Dynamic Of Noncoding RNA In Polyploidization Of Cotton

Hybridization and polyploidization are driving forces in species formation and evolution.The common occurrence of plant polyploids,generate instantaneous genome-scale genetic and epigenetic changes,and often lead to novel agronomic traits and improved adaptation to environment.Cotton,belonging to Malvaceae,Gossypium spp.is the world’s most important natural textile fiber and a significant oilseed crop.Cotton is one of high quality plant protein source next to soybean.G.hirsutum originated from inter-genomic hybridization between an A-genome-like ancestral G.arboreum（A2）and a D-genome-like G.raimondii（D5）diploid.As the donor of the tetraploid,the diploid A genome species produce spinnable,whereas the D genome species do not.Cotton is also an efficient model of polyploid evolution.LncRNA and circRNA are two types of long,non-coding RNA widely prevalence in plant and animal genomes.They play important roles in plant development,response to stresses and maintenance of stability of genome.With the development of high-throughout sequencing technology,more and more long,non-coding RNA have been successfully sequenced and assembled.Non-coding trancriptome has been a hot area of research for several years,but the role of IncRNA in plant interspecies hybrid and polyploidization has not been investigated.Newly synthesized allopolyploids have been used to investigate early changes of interspecies and polyploidization.For our study,we crossed the G.arboreum（A2）accession Shixiya with G.raimondii（D5）to generate an F1 hybrid.Using G.arboreum（A2）accession Shixiya,G.raimondii（D5）,（G.arboreum x G,raimondii）F1 and TM-1（G.hirsutum,（AADD）1）,we constructed a model system to mimic the lncRNA evolution.of Gossypium spp.from diploid to allotetraploid.The main results are as the followings.Part I The dynamic change of IncRNA in interspecies hybrid and polyploidization1.Profied the IncRNA map in G.arboreum（Ga,accession Shixiya）,G.raimondii（Gr）,（G.arboreum × G.raimondii）F1 and G.hirsutum（Gh,accession TM-1）.Throught IncRNA sequencing,we ultimately obtained 4,107 IncRNAs in Ga,2,767 in Gr,8,126 in F1,and 8,514 in Gh.Among the predicted lncRNAs,12,316（90.63%）were lincRNA（Large intergenic non-coding RNAs）,1,451（6.17%）were NATs（Nature antisense transcript）and 753 intronic RNAs（3.20%）.2.LncRNA showed high primary sequence conservation in Gossypium species.Most IncRNA loci（86%,n=13,282）were genomic constitutional in three sequenced cotton species（Ga,Gr and Gh）.Less than 14.64%（n=2,253）of IncRNA showed homoloy to other model plants like Arabidopsis thaliana and Oryza sativa.In contrast to protein coding genes,1ncRNA exhibits lower sequence conservation.3.LncRNA showed position conservation in three cotton species.4,182-5,616 syntenic blocks were identified in the comparison of the four genomes.These blocks covered 60.51-161 78.32%（n from 47,472 to 60,680）of the transcripts of each genome with chromosome collinearity.We found 71.66-86%IncRNA were allelic-expressed.Especially in the comparison between GhAT and GhDT from identical genome and tissues,the allelic-expressed IncRNA comprised over 80%（1,269 out of 1,517）of syntenic IncRNA loci.The allelic-expressed pattern can explain the species specific of IncRNA.4.LncRNA were reprogramed in the interspecies hybrid F1.Two near isogenic lines（NILs）from inbred lines of cotton,upland cotton（Gh,accession Zhongl2）and Zhongl2 GL（a glandless line produced by multiple generations of backcrossing Zhongl2）were selected as control.In F1,79.38%IncRNA were reprogramed,while 1.72%IncRNA were reprogramed in NILs,indicating IncRNA can be legacy in breeding but reprogramed in interspecies hybrid.5.Legacy lncRNAs were stably expressed in Gossypium spp..Among the IncRNAs obtained in F1,only 2,395 loci（20.61%,out of 11,618）were also expressed in parent genomes.Accordingly,these lncRNAs were classified as parent legacies.5,731 IncRNA loci were expressed exclusively in F1.It is likely these IncRNA transcripts were activated by the genomic shock of hybridization,so we classified them as genome-shock response positive（GSR+）transcripts.We found legacy lncRNA usually with higher expression levels,longer transcripts,rich in repetitive sequence.Legacy lncRNA loci hold high potential to survive the genomic shock of hybridization and polyploidization,6.Transposon elements（TEs）made different contributions to lncRNAs.About 60%lncRNA contained TEs.We found that-40%exons of lncRNA were LINE/Gypsy origin.LncRNA which were constantly expressed from interspecies hybrid and polyploidization were significantly rich in TEs regions.Non-TE sequences in lncRNAs evolve under modest yet greater selection pressure than their TE sequences.LINE might contribute to main the stability transcription of IncRNA,while Gypsy may drive the expression variance of lncRNA.7.lncRNA might originate from the transposon region during hybridization.Transposon region generate the major body of siRNA in genome.While the lncRNA derived siRNA were not from the lncRNA-overlaped TEs.The genomic DNA methylation analysis also showed the lncRNA-overlaped TEs were lower with CG,CHG and CHH methylation.The differently expressed lncRNA were associated with the differently DNA methylation.XLOC₄09583 were transcriptionally activated by plant LINE transposable element after polyploidization.The suppression of XLOC₄09583 in TM-1 using virus induced gene silencing（VIGS）increased the plant height.We hypothysized that the activated lncRNA in F1 were originated from the TEs released from siRNA dependent DNA methylation（RdDM）.8 Integrating previous genome-wide association studies（GWAS）,we found 42 lncRNAs were closed to trait-associated SNPs loci.LncRNA XLOC₂77040 and XLOC₃19336 contained micronaire and fiber uniformity trait-associated SNPs suggesting their putative contributions to developmental and agriculture traits.We found 62%（n=26）of lncRNA were novel transcription after polyploidization.Part Ⅱ Characterization of conserved circular RNA in polyploid and its ancestors,Gossypium spp.1.We identified a total of 49,724 back-spliced reads.These reads represent 4,326 circular RNAs（Ga 1,041,Gr 1,478,F1 1,311 and Gh 499）with a minimum of two back-splice reads in ovule and leaf tissues.CircRNA were mainly transcribed from exons,next to intergentic regions.CircRNA were prefer to express in ovule tissues compared to leaf.2.Cotton circRNA parent gene have species structure features.CircRNA parent genes usuallay contain more than six exons,long flanking introns and are enriched with repeat sequences and with more complex alternative splicing events compared to liner genes.3.Cotton circRNAs are independent of GT/AG signals and flanking intronic complementary sequences.A prevalence of～80%of exon-circRNA are non-canonical GT/AG signals,and only～10%of exon-circRNA are associated with reverse complementary intronic sequences.4.A proportion of circRNA were conserved in plant species.We identified a total of 432（19.97%）conserved circRNAs and 961（44.43%）specific circRNAs.We found that circRNA of Gr shared 18.32%homology to Arabidopsis thaliana and 15.6%to Oriza sativa.5.Complementary sequences and repeated sequences on the flanking ends of parent genes could be critical for the generation of conserved circRNA.