Genetic and Genomic Explorations of Flowering Time Components in Lettuce

Lettuce (Lactuca sativa) is one of the most popular leafy vegetables in the United States. A popular leafy vegetable harvested in its vegetative stage, cultivated lettuce has been selected for delayed flowering and insensitivity to photoperiod, both intentionally and unintentionally. In the past decades, the advancement in functional genetics in model organisms and the development of marker technologies have allowed fast and accurate mapping of quantitative trait loci (QTL) that control flowering time in lettuce. These past endeavors have been summarized in Chapter Two of this dissertation. In Chapters Three and Four of this dissertation, I mapped QTLs for two distinct and understudied components of flowering time in lettuce: daily floral opening time and photoperiod sensitivity. Chapter Three reported the discovery of two QTLs, qDFO2.1 and 8.1, that controlled daily floral opening time in lettuce. These are the first genetic loci reported to regulate floral opening. The study in Chapter Four revealed five QTLs on Chromosomes 1, 2 and 4 that explained photoperiod sensitivity in lettuce; these QTLs were distinct from loci associated with long-day flowering-time in lettuce. Chapter Four also identified a candidate gene as the functional ortholog of Arabidopsis CONSTANS in lettuce. These results supported the complex, networked architecture of the flowering time regulatory pathways in lettuce. Chapter Five highlighted the importance of microRNAs (miRNAs) in floral development. This chapter provided updated, tissue-specific miRNA and miRNA target annotations for lettuce. The diversity of DNA sequence variations at miRNA regions within the genus Lactuca was also characterized. The differential expression pattern of miRNAs between vegetative and floral tissues supported a model where archetypal miRNAs, miRNA156 and miRNA172, and their regulatory functions were conserved between Arabidopsis and lettuce. Chapter Six of the dissertation proposed promising future experiments to extend the novel methodologies developed in this dissertation and to further elucidate the molecular regulatory network for flowering time in lettuce. Overall, this dissertation found new angles to provide knowledge in the realms of genetics and genomics of flowering time regulation in lettuce. Results from these studies can be used to effectively accelerate vegetable breeding efforts

Variance of allele balance calculated from low coverage sequencing data infers departure from a diploid state

BackgroundPolyploidy and heterokaryosis are common and consequential genetic phenomena that increase the number of haplotypes in an organism and complicate whole-genome sequence analysis. Allele balance has been used to infer polyploidy and heterokaryosis in diverse organisms using read sets sequenced to greater than 50× whole-genome coverage. However, sequencing to adequate depth is costly if applied to multiple individuals or large genomes.ResultsWe developed VCFvariance.pl to utilize the variance of allele balance to infer polyploidy and/or heterokaryosis at low sequence coverage. This analysis requires as little as 10× whole-genome coverage and reduces the allele balance profile down to a single value, which can be used to determine if an individual has two or more haplotypes. This approach was validated using simulated, synthetic, and authentic read sets from the oomycete species Bremia lactucae and Phytophthora infestans, the fungal species Saccharomyces cerevisiae, and the plant species Arabidopsis arenosa. This approach was deployed to determine that nine of 21 genotyped European race-type isolates of Bremia lactucae were inconsistent with diploidy and therefore likely heterokaryotic.ConclusionsVariance of allele balance is a reliable metric to detect departures from a diploid state, including polyploidy, heterokaryosis, a mixed sample, or chromosomal copy number variation. Deploying this strategy is computationally inexpensive, can reduce the cost of sequencing by up to 80%, and used to test any organism

Quantitative Trait Loci and Candidate Genes Associated with Photoperiod Sensitivity in Lettuce (Lactuca spp.)

Key messageA population of lettuce that segregated for photoperiod sensitivity was planted under long-day and short-day conditions. Genetic mapping revealed two distinct sets of QTLs controlling daylength-independent and photoperiod-sensitive flowering time. The molecular mechanism of flowering time regulation in lettuce is of interest to both geneticists and breeders because of the extensive impact of this trait on agricultural production. Lettuce is a facultative long-day plant which changes in flowering time in response to photoperiod. Variations exist in both flowering time and the degree of photoperiod sensitivity among accessions of wild (Lactuca serriola) and cultivated (L. sativa) lettuce. An F6 population of 236 recombinant inbred lines (RILs) was previously developed from a cross between a late-flowering, photoperiod-sensitive L. serriola accession and an early-flowering, photoperiod-insensitive L. sativa accession. This population was planted under long-day (LD) and short-day (SD) conditions in a total of four field and screenhouse trials; the developmental phenotype was scored weekly in each trial. Using genotyping-by-sequencing (GBS) data of the RILs, quantitative trait loci (QTL) mapping revealed five flowering time QTLs that together explained more than 20% of the variation in flowering time under LD conditions. Using two independent statistical models to extract the photoperiod sensitivity phenotype from the LD and SD flowering time data, we identified an additional five QTLs that together explained more than 30% of the variation in photoperiod sensitivity in the population. Orthology and sequence analysis of genes within the nine QTLs revealed potential functional equivalents in the lettuce genome to the key regulators of flowering time and photoperiodism, FD and CONSTANS, respectively, in Arabidopsis

Drought and Competition With Ivyleaf Morningglory (Ipomoea hederacea) Inhibit Corn and Soybean Growth

National audienceAgricultural impacts of climate change include direct effects on crop plants and indirect effects, such as changes to the distributions and competitiveness of weed species. In the northeastern United States, warming temperatures are likely to result in periods of soil moisture deficit and changes to weed communities. Ivyleaf morningglory (IMG, Ipomoea hederacea Jacq.) is a summer annual vine that competes with field crops and interferes with harvesting. Climate change may increase the competitive effects of IMG on northeastern U.S. field crops. We conducted a greenhouse study to evaluate the effects of IMG on corn ( Zea mays L.) and soybean [ Glycine max (L.) Merr.] under drought and non-drought conditions. The drought treatment was crossed against an IMG competition treatment with five levels: one crop plant without IMG plants, one crop plant with one, two, or three IMG plants, and one IMG plant without crop plants. Both drought and IMG (presence or biomass) reduced the biomass of corn and soybean ( P < 0.05). Drought and IMG (presence) reduced soybean pod production ( P < 0.001). IMG biomass was reduced by drought and the presence of corn ( P < 0.001). Across all competition treatments, drought reduced IMG biomass by 71% in the corn experiment and 79% in the soybean experiment, compared with a corn biomass reduction of 50% and a soybean biomass reduction of 58%. Well-designed management programs should mitigate the risks associated with stressors such as IMG and drought, which may threaten northeastern U.S. field crop production under climate change

Genome-Wide Analysis of Cyclophilin Proteins in 21 Oomycetes

Cyclophilins (CYPs), a highly-conserved family of proteins, belong to a subgroup of immunophilins. Ubiquitous in eukaryotes and prokaryotes, CYPs have peptidyl-prolyl cis-trans isomerase (PPIase) activity and have been implicated as virulence factors in plant pathogenesis by oomycetes. We identified 16 CYP orthogroups from 21 diverse oomycetes. Each species was found to encode 15 to 35 CYP genes. Three of these orthogroups contained proteins with signal peptides at the N-terminal end, suggesting a role in secretion. Multidomain analysis revealed five conserved motifs of the CYP domain of oomycetes shared with other eukaryotic PPIases. Expression analysis of CYP proteins in different asexual life stages of the hemibiotrophic Phytophthora infestans and the biotrophic Plasmopara halstedii demonstrated distinct expression profiles between life stages. In addition to providing detailed comparative information on the CYPs in multiple oomycetes, this study identified candidate CYP effectors that could be the foundation for future studies of virulence

AFLAP: assembly-free linkage analysis pipeline using k-mers from genome sequencing data.

Our assembly-free linkage analysis pipeline (AFLAP) identifies segregating markers as k-mers in the raw reads without using a reference genome assembly for calling variants and provides genotype tables for the construction of unbiased, high-density genetic maps without a genome assembly. AFLAP is validated and contrasted to a conventional workflow using simulated data. AFLAP is applied to whole genome sequencing and genotype-by-sequencing data of F1, F2, and recombinant inbred populations of two different plant species, producing genetic maps that are concordant with genome assemblies. The AFLAP-based genetic map for Bremia lactucae enables the production of a chromosome-scale genome assembly

A Composite Analysis of Flowering Time Regulation in Lettuce

Plants undergo profound physiological changes when transitioning from vegetative to reproductive growth. These changes affect crop production, as in the case of leafy vegetables. Lettuce is one of the most valuable leafy vegetable crops in the world. Past genetic studies have identified multiple quantitative trait loci (QTLs) that affect the timing of the floral transition in lettuce. Extensive functional molecular studies in the model organism Arabidopsis provide the opportunity to transfer knowledge to lettuce to explore the mechanisms through which genetic variations translate into changes in flowering time. In this review, we integrated results from past genetic and molecular studies for flowering time in lettuce with orthology and functional inference from Arabidopsis. This summarizes the basis for all known genetic variation underlying the phenotypic diversity of flowering time in lettuce and how the genetics of flowering time in lettuce projects onto the established pathways controlling flowering time in plants. This comprehensive overview reveals patterns across experiments as well as areas in need of further study. Our review also represents a resource for developing cultivars with delayed flowering time

High resolution genetic dissection of the major QTL for tipburn resistance in lettuce, Lactuca sativa

Tipburn is an important physiological disorder of lettuce, Lactuca sativa L., related to calcium deficiency that can result in leaf necrosis and unmarketable crops. The major quantitative trait locus (QTL), qTPB5.2, can account for up to 70% of the phenotypic variance for tipburn incidence in the field. This QTL was genetically dissected to identify candidate genes for tipburn by creating lines with recombination events within the QTL and assessing their resistance to tipburn. By comparing lines with contrasting haplotypes, the genetic region was narrowed down to ∼877 Kb that was associated with a reduction of tipburn by ∼60%. Analysis of the lettuce reference genome sequence revealed 12 genes in this region, one of which is a calcium transporter with a single nucleotide polymorphism in an exon between haplotypes with contrasting phenotypes. RNA-seq analysis of recombinants revealed two genes that were differentially expressed between contrasting haplotypes consistent with the tipburn phenotype. One encodes a Teosinte branched1/Cycloidea/Proliferating Cell factor transcription factor; however, differential expression of the calcium transporter was detected. The phenotypic data indicated that there is a second region outside of the ∼877 Kb region but within the QTL, at which a haplotype from the susceptible parent decreased tipburn by 10-20%. A recombinant line was identified with beneficial haplotypes in each region from both parents that showed greater tipburn resistance than the resistant parent; this line could be used as the foundation for breeding cultivars with more resistance than is currently available

AFLAP: assembly-free linkage analysis pipeline using k-mers from genome sequencing data.

Our assembly-free linkage analysis pipeline (AFLAP) identifies segregating markers as k-mers in the raw reads without using a reference genome assembly for calling variants and provides genotype tables for the construction of unbiased, high-density genetic maps without a genome assembly. AFLAP is validated and contrasted to a conventional workflow using simulated data. AFLAP is applied to whole genome sequencing and genotype-by-sequencing data of F1, F2, and recombinant inbred populations of two different plant species, producing genetic maps that are concordant with genome assemblies. The AFLAP-based genetic map for Bremia lactucae enables the production of a chromosome-scale genome assembly