Mengjun Liu

The complex jujube genome provides insights into fruit tree biology

The jujube (Ziziphus jujuba Mill.), a member of family Rhamnaceae, is a major dry fruit and a traditional herbal medicine for more than one billion people. Here we present a high-quality sequence for the complex jujube genome, the first genome sequence of Rhamnaceae, using an integrated strategy. The final assembly spans 437.65 Mb (98.6% of the estimated) with 321.45 Mb anchored to the 12 pseudo-chromosomes and contains 32,808 genes. The jujube genome has undergone frequent inter-chromosome fusions and segmental duplications, but no recent whole-genome duplication. Further analyses of the jujube-specific genes and transcriptome data from 15 tissues reveal the molecular mechanisms underlying some specific properties of the jujube. Its high vitamin C content can be attributed to a unique high level expression of genes involved in both biosynthesis and regeneration. Our study provides insights into jujube-specific biology and valuable genomic resources for the improvement of Rhamnaceae plants and other fruit trees.

Genome-Wide Characterization of Simple Sequence Repeat (SSR) Loci in Chinese Jujube and Jujube SSR Primer Transferability.

Chinese jujube (Ziziphus jujuba), an economically important species in the Rhamnaceae family, is a popular fruit tree in Asia. Here, we surveyed and characterized simple sequence repeats (SSRs) in the jujube genome. A total of 436,676 SSR loci were identified, with an average distance of 0.93 Kb between the loci. A large proportion of the SSRs included mononucleotide, dinucleotide and trinucleotide repeat motifs, which accounted for 64.87%, 24.40%, and 8.74% of all repeats, respectively. Among the mononucleotide repeats, A/T was the most common, whereas AT/TA was the most common dinucleotide repeat. A total of 30,565 primer pairs were successfully designed and screened using a series of criteria. Moreover, 725 of 1,000 randomly selected primer pairs were effective among 6 cultivars, and 511 of these primer pairs were polymorphic. Sequencing the amplicons of two SSRs across three jujube cultivars revealed variations in the repeats. The transferability of jujube SSR primers proved that 35/64 SSRs could be transferred across family boundary. Using jujube SSR primers, clustering analysis results from 15 species were highly consistent with the Angiosperm Phylogeny Group (APGIII) System. The genome-wide characterization of SSRs in Chinese jujube is very valuable for whole-genome characterization and marker-assisted selection in jujube breeding. In addition, the transferability of jujube SSR primers could provide a solid foundation for their further utilization.

Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill.) under a Variety of Conditions

Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selection of reference genes. Indeed, suitable reference gene selection for RT-qPCR is critical for accurate normalization of target gene expression. In this study, the software packages geNorm and NormFinder were employed to examine the expression stabilities of nine candidate reference genes under a variety of conditions. Actin-depolymerizing factor 1 (ACT1), Histone-H3 (His3), and Polyadenylate-binding protein-interacting protein (PAIP) were determined to be the most stably expressed genes during five stages of fruit development and ACT1, SiR-Fd, BTF3, and Tubulin alpha chain (TUA) across different tissues/organs. Whereas ACT1, Basic Transcription factor 3 (BTF3), Glyceraldehyde-3-phosphate dehydrogenase (GADPH), and PAIP were the most stable under dark conditions. ACT1, PAIP, BTF3, and Elongation factor 1- gamma (EF1γ) were the most stably expressed genes under phytoplasma infection. Among these genes, SiR-Fd and PAIP are here first reported as stable reference genes. When normalized using these most stable reference genes, the expression patterns of four target genes were found to be in accordance with physiological data, indicating that the reference genes selected in our study are suitable for use in such analyses. This study provides appropriate reference genes and corresponding primers for further RT-qPCR studies in Chinese jujube and emphasizes the importance of validating reference genes for gene expression analysis under variable experimental conditions.

Identification of two Bacillus amyloliquefaciens strains with high suppression to the key fruit pathogens of Chinese jujube

Two bacterial strains (ZJ01 and ZJ02) isolated from the phyllosphere of Chinese jujube were tested for antifungal activities and morphological mechanisms against Phoma destructiva, Alternaria alternata and Fusicoccum spp., three key pathogens of jujube fruits. Based on their physiological characteristics and 16S rDNA gene sequences, both strains were identified as Bacillus amyloliquefaciens. The disease incidence of jujube fruits sprayed with the fermentation liquor of ZJ01 and ZJ02 on the puncture wounds before inoculation with the spores of those three key pathogenic fungi was 10% or 66.7%, significantly lower than that of Nutrient Broth medium treatment (100%). These results suggest that strains ZJ01 may be a potential biological control agent for Chinese jujube fruit diseases. The antagonistic bacteria could cause five kinds of hyphal alterations of the causal agents, the maximum number ever observed. Of them, hyphal swelling and excessive branching were the dominant phenomenon, the other three varied with the pathogens. The diverse morphological malformation might be helpful to understand the broad antifungal activity of Bacillus spp..

Genome-wide identification, characterization of the MADS-box gene family in Chinese jujube and their involvement in flower development

MADS-box genes encode transcription factors that are involved in plant development control (particularly in floral organogenesis) and signal transduction pathways, though a comprehensive analysis of MADS-box family proteins in Chinese jujube (Ziziphus jujuba Mill.) is still missing. Here, we report a genome-wide analysis of the MADS-box gene family in Chinese jujube. Based on phylogenetic analyses, 52 jujube MADS-box genes were classified into 25 MIKCC-type, 3 MIKC*-type, 16 Mα, 5 Mβ and 3 Mγ genes. 37 genes were randomly distributed across all 12 putative chromosomes. We found that the type II genes are more complex than the type I genes and that tandem duplications have occurred in three groups of MADS-box genes. Meanwhile, some gene pairs in the same clade displayed similar or distinct expression profiles, suggesting possible functional redundancy or divergence. MIKCC-type genes exhibited typical temporal and spatial expression patterns in the four whorls of floral tissues. The expressions of B, C/D and E-type genes were significantly suppressed in phyllody as compared to flower, providing valuable evidence for their involvement in flower development. This study is the first comprehensive analysis of the MADS-box family in jujube, and provides valuable information for elucidating molecular regulation mechanism of jujube flower development.

Phylogenetic analysis of 25 plant species representing 19 angiosperm families and one gymnosperm family based on 390 orthologous genes

High-throughput sequencing is fundamentally altering traditional phylogenetic classifications. While the Angiosperm Phylogeny Group (APG) III system based on chloroplast sequences has opened up a new era of angiosperm molecular classification, the use of nuclear genome sequences is more helpful for a precise phylogenetic analysis. However, such attempts have so far been applied to only a very limited number of angiosperm plant families. We constructed a phylogenetic tree of 25 plant species with well assembled genome sequences representing 19 angiosperm families and one gymnosperm family (as out group) using 390 orthologous genes. Our results strongly support the moving of Rhamnaceae to order Rosales from Rhamnales, abolishing Rhamnales, and establishing the new order Malpighiales in the APG III system. Our data also showed some characteristics inconsistent with the APG III classification and provided a reinterpretation of phylogeny for some of the families involved. We propose that Malpighiales should be placed in Malvids, not Fabids. The four monocot species representing four families were clustered together, indicating that monocot is a natural taxon. In summary, our results support almost all the APGIII treatments of the orders involved in this study and provided some surprising reinterpretations at levels beyond order.

Genome-wide identification and analysis of MAPK and MAPKK gene family in Chinese jujube (Ziziphus jujuba Mill.)

BackgroundChinese jujube (Ziziphus jujuba Mill.) is one of the most important members in the Rhamnaceae family. The whole genome sequence and more than 30,000 proteins of Chinese jujube have been obtained in 2014. Mitogen-activated protein kinase cascades are universal signal transduction modules in plants, which is rapidly activated under various biotic and abiotic stresses. To date, there has been no comprehensive analysis of the MAPK and MAPKK gene family in Chinese jujube at the whole genome level.ResultsBy performing a series of bioinformatics analysis, ten MAPK and five MAPKK genes were identified from the genome database of Chinese jujube, and then compared with the homologous genes from Arabidopsis. Phylogenetic analysis showed that ZjMAPKs was classified into four known groups, including A, B, C and D. ZjMAPKs contains five members of the TEY phosphorylation site and five members with the TDY motif. The ZjMAPKK family was subsequently divided into three groups, A, B and D. The gene structure, conserved motifs, functional annotation and chromosome distribution of ZjMAPKs and ZjMAPKKs were also predicted. ZjMAPKs and ZjMAPKKs were distributed on nine pseudo-chromosomes of Chinese jujube. Subsequently, expression analysis of ZjMAPK and ZjMAPKK genes using reverse transcription PCR and quantitative real-time PCR was carried out. The majority of ZjMAPK and ZjMAPKK genes were expressed in all tested organs/tissues with considerable differences in transcript levels indicating that they might be constitutively expressed. Moreover, ZjMKK5 was specific expressed in early development stage of jujube flower bud, indicating it plays some roles in reproductive organs development. The transcript expression of most ZjMAPK and ZjMAPKK genes was down-regulated in response to plant growth regulators, darkness treatment and phytoplasma infection.ConclusionsWe identified ten ZjMAPK and five ZjMAPKK genes from the genome database of Chinese jujube, the research results shown that ZjMPKs and ZjMKKs have the different expression patterns, indicating that they might play different roles in response to various treatments. The results provide valuable information for the further elucidation of physiological functions and biological roles of jujube MAPKs and MAPKKs.