Daniel C. Ilut

Floral gene resources from basal angiosperms for comparative genomics research

BackgroundThe Floral Genome Project was initiated to bridge the genomic gap between the most broadly studied plant model systems. Arabidopsis and rice, although now completely sequenced and under intensive comparative genomic investigation, are separated by at least 125 million years of evolutionary time, and cannot in isolation provide a comprehensive perspective on structural and functional aspects of flowering plant genome dynamics. Here we discuss new genomic resources available to the scientific community, comprising cDNA libraries and Expressed Sequence Tag (EST) sequences for a suite of phylogenetically basal angiosperms specifically selected to bridge the evolutionary gaps between model plants and provide insights into gene content and genome structure in the earliest flowering plants.ResultsRandom sequencing of cDNAs from representatives of phylogenetically important eudicot, non-grass monocot, and gymnosperm lineages has so far (as of 12/1/04) generated 70,514 ESTs and 48,170 assembled unigenes. Efficient sorting of EST sequences into putative gene families based on whole Arabidopsis/rice proteome comparison has permitted ready identification of cDNA clones for finished sequencing. Preliminarily, (i) proportions of functional categories among sequenced floral genes seem representative of the entire Arabidopsis transcriptome, (ii) many known floral gene homologues have been captured, and (iii) phylogenetic analyses of ESTs are providing new insights into the process of gene family evolution in relation to the origin and diversification of the angiosperms.ConclusionInitial comparisons illustrate the utility of the EST data sets toward discovery of the basic floral transcriptome. These first findings also afford the opportunity to address a number of conspicuous evolutionary genomic questions, including reproductive organ transcriptome overlap between angiosperms and gymnosperms, genome-wide duplication history, lineage-specific gene duplication and functional divergence, and analyses of adaptive molecular evolution. Since not all genes in the floral transcriptome will be associated with flowering, these EST resources will also be of interest to plant scientists working on other functions, such as photosynthesis, signal transduction, and metabolic pathways.

Defining Loci in Restriction-Based Reduced Representation Genomic Data from Nonmodel Species: Sources of Bias and Diagnostics for Optimal Clustering

Next generation sequencing holds great promise for applications of phylogeography, landscape genetics, and population genomics in wild populations of nonmodel species, but the robustness of inferences hinges on careful experimental design and effective bioinformatic removal of predictable artifacts. Addressing this issue, we use published genomes from a tunicate, stickleback, and soybean to illustrate the potential for bioinformatic artifacts and introduce a protocol to minimize two sources of error expected from similarity-based de-novo clustering of stacked reads: the splitting of alleles into different clusters, which creates false homozygosity, and the grouping of paralogs into the same cluster, which creates false heterozygosity. We present an empirical application focused on Ciona savignyi, a tunicate with very high SNP heterozygosity (~0.05), because high diversity challenges the computational efficiency of most existing nonmodel pipelines while also potentially exacerbating paralog artifacts. The simulated and empirical data illustrate the advantages of using higher sequence difference clustering thresholds than is typical and demonstrate the utility of our protocol for efficiently identifying an optimum threshold from data without prior knowledge of heterozygosity. The empirical Ciona savignyi data also highlight null alleles as a potentially large source of false homozygosity in restriction-based reduced representation genomic data.

An EST database for Liriodendron tulipifera L. floral buds: the first EST resource for functional and comparative genomics in Liriodendron

Liriodendron tulipifera L. was selected by the Floral Genome Project for identification of new genes related to floral diversity in basal angiosperms. A large, non-normalized cDNA library was constructed from premeiotic and meiotic floral buds and sequenced to generate a database of 9,531 high-quality expressed sequence tags. These sequences clustered into 6,520 unigenes, of which 5,251 were singletons, and 1,269 were in contigs. Homologs of genes regulating many aspects of flower development were identified, including those for organ identity and development, cell and tissue differentiation, and cell-cycle control. Almost 5% of the transcriptome consisted of homologs to known floral gene families. Homologs of most of the genes involved in cell-wall construction were also recovered. This provides a new opportunity for comparative studies in lignin biosynthesis, a trait of key importance in the evolution of land plants and in the utilization of fiber from economically important tree species, such as Liriodendron. Also of note is that 1,089 unigenes did not match any sequence in the public databases, including the complete genomes of Arabidopsis, rice, and Populus. Some of these novel genes might be unique in basal angiosperm species and, when better characterized, may be informative for understanding the origins of diverged gene families. Thus, the Liriodendron expressed sequence tag database and library will help bridge our understanding of the mechanisms of flower initiation and development that are shared among basal angiosperms, eudicots, and monocots, and provide new opportunities for comparative analysis of gene families across angiosperm species.

Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium

BackgroundThe majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterize the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation.ResultsEleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species (G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes (FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes (FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings.ConclusionsThe omega-3 FAD gene family in cotton was characterized at the genome-wide level in three species, showing relatively ancient establishment of the gene family prior to the split of A and D diploid progenitor species. The FAD genes are differentially expressed in various organs and cell types, including fiber, and expression of the FAD7/8-1 gene was induced by cold temperature. Collectively, these data define the genetic and functional genomic properties of this important gene family in cotton and provide a foundation for future efforts to improve cotton abiotic stress tolerance through molecular breeding approaches.

rAmpSeq: Using repetitive sequences for robust genotyping

Repetitive sequences have been used for DNA fingerprinting and genotyping for more than a quarter century. Now, with our knowledge of whole genome sequences, repetitive sequences can be used to identify polymorphisms that can be mapped and scored in a systematic manner. We have developed a simple, robust platform for designing primers, PCR amplification, and high throughput cloning that allows hundreds to thousands of markers to be scored for less than

Selecting Nuclear Sequences for Fine Detail Molecular Phylogenetic Studies in Plants: A Computational Approach and Sequence Repository

5 per sample. Conserved regions were used to design PCR primers for amplifying thousands of middle repetitive regions of the maize (Zea mays ssp. mays) genome. Bioinformatic scans were then used to identify DNA sequence polymorphisms in the low copy intervening sequences. When used in conjunction with simple DNA preps, optimized PCR conditions, high multiplex Illumina indexing and a bioinformatic marker calling platform tailored for repetitive sequences, this methodology provides a cost effective genotyping strategy for large-scale genomic selection projects. We show detailed results from four maize primer sets that produced between 1,335-3,225 good coverage loci with 1056 that segregated appropriately in a bi-parental family. This approach could have wide applicability to breeding and conservation biology, where hundreds of thousands of samples need to be genotyped for very minimal cost.

Population Genomic Analysis Reveals Differential Evolutionary Histories and Patterns of Diversity across Subgenomes and Subpopulations of Brassica napus L.

Abstract When studying the phylogenetic relationships of closely related species, it is often difficult to find nuclear DNA regions that are both easy to amplify across taxa and contain informative characters. To address this problem, we have created a database of gene sequences specifically selected to greatly increase both the likelihood of amplification across the species of interest and the likelihood of retrieving nuclear regions that are variable among these species. This result is achieved by designing primers flanking putative intron splicing sites within highly conserved genes. Over 40 species were sampled spanning rosids (14 taxa), asterids (12 taxa), grasses (seven taxa) and other angiosperms, as well as several gymnosperms, a moss, and a green alga.

Novel Loci Underlie Natural Variation in Vitamin E Levels in Maize Grain

The allotetraploid species Brassica napus L. is a global crop of major economic importance, providing canola oil (seed) and vegetables for human consumption and fodder and meal for livestock feed. Characterizing the genetic diversity present in the extant germplasm pool of B. napus is fundamental to better conserve, manage and utilize the genetic resources of this species. We used sequence-based genotyping to identify and genotype 30,881 SNPs in a diversity panel of 782 B. napus accessions, representing samples of winter and spring growth habits originating from 33 countries across Europe, Asia, and America. We detected strong population structure broadly concordant with growth habit and geography, and identified three major genetic groups: spring (SP), winter Europe (WE), and winter Asia (WA). Subpopulation-specific polymorphism patterns suggest enriched genetic diversity within the WA group and a smaller effective breeding population for the SP group compared to WE. Interestingly, the two subgenomes of B. napus appear to have different geographic origins, with phylogenetic analysis placing WE and WA as basal clades for the other subpopulations in the C and A subgenomes, respectively. Finally, we identified 16 genomic regions where the patterns of diversity differed markedly from the genome-wide average, several of which are suggestive of genomic inversions. The results obtained in this study constitute a valuable resource for worldwide breeding efforts and the genetic dissection and prediction of complex B. napus traits.

A Century of Guayule: Comprehensive Genetic Characterization of the Guayule (Parthenium argentatum A. Gray) USDA Germplasm Collection

This joint linkage and genome-wide association study comprehensively investigates natural variation in maize grain vitamin E levels using the 5000-line U.S. nested association-mapping panel. Tocopherols, tocotrienols, and plastochromanols (collectively termed tocochromanols) are lipid-soluble antioxidants synthesized by all plants. Their dietary intake, primarily from seed oils, provides vitamin E and other health benefits. Tocochromanol biosynthesis has been dissected in the dicot Arabidopsis thaliana, which has green, photosynthetic seeds, but our understanding of tocochromanol accumulation in major crops, whose seeds are nonphotosynthetic, remains limited. To understand the genetic control of tocochromanols in grain, we conducted a joint linkage and genome-wide association study in the 5000-line U.S. maize (Zea mays) nested association mapping panel. Fifty-two quantitative trait loci for individual and total tocochromanols were identified, and of the 14 resolved to individual genes, six encode novel activities affecting tocochromanols in plants. These include two chlorophyll biosynthetic enzymes that explain the majority of tocopherol variation, which was not predicted given that, like most major cereal crops, maize grain is nonphotosynthetic. This comprehensive assessment of natural variation in vitamin E levels in maize establishes the foundation for improving tocochromanol and vitamin E content in seeds of maize and other major cereal crops.

A comparative transcriptomic study of an allotetraploid and its diploid progenitors illustrates the unique advantages and challenges of RNA-seq in plant species

The fragility of a single-source, geographically concentrated supply of natural rubber, a critical material of the modern economy, has brought guayule (Parthenium argentatum A. Gray) to the forefront as an alternative source of natural rubber. The improvement of guayule for commercial-scale production has been limited by the lack of genomic tools and well-characterized genetic resources required for genomics-assisted breeding. To address this issue, we developed nearly 50,000 single nucleotide polymorphism (SNP) genetic markers and genotyped 69 accessions of guayule and its sister taxa mariola (Parthenium incanum Kunth), representing the entire available NALPGRU germplasm collection. We identified multiple interspecific hybrid accessions previously considered guayule, including six guayule-mariola hybrids and non-mariola interspecific hybrid accessions AZ-2 and AZ-3, two commonly used high-yielding cultivars. We dissected genetic diversity within the collection to identify a highly diverse subset of guayule accessions, and showed that wild guayule stands in Big Bend National Park, Texas, USA have the potential to provide hitherto untapped guayule genetic diversity. Together, these results provide the most thorough genetic characterization of guayule germplasm to date and lay the foundation for rapid genetic improvement of commercial guayule germplasm. Key Results Six guayule accessions are guayule-mariola hybrids Guayule collections from Big Bend National Park contain novel guayule genotypes not present in collections from Mexico Commonly cultivated accessions AZ2 and AZ3 contain introgressions from other Parthenium species The triploid accessions 11591, 11646, N576, N565, N565II, and RICHARDSON are generally indistinguishable from each other with respect to genetic background and likely represent the 4265-I source genotype (Johnson, 1950) Open pollinated and purposefully outcrossed tetraploid selections derived from 4265-I incorporate further genetic diversity and form distinct genotypes