Selection of Stable Reference Genes for QRT-PCR in Tree Peony 'Doulv' and Functional Analysis of PsCUC3.

(1) Background: Tree peonies display extensive cultivar diversity due to widespread hybridization, resulting in a complex genetic architecture. This complexity complicates the selection of universal reference genes across different cultivars for qRT-PCR analyses. Paeonia suffruticosa 'Doulv', notable for its unique green blooms in China, exhibits chlorosis post-flowering and features petaloid stamens and pistils. (2) Methods: Based on published literature and RNA-seq data from 'Doulv', nine candidate reference genes-ACT (Actin), TUB (ß-Tubulin), UBC (Ubiquitin Conjugating Enzyme), UBQ (Ubiquitin), UPL (Ubiquitin Protein Ligase), PP2A (Protein Phosphatase 2A), PP2C (Protein Phosphatase 2C), MBF1A (Multiprotein Bridging Factor 1A), and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase)-were selected. Their expression stability was assessed across various tissues and developmental stages of 'Doulv' flowers using qRT-PCR, with evaluations conducted via GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0. Gene cloning and expression analyses of PsCUC3, including its subcellular localization, were performed. (3) Results: GAPDH and ACT were identified as the most stable reference genes in petaloid stamens across various developmental stages of 'Doulv', whereas UBC and MBF1A were optimal across different tissues. Notably, specific conserved amino acids in PsCUC3 from 'Doulv' diverged from those in NAM/CUC3 proteins of other species, impacting its protein structure. PsCUC3 expression analysis revealed no correlation with chlorophyll content in petaloid stamens but an association with petaloid organ development. Furthermore, PsCUC3 was predominantly localized in the nucleus. (4) Conclusions: This study comprehensively evaluated suitable reference genes using GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0 software, establishing a robust qRT-PCR detection system for 'Doulv' peony. These results provide a solid experimental foundation for further research on 'Doulv' peony. Building on this experimental foundation, the functional analysis of the PsCUC3 gene was conducted. The findings suggest a potential association between the PsCUC3 gene and floral morphology alterations in 'Doulv', identifying PsCUC3 as crucial for understanding the molecular mechanisms influencing floral structure in tree peonies.

Identification and characterization of miRNAs in two closely related C4 and C3 species of Cleome by high-throughput sequencing.

Cleome gynandra and Cleome hassleriana, which are C4 and C3 plants, respectively, are two species of Cleome. The close genetic relationship between C. gynandra and C. hassleriana provides advantages for discovering the differences in leaf development and physiological processes between C3 and C4 plants. MicroRNAs (miRNAs) are a class of important regulators of various biological processes. In this study, we investigate the differences in the characteristics of miRNAs between C. gynandra and C. hassleriana using high-throughput sequencing technology. In total, 94 and 102 known miRNAs were identified in C. gynandra and C. hassleriana, respectively, of which 3 were specific for C. gynandra and 10 were specific for C. hassleriana. Ninety-one common miRNAs were identified in both species. In addition, 4 novel miRNAs were detected, including three in C. gynandra and three in C. hassleriana. Of these miRNAs, 67 were significantly differentially expressed between these two species and were involved in extensive biological processes, such as glycol-metabolism and photosynthesis. Our study not only provided resources for C. gynandra and C. hassleriana research but also provided useful clues for the understanding of the roles of miRNAs in the alterations of biological processes in leaf tissues during the evolution of the C4 pathway.

The wheat MYB-related transcription factor TaMYB72 promotes flowering in rice.

Through large-scale transformation analyses, TaMYB72 was identified as a flowering time regulator in wheat. TaMYB72 is a MYB family transcription factor localized to the nucleus. Three TaMYB72 homologs, TaMYB72-A, TaMYB72-B and TaMYB72-D, cloned from hexaploid wheat were mapped to the short arm of the group 6 chromosomes. Under the long-day conditions, over-expression of the TaMYB72 in rice shortened the flowering time by approximately 12 d. Expression analyses suggest that TaMYB72 may function through up-regulation of florigen genes Hd3a and RFT1.

Foliar application with nano-silicon alleviates Cd toxicity in rice seedlings.

Nanofertilizers may be more effective than regular fertilizers in improving plant nutrition, enhancing nutrition use efficiency, and protecting plants from environmental stress. A hydroponic pot experiment was conducted to study the role of foliar application with 2.5 mM nano-silicon in alleviating Cd stress in rice seedlings (Oryza sativa L. cv Youyou 128) grown in solution added with or without 20 µM CdCl2. The results showed that Cd treatment decreased the growth and the contents of Mg, Fe, Zn, chlorophyll a, and glutathione (GSH), accompanied by a significant increase in Cd accumulation. However, foliar application with nano-Si improved the growth, Mg, Fe, and Zn nutrition, and the contents of chlorophyll a of the rice seedlings under Cd stress and decreased Cd accumulation and translocation of Cd from root to shoot. Cd treatment produced oxidative stress to rice seedlings indicated by a higher lipid peroxidation level (as malondialdehyde (MDA)) and higher activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and a lower GSH content. However, those nano-Si-treated plants had lower MDA but higher GSH content and different antioxidant enzyme activities, indicating a higher Cd tolerance in them. The results suggested that nano-Si application alleviated Cd toxicity in rice by decreasing Cd accumulation, Cd partitioning in shoot and MDA level and by increasing content of some mineral elements (Mg, Fe, and Zn) and antioxidant capacity.

Rapid evolution and complex structural organization in genomic regions harboring multiple prolamin genes in the polyploid wheat genome.

Genes encoding wheat prolamins belong to complicated multi-gene families in the wheat genome. To understand the structural complexity of storage protein loci, we sequenced and analyzed orthologous regions containing both gliadin and LMW-glutenin genes from the A and B genomes of a tetraploid wheat species, Triticum turgidum ssp. durum. Despite their physical proximity to one another, the gliadin genes and LMW-glutenin genes are organized quite differently. The gliadin genes are found to be more clustered than the LMW-glutenin genes which are separated from each other by much larger distances. The separation of the LMW-glutenin genes is the result of both the insertion of large blocks of repetitive DNA owing to the rapid amplification of retrotransposons and the presence of genetic loci interspersed between them. Sequence comparisons of the orthologous regions reveal that gene movement could be one of the major factors contributing to the violation of microcolinearity between the homoeologous A and B genomes in wheat. The rapid sequence rearrangements and differential insertion of repetitive DNA has caused the gene islands to be not conserved in compared regions. In addition, we demonstrated that the i-type LMW-glutenin originated from a deletion of 33-bps in the 5' coding region of the m-type gene. Our results show that multiple rounds of segmental duplication of prolamin genes have driven the amplification of the omega-gliadin genes in the region; such segmental duplication could greatly increase the repetitive DNA content in the genome depending on the amount of repetitive DNA present in the original duplicate region.

Rhodococcus equi in the soil environment of horses in Inner Mongolia, China.

Little is known about the distribution of Rhodococcus equi in the soil environment of native horses in China. One hundred and eight soil samples were collected from native-horse farms in the Hulun Beier grasslands of eastern Mongolia, the Xilin Goler grasslands of southern Mongolia, and Tongliao City in Inner Mongolia, China. The isolation rates of R. equi from soil samples from the Hulun Beier and Xilin Goler grasslands ranged from 25.9% to 30.0%. In contrast, isolation rates from soil samples from Tongliao City were as high as 82.3% and the mean number of R. equi in soil samples from Tongliao City was 10 times more than those of samples from the grasslands. The 488 isolates were examined using PCR for the presence of genes that encode virulence-associated 15-17 kDa antigen protein (VapA) and the 20 kDa antigen protein (VapB). All isolates were negative for virulence-associated proteins. Plasmid profiles of these avirulent isolates showed that cryptic plasmids of various sizes were present with an incidence of 13.3% to 21.5%. The results of the present study contrast with those of our recent study (J. Vet. Med. Sci. 67:611-613, 2005), in which we reported that R. equi was absent from Mongolian horses in Ulaanbaatar, Mongolia. It is suggested that the difference between the results of these two studies is due to the mobile pasturing system in Mongolia and nonmobile pasturing system in Inner Mongolia.