- Functional examination of lncRNAs in allotetraploid Gossypium hirsutum. - The function of the lncRNAs activated in allotetraploid cotton remain largely unknown.. - LncRNA expression was preferentially associated with that of the flanking PCGs. - A comparative analysis of the human and mouse genomes determined that lncRNAs are under positive selection rather than neutral mutation [2]. - altered expression of lncRNAs during domestication has likewise been identified in rice [4].. - Therefore, lncRNAs actively take part in the evolution of species just as coding genes do. - 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. - Full list of author information is available at the end of the article Wang et al. - The roles of lncRNAs in plants are of considerable interest, having emerged as new epigenetic regulators of coding gene expression in biological activities and as specifically affecting plant responses to abiotic stress [10–13]. - Although a number of lncRNAs have been identi- fied, only a few are functionally well characterized.. - Further- more, global genomic comparisons have revealed that lncRNAs burst in the process of polyploidization [31].. - The diploid ancestors of the cotton A and D subgenomes underwent interspecific hybridization and polyploidy to form allotetraploid cot- ton one to two million years ago [33]. - As research re- garding the evolutionary genomics, population genetics, and epigenetics of allotetraploid cotton has developed rapidly [34–38], this provides a useful model system in which to further examine the rapid evolution of lncRNAs.. - The source of lncRNAs activated in Gossypium hirsutum The function of a lncRNA may be related to its sequence conservation, location on the chromosome, and tran- scriptional activity [39]. - In our previous study, we re- spectively identified and 8,514 lncRNAs in the G. - We then classified conserved lncRNAs (C-lncRNAs) and non-conserved lncRNAs (NC-lncRNAs) in Gh based on (1) the sequence similar- ity (blastn -evalue 10^ −10 -max_target_seqs 1), (2) collin- earity on the chromosome (MCscanX -b 2, -s 5) of the lncRNA for downstream functional analysis. - Expression pattern analysis of lncRNAs under abiotic stresses. - Plant lncRNAs are reported to be actively involved in the molecular regulation of responses to environmental stimuli and stresses. - The leaves of the treated seedlings were harvested for RNA-seq profiling (Fig. - Finally, biological replicates of the RNA-seq results showed high consistency (Fig S1). - Co-expression of lncRNAs with adjacent PCGs. - To investigate potential patterns in the mode by which lncRNAs regulate PCGs, we examined the association of their expression profiles in the context of abiotic stress. - Here we defined a 1:1 orthologous adjacent PCG that co-expressed with a lncRNA within 50 kb as being subject to a cis effect of the lncRNA, consistent with the latest literature [5, 32].. - Functional examination of lncRNAs in allotetraploid cotton. - Therefore, in this study, we carried out a functional evaluation of lncRNAs in upland cotton (G. - Wang et al. - 2 Expression patterns of lncRNAs and PCGs under four abiotic stressors. - F: Density distributions of the Pearson correlation coefficients between adjacent PCG-lncRNA pairs. - G: Density distributions of the Pearson correlation coefficients between adjacent C-lncRNA-PCG and NC- lncRNA-PCG pairs. - Overall, most of the potential functional lncRNAs that were screened were associated with abiotic stress.. - 3C-H, with transcription suppression of the. - Detailed information and phenotypic observations of lncRNAs after VIGS silencing are given in Table S6. - 3 Results of the low-throughput VIGS screening for functional lncRNAs in G. - A: Bar chart showing the numbers of functional and non-functional lncRNAs that exhibited differences in the low-throughput VIGS screening. - Red dots: lncRNA candidates in the preselection for VIGS. - I: Histogram showing relative expression of lncRNAs compared with the TRV2 vector control after VIGS. - Functional validation of lncRNAs identified in the low- throughput screen. - To validate the accuracy of the low-throughput func- tional examination, representative functional lncRNAs were further assessed. - ABA plays an important role in plant drought stress response, and some specific genes in the ABA pathway have been singled out as involved, such as ABSCISIC ACID RESPONSIVE ELEMENT-BINDING FACTOR 1 (ABF1), PYRABACTIN RESISTANCE 1-LIKE 9 (PYL9), and DEHYDRATION-RESPONSIVE PROTEIN RD22 (RD22) [48]. - The rapid evolution of lncRNAs to obtain new functions Rapid evolution of lncRNAs is commonly observed in both plant and animal kingdoms in the form of interspe- cies polymorphisms and epigenomic modifications [39, 50]. - For example, diverged Arabidopsis ecotypes show polymorphisms in the promoter region of the flowering. - Our study on functional lncRNAs in allotetraploid cotton confirms that the rapid evolution of lncRNAs introduces new, functional non- coding genes.. - Among lncRNAs, no statistical difference in the re- sponse to stimuli was observed for C-lncRNAs com- pared with either all lncRNAs or NC-lncRNAs.. - Furthermore, our primary functional screening indicated that in the cotton lineage, both C-lncRNAs and NC- lncRNAs have biological roles. - However, the trend observed in our study also supports that NC-lncRNAs have the chance to gain a new regulatory role and, for cotton, were selected in the cultivated population. - We therefore speculate that inherit- able and functional epigenetic modifications can play a role in the rise of domesticated traits. - LncRNAs are abundantly present and transcribed in the genome [57]. - Utilization of cell lines may aid in achiev- ing high-throughput screening with strong transcrip- tional signal, but lncRNA function is often cell-type specific, thus the cultured cells may not reflect the real functions of the lncRNAs [60]. - The lower graph shows a stack view of the locus with leaf RNA-seq reads from Ga and Gh. - Error bar: standard error of the mean. - I: Scatterplot showing the linear relationship between expression of XLOC_227558 and that of the adjacent PCG, Gh_A08G1105 , under different abiotic stressors. - The lower graph shows a stack view of the locus in Gh. - I: Scatterplot showing the linear relationship between expression of XLOC_107738 and that of the adjacent PCG, Gh_A05G0714 , under different abiotic stressors. - The reported low conservation of lncRNAs across species and the screening-out of conserved lncRNAs among functional candidates in this study suggest that the potential func- tionality of each conserved non-coding gene needs to be considered as an individual case. - The results of the present study might be skewed due to the small number of conserved lncRNAs, but that the functional lncRNA profile is unique to each organism is in agree- ment with most of the reported biological impacts of lncRNAs.. - The development of in- heritable and functional lncRNAs over evolution can participate in the emergence of adaptive traits.. - Plants of the upland cotton (G. - Unique 200- to 400-bp fragments of lncRNAs were amplified from TM-1 cDNA by polymerase chain reac- tion (PCR) using Ex Taq (Takara, Code No.: RR01AM).. - A 1:100 dilu- tion of the primary culture was then inoculated into a secondary liquid induction media culture with 50 µg/mL of kanamycin and 25 µg/mL of rifampicin, which was in- cubated with shaking at 28 °C for 14 to 16 h at 200 rpm until the OD 600 was 1.5 to 2.0. - For each culture, a bacterial suspension with an OD 600 of 2.0 was prepared and incubated in the dark at 28 °C for three to five hours. - The bacterial suspen- sions were infiltrated into the cotyledons of the seed- lings, and the plants were kept at 21 °C ± 1 °C in a growth chamber with a 16-hour day length and 50 % relative humidity for at least three weeks before use in assays.. - Individual transcript expression levels were quantified in terms of the fragments per kilobase of exon per million fragments (FPKM) by StringTie [67].. - salt with 200 mmol NaCl, and drought in the form of water restriction. - 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