The objective of this study was to increase understanding about genetic mechanisms affecting calyx persistence in Korla fragrant pear (Pyrus brestschneideri Rehd). Flowers were collected at early bloom, full bloom, and late bloom. The RNA was extracted from the flowers and then combined according to calyx type. Transcriptome and digital gene expression (DGE) profiles of flowers, ovaries, and sepals with persistent calyx (SC_hua, SC_ep, and SC_zf, respectively) were compared with those of flowers, ovaries, and sepals with deciduous calyx (TL_hua, TL_ep, and TL_zf, respectively). Temporal changes in the expression of selected genes in floral organs with either persistent or deciduous calyx were compared using real-time quantitative PCR (qRT-PCR). Comparison of the transcriptome sequences for SC_hua and TL_hua indicated 26 differentially expressed genes (DEGs) with known relationship to abscission and 10 DEGs with unknown function. We identified 98 MYB and 21 SPL genes from the assembled unigenes. From SC_zf vs TL_zf, we identified 21 DEGs with known relationship to abscission and 18 DEGs with unknown function. From SC_ep vs TL_ep, 12 DEGs with known relationship to abscission were identified along with 11 DEGs with unknown function. Ten DEGs were identified by both transcriptome sequencing and DGE sequencing. More than 50 DEGs were observed that were related to calyx persistence in Korla fragrant pear. Some of the genes were related to cell wall degradation, plant hormone signal transduction, and stress response. Other DEGs were identified as zinc finger protein genes and lipid transfer protein genes. Further analysis showed that calyx persistence in Korla fragment pear was a metabolic process regulated by many genes related to cell wall degradation and plant hormones.

Fifty-two GRAS genes are identified in walnut genome. Based on the evolutionary relationship and motif analysis, the walnut GRAS gene family was divided into eight subfamilies, and the sequence features analysis of JrGRAS proteins showed that the JrGRAS protein sequences were both conserved and altered during the evolutionary process. Gene duplication analysis indicated that seven GRAS genes in walnut have orthologous genes in other species, and five of them occurred duplicated events in walnut genome. Expression pattern analysis of the GRAS family genes in walnut showed that two JrGRAS genes (JrCIGRa-b and JrSCL28a) were differentially expressed between flower bud and leaf bud (p < 0.01), and two JrGRAS genes (JrCIGRa-b and JrSCL13b-d) were differentially expressed between the different development stages of flower buds transition (p < 0.01), besides, three hub genes (JrGAIa, JrSCL3f and JrSHRc) were identified by co-expression analysis, which suggested these GRAS genes may play an important role in regulating the development of apical meristem in walnut. This study laid a foundation for further understanding of the function of GRAS family genes in walnut.

Using paraffin sections, the stages of walnut female flower bud differentiation were divided into the predifferentiation period (F_1), initial differentiation period (F_2) and flower primordium differentiation period (F_3). Leaf buds collected at the same stage as F_2 were designated JRL. Transcriptomic profiling was performed, and a total of 132,154 unigenes were obtained with lengths ranging from 201 bp to 16,831 bp. The analysis of differentially expressed genes (DEGs) showed that there were 597, 784 and 532 DEGs in the three combinations F_1vsF_2, F_1vsF_3, and F_2vsF_3, respectively. The comparison F_2vsJRL showed that 374 DEGs were differentially expressed between female buds and leaf buds. Thirty-one DEGs related to flowering time were further used to construct coexpression networks, and CRY2 and NF-YA were identified as core DEGs in flowering time regulation. Eighteen DEGs related to flowering time were subjected to real-time quantitative analysis. Our work provides a foundation for further research on the walnut floral transition and provides new resources for future research on walnut biology and biotechnology.

The eukaryotic transcription factor NF-Y (which consists of NF-YA, NF-YB and NF-YC subunits) is involved in many important plant development processes. There are many reports about the NF-Y family in Arabidopsis and other plant species. However, there are no reports about the NF-Y family in walnut (Juglans regia L.). Thirty-three walnut NF-Y genes (JrNF-Ys) were identified and mapped on the walnut genome. The JrNF-Y gene family consisted of 17 NF-YA genes, 9 NF-YB genes, and 7 NF-YC genes. The structural features of the JrNF-Y genes were investigated by comparing their evolutionary relationship and motif distributions. The comparisons indicated the NF-Y gene structure was both conserved and altered during evolution. Functional prediction and protein interaction analysis were performed by comparing the JrNF-Y protein structure with that in Arabidopsis. Two differentially expressed JrNF-Y genes were identified. Their expression was compared with that of three JrCOs and two JrFTs using quantitative real-time PCR (qPCR). The results revealed that the expression of JrCO2 was positively correlated with the expression of JrNF-YA11 and JrNF-YA12. In contrast, JrNF-CO1 and JrNF-YA12 were negatively correlated. Thirty-three JrNF-Ys were identified and their evolutionary, structure, biological function and expression pattern were analyzed. Two of the JrNF-Ys were screened out, their expression was differentially expressed in different development periods of female flower buds, and in different tissues (female flower buds and leaf buds). Based on prediction and experimental data, JrNF-Ys may be involved in flowering regulation by co-regulate the expression of flowering genes with other transcription factors (TFs). The results of this study may make contribution to the further investigation of JrNF-Y family.