High Throughput Sequencing of Small RNAs in the Two Cucurbita Germplasm with Different Sodium Accumulation Patterns Identifies Novel MicroRNAs Involved in Salt Stress Response

Abstract

MicroRNAs (miRNAs), a class of small non-coding RNAs, recognize their mRNA targets based on perfect sequence complementarity. MiRNAs lead to broader changes in gene expression after plants are exposed to stress. High-throughput sequencing is an effective method to identify and profile small RNA populations in non-model plants under salt stresses, significantly improving our knowledge regarding miRNA functions in salt tolerance. Cucurbits are sensitive to soil salinity, and the Cucurbita genus is used as the rootstock of other cucurbits to enhance salt tolerance. Several cucurbit crops have been used for miRNA sequencing but salt stress-related miRNAs in cucurbit species have not been reported. In this study, we subjected two Cucurbita germplasm, namely, N12 (Cucurbita. maxima Duch.) and N15 (Cucurbita. moschata Duch.), with different sodium accumulation patterns, to Illumina sequencing to determine small RNA populations in root tissues after 4 h of salt treatment and control. A total of 21,548,326 and 19,394,108 reads were generated from the control and salt-treated N12 root tissues, respectively. By contrast, 19,108,240 and 20,546,052 reads were obtained from the control and salt-treated N15 root tissues, respectively. Fifty-eight conserved miRNA families and 33 novel miRNAs were identified in the two Cucurbita germplasm. Seven miRNAs (six conserved miRNAs and one novel miRNAs) were up-regulated in salt-treated N12 and N15 samples. Most target genes of differentially expressed novel miRNAs were transcription factors and salt stress-responsive proteins, including dehydration-induced protein, cation/H+ antiporter 18, and CBL-interacting serine/threonine-protein kinase. The differential expression of miRNAs between the two Cucurbita germplasm under salt stress conditions and their target genes demonstrated that novel miRNAs play an important role in the response of the two Cucurbita germplasm to salt stress. The present study initially explored small RNAs in the response of pumpkin to salt stress, and provided valuable information on novel miRNAs and their target genes in Cucurbita.

Fig 1. Na⁺ concentrations in the leaves, stems and roots (A) and Na⁺ distribution (B) in N12 and N15 treated with 100 mM NaCl for 10d.

Data represent the mean ± SE (n = 6). Different letters indicate significant differences between treatments using Duncan’s multiple range test at p<0.05.

Fig 2. Length distribution of small RNAs in the four libraries.

X-axis, length of sRNA distribution; Y-axis, percent frequency of raw reads. 24hR, N12 root under control library; 24hNR, N12 root under salt stress treatment library; 54hR, N15 root under control library; 54hNR, N15 root under salt stress treatment library.

Fig 3. Numbers of identical miRNA members in each conserved miRNA family in the four libraries.

24hR, N12 root under control library; 24hNR, N12 root under salt stress treatment library; 54hR, N15 root under control library; 54hNR, N15 root under salt stress treatment library.

Fig 4. Gene Ontology classifications of the target genes of conserved miRNAs in the two Cucurbita germplasm.

Fig 5. Expression analysis of miRNAs in N12 by qRT-PCR.

Column above the X-axis indicates up-regulated miRNAs; column below the X-axis represents down-regulated miRNAs.

Fig 6. Expression analysis of miRNAs in N15 by qRT-PCR.

Column above the X-axis indicates up-regulated miRNAs; column below the X-axis represents down-regulated miRNAs.

Fig 7. Expression analysis of the targets by qRT-PCR in N12 and N15.

Column above the X-axis indicates up-regulated mRNAs; column below the X-axis represents down-regulated mRNAs.