David Kudrna

The genome of Eucalyptus grandis

Eucalypts are the world’s most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

Oil palm genome sequence reveals divergence of interfertile species in Old and New worlds

Oil palm is the most productive oil-bearing crop. Although it is planted on only 5% of the total world vegetable oil acreage, palm oil accounts for 33% of vegetable oil and 45% of edible oil worldwide, but increased cultivation competes with dwindling rainforest reserves. We report the 1.8-gigabase (Gb) genome sequence of the African oil palm Elaeis guineensis, the predominant source of worldwide oil production. A total of 1.535 Gb of assembled sequence and transcriptome data from 30 tissue types were used to predict at least 34,802 genes, including oil biosynthesis genes and homologues of WRINKLED1 (WRI1), and other transcriptional regulators, which are highly expressed in the kernel. We also report the draft sequence of the South American oil palm Elaeis oleifera, which has the same number of chromosomes (2n = 32) and produces fertile interspecific hybrids with E. guineensis but seems to have diverged in the New World. Segmental duplications of chromosome arms define the palaeotetraploid origin of palm trees. The oil palm sequence enables the discovery of genes for important traits as well as somaclonal epigenetic alterations that restrict the use of clones in commercial plantings, and should therefore help to achieve sustainability for biofuels and edible oils, reducing the rainforest footprint of this tropical plantation crop.

Aluminum tolerance in maize is associated with higher MATE1 gene copy number

Genome structure variation, including copy number variation and presence/absence variation, comprises a large extent of maize genetic diversity; however, its effect on phenotypes remains largely unexplored. Here, we describe how copy number variation underlies a rare allele that contributes to maize aluminum (Al) tolerance. Al toxicity is the primary limitation for crop production on acid soils, which make up 50% of the world’s potentially arable lands. In a recombinant inbred line mapping population, copy number variation of the Al tolerance gene multidrug and toxic compound extrusion 1 (MATE1) is the basis for the quantitative trait locus of largest effect on phenotypic variation. This expansion in MATE1 copy number is associated with higher MATE1 expression, which in turn results in superior Al tolerance. The three MATE1 copies are identical and are part of a tandem triplication. Only three maize inbred lines carrying the three-copy allele were identified from maize and teosinte diversity panels, indicating that copy number variation for MATE1 is a rare, and quite likely recent, event. These maize lines with higher MATE1 copy number are also Al-tolerant, have high MATE1 expression, and originate from regions of highly acidic soils. Our findings show a role for copy number variation in the adaptation of maize to acidic soils in the tropics and suggest that genome structural changes may be a rapid evolutionary response to new environments.

The stem rust resistance gene Rpg5 encodes a protein with nucleotide-binding-site, leucine-rich, and protein kinase domains.

We isolated the barley stem rust resistance genes Rpg5 and rpg4 by map-based cloning. These genes are colocalized on a 70-kb genomic region that was delimited by recombination. The Rpg5 gene consists of an unusual structure encoding three typical plant disease resistance protein domains: nucleotide-binding site, leucine-rich repeat, and serine threonine protein kinase. The predicted RPG5 protein has two putative transmembrane sites possibly involved in membrane binding. The gene is expressed at low but detectable levels. Posttranscriptional gene silencing using VIGS resulted in a compatible reaction with a normally incompatible stem rust pathogen. Allele sequencing also validated the candidate Rpg5 gene. Allele and recombinant sequencing suggested that the probable rpg4 gene encoded an actin depolymerizing factor-like protein. Involvement of actin depolymerizing factor genes in nonhost resistance has been documented, but discovery of their role in gene-for-gene interaction would be novel and needs to be further substantiated.

The physical and genetic framework of the maize b73 genome

Maize is a major cereal crop and an important model system for basic biological research. Knowledge gained from maize research can also be used to genetically improve its grass relatives such as sorghum, wheat, and rice. The primary objective of the Maize Genome Sequencing Consortium (MGSC) was to generate a reference genome sequence that was integrated with both the physical and genetic maps. Using a previously published integrated genetic and physical map, combined with in-coming maize genomic sequence, new sequence-based genetic markers, and an optical map, we dynamically picked a minimum tiling path (MTP) of 16,910 bacterial artificial chromosome (BAC) and fosmid clones that were used by the MGSC to sequence the maize genome. The final MTP resulted in a significantly improved physical map that reduced the number of contigs from 721 to 435, incorporated a total of 8,315 mapped markers, and ordered and oriented the majority of FPC contigs. The new integrated physical and genetic map covered 2,120 Mb (93%) of the 2,300-Mb genome, of which 405 contigs were anchored to the genetic map, totaling 2,103.4 Mb (99.2% of the 2,120 Mb physical map). More importantly, 336 contigs, comprising 94.0% of the physical map (∼1,993 Mb), were ordered and oriented. Finally we used all available physical, sequence, genetic, and optical data to generate a golden path (AGP) of chromosome-based pseudomolecules, herein referred to as the B73 Reference Genome Sequence version 1 (B73 RefGen_v1).

Construction, alignment and analysis of twelve framework physical maps that represent the ten genome types of the genus Oryza

We describe the establishment and analysis of a genus-wide comparative framework composed of 12 bacterial artificial chromosome fingerprint and end-sequenced physical maps representing the 10 genome types of Oryza aligned to the O. sativa ssp. japonica reference genome sequence. Over 932 Mb of end sequence was analyzed for repeats, simple sequence repeats, miRNA and single nucleotide variations, providing the most extensive analysis of Oryza sequence to date.

Evolution of a Complex Locus for Terpene Biosynthesis in Solanum

A region on chromosome 8 of several Solanum species contains genes for terpene synthases and cis-prenyl transferases, the latter encoding the enzymes that catalyze the formation of the substrates used by enzymes encoded by the former. Detailed sequence and biochemical analyses identify molecular events that gave rise to distinct gene composition and function in the different Solanum species. Functional gene clusters, containing two or more genes encoding different enzymes for the same pathway, are sometimes observed in plant genomes, most often when the genes specify the synthesis of specialized defensive metabolites. Here, we show that a cluster of genes in tomato (Solanum lycopersicum; Solanaceae) contains genes for terpene synthases (TPSs) that specify the synthesis of monoterpenes and diterpenes from cis-prenyl diphosphates, substrates that are synthesized by enzymes encoded by cis-prenyl transferase (CPT) genes also located within the same cluster. The monoterpene synthase genes in the cluster likely evolved from a diterpene synthase gene in the cluster by duplication and divergence. In the orthologous cluster in Solanum habrochaites, a new sesquiterpene synthase gene was created by a duplication event of a monoterpene synthase followed by a localized gene conversion event directed by a diterpene synthase gene. The TPS genes in the Solanum cluster encoding cis-prenyl diphosphate–utilizing enzymes are closely related to a tobacco (Nicotiana tabacum; Solanaceae) diterpene synthase encoding Z-abienol synthase (Nt-ABS). Nt-ABS uses the substrate copal-8-ol diphosphate, which is made from the all-trans geranylgeranyl diphosphate by copal-8-ol diphosphate synthase (Nt-CPS2). The Solanum gene cluster also contains an ortholog of Nt-CPS2, but it appears to encode a nonfunctional protein. Thus, the Solanum functional gene cluster evolved by duplication and divergence of TPS genes, together with alterations in substrate specificity to utilize cis-prenyl diphosphates and through the acquisition of CPT genes.

Mapping and pyramiding of qualitative and quantitative resistance to stripe rust in barley

Abstract The identification and location of sources of genetic resistance to plant diseases are important contributions to the development of resistant varieties. The combination of different sources and types of resistance in the same genotype should assist in the development of durably resistant varieties. Using a doubled haploid (DH), mapping population of barley, we mapped a qualitative resistance gene (Rpsx) to barley stripe rust in the accession CI10587 (PI 243183) to the long arm of chromosome 1(7H). We combined the Rpsx gene, through a series of crosses, with three mapped and validated barley stripe rust resistance QTL alleles located on chromosomes 4(4H) (QTL4), 5(1H) (QTL5), and 7(5H) (QTL7). Three different barley DH populations were developed from these crosses, two combining Rpsx with QTL4 and QTL7, and the third combining Rpsx with QTL5. Disease severity testing in four environments and QTL mapping analyses confirmed the effects and locations of Rpsx, QTL4, and QTL5, thereby validating the original estimates of QTL location and effect. QTL alleles on chromosomes 4(4H) and 5(1H) were effective in decreasing disease severity in the absence of the resistance allele at Rpsx. Quantitative resistance effects were mainly additive, although magnitude interactions were detected. Our results indicate that combining qualitative and quantitative resistance in the same genotype is feasible. However, the durability of such resistance pyramids will require challenge from virulent isolates, which currently are not reported in North America.

Barley putative hypersensitive induced reaction genes: genetic mapping, sequence analyses and differential expression in disease lesion mimic mutants

Abstract The hypersensitive response (HR) is one of the most-efficient forms of plant defense against biotrophic pathogens, and results in localized cell death and the formation of necrotic lesions; however, the molecular components of pathways leading to HR remain largely unknown. Barley (Hordeum vulgare ssp. vulgare L.) cDNAs for putative hypersensitive-induced reaction (HIR) genes were isolated based on DNA and amino-acid homologies to maize HIR genes. Analyses of the cDNA and genomic sequences and genetic mapping found four distinct barley HIR genes, Hv-hir1, Hv-hir2, Hv-hir3 and Hv-hir4, on chromosomes 4(4H) bin10, 7(5H) bin04, 7(5H) bin07 and 1(7H) bin03, respectively. Hv-hir1, Hv-hir2 and Hv-hir3 genes were highly homologous at both DNA and the deduced amino-acid level, but the Hv-hir4 gene was similar to the other genes only at the amino-acid sequence level. Amino-acid sequence analyses of the barley HIR proteins indicated the presence of the SPFH protein-domain characteristic for the prohibitins and stomatins which are involved in control of the cell cycle and ion channels, as well as in other membrane-associated proteins from bacteria, plants and animals. HIR genes were expressed in all organs and developement stages analyzed, indicating a vital and non-redundant function. Barley fast-neutron mutants exhibiting spontaneous HR (disease lesion mimic mutants) showed up to a 35-fold increase in Hv-hir3 expression, implicating HIR genes in the induction of HR.

RL-SAGE and microarray analysis of the rice transcriptome after Rhizoctonia solani infection

Sheath blight caused by the fungal pathogen Rhizoctonia solani is an emerging problem in rice production worldwide. To elucidate the molecular basis of rice defense to the pathogen, RNA isolated from R. solani-infected leaves of Jasmine 85 was used for both RL-SAGE library construction and microarray hybridization. RL-SAGE sequence analysis identified 20,233 and 24,049 distinct tags from the control and inoculated libraries, respectively. Nearly half of the significant tags (≥2 copies) from both libraries matched TIGR annotated genes and KOME full-length cDNAs. Among them, 42% represented sense and 7% antisense transcripts, respectively. Interestingly, 60% of the library-specific (≥10 copies) and differentially expressed (>4.0-fold change) tags were novel transcripts matching genomic sequence but not annotated genes. About 70% of the genes identified in the SAGE libraries showed similar expression patterns (up or down-regulated) in the microarray data obtained from three biological replications. Some candidate RL-SAGE tags and microarray genes were located in known sheath blight QTL regions. The expression of ten differentially expressed RL-SAGE tags was confirmed with RT-PCR. The defense genes associated with resistance to R. solani identified in this study are useful genomic materials for further elucidation of the molecular basis of the defense response to R. solani and fine mapping of target sheath blight QTLs.