- In addition, six high-copy retrotransposons were identified according to their reads in the sequenced raw data. - The 12-kb Orchid-rt1 contains 18,000 copies representing 220 Mbp of the P. - Southern blot and slot blot assays showed that these four retrotransposons Gypsy1, Gypsy2, Gypsy3, and Orchid-rt1 contained high copies in the large-genome-size/large-chromosome species P. - Both Orchid-rt1 and Gypsy1 displayed various ratios of copy number for the LTR sequences versus coding sequences among four Phalaenopsis species, including P. - formosana, which suggests that Orchid-rt1 and Gypsy1 have been through various mutations and. - FISH results showed amplification of Orchid-rt1 in the euchromatin regions among the four Phalaenopsis species. - In addition, insertion of Orchid-rt1 in these three genes are all in their intron regions.. - Conclusion: Orchid-rt1 and Gypsy1 – 3 have amplified within Phalaenopsis orchids concomitant with the expanded genome sizes, and Orchid-rt1 and Gypsy1 may have gone through various mutations and homologous. - Insertion of Orchid-rt1 is in the introns and affects gene expression levels.. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - The amount of constitutive heterochromatin in the interphase nucleus indicates that Phalaenopsis species with large chromo- somes contain more heterochromatin and repetitive sequences than those with small chromosomes [5].. - are the most common and ubiquitous in the plant king- dom [13, 14]. - The main TE groups are ancient and are present in all plant kingdoms, yet TE contents show ex- treme diversity among species in the classes of TEs present, their fraction in the genome and level of trans- position activity. - A retrotransposon, Harlequin Orchid RetroTransposon 1 (HORT1), is inserted in the upstream regulatory sequence. - Second, the high-copy num- bers of retrotransposons with high similarities were pre- dicted from the sequencing reads, then those with the highest copies in the sequenced raw data were selected.. - Considering that the new amplified retrotransposons with very similar sequence would be assembled into the same contig after next-generation sequencing and could not be identified as a high-copy element by LTRfinder and Gepard, we used a second approach of high-copy retrotransposons with high similarity predicted as the most sequenced reads in the sequenced raw data. - Among them, Orchid-rt1 is a Gypsy-like retrotransposon and contains 12,671-bp sequences with 707-bp LTR se- quences and a 5121-bp protein-coding region (Fig. - Based on the numbers of these sequenced reads, Orchid- rt1 is predicted to be present with 18,000 copies in the P. - retrotransposition could induce genome size changes with different evolutionary effects in the same genus, as reported for Oryza, Nicotiana, and Genlisea [29]. - A 10-ng plasmid DNA, containing a 707-bp LTR se- quence of Orchid-rt1 and a 3015-bp sequence of T-easy vector, was used as a standard containing the Orchid- rt1-LTR sequence for 1.9 ng (Fig. - The slot blot results showed that 3 −2 x diluted 10-ng Orchid-rt1-LTR plasmid DNA revealed a similar intensity of hybridized spots as 3 −1 x diluted genomic DNA of P. - 2a, ar- rowheads), which suggests an approximately 0.63-ng (1.9 ng ÷ 3) LTR sequence for Orchid-rt1 within 1 μg genomic DNA of P. - equestris of bp [11], the LTR sequence for Orchid-rt1 was bp, for about 1426 copies. - the estimated copy numbers of LTR sequences for Orchid-rt1 were and 19,000 in the genomes of P. - Similarly, a 10-ng plasmid DNA, having a 5121-bp protein-coding sequence (CDS) of Orchid-rt1 and a 3015-bp sequence of T-easy vector, was used as a stand- ard with the Orchid-rt1-CDS of 6.3 ng (Fig. - Slot blot results showed that 3 − 3 x diluted 10-ng Orchid-rt1-CDS plasmid DNA revealed a similar intensity of hybridized spots as 3 −3 x diluted genomic DNA of P. - 2b, arrowheads), which suggests an approximate 6.3-ng CDS for Orchid-rt1 within 1 μg genomic DNA of P. - So the CDS for Orchid-rt1 was bp, for about 1968 copies in the P. - equestris genome (Table 3), and the estimated copy numbers of the CDS for Orchid-rt1 were and 8698 within the ge- nomes of P. - 1 A diagram for the sequence structures of the Gypsy1 to Gypsy3 and Orchid-rt1. - LTR versus CDS in the genomes of P. - bellina, so most of Orchid-rt1 involved full-length copies flanked by two LTR sequences in these genomes. - In the “increase/decrease model”, whereby following the retrotransposon amplifi- cation, these sequences without selection pressure are eliminated by homologous or illegitimate recombination and lead to the solo-LTR formation and deletion [15], the copies of Orchid-rt1 in P. - All retrotransposons contained high copy numbers in the large-chromosome P. - ahprodite have similar genome size, we found a prevalence for Gypsy2 or Gypsy3 in the P. - Chromosomal localization of Orchid-rt1 in Phalaenopsis genomes. - genomic DNA (ng/ μ l) Orchid-rt1-LTR/CDS . - Mb/genome Orchid-rt1-LTR/CDS . - /genome Orchid-rt1-LTR/CDS . - FISH re- sults showed Orchid-rt1 dispersed through all chromosomes in P. - 3g) have more Orchid-rt1 as compared with P.. - 3g), Orchid-rt1 repeats distrib- uted in the intercalary regions of medium and large chromo- somes and throughout all small chromosomes. - Gene expression affected by Orchid-rt1 insertion. - Unlike other plants, these TEs are enriched in the intron regions [11]. - insertion of Orchid-rt1 may affect gene expressions. - For this, whole genome alignment was performed by using the full length of Orchid-rt1 against the P. - To confirm whether these genes do contain portions of Orchid-rt1 sequence, primers around the predicted Orchid-rt1 insertion sites were designed from three pre- dicted Orchid-rt1 inserted genes, Peq018599, Peq009948, and Peq014239. - The amplified regions were sequenced and aligned to the Orchid-rt1 sequence. - A 600-bp frag- ment from Orchid-rt1 was found within the predicted insertion region from Peq009948 (Additional file 3: Fig.. - This suggests that some predicted gene do harbor the Orchid-rt1 region.. - 3 Fluorescence in situ hybridization (FISH) mapped the LTR sequence of Orchid-rt1 on chromosomes of P. - a, c, e and g The distribution of Orchid-rt1 (red) on chromosomes (blue). - qRT-PCR was applied to validate the expres- sion level of the predicted Orchid-rt1 inserted genes and its paralogous genes (Fig. - The predicted Orchid-rt1 inserted gene Peq018599 showed up-regulated expres- sion as compared to its paralogous genes Peq025947 and Peq023950 in RNA-seq data and confirmed with qRT- PCR. - In contrast, the other two predicted Orchid-rt1 inserted genes Peq009948 and Peq014239 showed down- regulated expression compared to their paralogous genes Peq019291 and Peq000381, respectively, in RNA-seq analysis, and this was confirmed by using qRT-PCR (Fig.. - Expression levels of four other genes were not signifi- cantly affected with the insertion of Orchid-rt1 as com- pared to their paralogs (Additional file 4: Table S2).. - Intriguingly, the above three genes (Peq018599, Peq009948, and Peq014239) with their expression levels been traumatized all with the insertion of Orchid-rt1 in the intron regions with various size of 765 bp, 473 bp and 297 bp, respectively (Fig. - Among them, four Gypsy-like LTR retrotransposons, Or- chid-rt1, Gypsy1, Gypsy2, and Gypsy3, represented a large amount of the Phalaenopsis genomes, while the in- creased copies of both Orchid-rt1 and Gypsy1 in two species with large genome size/chromosome size, P. - The orange arrow indicates the Orchid-rt1 partially inserted genes, green and blue arrows displayed the paralogous genes. - Two approaches have been performed for the identifica- tion of the high-copy LTR retrotransposons from the Phalaenopsis genome, including LTRfinder for the full- length LTR retrotransposons from the assembled se- quences, and the prediction of high-copy retrotransposons with high similarity from the most sequenced reads in the sequenced raw data. - To avoid this, the second approach was performed via the predic- tion of high-copy retrotransposons with high similarities from the most sequenced reads in the sequenced raw data. - Therefore, Orchid-rt1 has been identified and pre- dicted to be present with 18,000 copies in the P. - The fact of high-copy numbers of Orchid-rt1 present in Phalaenop- sis genomes was then confirmed by using Southern blot, slot blot, and FISH results. - It indicated that each copy of Orchid-rt1 was similar to each other, while the amplified. - 5 Insertion site of Orchid-rt1 in the three genes with their gene expression levels been affected with the TE insertion. - copies of Orchid-rt1 were recently occurred without many mutations accumulated. - FISH results showed Orchid-rt1 dispersed throughout all chromosomes in P. - aphrodite, while only in the intercalary regions of medium and large chromosomes and throughout all small chromosomes in P. - Therefore, FISH results showed amplification of Orchid- rt1 in the euchromatin regions also contributing to the expanded genome sizes. - Similar situation was also found in the cytogenetic analysis on Brachiaria genus, while an Athila retrotransposon was detected mainly in the centromeric-pericentromeric regions of chromosomes of diploids B. - brizantha, but differences in location and signal strength in the polyploid B. - Expression levels of genes inserted with Orchid-rt1 in P. - We found 3 genes that have their expression levels regulated with the Orchid-rt1 in- sertion. - Peq018599 encoding for copper transporter 1 with the insertion of Orchid-rt1 showed highly upregu- lated gene expression in root and seed development as compared to its paralogs. - Expression level of Peq009948 is reduced to half amount of its paralog in most organs, and abolished in the floral stalk and leaf.. - In the Peq014239, its. - expression is almost abolished in the floral stalk, leaf and root, trace amount in sepal and seed development, and a substantial amount in petal, labellum, pollinium and gynostemium. - All these three genes are involved in growth and development, and further analysis will be re- quired to detail the significance of regulation of gene expression upon the insertion of Orchid-rt1 into these genes.. - Both Orchid-rt1 and Gypsy1 showed increased copies in two species with large genome size/chromosome size, P. - FISH results revealed that abundant Orchid-rt1 mainly distributed in the inter- calary euchromatin regions of large chromosomes in these two species. - Insertion of Orchid-rt1 is found in the intron regions and gene expression are affected upon the insertion of Orchid-rt1.. - All plant ma- terials were grown in the greenhouse at National Cheng Kung University (Tainan, Taiwan) under natural light and controlled temperature from 23 °C to 27 °C.. - Each predicted TE was verified for its terminal repeats on both ends and identified for other copies in the Phalaenopsis genome by using NCBI-BLAST2 Sequences (http://www.ncbi.nlm.. - Each TE family was identified and characterized with its sequence structure and copy num- ber in the Phalaenopsis genome. - (Onozuka R-10, Yakult Honsha, Tokyo) and 2% pecti- nase (Sigma Chemical Co.) in 10 mM citrate buffer, pH 4.0, at 37 °C for 90 min, then squashed on slides in the same fixative. - Expression levels of genes inserted by Orchid-rt1 and their paralogs. - To find out the genes that have insertions of Orchid-rt1, whole genome alignment was performed by using the full length of Orchid-rt1 against the P. - To confirm the insertion of Orchid-rt1 in the predicted genes, primers were design from outside of pre- dicted insertion sites by primer3plus (http://www.bioinfor- matics.nl/cgi-bin/primer3plus/ primer3plus.cgi. - The amplified fragments were sequenced and BLAST against Orchid-rt1 full-length sequence using the global alignment in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi).. - The relative gene expression levels of those genes inserted with Orchid-rt1 and their paralogous genes in several organs were found in the Orchidbase as well and presented as heatmap. - The up-regulated or down- regulated gene expression of the inserted genes com- pared to their paralogs identified in the RAN-seq data were further confirmed by using qRT-PCR. - Four native Phalaenopsis species analyzed in the study, including P. - The global alignment result between Orchid-rt1 full length and predicted insertion region from Peq009948.. - The expression levels of Orchid-rt1 inserted gene and their paralogs genes among different organs.. - Molecular paleontology of transposable elements in the Drosophila melanogaster genome. - Abundance, distribution, and transcriptional activity of repetitive elements in the maize genome. - Nested retrotransposons in the intergenic regions of the maize genome. - A HORT1 retrotransposon insertion in the PeMYB11 promoter causes harlequin/black flowers in Phalaenopsis orchids. - Identification, characterization and distribution of transposable elements in the flax (Linum usitatissimum L.) genome
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