Deep-Sequencing Reveals Differential Expressions of microRNAs in Different Developmental Stages of Meloidogyne Incognita Infection of Cotton Plant

The Southern root-knot nematode (RKN) (Meloidogyne incognita) is among the most damaging plant-parasitic pests of various commercial crops including cotton (Gossypium hirsutum). At the second juvenile stage (J2), M. incognita acquires a parasitic character. In previous report we identified miRNAs from the small RNA database of M. incognita (GSM611102) generated from deep sequencing of J2 M. incognita infection of pepper (Solanum capsicum) plants. In this study we identified the differentially expressed miRNAs in the parasitic J2 stage after they infected cotton plants compared to those miRNAs expressed in their embryos. All small RNAs from RKN at two developmental stages (eggs and J2) were sequenced using the Illumina HiSeq X-ten sequencing platform. A total of 33.83 million clean reads were obtained, and with each sample no less than 15 million clean reads were found.  Categorization of the small RNAs was done using Bowtie software, which aligns clean reads to the Silva, GtRNAdb, Rfam, and Repbase databases. After excluding repeats and other types of small RNAs, the remaining sRNA sequences were mapped to the unigene of the M. incognita genome.  A genome-wide chromosome distribution of mapped reads was obtained.  When comparing the unique sRNA sequence reads, the great majority (~ 90%) of the unique sequences differed between the eggs and J2 stages, with 56% unique sequences expressed in eggs but not in J2, and 34% expressed in J2s but not in eggs. Unique sequences expressed in only one or the other stages constituted only 3.5% of the total reads, but these might be of special interest to identify miRNAs that are important in parasitism. Based on two deep sequence databases, we have identified a total of 45 miRNA families from RKN eggs and J2. A total of 547 miRNA targeted protein-coding genes were predicted, and their regulated gene network was analyzed.​