Polyploidy underlies co-option and diversification of biosynthetic triterpene pathways in the apple tribe.

Whole-genome duplication (WGD) plays important roles in plant evolution and function, yet little is known about how WGD underlies metabolic diversification of natural products that bear significant medicinal properties, especially in nonmodel trees. Here, we reveal how WGD laid the foundation for co-option and differentiation of medicinally important ursane triterpene pathway duplicates, generating distinct chemotypes between species and between developmental stages in the apple tribe. After generating chromosome-level assemblies of a widely cultivated loquat variety and Gillenia trifoliata, we define differentially evolved, duplicated gene pathways and date the WGD in the apple tribe at 13.5 to 27.1 Mya, much more recent than previously thought. We then functionally characterize contrasting metabolic pathways responsible for major triterpene biosynthesis in G. trifoliata and loquat, which pre- and postdate the Maleae WGD, respectively. Our work mechanistically details the metabolic diversity that arose post-WGD and provides insights into the genomic basis of medicinal properties of loquat, which has been used in both traditional and modern medicines.

Genome-Wide Analysis of SAUR Gene Family Identifies a Candidate Associated with Fruit Size in Loquat (Eriobotrya japonica Lindl.).

Fruit size is an important fruit quality trait that influences the production and commodity values of loquats (Eriobotrya japonica Lindl.). The Small Auxin Upregulated RNA (SAUR) gene family has proven to play a vital role in the fruit development of many plant species. However, it has not been comprehensively studied in a genome-wide manner in loquats, and its role in regulating fruit size remains unknown. In this study, we identified 95 EjSAUR genes in the loquat genome. Tandem duplication and segmental duplication contributed to the expansion of this gene family in loquats. Phylogenetic analysis grouped the SAURs from Arabidopsis, rice, and loquat into nine clusters. By analyzing the transcriptome profiles in different tissues and at different fruit developmental stages and comparing two sister lines with contrasting fruit sizes, as well as by functional predictions, a candidate gene (EjSAUR22) highly expressed in expanding fruits was selected for further functional investigation. A combination of Indoleacetic acid (IAA) treatment and virus-induced gene silencing revealed that EjSAUR22 was not only responsive to auxin, but also played a role in regulating cell size and fruit expansion. The findings from our study provide a solid foundation for understanding the molecular mechanisms controlling fruit size in loquats, and also provide potential targets for manipulation of fruit size to accelerate loquat breeding.

Method for fast staining and obtaining high-magnification and high-resolution cell images of Nicotiana benthamiana.

As tools of plant molecular biology, fluorescence microscopy and Nicotiana benthamiana have been used frequently to study the structure and function of plant cells. However, it is difficult to obtain ideal micrographs; for example, the images are typically unclear, the inner cell structure cannot be observed under a high-power lens by fluorescence microscopy, etc. Here, we describe a method for observing the cell structure of N. benthamiana. This method significantly improves imaging by fluorescence microscopy and allows clear images to be obtained under a high-power lens. This method is easy to perform with good stability, and the stomatal structure, nucleus, nucleolus, chloroplast and other organelles in N. benthamiana cells as well as protein localizations and the locations of protein-protein interactions have been observed clearly. Furthermore, compared with traditional methods, fluorescent dye more efficiently dyes cells with this method. The applicability of this method was verified by performing confocal scanning laser microscopy (CSLM), and CSLM imaging was greatly improved. Thus, our results provided a method to visualize the subcellular structures of live cells in the leaves of N. benthamiana by greatly improving imaging under a fluorescence microscope and provided new insights and references for the study of cell structures and functions in other plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00931-5.

Functional characterization of GI and CO homologs from Eriobotrya deflexa Nakai forma koshunensis.

KEY MESSAGE: The first report of the cloning and characterization of the flowering time-regulating genes GI and CO homologs from loquat. Flowering time is critical for successful reproduction in plants. In fruit trees, it can also influence the fruit yield and quality. In the previous work, we cloned the important florigen one EdFT and two EdFDs from wild loquat (Eriobotrya deflexa Nakai forma koshunensis); however, the upstream transcription factors are still unknown. The photoperiod pathway genes GIGANTEA (GI) and CONSTANS (CO) have been reported to mainly regulate FT expression in model plants. In this work, we first cloned photoperiod pathway orthologs EdGI and EdCO from E. deflexa Nakai f. koshunensis. Phylogenetic analysis showed they are highly conserved to those from Arabidopsis. They are mainly expressed in the leaves. The EdGI and EdCO were localized in the nucleus. Their expression showed in photoperiodic regulation, while the EdCO transcripts reached the peak at different periods from that of CO in Arabidopsis. Moreover, EdCO significantly activated the EdFT promoter activity. In the transgenic Arabidopsis, downstream-flowering genes like FT and AP1 were obviously upregulated, and consequently resulted in early-flowering phenotype compared to the wild type. These data revealed that the EdGI and EdCO may play a similar role as GI and CO in Arabidopsis, and regulate flower initiation in loquat.

The Role of EjSVPs in Flower Initiation in Eriobotrya japonica.

Flowering plants have evolved different flowering habits to sustain long-term reproduction. Most woody trees experience dormancy and then bloom in the warm spring, but loquat blooms in the cold autumn and winter. To explore its mechanism of flowering regulation, we cloned two SHORT VEGETATIVE PHASE (SVP) homologous genes from 'Jiefanzhong' loquat (Eriobotrya japonica Lindl.), namely, EjSVP1 and EjSVP2. Sequence analysis revealed that the EjSVPs were typical MADS-box transcription factors and exhibited a close genetic relationship with other plant SVP/DORMANCY-ASSOCIATED MADS-BOX (DAM) proteins. The temporal and spatial expression patterns showed that EjSVP1 and EjSVP2 were mainly expressed in the shoot apical meristem (SAM) after the initiation of flowering; after reaching their highest level, they gradually decreased with the development of the flower until they could not be detected. EjSVP1 expression levels were relatively high in young tissues, and EjSVP2 expression levels were relatively high in young to mature transformed tissues. Interestingly, EjSVP2 showed relatively high expression levels in various flower tissues. We analyzed the EjSVP promoter regions and found that they did not contain the C-repeat/dehydration-responsive element. Finally, we overexpressed the EjSVPs in wild-type Arabidopsis thaliana Col-0 and found no significant changes in the number of rosette leaves of Arabidopsis thaliana; however, overexpression of EjSVP2 affected the formation of Arabidopsis thaliana flower organs. In conclusion, EjSVPs were found to play an active role in the development of loquat flowering. These findings may provide a reference for exploring the regulation mechanisms of loquat flowering and the dormancy mechanisms of other plants.

The Role of EjSPL3, EjSPL4, EjSPL5, and EjSPL9 in Regulating Flowering in Loquat (Eriobotrya japonica Lindl.).

The age pathway is important for regulating flower bud initiation in flowering plants. The major regulators in this pathway are miR156 and SPL transcription factors. To date, SPL genes have been identified in many species of plants. Loquat, as a woody fruit tree of Rosaceae, is unique in flowering time as it blooms in winter. However, the study of its SPL homologous genes on the regulation mechanism of flowering time is still limited. In this study, four SPL homologs-EjSPL3, EjSPL4, EjSPL5, and EjSPL9-are cloned from loquat, and phylogenetic analysis showed that they share a high sequence similarity with the homologues from other plants, including a highly conserved SQUAMOSA promoter binding protein (SBP)-box domain. EjSPL3, EjSPL4, EjSPL5 are localized in the cytoplasm and nucleus, and EjSPL9 is localized only in the nucleus. EjSPL4, EjSPL5, and EjSPL9 can significantly activate the promoters of EjSOC1-1, EjLFY-1, and EjAP1-1; overexpression of EjSPL3, EjSPL4, EjSPL5, and EjSPL9 in wild-type Arabidopsis thaliana can promote flowering obviously, and downstream flowering genes expression were upregulated. Our work indicated that the EjSPL3, EjSPL4, EjSPL5, and EjSPL9 transcription factors are speculated to likely participate in flower bud differentiation and other developmental processes in loquat. These findings are helpful to analyze the flowering regulation mechanism of loquat and provide reference for the study of the flowering mechanism of other woody fruit trees.

Footprints of domestication revealed by RAD-tag resequencing in loquat: SNP data reveals a non-significant domestication bottleneck and a single domestication event.

BACKGROUND: The process of crop domestication has long been a major area of research to gain insights into the history of human civilization and to understand the process of evolution. Loquat (Eriobotrya japonica Lindl.) is one of the typical subtropical fruit trees, which was domesticated in China at least 2000 years ago. In the present study, we re-sequenced the genome of nine wild loquat accessions collected from wide geographical range and 10 representative cultivated loquat cultivars by using RAD-tag tacit to exploit the molecular footprints of domestication. RESULTS: We obtained 26.4 Gb clean sequencing data from 19 loquat accessions, with an average of 32.64 M reads per genotype. We identified more than 80,000 SNPs distributed throughout the loquat genome. The SNP density and numbers were slightly higher in the wild loquat populations than that in the cultivated populations. All cultivars were clustered together by structure, phylogenetic and PCA analyses. CONCLUSION: The modern loquat cultivars have experienced a non-significant genetic bottleneck during domestication, and originated from a single domesticated event. Moreover, our study revealed that Hubei province of China is probably the origin center of cultivated loquat.

Characterization of the third SERK gene in pineapple (Ananas comosus) and analysis of its expression and autophosphorylation activity in vitro.

Two somatic embryogenesis receptor-like kinase genes (identified as AcSERK1 and AcSERK2) have previously been characterized from pineapple (Ananas comosus). In this work, we describe the characterization of a third gene (AcSERK3) in this family. AcSERK3 had all the characteristic domains and shared extensive sequence homology with other plant SERKs. AcSERK3 expression was studied by in situ hybridization and quantitative real-time PCR to analyze its function. Intense in situ hybridization signals were observed only in single competent cells and competent cell clusters; no hybridization signal was detected in the subsequent stages of somatic embryogenesis. AcSERK3 was highly expressed in embryogenic callus compared to other organs, e.g., 20-80 fold more than in anther but similar to that of non-embryogenic callus, which was 20-50 fold that of anther. AcSERK3 expression in root was 80 fold higher than in anther and the highest amongst all organs tested. These results indicate that AcSERK3 plays an important role in callus proliferation and root development. His-tagged AcSERK3 protein was successfully expressed and the luminescence of His6-AcSERK3 protein was only ∼5% of that of inactivated AcSERK3 protein and reaction buffer without protein, and 11.3% of that of an extract of host Escherichia coli pET-30a. This finding confirmed that the AcSERK3 fusion protein had autophosphorylation activity.

Integration of genomics, transcriptomics and metabolomics identifies candidate loci underlying fruit weight in loquat.

Fruit weight is an integral part of fruit-quality traits and directly influences commodity values and economic returns of fruit crops. Despite its importance, the molecular mechanisms underlying fruit weight remain understudied, especially for perennial fruit tree crops such as cultivated loquat (Eriobotrya japonica Lindl.). Auxin is known to regulate fruit development, whereas its role and metabolism in fruit development remain obscure in loquat. In this study, we applied a multi-omics approach, integrating whole-genome resequencing-based quantitative trait locus (QTL) mapping with an F1 population, population genomics analysis using germplasm accessions, transcriptome analysis, and metabolic profiling to identify the genomic regions potentially associated with fruit weight in loquat. We identified three major loci associated with fruit weight, supported by both QTL mapping and comparative genomic analysis between small- and big-fruited loquat cultivars. Comparison between two genotypes with contrasting fruit weight performance through transcriptomic and metabolic profiling revealed an important role of auxin in regulating fruit development, especially at the fruit enlarging stage. The multi-omics approach identified two homologs of ETHYLENE INSENSITIVE 4 (EjEIN4) and TORNADO 1 (EjTRN1) as promising candidates controlling fruit weight. Moreover, three single nucleotide polymorphism (SNP) markers were closely associated with fruit weight. Results from this study provided insights from multiple perspectives into the genetic and metabolic controls of fruit weight in loquat. The candidate genomic regions, genes, and sequence variants will facilitate understanding the molecular basis of fruit weight and lay a foundation for future breeding and manipulation of fruit weight in loquat.

EjBZR1 represses fruit enlargement by binding to the EjCYP90 promoter in loquat.

Loquat (Eriobotrya japonica) is a subtropical tree that bears fruit that ripens during late spring. Fruit size is one of the dominant factors inhibiting the large-scale production of this fruit crop. To date, little is known about fruit size regulation. In this study, we first discovered that cell size is more important to fruit size than cell number in loquat and that the expression of the EjBZR1 gene is negatively correlated with cell and fruit size. Virus-induced gene silencing (VIGS) of EjBZR1 led to larger cells and fruits in loquat, while its overexpression reduced cell and plant size in Arabidopsis. Moreover, both the suppression and overexpression of EjBZR1 inhibited the expression of brassinosteroid (BR) biosynthesis genes, especially that of EjCYP90A. Further experiments indicated that EjCYP90A, a cytochrome P450 gene, is a fruit growth activator, while EjBZR1 binds to the BRRE (CGTGTG) motif of the EjCYP90A promoter to repress its expression and fruit cell enlargement. Overall, our results demonstrate a possible pathway by which EjBZR1 directly targets EjCYP90A and thereby affects BR biosynthesis, which influences cell expansion and, consequently, fruit size. These findings help to elucidate the molecular functions of BZR1 in fruit growth and thus highlight a useful genetic improvement that can lead to increased crop yields by repressing gene expression.

Gibberellin Induced Transcriptome Profiles Reveal Gene Regulation of Loquat Flowering.

Flowering is an integral part of the life cycle of flowering plants, which is essential for plant survival and crop production. Most woody fruit trees such as apples and pears bloom in spring, but loquat blooms in autumn and winter. Gibberellin (GA) plays a key role in the regulation of plant flower formation. In this study, we sprayed loquat plants with exogenous GA3, which resulted in vigorous vegetative growth rather than floral bud formation. We then performed a comprehensive RNA-seq analysis on GA3-treated and control-treated leaves and buds over three time periods to observe the effects of exogenous GA3 application on floral initiation and development. The results showed that 111 differentially expressed genes (DEGs) and 563 DEGs were down-regulated, and 151 DEGs and 506 DEGs were up-regulated in buds and leaves, respectively, upon treatment with GA3. Among those that are homologs of the DELLA-mediated GA signal pathway genes, some may be involved in the positive regulation of flower development, including EjWRKY75, EjFT, EjSOC1, EjAGL24, EjSPL, EjLFY, EjFUL, and EjAP1; while some may be involved in the negative regulation of flower development, including EjDELLA, EjMYC3, EjWRKY12, and EjWRKY13. Finally, by analyzing the co-expression of DEGs and key floral genes EjSOC1s, EjLFYs, EjFULs, EjAP1s, 330 candidate genes that may be involved in the regulation of loquat flowering were screened. These genes belong to 74 gene families, including Cyclin_C, Histone, Kinesin, Lipase_GDSL, MYB, P450, Pkinase, Tubulin, and ZF-HD_dimer gene families. These findings provide new insights into the regulation mechanism of loquat flowering.

Identification of key taste components in loquat using widely targeted metabolomics.

Loquats can be divided into white- and yellow-fleshed cultivars. Generally, white-fleshed cultivars taste better than yellow-fleshed cultivars. Currently, metabolic causes of differences in taste are unknown, due to the lack of a large-scale and comprehensive investigation of metabolites in loquat fruit. Here, we performed a LC-MS/MS-based widely targeted metabolome analysis on two cultivars, 'Baiyu' (white-fleshed) and 'ZaozhongNo. 6' (yellow-fleshed). A total of 536 metabolites were identified, 193 of which (including 7 carbohydrates, 12 organic acids and 8 amino acids) were different between the cultivars. Pathway enrichment analysis also identified significant differences in phenolic pathways between the cultivars. Our results suggest that taste differences between the cultivars can be explained by variations in composition and abundance of carbohydrates, organic acids, amino acids, and phenolics. This study provides new insights into the underlying metabolic causes of taste variation in loquat.

EjTFL1 Genes Promote Growth but Inhibit Flower Bud Differentiation in Loquat.

TERMINAL FLOWER1 (TFL1), a key factor belonging to the phosphatidyl ethanolamine-binding protein (PEBP) family, controls flowering time and inflorescence architecture in some plants. However, the role of TFL1 in loquat remains unknown. In this study, we cloned two TFL1-like genes (EjTFL1-1 and EjTFL1-2) with conserved deduced amino acid sequences from cultivated loquat (Eriobotrya japonica Lindl.). First, we determined that flower bud differentiation occurs at the end of June and early July, and then comprehensively analyzed the temporal and spatial expression patterns of these EjTFL1s during loquat growth and development. We observed the contrasting expression trends for EjTFL1s and EjAP1s (APETALA 1) in shoot apices, and EjTFL1s were mainly expressed in young tissues. In addition, short-day and exogenous GA3 treatments promoted the expression of EjTFL1s, and no flower bud differentiation was observed after these treatments in loquat. Moreover, EjTFL1s were localized to the cytoplasm and nucleus, and both interacted with another flowering transcription factor, EjFD, in the nucleus, and EjTFL1s-EjFD complex significantly repressed the promoter activity of EjAP1-1. The two EjTFL1s were overexpressed in wild-type Arabidopsis thaliana Col-0, which delayed flowering time, promoted stem elongation, increased the number of branches, and also affected flower and silique phenotypes. In conclusion, our results suggested that EjTFL1-1 and EjTFL1-2 do not show the same pattern of expression whereas both are able of inhibiting flower bud differentiation and promoting vegetative growth in loquat by integrating GA3 and photoperiod signals. These findings provide useful clues for analyzing the flowering regulatory network of loquat and provide meaningful references for flowering regulation research of other woody fruit trees.

Transcriptional analysis for the difference in carotenoids accumulation in flesh and peel of white-fleshed loquat fruit.

Loquat (Eriobotrya japonica Lindl.) is divided into yellow- and white-fleshed based on the difference in fruit color, and the variations in carotenoids accumulation are considered as the main reasons for this difference. Using RNA-seq technology, a transcriptome analysis was carried out on the flesh and peel of 'Baiyu' fruit during four different fruit development stages. A total of 172.53 Gb clean reads with an average of 6.33 Gb reads were detected for each library, and the percentage of Q30 was higher than 90.84%. We identified 16 carotenogenic and 13 plastid-lipid-associated protein (PAP) genes through RNA-seq. Of these, five carotenogenic and four PAP related genes exhibited remarkable differences in the expression patterns. Carotenoids biosynthetic genes, including DXS, PSY1 and VDE displayed higher expression levels in peel than that in the flesh. However, carotenoids decomposition gene, such as NCDE1, exhibited higher expression in flesh than that in the peel. Notably, all differentially expressed PAP genes showed higher expression levels in peel than flesh. We inferred that the differential accumulation of carotenoids in flesh and peel of 'Baiyu' is caused by the up- or down-regulation of the carotenogenic and PAP related genes. The functional analysis of these important genes will provide valuable information about underlying molecular mechanism of carotenoids accumulation in loquat.

The Role of EjSOC1s in Flower Initiation in Eriobotrya japonica.

The MADS-box transcription factor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) integrates environmental and endogenous signals to promote flowering in Arabidopsis. However, the role of SOC1 homologs in regulating flowering time in fruit trees remains unclear. To better understand the molecular mechanism of flowering regulation in loquat (Eriobotrya japonica Lindl.), two SOC1 homologs (EjSOC1-1 and EjSOC1-2) were identified and characterized in this work. Sequence analysis showed that EjSOC1-1 and EjSOC1-2 have conserved MADS-box and K-box domains. EjSOC1-1 and EjSOC1-2 were clearly expressed in vegetative organs, and high expression was detected in flower buds. As observed in paraffin-embedded sections, expression of the downstream flowering genes EjAP1s and EjLFYs started to increase at the end of June, a time when flower bud differentiation occurs. Additionally, high expression of EjSOC1-1 and EjSOC1-2 began 10 days earlier than that of EjAP1s and EjLFYs in shoot apical meristem (SAM). EjSOC1-1 and EjSOC1-2 were inhibited by short-day (SD) conditions and exogenous GA3, and flower bud differentiation did not occur after these treatments. EjSOC1-1 and EjSOC1-2 were found to be localized to the nucleus. Moreover, ectopic overexpression of EjSOC1-1 and EjSOC1-2 in wild-type Arabidopsis promoted early flowering, and overexpression of both was able to rescue the late flowering phenotype of the soc1-2 mutant. In conclusion, the results suggest that cultivated loquat flower bud differentiation in southern China begins in late June to early July and that EjSOC1-1 and EjSOC1-2 participate in the induction of flower initiation. These findings provide new insight into the artificial regulation of flowering time in fruit trees.

Selection of the optimal reference genes for expression analyses in different materials of Eriobotrya japonica.

BACKGROUND: Loquat (Eriobotrya japonica) is a subtropical tree bearing fruit that ripens during late spring and early summer, which is the off-season for fruit production. The specific flowering habit of loquat, which starts in fall and ends in winter, has attracted an increasing number of researchers who believe that it may represent an ideal model for studying flowering shift adaptations to climate change in Rosaceae. These studies require an understanding of gene expression patterns within the fruit and other tissues of this plant. Although ACTINs (ACTs) have previously been used as reference genes (RGs) for gene expression studies in loquats, a comprehensive analysis of whether these RGs are optimal for normalizing RT-qPCR data has not been performed. RESULTS: In this study, 11 candidate RGs (RIBOSOMAL-LIKE PROTEIN4 (RPL4), RIBOSOMAL-LIKE PROTEIN18 (RPL18), Histone H3.3 (HIS3), Alpha-tubulin-3 (TUA3), S-Adenosyl Methionine Decarboxylase (SAMDC), TIP41-like Family Protein (TIP41), (UDP)-glucose Pyrophosphorylase (UGPase), 18S ribosomal RNA (18S), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Plasma Intrinsic Protein 2 (PIP2) and ACTIN(ACT)) were assessed to determine their expression stability in 23 samples from different tissues or organs of loquat. Integrated expression stability evaluations using five computational statistical methods (GeNorm, NormFinder, ΔCt, BestKeeper, and RefFinder) suggested that a RG set, including RPL4, RPL18, HIS3 and TUA3, was the most stable one across all of the tested loquat samples. The expression pattern of EjCDKB1;2 in the tested loquat tissues normalized to the selected RG set demonstrated its reliability. CONCLUSIONS: This study reveals the reliable RGs for accurate normalization of gene expression in loquat. In addition, our findings demonstrate an efficient system for identifying the most effective RGs for different organs, which may be applied to related rosaceous crops.

Effects of trimethylamine oxide (TMAO) and loading duration on the shoot tip cryopreservation of loquat (Eriobotrya japonica).

Two cryoprotectant solutions, including trimethylamine oxide (TMAO) and dimethyl sulfoxide (DMSO), and several loading durations were used to evaluate the cryopreservation of the shoot tip of Eriobotrya plants. The best results for regrowth (59.91%) were obtained from 10% TMAO compared to 10% DMSO as cryoprotectant, although nonsignificant differences were found for survival between the two cryoprotectants. We detected pronounced effects of loading duration on survival and regrowth rates of shoot tips. The maximum regrowth (56.36%) was observed at 9 h of loading duration. The cryoprotectants and loading durations greatly affected the regrowth of Eriobotrya shoot tips, and TMAO could be introduced as a nontoxic and efficient cryoprotectant. These results could lay a foundation for the cryopreservation of Eriobotrya.

Over-expression of EjLFY-1 Leads to an Early Flowering Habit in Strawberry (Fragaria × ananassa) and Its Asexual Progeny.

As a master regulator involved in flower development, LEAFY-like gene has been demonstrated to play a key role in the flowering process regulation of angiosperms. Expression analysis of EjLFY-1, a LEAFY (LFY) homolog of loquat (Eriobotrya japonica Lindl.), indicated its participation in the regulation of flowering in loquat. To verify its function and potential value in the genetic engineering to shorten the juvenile phase, ectopic expression of EjLFY-1 in strawberry (Fragaria × ananassa) was achieved using Agrobacterium-mediated gene transfer of a plant expression vector with the loquat EjLFY-1 gene driven by the CaMV 35S promoter. Totally 59 plantlets were verified to be the transformants. The presence, expression and integration of EjLFY-1 in the transformants were assessed by PCR, quantitative real-time PCR and Southern blot, respectively. Constitutive expression of EjLFY-1 in strawberry accelerated the flowering process in strawberry with the shorten necessary period for flowering induction, development of flower and fruit set. While vegetative growth habits of the transformants in the first cropping season were consistent with the WT ones. Meanwhile, both the flowers and fruits of the transformants were also as same as those of the WT ones. Furthermore, the early-flowering habit was maintained in their asexual progeny, the runner plants. While with continuous asexual propagation, the clones showed a more strengthen early-flowering phenotype, such as the reduced vegetative growth and the abnormal floral organs in individual plantlets. These results demonstrated the function of this gene and at the same time provided us new insights into the utilization potential of such genes in the genetic engineering of perennial fruits.

Genetic relationships among Eriobotrya species revealed by genome-wide RAD sequence data.

Restriction site-associated DNA sequencing (RAD-seq) was used to illuminate the genetic relationships among Eriobotrya species. The raw data were filtered, and 221 million clean reads were used for further analysis. A total of 1,983,332 SNPs were obtained from 23 Eriobotrya species and two relative genera. We obtained similar results by neighbor-joining and maximum likelihood phylogenetic trees. All Eriobotrya plants grouped together into a big clade, and two out-groups clustered together into a single or separate clade. Chinese and Vietnam accessions were distributed throughout the dendrogram. There was nonsignificant correlation between genotype and geographical distance. However, clustering results were correlated with leaf size to some extent. The Eriobotrya species could be divided into following three groups based on leaf size and phylogenetic analysis: group A and group B comprised of small leaves with <10 cm length except E. stipularis (16.76 cm), and group C can be further divided into two subgroups, which contained medium-size leaves with a leaf length ranged from 10 to 20 cm and a leaf length bigger than 20 cm.

Molecular Characterization of FT and FD Homologs from Eriobotrya deflexa Nakai forma koshunensis.

In angiosperms, regulation of flowering is a vital process for successful reproduction. To date, the molecular mechanism of flowering is well-studied in the model plant, Arabidopsis, in which key genes such as FLOWERING LOCUST (FT) or FD have been identified to regulate flowering. However, the flowering mechanisms are still largely unknown in fruit trees like loquat. To this end, we first cloned one FT- and two FD-like genes from the loquat (Eriobotrya deflexa Nakai f. koshunensis) and referred to as EdFT, EdFD1, and EdFD2, respectively. Phylogenetic analysis has shown that EdFT, EdFD1, and EdFD2 are conserved during the evolution process. EdFT is mainly expressed in reproductive tissues (e.g., flower buds, flowers, and fruits), while EdFD1 and EdFD2 are mainly expressed in apical buds including leaf buds and flower buds. EdFT is localized in the whole cell, while EdFD1 or EdFD2 is localized in the nucleus. Ectopic expression of EdFT, EdFD1, and EdFD2 in Arabidopsis results in early flowering. In addition, we have also revealed that the EdFT interacts with both EdFD1 and EdFD2. Overall, these data suggest that the EdFT, EdFD1, and EdFD2 are the functional homologs of FT and FD, respectively, which might act together to regulate loquat flowering through a similar mechanism found in Arabidopsis.