutionary processes responsible for considerable genome reduction.Our study is the first comparison of two global sequence data sets in Utricularia.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The species of Utricularia attract attention not only owing to their carnivorous lifestyle, but also due to an elevated substitution rate and a dynamic evolution of genome size leading to its dramatic reduction. To better understand the evolutionary dynamics of genome size and content as well as the great physiological plasticity in this mostly aquatic carnivorous genus, we analyzed the transcriptome of Utricularia vulgaris, a temperate species with well characterized physiology and ecology. We compared its transcriptome, namely gene content and overall transcript profile, with a previously described transcriptome of Utricularia gibba, a congener possessing one of the smallest angiosperm genomes.

Results: We sequenced a normalized cDNA library prepared from total RNA extracted from shoots of U. vulgaris including leaves and traps, cultivated under sterile or outdoor conditions. 454 pyrosequencing resulted in more than 1,400,000 reads which were assembled into 41,407 isotigs in 19,522 isogroups. We observed high transcript variation in several isogroups explained by multiple loci and/or alternative splicing. The comparison of U. vulgaris and U. gibba transcriptomes revealed a similar distribution of GO categories among expressed genes, despite the differences in transcriptome preparation. We also found a strong correspondence in the presence or absence of root-associated genes between the U. vulgaris transcriptome and U. gibba genome, which indicated that the loss of some root-specific genes had occurred before the divergence of the two rootless species.

Conclusions: The species-rich genus Utricularia offers a unique opportunity to study adaptations related to the environment and carnivorous habit and also evolutionary processes responsible for considerable genome reduction. We show that a transcriptome may approximate the genome for gene content or gene duplication estimation. Our study is the first comparison of two global sequence data sets in Utricularia.

Show MeSH
Related in: MedlinePlus