Comparative genomic and phylogenetic analyses of mitochondrial genomes of hawthorn (Crataegus spp.) in Northeast China.

Hawthorn (Crataegus spp.) plants are major sources of health food and medicines. Twenty species and seven variations of Crataegus are present in China. A variety of unique Crataegus species was found in their natural distribution in northeast China. In the present study, we assembled and annotated the mitochondrial genomes of five Crataegus species from northeastern China. The sizes of the newly sequenced mitochondrial genomes ranged from 245,907 bp to 410,837 bp. A total of 45-55 genes, including 12-19 transfer RNA genes, three ribosomal RNA genes, and 29-33 protein-coding genes (PCGs) were encoded by these mitochondrial genomes. Seven divergent hotspot regions were identified by comparative analyses: atp6, nad3, ccmFN, matR, nad1, nad5, and rps1. The most conserved genes among the Crataegus species, according to the whole-genome correlation analysis, were nad1, matR, nad5, ccmFN, cox1, nad4, trnQ-TTG, trnK-TTT, trnE-TTC, and trnM-CAT. Horizontal gene transfer between organellar genomes was common in Crataegus plants. Based on the phylogenetic trees of mitochondrial PCGs, C. maximowiczii, C. maximowiczii var. ninganensis, and C. bretschneideri shared similar maternal relationships. This study improves Crataegus mitochondrial genome resources and offers important insights into the taxonomy and species identification of this genus.

Genome-Wide Identification, Characterization, and Expression Profile Analysis of CONSTANS-like Genes in Woodland Strawberry (Fragaria vesca).

CONSTANS-like (CO-like) gene is one of the most important regulators in the flowering process of the plant, playing a core role in the photoperiodic flowering induction pathway. In this study, we identified 10 distinct CO-like genes (FveCOs) in woodland strawberry (Fragaria vesca). They were classified into three groups with specific gene structure characteristics or protein domains in each group. The effect of selection pressure on the FveCOs in the woodland strawberry was tested by Ka/Ks, and it was shown that the evolution rate of FveCOs was controlled by purification selection factors. Intraspecific synteny analysis of woodland strawberry FveCOs showed that at least one duplication event existed in the gene family members. Collinearity analysis of woodland strawberry genome with genomes of Arabidopsis, rice (Oryza sativa), and apple (Malus × domestica) showed that CO-like genes of F. vesca and Malus × domestica owned higher similarity for their similar genomes compared with those of other two species. The FveCOs showed different tissue-specific expression patterns. Moreover, real-time quantitative PCR results revealed that the expressions of the most FveCOs followed a 24-h rhythm oscillation under both long-day (LD) and short-day (SD) conditions. Further expression analysis showed that the individual expression changing profile of FveCO3 and FveCO5 was opposite to each other under both LD and SD conditions. Moreover, the expression of FveCO3 and FveCO5 was both negatively correlated with the flowering time variation of the woodland strawberry grown under LD and SD conditions, indicating their potential vital roles in the photoperiodic flowering regulation. Further protein interaction network analysis also showed that most of the candidate interaction proteins of FveCO3 and FveCO5 were predicted to be the flowering regulators. Finally, LUC assay indicated that both FveCO3 and FveCO5 could bind to the promoter of FveFT1, the key regulator of flowering regulation in the woodland strawberry, and thus activate its expression. Taken together, this study laid a foundation for understanding the exact roles of FveCOs in the reproductive development regulation of the woodland strawberry, especially in the photoperiodic flowering process.

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.

The high-quality genome of diploid strawberry (Fragaria nilgerrensis) provides new insights into anthocyanin accumulation.

Fragaria nilgerrensis is a wild diploid strawberry species endemic to east and southeast region in Asia and provides a rich source of genetic variations for strawberry improvement. Here, we present a chromosome-scale assembly of F. nilgerrensis using single-molecule real-time (SMRT) Pacific Biosciences sequencing and chromosome conformation capture (Hi-C) genome scaffolding. The genome assembly size was 270.3 Mb, with a contig N50 of ∼8.5 Mb. A total of 28 780 genes and 117.2 Mb of transposable elements were annotated for this genome. Next, detailed comparative genomics with the high-quality F. vesca reference genome was conducted to obtain the difference among transposable elements, SNPs, Indels, and so on. The genome size of F. nilgerrensis was enhanced by around 50 Mb relatively to F. vesca, which is mainly due to expansion of transposable elements. In comparison with the F. vesca genome, we identified 4 561 825 SNPs, 846 301 Indels, 4243 inversions, 35 498 translocations and 10 099 relocations. We also found a marked expansion of genes involved in phenylpropanoid biosynthesis, starch and sucrose metabolism, cyanoamino acid metabolism, plant-pathogen interaction, brassinosteroid biosynthesis and plant hormone signal transduction in F. nilgerrensis, which may account for its specific phenotypes and considerable environmental adaptability. Interestingly, we found sequence variations in the upstream regulatory region of FnMYB10, a core transcriptional activator of anthocyanin biosynthesis, resulted in the low expression level of the FnMYB10 gene, which is likely responsible for white fruit phenotype of F. nilgerrensis. The high-quality F. nilgerrensis genome will be a valuable resource for biological research and comparative genomics research.

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.

FveRGA1, encoding a DELLA protein, negatively regulates runner production in Fragaria vesca.

MAIN CONCLUSION: FveRGA1 was highly expressed in tender tissues such as young leaves and stem apices and was localized in the nucleus. RNAi silencing of FveRGA1 in non-runnering woodland strawberry produced many runners. FveRGA1 is thus a key gene controlling strawberry runner formation. The propagation of strawberry is mainly based on runners, while the genes controlling runner production have not been well characterized. Exogenous applications of optimum concentration gibberellins (GAs) promote runner formation in strawberry cultivation and GA can accelerate the degradation of DELLA proteins. To investigate whether DELLA proteins are responsible for runner production, we analyzed all the DELLA genes in Fragaria vesca and cloned a DELLA protein-encoding gene FveRGA1 in woodland strawberry using RT-PCR. Subcellular localization analysis indicated that FveRGA1 was localized in the nucleus. A transcription analysis suggested that FveRGA1 was expressed ubiquitously in all examined strawberry organs, especially in young leaves, petioles, and stem apices. RNA interference (RNAi) technology was carried out to investigate the function of FveRGA1 in woodland strawberry 'Yellow Wonder' (YW) and 'Ruegen' (RG) via an Agrobacterium-mediated transformation. Interestingly, the RNAi silencing transgenic plants in the naturally non-runnering YW and RG strains produced many runners, suggesting FveRGA1 as a key gene controlling strawberry runner formation. Our study lays a solid basis for unraveling the detailed molecular mechanism of runner formation in strawberry.

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.

Transcript Quantification by RNA-Seq Reveals Differentially Expressed Genes in the Red and Yellow Fruits of Fragaria vesca.

Fragaria vesca (2n = 2x = 14), the woodland strawberry, is a perennial herbaceous plant with a small sequenced genome (240 Mb). It is commonly used as a genetic model plant for the Fragaria genus and the Rosaceae family. Fruit skin color is one of the most important traits for both the commercial and esthetic value of strawberry. Anthocyanins are the most prominent pigments in strawberry that bring red, pink, white, and yellow hues to the fruits in which they accumulate. In this study, we conducted a de novo assembly of the fruit transcriptome of woodland strawberry and compared the gene expression profiles with yellow (Yellow Wonder, YW) and red (Ruegen, RG) fruits. De novo assembly yielded 75,426 unigenes, 21.3% of which were longer than 1,000 bp. Among the high-quality unique sequences, 45,387 (60.2%) had at least one significant match to an existing gene model. A total of 595 genes, representing 0.79% of total unigenes, were differentially expressed in YW and RG. Among them, 224 genes were up-regulated and 371 genes were down-regulated in the fruit of YW. Particularly, some flavonoid biosynthetic pathway genes, including C4H, CHS, CHI, F3H, DFR and ANS, as well as some transcription factors (TFs), including MYB (putative MYB86 and MYB39), WDR and MADS, were down-regulated in YW fruit, concurrent with a reduction in anthocyanin accumulation in the yellow pigment phenotype, whereas a putative transcription repressor MYB1R was up-regulated in YW fruit. The altered expression levels of the genes encoding flavonoid biosynthetic enzymes and TFs were confirmed by quantitative RT-PCR. Our study provides important insights into the molecular mechanisms underlying the yellow pigment phenotype in F. vesca.

Transcript assembly and quantification by RNA-Seq reveals differentially expressed genes between soft-endocarp and hard-endocarp hawthorns.

Hawthorn (Crataegus spp.) is an important pome with a long history as a fruit, an ornamental, and a source of medicine. Fruits of hawthorn are marked by hard stony endocarps, but a hawthorn germplasm with soft and thin endocarp was found in Liaoning province of China. To elucidate the molecular mechanism underlying the soft endocarp of hawthorn, we conducted a de novo assembly of the fruit transcriptome of Crataegus pinnatifida and compared gene expression profiles between the soft-endocarp and the hard-endocarp hawthorn varieties. De novo assembly yielded 52,673 putative unigenes, 20.4% of which are longer than 1,000 bp. Among the high-quality unique sequences, 35,979 (68.3%) had at least one significant match to an existing gene model. A total of 1,218 genes, represented 2.31% total putative unigenes, were differentially expressed between the soft-endocarp hawthorn and the hard-endocarp hawthorn. Among these differentially expressed genes, a number of lignin biosynthetic pathway genes were down-regulated while almost all the flavonoid biosynthetic pathway genes were strongly up-regulated, concomitant with the formation of soft endocarp. In addition, we have identified some MYB and NAC transcription factors that could potentially control lignin and flavonoid biosynthesis. The altered expression levels of the genes encoding lignin biosynthetic enzymes, MYB and NAC transcription factors were confirmed by quantitative RT-PCR. This is the first transcriptome analysis of Crataegus genus. The high quality ESTs generated in this study will aid future gene cloning from hawthorn. Our study provides important insights into the molecular mechanisms underlying soft endocarp formation in hawthorn.

Deep sequencing discovery of novel and conserved microRNAs in wild type and a white-flesh mutant strawberry.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by base pairing to mRNA target sequences, and play crucial roles in plant development and stress responses. The knowledge on post-transcriptional regulation by miRNAs in strawberry is rather limited so far. In order to understand the role of miRNA in the molecular control during strawberry fruit development, small RNA libraries were constructed from fruits at the turning stage of strawberry cultivar 'Sachinoka' and its white-flesh mutant by using the Solexa platform. One hundred and twenty conserved miRNAs belonging to 27 miRNA families and 33 putative novel strawberry miRNAs were identified in both libraries. Their target genes were predicted using the Fragaria vesca genome. Nine of all miRNAs showed significant expression differences between two types of samples. Four miRNAs were up-regulated and five were down-regulated in white-flesh mutant. The sequencing results were partially validated by quantitative RT-PCR. Among them, the expression of miR399a shows the biggest change between the two samples. The prediction of its target gene showed that miR399 may play an important role in phosphate homeostasis of strawberry fruits. Furthermore, we deduce that the expression of miR399 has negative correlation with the content of sugars.