Comparative Analysis Of MiRNA Among The Sugarcane Progenitors And Cultivate Hybrid

Sugarcane(Saccharum spp.)is a major crop in tropical and subtropical regions,contributing to?80%of the world sugar and?40%of ethanol production worldwide.Modern sugarcane cultivar has one of the most complex genome being both aneuploid and autopolyploid among all the crops,which makes it extremely difficult to sequence the sugarcane genome.The sugarcane genome sequencing have not been completed.MicroRNAs,which are widely considered as master regulators in plants due to their functions/roles as the cores of gene regulatory networks targeting genes that encode transcription factors,is endogenous 21-24 nt,small no-coding RNA molecule derived from longer primary miRNA transcripts with capability of adopting hairpin-like structure by dicer-like enzymes.Previous studies about small RNA have mainly focused on Saccharum hybrids.Elucidating the function of microRNAs in sugarcane progenitors will help reveal microRNA origins and assist functional study for sugarcane regulatory network.In this study,we are using the combination of molecular biology and bioinformatics method to predict and verify the novel miRNA and their target genes in sugarcane.The main findings are as follows:(1)We constructed a small RNA library from the 9-month-old Saccharum officinarum leaves.In this study,through deep sequencing and bioinformatics tools,267 miRNA candidates were predicted in S.officinarum,and 52 of them were assessed to be likely based on our critical criteria.Among these 52 miRNAs,43 miRNAs from 26 miRNA families were found to have homologous miRNAs in the database,and 9 miRNA were identified to be novel in S.officinarum.These 52 miRNA were predicted to have 237 and 76 targets in sugarcane and sorghum,respectively,including Auxin response factor,MADS-box transcription factor,Zinc finger protein-like(like protein)as well.Six of the miRNAs were randomly verified by stem-loop RT-PCR.We drew a conclusion through observation and comparison that an opposite expression pattern between miRNA and targets;conversely,it is also possible that miRNA played a little role in the reg?Lation of target gene and the target gene may be regulated by other genes.(2)We constructed small RNA libraries from different tissues of the sugarcane progenitor wild species and cultivate hybrid.In this study,we identified 60 homologous miRNA belong to 36 families and 168 novel miRNA.In order to survey the expression of miRNA among different sugarcane varieties,we carried out clustering analysis for miRNA.The results showed that miRNA are timing and tissue-specific,such as Ss-miR396 have high expression in nearly all the sample.Besides the roll leaves of Saccharum officinarum under sugar accumulation stage and the stem of Saccharum hybrids under the seeding stage;Ss-miR5564seq2 was no expression in the stem of Saccharum spontaneum at the seeding stage under drought stress and had high expression in other tissues.Few miRNA may be related to drought stress,such as Ss-miR166seql can participate in drought stress.The expression level of Ss-miR166seql increased significantly under drought stress.(3)We predicted miRNA targets for 60 homologous miRNA belongs to 36 families and 168 novel miRNA from the sugarcane progenitor wild species and cultivated hybrids.Further analysis suggested that there are 4 miRNA showing patterns of expression in contrast to corresponding target gene,and may be involved in drought stress,growth and development.