Small RNA Regulatory Networks in Susceptible, Hypersensitive, and Resistant Genotypes of Cotton (Gossypium hirsutum) During Reniform Nematode (Rotylenchulus reniformis) Infestation

Reniform nematode (RN, Rotylenchulus reniformis) is a major soil-born pathogen affecting cotton. Most commercial cotton germplasm is susceptible to RN, but germplasm that appears to be resistant to RN including the RN hypersensitive lines, LONREN1 and LONREN2 and resistant line BARBAREN713 have emerged as promising sources of resistance to this important pathogen.   The objective of this investigation is to examine the small RNA regulatory networks during RN investation of susceptible, hypersensitive, and resistant genotypes, and based on changes in the expression of genes in miRNAome, tasiRNAome, and the transcriptome of these respective genotypes, define the role that specific networks may play in resistance and hypersensitivity to RN infestation. Eight smallRNA libraries were generated from the roots of  susceptible genotypes (DPL90 & SG747, combined to make 1 susceptible sample), two hypersensitive genotypes (LONREN1 & LONREN2), and the resistant BARBAREN713 genotype either pre- or post-infestation (combined 1,2,5, & 10 dpi) with reniform nematodes. Illumina sequencing was utilized to reveal a total of over 282,435,835 million raw reads in the 8 sequenced sRNA libraries. From these 104,326,160 reads remained in the 8 sRNA libraries after primer trimming and filtering out low quality reads and reads less than 17 and greater than 28 nt in length.  These reads were mapped to 21,613,650 unique sequences in the total of 8 sRNA libaries, and unique reads in each library were mapped to the unique sequences in all libraries, and those reads in each library with read counts greater than 2 were filtered leaving a total of 2,068,515 unique reads in the 8 libraries.  Based on similarity to previously reported Viridiplantae miRNAs in miRBase19, 1089 small RNA sequence reads belonging to 281 distinct miRNA families were identified, leaving with RR reads belonging to 915,146 unique sequences as unanotated sRNAs. The expression of 553 conserved microRNA sequences were determined, and 83 conserved miRNA sequences were identified as differentially expressed microRNAs amongst different genotypes and treaments in the experiment. Finally, 259 phased small RNA clusters were computed using unannotated sRNA sequences in each library with the miRNAs used as phase initiators to predict valid cleavage sites directed by phase initiators.  Eighteen differentially expressed conserved miRNA sequences were predicted to be phase initiators, and the expression of the these phasing miRNAs and their predicted tasi-RNAs were examined in the libraries and will be compared between genotypes and treaments.  The differential expression following nematode infestationn in each genotype will be to the reported.