Mei (Prunus mume) is widely cultivated in eastern Asia owing to its favored ornamental characteristics and its tolerance for low temperatures. Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for gene expression analysis, requiring carefully selected reference genes to ensure data reliability. The aim of this study was to identify and evaluate reference genes for qRT-PCR in mei. Ten candidate reference genes were chosen, and their expression levels were assessed by qRT-PCR in four sample sets: 1) flowering mei; 2) mei undergoing abiotic stress; 3) different genotypes of Prunus species; and 4) all mei samples. The stability and suitability of the candidate reference genes were validated using commercially available software. We found that protein phosphatase 2A-1 (PP2A-1) and PP2A-2 were suitable reference genes for flowering with ubiquitin-conjugating enzyme E2 (UBC) also being suitable for different genotypes of Prunus species. UBC and actin (ACT) were most stably expressed under abiotic stress. Finally, the expression of an AGAMOUS homolog of Arabidopsis thaliana (PmAG) and a putative homolog of Group 2 late embryogenesis abundant protein gene in A. thaliana (PmLEA) were assessed to allow comparisons between selected candidate reference genes, highlighting the importance of careful reference gene selection.
The MADS-box gene SOC1/TM3 (suppressor of overexpression of constans 1/tomato MADS-box gene 3) integrates multiple flowering signals to regulate the transition from vegetative to reproductive development in arabidopsis (Arabidopsis thaliana). Although SOC1-like genes have been isolated from a wide range of plant species, their orthologs are not well characterized in mei (Prunus mume), an important ornamental and fruit plant in east Asia. To better understand the molecular regulation of flower development in mei, we isolated and characterized three putative orthologs of arabidopsis SOC1, including PmSOC1-1, PmSOC1-2, and PmSOC1-3. The phylogenetic tree revealed that these genes fall into different subgroups within the SOC1-like gene group, suggesting distinct functions. PmSOC1-1 and PmSOC1-3 were mainly expressed in vegetative organs and at low expression levels in floral parts of the plants, whereas PmSOC1-2 was expressed only in vegetative organs. Furthermore, the expression level decreased significantly during flower bud differentiation development, suggesting a role for these genes in the transition from the vegetative to the reproductive phase. Overexpression of PmSOC1-1, PmSOC1-2, and PmSOC1-3 in arabidopsis caused early flowering. Early flowering also increased expression levels of four other flowering promoters, agamous-like 24 (AGL24), leafy (LFY), apetala 1 (AP1), and fruitfull (FUL). Moreover, the overexpression of PmSOC1-1 and PmSOC1-2 resulted in a range of floral phenotype changes such as sepals into leaf-like structures, petal color into green, and petal into filament-like structures. These results suggested that the genes PmSOC1-1, PmSOC1-2, and PmSOC1-3 play an evolutionarily conserved role in promoting flowering in mei, and may have distinct roles during flower development. Our findings will help elucidate the molecular mechanisms involved in the transition from vegetative to reproductive development in mei.
Heterostylous Primula forbesii is an important ornamental flower in China because of its long-lasting flowers and winter bloom. This study aimed to develop markers of expressed sequence tag–simple sequence repeats (EST-SSRs) that are associated with heterostyly and that can be used for molecular-assisted selective breeding in P. forbesii. We investigated 114,474 unigenes and identified 25,095 SSRs in P. forbesii. Dinucleotide repeats (46.14%), mononucleotide repeats (44.65%), and trinucleotide repeats (8.27%) were the most abundant SSRs. Among the 25,095 SSRs, 10,645 SSR primer pairs were successfully designed, of which 130 primer pairs were randomly selected for further amplification validation using eight accessions of P. forbesii; 98 pairs produced clear and stable polymerase chain reaction (PCR) products, and 28 pairs showed polymorphism. Bulked segregant analysis (BSA) was conducted for the F1 population with respect to thrum style and pin style by scanning 28 polymorphic SSR primer combinations. One SSR marker, c64326, linked to the heterostyly trait at a genetic distance of ≈3.70 cM was identified. The marker c64326 was further validated in two populations with an accuracy of 97.92% and 90.63%. The novel and linked EST-SSR markers can be valuable resources for genetic diversity analysis, mapping, and marker-assisted breeding in P. forbesii.