Hongyan Dai

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.

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.

Genome-wide Characterization of Long Terminal Repeat -retrotransposons in Apple Reveals the Differences in Heterogeneity and Copy Number between Ty1-copia and Ty3-gypsy Retrotransposons

The conserved domains of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy groups of long terminal repeat (LTR) retrotransposons were isolated from the Malus domestica genome using degenerate oligonucleotide primers. Sequence analysis showed that 45% of Ty1-copia and 63% of Ty3-gypsy RT sequences contained premature stop codons and/or indels disrupting the reading frame. High heterogeneity among RT sequences of both Ty1-copia and Ty3-gypsy group retrotransposons was observed, but Ty3-gypsy group retrotransposons in the apple genome are less heterogeneous than Ty1-copia elements. Retrotransposon copy number was estimated by dot blot hybridizations for Ty1-copia (approximately 5,000) and Ty3-gypsy (approximately 26,000). All elements of the two types of LTR retrotransposons comprise approximately 38% of the M. domestica genome, with the Ty3-gypsy group contribution being higher (33.5%) than the Ty1-copia one (4.6%). Transcription was not detected by reverse transcription-polymerase chain reaction for either Ty1-copia or Ty3-gypsy retrotransposons in the leaves of plants in vitro or in leaf explants cultured on medium supplemented with high concentration benzylaminopurine. This research reveals the differences in heterogeneity and copy number between Ty1-copia and Ty3-gypsy retrotransposons in the apple genome. Ty1-copia retrotransposon has higher heterogeneity than Ty3-gypsy retrotransposon, but the latter has a higher copy number, which implies that Ty3-gypsy retrotransposons may play a more important role in the apple genome evolution.

Genome Sequences of Three Apple chlorotic leaf spot virus Isolates from Hawthorns in China

The genome sequences of Apple chlorotic leaf spot virus (ACLSV) isolates from three accessions of hawthorns (Crataegus pinnatifida) grown at Shenyang Agricultural University were determined using Illumina RNA-seq. To confirm the assembly data from the de novo sequencing, two ACLSV genomic sequences (SY01 and SY02) were sequenced using the Sanger method. The SY01 and SY02 sequences obtained with the Sanger method showed 99.5% and 99.7% nucleotide identity with the transcriptome data, respectively. The genome sequences of the hawthorn isolates SY01, SY02 and SY03 (GenBank accession nos. KM207212, KU870524 and KU870525, respectively) consisted of 7,543, 7,561 and 7,545 nucleotides, respectively, excluding poly-adenylated tails. Sequence analysis revealed that these hawthorn isolates shared an overall nucleotide identity of 82.8–92.1% and showed the highest identity of 90.3% for isolate YH (GenBank accession no. KC935955) from pear and the lowest identity of 67.7% for isolate TaTao5 (GenBank accession no. EU223295) from peach. Hawthorn isolate sequences were similar to those of ‘B6 type’ ACLSV. The relationship between ACLSV isolates largely depends upon the host species. This represents the first comparative study of the genome sequences of ACLSV isolates from hawthorns.

Characterization of the Hormone and Stress-Induced Expression of FaRE1 Retrotransposon Promoter in Strawberry

Retrotransposons are the most abundant mobile elements in the plant genome and seem to play an important role in genome reorganization induced by environmental challenges. Their success in this function depends on the ability of their promoters to regulate plant adaptation to biotic and abiotic stresses. In this study, the promoter region of FaRE1 was amplified in the strawberry genome, and promoter::GUS fusion was constructed. We produced transgenic strawberry plants carrying FaRE1 promoter::GUS-fusion genes, and monitored GUS reporter activity. Histochemical and fluorimetric GUS analysis these plants showed the characteristics of the FaRE1 promoter were activated by either hormones treatments with ABA, NAA, and 2,4-D or cold stress. In addition, we found the GUS reporter was activated in the leaves of transgenic strawberry plants using 5-azaC. These results suggest that the promoter of FaRE1 may act as different signal transduction pathways, allowing FaRE1 retrotransposon to be activated in response to multiples challenges.

Isolation and Characterization of Transcriptionally Active Ty1-copia Retrotransposons in Fragaria × ananassa

One possible mechanism suggested for somaclonal variation is the activation of transposable elements. The activation of retrotransposons by stresses and external changes is commonly observed in plants. In previous study, we isolated the reverse transcriptase (RT) gene sequences of Ty1-copia retrotransposons from tissue culture strawberry (Fragaria×ananassa) plant, but not the transcriptionally active sequence. For further understanding the relationship between retrotransposon and somaclonal varation, in this study, we isolated the transcriptionally active RT gene sequences from strawberry plants subjected to different abiotic stresses. These retrotransposons were activated by spraying strawberry leaves with 2 mmol L^(-1) salicylic acid (SA), 50 mmol L^(-1) methyl jasmonate (MeJA), 50 mmol L^(-1) abscisic acid (ABA), 50 mmol L^(-1) 2,4-dichlorophenoxyacetic acid (2,4-D) or by inducing callus growth in 2 types of MS media: first medium supplemented with 0.5 mg L^(-1) 6-benzylaminopurine (6-BA), 0.5 mg L^(-1) gibberellic acid (GA3), 1.0 mg L^(-1) thidiazuron (TDZ), and 0.1 mg L^(-1) 2, 4-D, and the second medium supplemented with 0.5 mg L^(-1) 6-BA, 0.5 mg L^(-1) GA3, 2.0 mg L^(-1) TDZ, and 0.02 mg L^(-1) indole butyric acid (IBA). Analysis of gene sequences of 17 RTs revealed that none of them contained stop codons and/or indels disrupting the reading frame. These different stress-origin transcriptionally active RTs were remarkably similar to each other-FATEXP2-8 and FATEYS9-7 showed 100% sequence identity. Analysis of pylogenetic of these transcriptionally active RTs and the RT sequences from genome showed that there were close phylogenetic relationships of most of the transcriptionally active RTs. The results of this study have contributed to the background information necessary for future studies for evaluating the relationship between retrotransposons and somaclonal variation.

Isolation and Identification of Virus dsRNA from Strawberry Plants

Abstract The analysis of virus genome is based on nucleic acid isolation. The aims of this study were to develop a method for isolation and identification of virus double-stranded ribonucleic acid (dsRNA) and to elucidate the nucleotide sequences of strawberry virus. Using the modified method, virus dsRNA was extracted from strawberry virus indicator plants and cultivated strawberry plants and detected using agarose gel electrophoresis with ethidium bromide staining and reverse transcription-polymerase chain reaction (RT-PCR). The quantity of virus dsRNA varied among strawberry cultivars. The quantity of dsRNA from in vitro plantlets was higher than that from the young leaves of field plants. For the field-grown plants, there was more dsRNA in the young leaves. Virus dsRNA extracted from strawberry plants was resistant to deoxyribonuclease I (DNase I), but evidently, it became resistant to ribonuclease A (RNase A) only in the presence of 0.5 M NaCl. Its bands in agarose gel could be readily recycled using an agarose gel DNA purification kit. With RT-PCR, the segments of both strawberry mottle virus and Strawberry mild yellow edge virus genomes were amplified by using the virus dsRNA recycled from gel or treated with DNase I/RNase A as templates. The system developed for dsRNA isolation and identification in strawberry plants laid a sound foundation for the work on genome analysis of strawberry virus isolates in China.

Study on the Molecular Variation and PCR Detection of Strawberry mottle virus

Strawberry mottle virus (SMoV) is an important viral pathogen infecting strawberry (Fragaria spp.). The study was conducted to analyze the characterization of the molecular variation of SMoV and develop the methods for detection of SMoV by nested PCR and transcriptional enhancement techniques. The 3′ non-coding region (NCR) and large coat protein (LCP) gene of SMoV genome were amplified by reverse transcription-polymerase chain reaction (RT-PCR). The specific segments were cloned and sequenced. The characterization of the molecular variation for some isolates of SMoV and phylogenetic analysis were studied. Based on the primers located in the conserved region of genome of SMoV, SMoV could be steadily detected using semi-nested PCR and transcriptional enhancement techniques. Both semi-nested PCR and transcriptional enhancement techniques were considerably more sensitive than the standard RT-PCR. The nucleotide sequences of NCR and partial LCP gene of Chinese isolates were obtained, and sequence analysis of the partial LCP gene of various SMoV isolates showed nucleotide identities ranging from 76.8 to 99.7%. There was a slight tendency for isolates to group according to their geographical origin. All 3 Polish isolates, 4 isolates of 7 Dutch isolates, and 3 isolates of 4 Chinese isolates formed a small separate branch, respectively. Two Germanic isolates had a far relationship with other isolates, and formed a separate clade. A high level of sequence variability was found among SMoV isolates, and the Germanic isolates were likely to a special strain group.

Expression and functional analysis of FaPH01;H9 gene of strawberry (Fragariaxananassa)

Abstract Although the phosphate 1 ( PHO1 ) gene family has been implicated in inorganic phosphate transport and homeostasis, the underlying mechanism of this gene in the strawberry has not yet been revealed. In the present study, we analyzed the expression of the PH01;H9 gene in the strawberry ( Fragaria x ananassa ), revealing the involvement of this gene in the regulation of phosphorus (P) content. The coding sequence (CDS) of the PH01;H9 gene, was isolated from the cultivated strawberry ‘Sachinoka’ and named as FaPH01;H9 . The full-length CDS of this gene was 2292 bp, encoding 763 amino acids, and the protein contained both SYG1/Pho81/XPR1 (SPX) and ERD1/XPR1/SYG1 (EXS) domains, which were involved in phosphate (Pi) signaling. Real-time reverse transcription-polymerase chain reaction (RT-PCR) data suggested that the level of FaPHO1;H9 expression was consistent with the P content in different organs, except for the petiole. Particularly, its expression level was also correlated with P content in fruits of different developmental stages. The expression of FaPHO1;H9 was also consistent with P content in leaves under different concentrations of P fertilizer application. Furthermore, transgenic Arabidopsis lines were generated, and the P content in Arabidopsis plants over-expressing FaPH01;H9 was significantly higher than that in wild-type plants. Therefore, we proposed that FaPH01;H9 functions in P transport.