Comparative phenotype and microRNAome in developing anthers of wild-type and male-sterile Lycium barbarum L.

Lycium barbarum L. (L. barbarum) is an economically important plant, as its fruit is highly marketable for its healthy nutrient content. In this study, we characterized the anther development of a major cultivar (Ningqi No. 1) and a male-sterile mutant (Ningqi No. 5) of L. barbarum. We initially investigated the phenotypes of Ningqi No. 1 and Ningqi No. 5 using microscopy and chemical staining, which showed that Ningqi No. 5 failed in the degradation of anther callose, leading to an absence of mature pollen grains and thus to male sterility. Then, to understand the dynamic profile of miRNA expression during the development of the anthers, we collected anther samples from both Ningqi No. 1 and Ningqi No. 5 throughout anther development, and we further identified 137 novel miRNAs from these anther samples by using next-generation deep sequencing technology. Of these 137 novel miRNAs, 96 miRNAs were conserved miRNAs classified into 65 miRNA families, including a few well-known miRNA families related to anther development, such as miR156, miR159 and miR172. In addition, the remaining 41 miRNAs were considered lineage-specific miRNAs, which had no orthologues in other species. The expression data showed that 45 of the 137 miRNAs were differentially expressed in the different samples, including 4 Ningqi No. 5-specific miRNAs and 15 stage-specific miRNAs. The expression patterns of six miRNAs and their predicted targets were verified by Q-PCR, and one of miRNAs and its target were chosen for transient co-expression in Nicotiana benthamiana leaves to verify the correlations between the miRNA and its predicted target. Overall, the identification of the miRNAs in the anther development of Ningqi No. 1 and Ningqi No. 5 provides a valuable resource for understanding the molecular mechanisms of male sterility in L. barbarum.