Michael E. Foley

Multiple Loci and Epistases Control Genetic Variation for Seed Dormancy in Weedy Rice (Oryza sativa)

Weedy rice has much stronger seed dormancy than cultivated rice. A wild-like weedy strain SS18-2 was selected to investigate the genetic architecture underlying seed dormancy, a critical adaptive trait in plants. A framework genetic map covering the rice genome was constructed on the basis of 156 BC1 [EM93-1 (nondormant breeding line)//EM93-1/SS18-2] individuals. The mapping population was replicated using a split-tiller technique to control and better estimate the environmental variation. Dormancy was determined by germination of seeds after 1, 11, and 21 days of after-ripening (DAR). Six dormancy QTL, designated as qSDS-4, -6, -7-1, -7-2, -8, and -12, were identified. The locus qSDS-7-1 was tightly linked to the red pericarp color gene Rc. A QTL × DAR interaction was detected for qSDS-12, the locus with the largest main effect at 1, 11, and 21 DAR (R2 = 0.14, 0.24, and 0.20, respectively). Two, three, and four orders of epistases were detected with four, six, and six QTL, respectively. The higher-order epistases strongly suggest the presence of genetically complex networks in the regulation of variation for seed dormancy in natural populations and make it critical to select for a favorable combination of alleles at multiple loci in positional cloning of a target dormancy gene.

Seed dormancy: an update on terminology, physiological genetics, and quantitative trait loci regulating germinability

Dormancy is a form of developmental arrest and is an adaptive trait that promotes the survival of many organisms. In flowering plants, dormancy occurs in seeds and vegetative propagules (Lang 1996). Seed dormancy increases the distribution of germination over time, thus enhancing the survival of plants in an ever-changing environment. Seed dormancy is of intrinsic interest to weed scientists because it is one of 12 adaptive characteristics associated with weeds (Baker 1974). The sporadic emergence of seedlings derived from populations of dormant and nondormant weed seeds in the soil (Benech-Arnold et al. 2000; Forcella et al. 2000) is a key factor that dictates the need to apply weed control measures repeatedly within, between, and across growing seasons. My objective in writing this paper is to provide weed scientists, advanced students, and others with limited background information, some recent findings concerning the physiological genetics of dormancy, and steps toward identifying genes that directly regulate seed dormancy and germination. Molecular aspects of dormancy and germination will not be covered here because they have been reviewed recently (Bewley 1997; Li and Foley 1997). Readers can obtain additional and more extensive information on the biology and ecology of seed dormancy and germination from several recent books and reviews (Baskin and Baskin 1998; Benech-Arnold et al. 2000; Bewley and Black 1994; Casal and Sanchez 1998; Cohn 1996, 1998; Fennell 1999; Forcella et al. 2000; Hilhorst 1995, 1998; Hilhorst and Toorop 1997; Kelley et al. 1992; Kigel and Galili 1995; Simpson 1990; Vleeshouwers et al. 1995).

Association between seed dormancy and pericarp color is controlled by a pleiotropic gene that regulates abscisic acid and flavonoid synthesis in weedy red rice.

Seed dormancy has been associated with red grain color in cereal crops for a century. The association was linked to qSD7-1/qPC7, a cluster of quantitative trait loci for seed dormancy/pericarp color in weedy red rice. This research delimited qSD7-1/qPC7 to the Os07g11020 or Rc locus encoding a basic helix-loop-helix family transcription factor by intragenic recombinants and provided unambiguous evidence that the association arises from pleiotropy. The pleiotropic gene expressed in early developing seeds promoted expression of key genes for biosynthesis of abscisic acid (ABA), resulting in an increase in accumulation of the dormancy-inducing hormone; activated a conserved network of eight genes for flavonoid biosynthesis to produce the pigments in the lower epidermal cells of the pericarp tissue; and enhanced seed weight. Thus, the pleiotropic locus most likely controls the dormancy and pigment traits by regulating ABA and flavonoid biosynthetic pathways, respectively. The dormancy effect could be eliminated by a heat treatment, but could not be completely overcome by gibberellic acid or physical removal of the seed maternal tissues. The dormancy-enhancing alleles differentiated into two groups basically associated with tropical and temperate ecotypes of weedy rice. Of the pleiotropic effects, seed dormancy could contribute most to the weed adaptation. Pleiotropy prevents the use of the dormancy gene to improve resistance of white pericarp cultivars against pre-harvest sprouting through conventional breeding approaches.

Potential model weeds to study genomics, ecology, and physiology in the 21st century

Abstract Plant model systems have contributed greatly to the dramatic progress in understanding the fundamental aspects of plant biology. Using model weeds will also help facilitate focused funding and research in the weed science community. Criteria for developing model weeds require attention to weedy characteristics that impart economic losses and a wide geographic distribution, attributes that present the potential for political and scientific support. Expressed sequence tag (EST) databases for model weeds are the most practical approach to identifying new genes and obtaining data on the gene expression underlying weedy characteristics. Weeds such as Canada thistle, eastern black nightshade, johnsongrass, jointed goatgrass, leafy spurge, waterhemp, and weedy rice are proposed as model systems. Nomenclature: Canada thistle, Cirsium arvense (L.) Scop CIRAR; common waterhemp, Amaranthus rudis Sauer AMATA; eastern black nightshade, Solanum ptycanthum Dun. SOLPT; johnsongrass, Sorghum halepense (L.) Pers SORHA; jointed goatgrass, Aegilops cylindrica Host. AEGCY; leafy spurge, Euphorbia esula L. EUPES; red rice (weedy rice), Oryza sativa L. ORYSA; tall waterhemp, Amaranthus tuberculatus (Moq.) J. D. Sauer AMATU.

Characterization of an EST Database for the Perennial Weed Leafy Spurge: An Important Resource for Weed Biology Research

Abstract Genomics programs in the weed science community have not developed as rapidly as that of other crop, horticultural, forestry, and model plant systems. Development of genomic resources for selected model weeds are expected to enhance our understanding of weed biology, just as they have in other plant systems. In this report, we describe the development, characteristics, and information gained from an expressed sequence tag (EST) database for the perennial weed leafy spurge. ESTs were obtained using a normalized cDNA library prepared from a comprehensive collection of tissues. During the EST characterization process, redundancy was minimized by periodic subtractions of the normalized cDNA library. A sequencing success rate of 88% yielded 45,314 ESTs with an average read length of 671 nucleotides. Using bioinformatic analysis, the leafy spurge EST database was assembled into 23,472 unique sequences representing 19,015 unigenes (10,293 clusters and 8,722 singletons). Blast similarity searches to the GenBank nonredundant protein database identified 18,186 total matches, of which 14,205 were nonredundant. These data indicate that 77.4% of the 23,472 unique sequences and 74.7% of the 19,015 unigenes are similar to other known proteins. Further bioinformatics analysis indicated that 2,950, or 15.5%, of the unigenes have previously not been identified suggesting that some may be novel to leafy spurge. Functional classifications assigned to leafy spurge unique sequences using Munich Information Center for Protein or Gene Ontology were proportional to functional classifications for genes of arabidopsis, with the exception of unclassified or unknowns and transposable elements which were significantly reduced in leafy spurge. Although these EST resources have been developed for the purpose of constructing high-density leafy spurge microarrays, they are already providing valuable information related to sugar metabolism, cell cycle regulation, dormancy, terpenoid secondary metabolism, and flowering. Nomenclature: Leafy spurge, Euphorbia esula L. EPHES, arabidopsis, Arabidopsis thaliana (L.) Heynh

Selection and validation of endogenous reference genes for qRT-PCR analysis in leafy spurge (Euphorbia esula).

Quantitative real-time polymerase chain reaction (qRT-PCR) is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in various experimental conditions for seed, adventitious underground bud, and other organs of leafy spurge. Eleven candidate reference genes (BAM4, PU1, TRP-like, FRO1, ORE9, BAM1, SEU, ARF2, KAPP, ZTL, and MPK4) were selected from among 171 genes based on expression stabilities during seed germination and bud growth. The other ten candidate reference genes were selected from three different sources: (1) 3 stably expressed leafy spurge genes (60S, bZIP21, and MD-100) identified from the analyses of leafy spurge microarray data; (2) 3 orthologs of Arabidopsis “general purpose” traditional reference genes (GAPDH_1, GAPDH_2, and UBC); and (3) 4 orthologs of Arabidopsis stably expressed genes (UBC9, SAND, PTB, and F-box) identified from Affymetrix ATH1 whole-genome GeneChip studies. The expression stabilities of these 21 genes were ranked based on the CT values of 72 samples using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCT method. Our analyses revealed SAND, PTB, ORE9, and ARF2 to be the most appropriate reference genes for accurate normalization of gene expression data. Since SAND and PTB were obtained from 4 orthologs of Arabidopsis, while ORE9 and ARF2 were selected from 171 leafy spurge genes, it was more efficient to identify good reference genes from the orthologs of other plant species that were known to be stably expressed than that of randomly testing endogenous genes. Nevertheless, the two newly identified leafy spurge genes, ORE9 and ARF2, can serve as orthologous candidates in the search for reference genes from other plant species.

Bud Dormancy in Perennial Plants: A Mechanism for Survival

Dormancy in vegetative buds of perennial plants plays an important role for surviving harsh environmental conditions. Identifying the genetic and physiological mechanisms regulating dormancy in these vegetative structures will allow manipulation of plant growth and development in both crops and weeds. Model plants have been used to study the physiological effects that photoperiod and temperature impart on dormancy regulation in perennial buds. At the molecular level, models derived through analysis of the transcriptome have shed new light on multiple cellular pathways and physiological processes associated with dormancy transitions and, in some cases, have revealed overlap with pathways regulating flowering and cold acclimation. In this chapter, we discuss proposed models based on advances to our understanding of physiological and molecular factors affecting dormancy regulation in vegetative buds of perennials.

A genetic model and molecular markers for wild oat (Avena fatua L.) seed dormancy.

Abstract Seed dormancy allows weed seeds to persist in agricultural soils. Wild oat (Avena fatua L.) is a major weed of cereal grains and expresses a range of seed dormancy phenotypes. Genetic analysis of wild oat dormancy has been complicated by the difficulty of phenotypic classification in segregating populations. Therefore, little is known about the nature of the genes that regulate dormancy in wild oat. The objectives of our studies were to develop methods to classify the germination responses of segregating wild oat populations and to find molecular markers linked to quantitative trait loci (QTL) that regulate seed dormancy in wild oat. RAPD markers OPX-06 and OPT-04 explained 12.6% and 6.8% respectively, of the F2 phenotypic variance. OPF-17 was not significant in a simple regression model, but it was linked in repulsion to OPT-04. A three-locus model of seed dormancy in wild oat is presented based on the 41-day germination profiles of F1, F2, F3, BC1P1F1, BC1P1F2, and BC1P2F1 generations, and the 113 day germination profile of 126 F7 recombinant inbred lines. Loci G1 and G2 promote early germination, and the D locus promotes late germination. If at least one copy of the dominant G1 or G2 alleles are present regardless of the genotype at D locus, then the individual will be nondormant. If the genotype is g1g1g2g2D_, then the phenotype will be dormant.

Isolation of three dormancy QTLs as Mendelian factors in rice

Seed dormancy is a key adaptive trait under polygenic control in many plants. We introduced the chromosomal regions containing the dormancy QTLs qSD1, qSD7-1, and qSD12 from an accession of weedy rice into a nondormant genetic background to examine component genetic effects and their interactions with time of afterripening (DAR). A BC4F2 plant, which was heterozygous for the three loci, was selected to develop the BC4F3 population. Single point analysis detected only qSD7-1 and qSD12 (R2=38–72%) at 10, 30, and 50 DAR in the population. However, multiple linear regression analysis detected genetic effects of the three QTLs and their trigenic epistasis, an environmental effect of DAR (E), and interactions of E with qSD12 and with the qSD1 × qSD7-1 and qSD7-1 × qSD12 epistases. The linear model demonstrates that QTL main effects varied with DAR, and that some epistasis or epistasis-by-DAR interactions partially counteract the main effects. The three QTLs were isolated as single Mendelian factors from the BC4F3 population and estimated for component genic effects based on the BC4F4 populations. Isolation improved estimation of the qSD1 effect and confirmed the major effect of qSD12. The qSD1 and qSD12 loci displayed a gene-additive effect. The qSD7-1, which was further narrowed to a chromosomal region encompassing the red pericarp color gene Rc, displayed gene additive and dominant effects.

Genetic diversity of Canada thistle (Cirsium arvense) in North Dakota

Abstract Canada thistle is a noxious weed that occurs in a wide range of habitats and is difficult to control because of its extensive root system and prolific seed production. Here, we focused on estimating the level of genetic diversity between populations in North Dakota as a first step in examining diversity across North America. Two types of genetic marker, intersimple sequence repeats (ISSRs) and microsatellites were used. Both marker types resulted in polymorphic alleles suitable for assessing diversity. Analysis of molecular variance (AMOVA), molecular diversity analyses, and cluster analysis were conducted. Highly significant variation was detected between populations (P < 0.01). The greatest variance recovered was between individuals within populations. Gene flow among populations in the Northern Great Plains was indicated by the presence of shared alleles between the North Dakota and Minnesota populations and in cluster formation. Multiple introductions and continued gene flow between populations has led to the continued success of Canada thistle as an invasive plant in North America. Nomenclature: Canada thistle, Cirsium arvense (L.) Scop. CIRAR.