Bi Ma

Draft genome sequence of the mulberry tree Morus notabilis.

Human utilization of the mulberry–silkworm interaction started at least 5,000 years ago and greatly influenced world history through the Silk Road. Complementing the silkworm genome sequence, here we describe the genome of a mulberry species Morus notabilis. In the 330-Mb genome assembly, we identify 128 Mb of repetitive sequences and 29,338 genes, 60.8% of which are supported by transcriptome sequencing. Mulberry gene sequences appear to evolve ~3 times faster than other Rosales, perhaps facilitating the species’ spread worldwide. The mulberry tree is among a few eudicots but several Rosales that have not preserved genome duplications in more than 100 million years; however, a neopolyploid series found in the mulberry tree and several others suggest that new duplications may confer benefits. Five predicted mulberry miRNAs are found in the haemolymph and silk glands of the silkworm, suggesting interactions at molecular levels in the plant–herbivore relationship. The identification and analyses of mulberry genes involved in diversifying selection, resistance and protease inhibitor expressed in the laticifers will accelerate the improvement of mulberry plants.

Identification of the Conserved and Novel miRNAs in Mulberry by High-Throughput Sequencing

miRNAs are a class of non-coding endogenous small RNAs. They play vital roles in plant growth, development, and response to biotic and abiotic stress by negatively regulating genes. Mulberry trees are economically important species with multiple uses. However, to date, little is known about mulberry miRNAs and their target genes. In the present study, three small mulberry RNA libraries were constructed and sequenced using high-throughput sequencing technology. Results showed 85 conserved miRNAs belonging to 31 miRNA families and 262 novel miRNAs at 371 loci. Quantitative real-time PCR (qRT-PCR) analysis confirmed the expression pattern of 9 conserved and 5 novel miRNAs in leaves, bark, and male flowers. A total of 332 potential target genes were predicted to be associated with these 113 novel miRNAs. These results provide a basis for further understanding of mulberry miRNAs and the biological processes in which they are involved.

New Insights into Nested Long Terminal Repeat Retrotransposons in Brassica Species

Long terminal repeat (LTR) retrotransposons, one of the foremost types of transposons, continually change or modify gene function and reorganize the genome through bursts of dramatic proliferation. Many LTR-TEs preferentially insert within other LTR-TEs, but the cause and evolutionary significance of these nested LTR-TEs are not well understood. In this study, a total of 1.52Gb of Brassica sequence containing 2020 bacterial artificial chromosomes (BACs) was scanned, and six bacterial artificial chromosome (BAC) clones with extremely nested LTR-TEs (LTR-TEs density: 7.24/kb) were selected for further analysis. The majority of the LTR-TEs in four of the six BACs were found to be derived from the rapid proliferation of retrotransposons originating within the BAC regions, with only a few LTR-TEs originating from the proliferation and insertion of retrotransposons from outside the BAC regions approximately 5-23Mya. LTR-TEs also preferably inserted into TA-rich repeat regions. Gene prediction by Genescan identified 207 genes in the 0.84Mb of total BAC sequences. Only a few genes (3/207) could be matched to the Brassica expressed sequence tag (EST) database, indicating that most genes were inactive after retrotransposon insertion. Five of the six BACs were putatively centromeric. Hence, nested LTR-TEs in centromere regions are rapidly duplicated, repeatedly inserted, and act to suppress activity of genes and to reshuffle the structure of the centromeric sequences. Our results suggest that LTR-TEs burst and proliferate on a local scale to create nested LTR-TE regions, and that these nested LTR-TEs play a role in the formation of centromeres.

MnTEdb, a collective resource for mulberry transposable elements

Mulberry has been used as an economically important food crop for the domesticated silkworm for thousands of years, resulting in one of the oldest and well-known plant-herbivore interactions. The genome of Morus notabilis has now been sequenced and there is an opportunity to mine the transposable element (TE) data. To better understand the roles of TEs in structural, functional and evolutionary dynamics of the mulberry genome, a specific, comprehensive and user-friendly web-based database, MnTEdb, was constructed. It was built based on a detailed and accurate identification of all TEs in mulberry. A total of 5925 TEs belonging to 13 superfamilies and 1062 families were deposited in this database. MnTEdb enables users to search, browse and download the mulberry TE sequences. Meanwhile, data mining tools, including BLAST, GetORF, HMMER, Sequence Extractor and JBrowse were also integrated into MnTEdb. MnTEdb will assist researchers to efficiently take advantage of our newly annotated TEs, which facilitate their studies in the origin, amplification and evolution of TEs, as well as the comparative analysis among the different species. Database URL: http://morus.swu.edu.cn/mntedb/

Genome‐wide identification and expression analyses of cytochrome P450 genes in mulberry (Morus notabilis)

Cytochrome P450s play critical roles in the biosynthesis of physiologically important compounds in plants. These compounds often act as defense toxins to prevent herbivory. In the present study, a total of 174 P450 genes of mulberry (Morus notabilis C.K.Schn) were identified based on bioinformatics analyses. These mulberry P450 genes were divided into nine clans and 47 families and were found to be expressed in a tissue-preferential manner. These genes were compared to the P450 genes in Arabidopsis thaliana. Families CYP80, CYP92, CYP728, CYP733, CYP736, and CYP749 were found to exist in mulberry, and they may play important roles in the biosynthesis of mulberry secondary metabolites. Analyses of the functional and metabolic pathways of these genes indicated that mulberry P450 genes may participate in the metabolism of lipids, other secondary metabolites, xenobiotics, amino acids, cofactors, vitamins, terpenoids, and polyketides. These results provide a foundation for understanding of the structures and biological functions of mulberry P450 genes.

Transposon variation by order during allopolyploidisation between Brassica oleracea and Brassica rapa

Although many studies have shown that transposable element (TE) activation is induced by hybridisation and polyploidisation in plants, much less is known on how different types of TE respond to hybridisation, and the impact of TE-associated sequences on gene function. We investigated the frequency and regularity of putative transposon activation for different types of TE, and determined the impact of TE-associated sequence variation on the genome during allopolyploidisation. We designed different types of TE primers and adopted the Inter-Retrotransposon Amplified Polymorphism (IRAP) method to detect variation in TE-associated sequences during the process of allopolyploidisation between Brassica rapa (AA) and Brassica oleracea (CC), and in successive generations of self-pollinated progeny. In addition, fragments with TE insertions were used to perform Blast2GO analysis to characterise the putative functions of the fragments with TE insertions. Ninety-two primers amplifying 548 loci were used to detect variation in sequences associated with four different orders of TE sequences. TEs could be classed in ascending frequency into LTR-REs, TIRs, LINEs, SINEs and unknown TEs. The frequency of novel variation (putative activation) detected for the four orders of TEs was highest from the F1 to F2 generations, and lowest from the F2 to F3 generations. Functional annotation of sequences with TE insertions showed that genes with TE insertions were mainly involved in metabolic processes and binding, and preferentially functioned in organelles. TE variation in our study severely disturbed the genetic compositions of the different generations, resulting in inconsistencies in genetic clustering. Different types of TE showed different patterns of variation during the process of allopolyploidisation.

Identification of the mulberry genes involved in ethylene biosynthesis and signaling pathways and the expression of MaERF-B2-1 and MaERF-B2-2 in the response to flooding stress

The phytohormone ethylene is essential to plant growth and development. It plays crucial roles in responses to biotic and abiotic stress. The mulberry tree is an important crop plant in countries in which people rear silkworms for silk production. The availability of the mulberry genome has made it possible to identify mulberry genes involved in ethylene biosynthesis and signal pathways. A total of 145 mulberry genes were identified by both homology-based and hidden Markov model (HMM) search, including 29 genes associated with ethylene biosynthesis and 116 genes in the AP2/ERF family. Studies on gene structure have provided a genetic basis for understanding the functions of these genes. The differences in gene expression were also observed in different tissues. The expression of two mulberry genes in the AP2/ERF family, MaERF-B2-1 and MaERF-B2-2, was found to be associated with the response to flooding stress.

Evolutionary and functional analysis of mulberry type III polyketide synthases

BackgroundType III polyketide synthases are important for the biosynthesis of flavonoids and various plant polyphenols. Mulberry plants have abundant polyphenols, but very little is known about the mulberry type III polyketide synthase genes. An analysis of these genes may provide new targets for genetic improvement to increase relevant secondary metabolites and enhance the plant tolerance to biotic and abiotic stresses.ResultsEighteen genes encoding type III polyketide synthases were identified, including six chalcone synthases (CHS), ten stilbene synthases (STS), and two polyketide synthases (PKS). Functional characterization of four genes representing most of the MnCHS and MnSTS genes by coexpression with 4-Coumaroyl-CoA ligase in Escherichia coli indicated that their products were able to catalyze p-coumaroyl-CoA and malonyl-CoA to generate naringenin and resveratrol, respectively. Microsynteny analysis within mulberry indicated that segmental and tandem duplication events contributed to the expansion of the MnCHS family, while tandem duplications were mainly responsible for the generation of the MnSTS genes. Combining the evolution and expression analysis results of the mulberry type III PKS genes indicated that MnCHS and MnSTS genes evolved mainly under purifying selection to maintain their original functions, but transcriptional subfunctionalization occurred during long-term species evolution. Moreover, mulberry leaves can rapidly accumulated oxyresveratrol after UV-C irradiation, suggesting that resveratrol was converted to oxyresveratrol.ConclusionsCharacterizing the functions and evolution of mulberry type III PKS genes is crucial for advancing our understanding of these genes and providing the basis for further studies on the biosynthesis of relevant secondary metabolites in mulberry plants.

Identification and characterization of Lateral Organ Boundaries Domain genes in mulberry, Morus notabilis.

Genes from the plant specific Lateral Organ Boundaries Domain (LBD) family encode transcriptional regulators that have a variety of functions in various physiological and developmental processes. In the present study, 31 LBD genes were identified in the mulberry genome. The genome features of all MnLBD genes and phylogenetic studies with Arabidopsis LBD protein sequences, accompanied by the expression analysis of each of the Morus LBD genes provide insights into the functional prediction of mulberry LBDs. The genome-wide surveys of the current mulberry genome have resulted in the identification of catalogs of MnLBD genes that may function in the development of leaf, root, and secondary metabolism in Morus sp.

Identification and characterization of genes involved in the jasmonate biosynthetic and signaling pathways in mulberry (Morus notabilis)

Jasmonate (JA) is an important phytohormone regulating growth, development, and environmental response in plants, particularly defense response against herbivorous insects. Recently, completion of the draft genome of the mulberry (Morus notabilis) in conjunction with genome sequencing of silkworm (Bombyx mori) provides an opportunity to study this unique plant-herbivore interaction. Here, we identified genes involved in JA biosynthetic and signaling pathways in the genome of mulberry for the first time, with the majority of samples showing a tissue-biased expression pattern. The analysis of the representative genes 12-oxophytodienoic acid reductase (OPRs) and jasmonate ZIM-domain (JAZs) was performed and the results indicated that the mulberry genome contains a relatively small number of JA biosynthetic and signaling pathway genes. A gene encoding an important repressor, MnNINJA, was identified as an alternative splicing variant lacking an ethylene-responsive element binding factor-associated amphiphilic repression motif. Having this fundamental information will facilitate future functional study of JA-related genes pertaining to mulberry-silkworm interactions.