Quantitative real-time reverse-transcriptase PCR (qRT-PCR) has been frequently used for detecting gene expression. To obtain reliable results, selection of suitable reference genes is a fundamental and necessary step. Garlic (Allium sativum), a member from Alliaceae family, has been used both as a food flavoring and as a traditional medicine. In the present study, garlic plants were exposed to salt stress (200 mM NaCl) for 0, 1, 4 and 12 h, and garlic roots, bulbs, and leaves were harvested for subsequent analysis. The expression stability of eight candidate reference genes, eukaryotic translation initiation factor 4α (eIF-4α), actin (ACTIN), tubulin β-7 (TUB7), TAP42-interacting protein of 41 kDa (TIP41), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SAND family protein (SAND), elongation factor 1 alpha (EF-1α), and protein phosphatase 2A (PP2A) were evaluated by geNorm, NormFinder, and BestKeeper. All genes tested displayed variable expression profiles under salt stress. In the leaf and root group, ACTIN was the best reference gene for normalizing gene expression. In garlic clove, ACTIN and SAND were the least variable, and were suitable for gene expression studies under salt stress; these two genes also performed well in all samples tested. Based on our results, we recommend that it is essential to use specific reference genes in different situations to obtain accurate results. Using a combination of multiple stable reference genes, such as ACTIN and SAND, to normalize gene expression is encouraged. The results from the study will be beneficial for accurate determination of gene expression in garlic and other plants.

Laccase, as a copper-containing polyphenol oxidase, primarily functions in the process of lignin, anthocyanin biosynthesis, and various abiotic/biotic stresses. In this study, forty-eight laccase members were identified in the eggplant genome. Only forty-two laccase genes from eggplant (SmLACs) were anchored unevenly in 12 chromosomes, the other six SmLACs were mapped on unanchored scaffolds. Phylogenetic analysis indicated that only twenty-five SmLACs were divided into six different groups on the basis of groups reported in Arabidopsis. Gene structure analysis revealed that the number of exons ranged from one to 13. Motif analysis revealed that SmLACs included six conserved motifs. In aspects of gene duplication analysis, twenty-one SmLACs were collinear with LAC genes from Arabidopsis, tomato or rice. Cis-regulatory elements analysis indicated many SmLACs may be involved in eggplant morphogenesis, flavonoid biosynthesis, diverse stresses and growth/development processes. Expression analysis further confirmed that a few SmLACs may function in vegetative and reproductive organs at different developmental stages and also in response to one or multiple stresses. This study would help to further understand and enrich the physiological function of the SmLAC gene family in eggplant, and may provide high-quality genetic resources for eggplant genetics and breeding.

Stress-associated proteins (SAP), a class of zinc-finger proteins, have been identified as novel stress regulatory proteins in stress responses. However, SAP genes in eggplant (SmSAP) have been little reported. It has important significance in identifying SAP members, understanding the molecular mechanisms underlying stress responses, and tolerance. We performed a comprehensive study of the A20/AN1 domains, motifs, gene structures, phylogenetic relationships, chromosomal locations, gene replications, collinearity, cis-acting elements, and expression pattern responses to various abiotic stresses. Twenty-one SAP genes were identified in eggplant (SmSAP) and were localized on 10 chromosomes. A phylogenetic analysis revealed that most of the SmSAP proteins showed a high homology with the tomato SAP members, and 21 members were divided into four groups based on the homology of the SAP members in eggplant, tomato, rice, and Arabidopsis. Further analysis revealed that SmSAP proteins contain the characteristic A20/AN1 domains, the A20 domain composed of motif 2 (ILCINNCGFFGSPATMNLCSKCYKDMJLK). Four pairs of tandem duplications were found in eggplant, and 10 SmSAP genes had collinearity with SAP genes from Arabidopsis, potato, or tomato, but only four SmSAP genes were collinear with SAP genes in the three species mentioned above. Moreover, the promoters of SmSAP genes were predicted to contain many cis-acting elements that respond to abiotic stress and hormones. A qRT-PCR analysis of the four selected SmSAP genes exhibited diverse expression levels in response to various environmental stresses. These results provided a comprehensive analysis of the SmSAP genes and lay a solid foundation for improving the understanding of the functional diversification of SAP genes under various environmental stresses in eggplant.

Laccase, a copper-containing polyphenol oxidase, is integral to plant growth and development, primarily due to its involvement in lignin and flavonoid bios