Comparative Analysis of Alternative Splicing in Two Contrasting Apple Cultivars Defense against Alternaria alternata Apple Pathotype Infection.

Alternaria blotch disease, caused by the Alternaria alternata apple pathotype (A. alternata AP), is one of the most serious fungal diseases in apples. Alternative splicing (AS), one of the pivotal post-transcriptional regulatory mechanisms, plays essential roles in various disease resistance responses. Here, we performed RNA-Seq for two apple cultivars (resistant cultivar 'Jonathan' (J) and susceptible cultivar 'Starking Delicious' (SD)) infected by A. alternata AP to further investigate their AS divergence. In total, 1454, 1780, 1367 and 1698 specifically regulated differential alternative splicing (DAS) events were detected in J36, J72, SD36 and SD72 groups, respectively. Retained intron (RI) was the dominant AS pattern. Conformably, 642, 764, 585 and 742 uniquely regulated differentially spliced genes (DSGs) were found during A. alternata AP infection. Comparative analysis of AS genes in differential splicing and expression levels suggested that only a small proportion of DSGs overlapped with differentially expressed genes (DEGs). Gene ontology (GO) enrichment analysis demonstrated that the DSGs were significantly enriched at multiple levels of gene expression regulation. Briefly, the specific AS was triggered in apple defense against A. alternata AP. Therefore, this study facilitates our understanding on the roles of AS regulation in response to A. alternata AP infection in apples.

Comparative transcriptomics analysis reveals MdGRAS53 contributes to disease resistance against Alternaria blotch of apple.

Alternaria blotch disease, caused by Alternaria alternata apple pathotype (AAAP), is one of the most prevalent diseases in apple production. To identify AAAP resistance-related genes and provide a theoretical basis for Alternaria blotch disease resistance breeding, we used two apple cultivars, 'Jonathan', a variety resistant to AAAP infection, and 'Starking Delicious', a variety susceptible to AAAP infection, as materials to perform transcriptome sequencing of apple leaves 72 h after AAAP infection. A Venn diagram showed that a total of 5229 DEGs of 'Jonathan' and 4326 DEGs of 'Starking Delicious' were identified. GO analysis showed that these DEGs were clustered into 25 GO terms, primarily "metabolic process" and "catalytic activity." Functional classification analyses of the DEGs indicated that "MAPK signaling pathway-plant pathway" is the most significant metabolic pathway among the top 15 KEGG pathways, followed by the "plant hormone signal transduction" pathway. There are more DEGs in 'Jonathan' that are significantly classified GO terms and KEGG pathways than in 'Starking Delicious'. Specifically, 13 DEGs were identified as involved in the GA-GID1-DELLA module, and the expression of MdGRAS53, a homologous gene of DELLA, was significantly upregulated in 'Jonathan' compared with 'Starking Delicious'. Phenotype analysis revealed that exogenous hormone GA3 suppressed apple resistance to AAAP infection and reduced the expression of MdGRAS53. The opposite result was observed for exogenous spraying of paclobutrazol (PAC), an inhibitor of gibberellin synthesis. Overexpression of MdGRAS53 in apple leaves by transient transformation decreased lesion area and the number of spores in leaves infected with AAAP, while silencing MdGRAS53 showed the opposite result. Meanwhile, SA/JA signaling pathway-related genes were upregulated significantly in MdGRAS53-overexpressed leaves and downregulated significantly in MdGRAS53-silenced leaves. The findings suggest that the GA-GID1-DELLA module is involved in apple resistance to AAAP, and MdGRAS53, a DELLA homologous gene, may play a positive role in this resistance by modulating cooperative JA- and SA-dependent pathways.

Identification of candidate genes for leaf scorch in Populus deltoids by the whole genome resequencing analysis.

Leaf scorch exists as a common phenomenon in the development of plant, especially when plants encounter various adversities, which leads to great losses in agricultural production. Both Jinhong poplar (JHP) and Caihong poplar (CHP) (Populus deltoids) are obtained from a bud sport on Zhonghong poplar. Compared with CHP, JHP always exhibits leaf scorch, poor growth, premature leaf discoloration, and even death. In this study, the candidate genes associated with leaf scorch between JHP and CHP were identified by the whole genome resequencing using Illumina HiSeqTM. There were 218,880 polymorphic SNPs and 46,933 indels between JHP and CHP, respectively. Among these, the candidate genes carrying non-synonymous SNPs in coding regions were classified into 6 groups. The expression pattern of these candidate genes was also explored in JHP and CHP among different sampling stages. Combined with the qRT-PCR analysis, the results showed that genes associated with transport of various nutritional elements, senescence and MYB transcription factor might play important roles during the process of leaf scorch in Populus deltoids. Four genes belonging to these three groups carried more than three SNPs in their coding sequence, which might play important roles in leaf scorch. The above results provided candidate genes involved in leaf scorch in Populus deltoids, and made us better understand the molecular regulation mechanism of leaf scorch in Populus deltoids.

The complete chloroplast genome sequence of strawberry (Fragaria × ananassa Duch.) and comparison with related species of Rosaceae.

Compared with other members of the family Rosaceae, the chloroplast genomes of Fragaria species exhibit low variation, and this situation has limited phylogenetic analyses; thus, complete chloroplast genome sequencing of Fragaria species is needed. In this study, we sequenced the complete chloroplast genome of F. × ananassa 'Benihoppe' using the Illumina HiSeq 2500-PE150 platform and then performed a combination of de novo assembly and reference-guided mapping of contigs to generate complete chloroplast genome sequences. The chloroplast genome exhibits a typical quadripartite structure with a pair of inverted repeats (IRs, 25,936 bp) separated by large (LSC, 85,531 bp) and small (SSC, 18,146 bp) single-copy (SC) regions. The length of the F. × ananassa 'Benihoppe' chloroplast genome is 155,549 bp, representing the smallest Fragaria chloroplast genome observed to date. The genome encodes 112 unique genes, comprising 78 protein-coding genes, 30 tRNA genes and four rRNA genes. Comparative analysis of the overall nucleotide sequence identity among ten complete chloroplast genomes confirmed that for both coding and non-coding regions in Rosaceae, SC regions exhibit higher sequence variation than IRs. The Ka/Ks ratio of most genes was less than 1, suggesting that most genes are under purifying selection. Moreover, the mVISTA results also showed a high degree of conservation in genome structure, gene order and gene content in Fragaria, particularly among three octoploid strawberries which were F. × ananassa 'Benihoppe', F. chiloensis (GP33) and F. virginiana (O477). However, when the sequences of the coding and non-coding regions of F. × ananassa 'Benihoppe' were compared in detail with those of F. chiloensis (GP33) and F. virginiana (O477), a number of SNPs and InDels were revealed by MEGA 7. Six non-coding regions (trnK-matK, trnS-trnG, atpF-atpH, trnC-petN, trnT-psbD and trnP-psaJ) with a percentage of variable sites greater than 1% and no less than five parsimony-informative sites were identified and may be useful for phylogenetic analysis of the genus Fragaria.

Transcriptomics Analysis of Apple Leaves in Response to Alternaria alternata Apple Pathotype Infection.

Alternaria blotch disease of apple (Malus × domestica Borkh.), caused by the apple pathotype of Alternaria alternata, is one of the most serious fungal diseases to affect apples. To develop an understanding of how apples respond to A. alternata apple pathotype (AAAP) infection, we examined the host transcript accumulation over the period between 0 and 72 h post AAAP inoculation. Large-scale gene expression analysis was conducted of the compatible interaction between "Starking Delicious" apple cultivar and AAAP using RNA-Seq and digital gene expression (DGE) profiling methods. Our results show that a total of 9080 differentially expressed genes (DEGs) were detected (>two-fold and FDR < 0.001) by RNA-Seq. During the early phase of infection, 12 h post inoculation (HPI), AAAP exhibited limited fungal development and little change in the transcript accumulation status (950 DEGs). During the intermediate phase of infection, the period between 18 and 36 HPI, increased fungal development, active infection, and increased transcript accumulation were detected (4111 and 3838 DEGs detected at each time point, respectively). The majority of DEGs were detected by 72 HPI, suggesting that this is an important time point in the response of apples' AAAP infection. Subsequent gene ontology (GO) and pathway enrichment analyses showed that DEGs are predominately involved in biological processes and metabolic pathways; results showed that almost gene associated with photosynthesis, oxidation-reduction were down-regulated, while transcription factors (i.e., WRKY, MYB, NAC, and Hsf) and DEGs involved in cell wall modification, defense signaling, the synthesis of defense-related metabolites, including pathogenesis-related (PRs) genes and phenylpropanoid/cyanoamino acid /flavonoid biosynthesis, were activated during this process. Our study also suggested that the cell wall defensive vulnerability and the down-regulation of most PRs and HSP70s in "Starking Delicious" following AAAP infection might interpret its susceptible to AAAP.

Characterization and Comparison of the CPK Gene Family in the Apple (Malus × domestica) and Other Rosaceae Species and Its Response to Alternaria alternata Infection.

As one of the Ca2+ sensors, calcium-dependent protein kinase (CPK) plays vital roles in immune and stress signaling, growth and development, and hormone responses, etc. Recently, the whole genome of apple (Malus × domestica), pear (Pyrus communis), peach (Prunus persica), plum (Prunus mume) and strawberry (Fragaria vesca) in Rosaceae family has been fully sequenced. However, little is known about the CPK gene family in these Rosaceae species. In this study, 123 CPK genes were identified from five Rosaceae species, including 37 apple CPKs, 37 pear CPKs, 17 peach CPKs, 16 strawberry CPKs, and 16 plum CPKs. Based on the phylogenetic tree topology and structural characteristics, we divided the CPK gene family into 4 distinct subfamilies: Group I, II, III, and IV. Whole-genome duplication (WGD) or segmental duplication played vital roles in the expansion of the CPK in these Rosaceae species. Most of segmental duplication pairs in peach and plum may have arisen from the γ triplication (~140 million years ago [MYA]), while in apple genome, many duplicated genes may have been derived from a recent WGD (30~45 MYA). Purifying selection also played a critical role in the function evolution of CPK family genes. Expression of apple CPK genes in response to apple pathotype of Alternaria alternata was verified by analysis of quantitative real-time RT-PCR (qPCR). Expression data demonstrated that CPK genes in apple might have evolved independently in different biological contexts. The analysis of evolution history and expression profile laid a foundation for further examining the function and complexity of the CPK gene family in Rosaceae.

Metabolic changes upon flower bud break in Japanese apricot are enhanced by exogenous GA4.

Gibberellin (GA4) has a significant effect on promoting dormancy release in flower buds of Japanese apricot (Prunus mume Sieb. et Zucc). The transcriptomic and proteomic changes that occur after GA4 treatment have been reported previously; however, the metabolic changes brought about by GA4 remain unknown. The present study was undertaken to assess changes in metabolites in response to GA4 treatment, as determined using gas chromatography-mass spectrometry and principal component analysis. Fifty-five metabolites that exhibited more than two-fold differences in abundance (P < 0.05) between samples collected over time after a given treatment or between samples exposed to different treatments were studied further. These metabolites were categorized into six main groups: amino acids and their isoforms (10), amino acid derivatives (7), sugars and polyols (14), organic acids (12), fatty acids (4), and others (8). All of these groups are involved in various metabolic pathways, in particular galactose metabolism, glyoxylate and dicarboxylate metabolism, and starch and sucrose metabolism. These results suggested that energy metabolism is important at the metabolic level in dormancy release following GA4 treatment. We also found that more than 10-fold differences in abundance were observed for many metabolites, including sucrose, proline, linoleic acid, and linolenic acid, which might play important roles during the dormancy process. The current research extends our understanding of the mechanisms involved in budburst and dormancy release in response to GA4 and provides a theoretical basis for applying GA4 to release dormancy.

Self-compatibility in 'Zaohong' Japanese apricot is associated with the loss of function of pollen S genes.

While most Japanese apricot (Prunus mume Sieb. et Zucc.) cultivars display typical S-RNase-based gametophytic self-incompatibility, some self-compatible (SC) cultivars have also been identified. In this study, we confirmed SC of 'Zaohong' through replicated self-pollination tests. Cross-pollination tests showed that SC of 'Zaohong' was caused by a loss of pollen function, so we determined that the S-genotype of 'Zaohong' was S 2 S 15 . Sequence analysis of the S-haplotypes of 'Zaohong' showed no mutations which were likely to alter gene function. Furthermore, expression analysis based on RT-PCR of S-locus genes revealed no differences at the transcript level when compared with 'Xiyeqing', a self-incompatible cultivar with the same S haplotypes. In addition, except for S-locus genes, a new type of F-box gene encoding a previously uncharacterised protein with high sequence similarity (61.03-64.65 %) to Prunus SFB genes was identified. Putative structural regions of PmF-box genes have been described, corresponding to regions in PmSFB alleles, but with some sequence variations. These results suggest that SC in 'Zaohong' occurs in pollen, and that other factors outside the S-locus, including PmF-box genes, might be associated with the loss of function of pollen S genes.

Stress-responsive gene ICE1 from Vitis amurensis increases cold tolerance in tobacco.

We report the identification of the inducer of CBF expression 1 (ICE1) from Vitis amurensis, an upstream transcription factor that regulates the transcription of CBF-like genes. The structure of the basic helix-loop-helix domain of VaICE1 is closely related to that of ICE1 in woody plants. This gene is strongly induced in leaves, roots, stems, and petioles by cold temperature. With longer duration of exposure to cold treatments, the expression patterns of organs exhibit differences, which are not observed in normal condition. Transgenic tobacco over-expressing VaICE1 has higher chilling tolerance and survival ability by improving the activities of superoxide dismutase, peroxidase, and catalase, as well as the chlorophyll yield.