Meizhong Luo

The Oryza map alignment project: The golden path to unlocking the genetic potential of wild rice species

The wild species of the genus Oryza offer enormous potential to make a significant impact on agricultural productivity of the cultivated rice species Oryza sativa and Oryza glaberrima. To unlock the genetic potential of wild rice we have initiated a project entitled the ‘Oryza Map Alignment Project’ (OMAP) with the ultimate goal of constructing and aligning BAC/STC based physical maps of 11 wild and one cultivated rice species to the International Rice Genome Sequencing Project’s finished reference genome – O. sativa ssp. japonica c. v. Nipponbare. The 11 wild rice species comprise nine different genome types and include six diploid genomes (AA, BB, CC, EE, FF and GG) and four tetrapliod genomes (BBCC, CCDD, HHKK and HHJJ) with broad geographical distribution and ecological adaptation. In this paper we describe our strategy to construct robust physical maps of all 12 rice species with an emphasis on the AA diploid O. nivara – thought to be the progenitor of modern cultivated rice.

An Improved Method for Plant BAC Library Construction

Large genomic DNA insert-containing libraries are required as critical tools for physical mapping, positional cloning, and genome sequencing of complex genomes. The bacterial artificial chromosome (BAC) cloning system has become a dominant system over others to clone large genomic DNA inserts. As the costs of positional cloning, physical mapping, and genome sequencing continuously decrease, there is an increasing demand for high-quality deep-coverage large insert BAC libraries. In our laboratory, we have constructed many high-quality deep-coverage large insert BAC libraries including arabidopsis, manocot and dicot crop plants, and plant pathogens. Here, we present the protocol used in our laboratory to construct BAC libraries.

Thioredoxin redox regulates ATPase activity of magnesium chelatase CHLI subunit and modulates redox-mediated signaling in tetrapyrrole biosynthesis and homeostasis of reactive oxygen species in pea plants.

The chloroplast thioredoxins (TRXs) function as messengers of redox signals from ferredoxin to target enzymes. In this work, we studied the regulatory impact of pea (Pisum sativum) TRX-F on the magnesium (Mg) chelatase CHLI subunit and the enzymatic activation of Mg chelatase in vitro and in vivo. In vitro, reduced TRX-F activated the ATPase activity of pea CHLI and enhanced the activity of Mg chelatase reconstituted from the three recombinant subunits CHLI, CHLD, and CHLH in combination with the regulator protein GENOMES UNCOUPLED4 (GUN4). Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that TRX-F physically interacts with CHLI but not with either of the other two subunits or GUN4. In vivo, virus-induced TRX-F gene silencing (VIGS-TRX-F) in pea plants did not result in an altered redox state of CHLI. However, simultaneous silencing of the pea TRX-F and TRX-M genes (VIGS-TRX-F/TRX-M) resulted in partially and fully oxidized CHLI in vivo. VIGS-TRX-F/TRX-M plants demonstrated a significant reduction in Mg chelatase activity and 5-aminolevulinic acid synthesizing capacity as well as reduced pigment content and lower photosynthetic capacity. These results suggest that, in vivo, TRX-M can compensate for a lack of TRX-F and that both TRXs act as important redox regulators of Mg chelatase. Furthermore, the silencing of TRX-F and TRX-M expression also affects gene expression in the tetrapyrrole biosynthesis pathway and leads to the accumulation of reactive oxygen species, which may also serve as an additional signal for the transcriptional regulation of photosynthesis-associated nuclear genes.

De Novo Next Generation Sequencing of Plant Genomes

The genome sequencing of all major food and bioenergy crops is of critical importance in the race to improve crop production to meet the future food and energy security needs of the world. Next generation sequencing technologies have brought about great improvements in sequencing throughput and cost, but do not yet allow for de novo sequencing of large repetitive genomes as found in most crop plants. We present a strategy that combines cutting edge next generation sequencing with “old school” genomics resources and allows rapid cost-effective sequencing of plant genomes.

Construction of a bacterial artificial chromosome library from the spikemoss Selaginella moellendorffii: a new resource for plant comparative genomics

BackgroundThe lycophytes are an ancient lineage of vascular plants that diverged from the seed plant lineage about 400 Myr ago. Although the lycophytes occupy an important phylogenetic position for understanding the evolution of plants and their genomes, no genomic resources exist for this group of plants.ResultsHere we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from the lycophyte Selaginella moellendorffii. Based on cell flow cytometry, this species has the smallest genome size among the different lycophytes tested, including Huperzia lucidula, Diphaiastrum digita, Isoetes engelmanii and S. kraussiana. The arrayed BAC library consists of 9126 clones; the average insert size is estimated to be 122 kb. Inserts of chloroplast origin account for 2.3% of the clones. The BAC library contains an estimated ten genome-equivalents based on DNA hybridizations using five single-copy and two duplicated S. moellendorffii genes as probes.ConclusionThe S. moellenforffii BAC library, the first to be constructed from a lycophyte, will be useful to the scientific community as a resource for comparative plant genomics and evolution.

Involvement of Linear Plasmids in Aerobic Biodegradation of Vinyl Chloride

ABSTRACT Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as the sole source of carbon and energy under aerobic conditions. Strains AJ and TD also use ethene and ethylene oxide as growth substrates. Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but it contained no circular plasmids. While strain AJ was growing on ethylene oxide, it was observed to contain a 100-kb linear plasmid, and its ability to use VC as a substrate was retained. The linear plasmids in strain AJ were cured, and the ability of strain AJ to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers. Strain TD contained three linear plasmids, ranging in size from approximately 90 kb to 320 kb, when growing on VC or ethene. As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria-Bertani broth and its ability to consume VC and ethene was lost. Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes. Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase. Their yields during growth on VC (0.15 to 0.20 mg of total suspended solids per mg of VC) are similar to the yields reported for other isolates (i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.).

Multiple transcription start sites of the carrot dihydrofolate reductase-thymidylate synthase gene, and sub-cellular localization of the bifunctional protein

The analysis of clones obtained by rapid amplification of the 5′ endand by primer extension of the mRNA for carrot bifunctionaldihydrofolate reductase-thymidylate synthase showed transcripts ofdiffering lengths that belonged to two sub-populations. The longertranscripts were found to contain a translation start site 147 ntupstream of, and in frame with, the one which is present in the shortertranscripts. The ORF that begins at this ATG codes for a protein of64714 Da, which is much larger than mature DHFR-TS subunit. TheN-terminusregion of this polypeptide shows features typical of plant transitpeptides. Immunogold labelling studies and immunorecognition of theplastid-containing sub-cellular fraction suggested a plastidiallocalisation of the bifunctional protein. Although plant cells wereshown to contain folate pools in plastids, in mitochondria and in thecytosol, few enzymes of the folate pathway have been associated with anysub-cellular compartment. Thus, this is the first indication for thepresence of an enzyme of the folate biosynthetic pathway in plastids.The longer transcripts revealed the presence of a TC microsatellite atthe 5′-untranslated end.

Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63

Significance Indica rice accounts for >70% of total rice production worldwide, is genetically highly diverse, and can be divided into two major varietal groups independently bred and widely cultivated in China and Southeast Asia. Here, we generated high-quality genome sequences for two elite rice varieties, Zhenshan 97 and Minghui 63, representing the two groups of indica rice and the parents of a leading rice hybrid. Comparative analyses uncovered extensive structural differences between the two genomes and complementarity in their hybrid transcriptome. These findings have general implications for understanding intraspecific variations of organisms with complex genomes. The availability of the two genomes will serve as a foundation for future genome-based explorations in rice toward both basic and applied goals. Asian cultivated rice consists of two subspecies: Oryza sativa subsp. indica and O. sativa subsp. japonica. Despite the fact that indica rice accounts for over 70% of total rice production worldwide and is genetically much more diverse, a high-quality reference genome for indica rice has yet to be published. We conducted map-based sequencing of two indica rice lines, Zhenshan 97 (ZS97) and Minghui 63 (MH63), which represent the two major varietal groups of the indica subspecies and are the parents of an elite Chinese hybrid. The genome sequences were assembled into 237 (ZS97) and 181 (MH63) contigs, with an accuracy >99.99%, and covered 90.6% and 93.2% of their estimated genome sizes. Comparative analyses of these two indica genomes uncovered surprising structural differences, especially with respect to inversions, translocations, presence/absence variations, and segmental duplications. Approximately 42% of nontransposable element related genes were identical between the two genomes. Transcriptome analysis of three tissues showed that 1,059–2,217 more genes were expressed in the hybrid than in the parents and that the expressed genes in the hybrid were much more diverse due to their divergence between the parental genomes. The public availability of two high-quality reference genomes for the indica subspecies of rice will have large-ranging implications for plant biology and crop genetic improvement.

A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni

To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (http://biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 x coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ~ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.

Molecular cloning and analysis of a cDNA coding for the bifunctional dihydrofolate reductase-thymidylate synthase of Daucus carota

Molecular cloning of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota was achieved by immunoscreening of a cDNA library obtaining a 2 kbp clone which contains an open reading frame of 1528 bp. Comparison of the deduced amino acid sequence with those from other sources revealed the presence of motifs typical of DHFR and TS thus confirming the bifunctional nature of the carrot protein. As in other organisms, a higher degree of conservation was observed in the TS domain. Analysis of the dhfr-ts gene content in carrot revealed the presence of several copies per diploid genome.