Performing Department
School of Plant and Environmental Sciences
Non Technical Summary
Florist's gloxinias (Sinningia speciosa) are attractive ornamental plants with devoted growers and consumers across America and Europe. Sinningia, with probably Saintpaula, are the most popular genus from the Gesneriaceae family. The popularity of S. speciosa as ornamental plant is based in two factors: (1) plants display a wide range of ornamental phenotypes, not only for flower color and morphology, but also for leaf color and shape including several types of double corolla phenotypes, and (2) they are easy to propagate by leaf or stem cuttings. The current wholesale prices for Sinningia are unknown, but some of the ornamental characteristics of this plant could make it a potentially attractive new crop for the fast growing nursery industry in the state of Virginia. Virginia is not one of the top fifteen ornamental crops producers in the US. The ornamental production is limited a several nurseries mainly in the Virginia Beach area, so most of the ornamentals consumed in Virginia come from other states, such as California, Florida and North Carolina. Nevertheless the development of new varieties with novel phenotypes such as double corollas could help to increase the production of floral crops in the state of Virginia, helping to the diversification of the economy with stable values such as flower production. The elucidation of molecular mechanisms involved in the production of double corolla phenotypes could be applied to develop new varieties more attractive to growers, retailers and consumers.Sinningia has a complex breeding history. We propose the genetic study of the domestication of Sinningia as an approach to search for genes involved in specific ornamental traits such as flower color and flower morphology. Additionally we will search for genes controlling the double corolla as a way to generate knowledge that could be applied to other floral crops (e.g. petunias). The development of new methods for massively-parallel DNA sequencing, such as the Illumina or PacBio sequencing platforms, make it possible to apply whole genome sequencing (WGS), genotyping-by-sequencing (GBS), and RNA-Seq methods to the genetic study of sinningia in a cost-effective manner. We will use two different complementary approaches to search candidate genes associated with these traits: (1) Quantitative Trait Loci (QTL) analysis to search for correlations between genomic regions and phenotypes using GBS genetic markers; (2) RNA-Seq analysis to search candidate genes based in differential expression between two or more samples. The result intersection between genome location (QTL analysis) and differentially expressed genes (RNA-Seq analysis) will reduce the list of candidate genes to a manageable size for a posterior functional validation. The results of the proposed study will have a favorable impact the breeding of this species, accelerating the development of more attractive varieties. Additionally, the use of Sinningia as a model for domestication can help us understand the impact of human manipulation on plant species, and how our search for specific phenotypes (e.g. double corolla) can modify the genetic information in these species, by selecting defective alleles for genes (e.g. in the flower development pathway as a way to increase the yield in crops).
Animal Health Component
0%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
ObjectivesThe main objective of this project is to produce a genetic description of the process of domestication and breeding in the species Sinningia speciosa (the florist's gloxinia), highlighting possible alleles and/or chromosome regions that have been selected during this process and that can be involved in specific ornamental traits. Specific objectives for this project are:Objective 1: Development of Sinningia speciosa reference genome and other genomic resources (e.g. gene expression atlas) to facilitate the use of approaches such as genomic selection in Sinningia speciosa breeding.Objective 2: Genetic characterization of wild and cultivated accessions to evaluate and describe: (a) the genetic diversity, (b) potential genetic bottlenecks that occurred during the breeding history, and (c) wild species introgressions into old and modern cultivars.Objective 3: Development and Quantitative Trait Loci (QTL) analysis of two mapping populations (S. speciosa var. "Buzios" x S. speciosa var. "Empress" for domesticated traits such as erect flowers and S. speciosa var. "Love Potion" x S. speciosa var. "Merry Christmas" for double corolla) to detect marker loci linked to Sinningia speciosa ornamental traits.Objective 4: Transcriptomic characterization of Sinningia speciosa, two mapping population samples and six to ten cultivars to search for candidate genes involved in begonia ornamental traits.
Project Methods
Plant ResourcesWe will use three different sources to get obtain material for this project:The Gesneriad Society (http://www.gesneriadsociety.org/) has a seed fund with >30 accessions.Different retail companies such as The Violet Barn (https://www.violetbarn.com/) where various commercial accessions can be purchased.A collaboration with Prof. Cassia Sakuragui (Universidade Federal do Rio de Janeiro) to explore the natural diversity of Sinningia speciosa in the wild.We will grow the plants, when needed, at the Virginia Tech greenhouses (http://www.hort.vt.edu/greenhouse/). Soil, pots, rooting hormone (for vegetative propagation), and fertilizers will be purchased locally. For objective 3, we will grow between three and six progenitor plants for the mapping population. We are planning to obtain F2 seeds in years 1 and 2. We will grow 150 plants per F2 mapping population for the QTL analysis for years 2 and 3. We will reproduce the different lines using vegetative propagation. For objective 4, we will grow 12-15 plants reproduced by cuttings from single plants of each accession during years 1 and 2, except for the bulk segregation analysis, where we will need to have segregating phenotypes for the mapping population, so we will grow the plants during years 3 and 4.DNA will be extracted using the DNeasy Plant Mini Kit® from Qiagen. RNA will be extracted using Qiagen RNeasy Plant Mini Kit®. DNA concentration and integrity will be evaluated with a Qubit® instrument and a Bioanalyzer 2100. Both instruments are available in the Bombarely laboratory.Objective 1: Development of a Sinningia speciosa reference genomeS. speciosa were developed through the domestication of one (or several) wild populations, although it is probable that some lines have been crossed with other species (e.g. S. macrophylla). Our approach will be focused on one species, S. speciosa (estimated genome size of 392 Mb), and completing the dataset with the re-sequencing of the three other wild species (S. macrophylla, S. helleri and S. pusilla) that may be participated in the Sinningia breeding. Additionally we will re-sequence three to six other varieties (wild types and cultivars). The design for the whole genome sequencing of these species will be:S. speciosa, hybrid assembly with Illumina and PacBio reads.75X of 2x125 Illumina pair ends (insert size 500 bp).80X of 2x100 Illumina mate pairs (insert size 2 Kb and 5Kb).20X of PacBio reads.S. macrophylla, S. helleriandS. pusilla, Illumina resequencing.20X of 2x150 Illumina pair ends (insert size 300 bp).Other cultivars (3-6), Illumina resequencing.20X of 2x150 Illumina pair ends (insert size 300 bp).Reads will be processed with Fastq-mcf. Illumina reads will be corrected with Musket and PacBio reads with LoRDEC. We will use SOAPdenovo2 to assemble the Illumina reads, and Sprai and the Celera Assembler to assemble the PacBio reads. Both assemblies will then be evaluated, and the best assembly will be used as the base, using the other read set to re-scaffold the assembly with SSPACE and to fill the gaps with GapCloser or PBJelly.We will annotate the different genomes using Maker-P, and also generate a reference transcriptome as evidence-based support for the annotation. RNA extracted from roots, leaves, stems, flowers will be sequenced on an Illumina instrument (2x150 bp, 300 bp insert size, 10-20 million reads/sample). Trinity will be used to assemble the reads. All the programs will be run on a 64-core server with 256 Gb of RAM and 7 Tb of disk space in the Bombarely laboratory. If more computational power is needed, the ARC resources at Virginia Tech will be used (https://www.arc.vt.edu/).Objective 2: Genetic characterization of wild and cultivated accessionsWe will use Genotyping-By-Sequencing (GBS) to characterize genetically wild and cultivated Sinningia accessions. GBS is a relatively cost-effective approach to rapidly generate thousand of polymorphic molecular markers. Based in the small genome size of these species, we will use a 5 bp restriction site enzyme (ApeKI), sequencing 0.5-5 million of reads per sample (Illumina; 1x100). Reads will be processed with Fastq-mcf and mapped to the reference genome using Bowtie2 or BWA depending of their performance. Sam files will be processed with Samtools. DNA sequence polymorphisms will be detected using GATK or FreeBayes with a minimum read depth of 10 and a minimum mapping score of 20. We will estimate the genetic diversity of each sample using a custom Perl script. The population structure will be analyzed using Structure and FineStructure. Because several species will be used, we will analyze their phylogenetic relations using SNAPP.Objective 3: QTL analysis for sinningia agronomical/ornamental traitsThere are several agronomical/ornamental traits that we will analyze in this project and that are related to the domestication and breeding of S. speciosa. The wild S. speciosa is a perennial herb with short stems and light to dark green. Flowers have bilateral symmetry and five petals. They are slipper shaped. There are a number of important traits that have been selected in the last hundred years during the breeding of S. speciosa, such as (1) Flower shape (erect flowers), (2) Flower color and patterning, (3) Flower size and petal number, (4) flower longevity (longer-lived. Additionally we will characterize other traits applicable to other floral crops such as (5) double corolla. Our approach to find genomic regions responsible for these traits is to generate two F2 mapping populations derived from a wild type accession ("Buzios") crossed with a cultivated accession ("Empress"). Additionally we will develop a population for the double corolla trait (staments to petals) crossing the single corolla accession "Love Potion" with the double corolla accession "Merry Christmas". We will analyze between 80 and 150 F2 lines (3 plants/each) for the traits described previously. Phenotypes such as flower and leaf color and morphology will be measured by scanning 3-5 flowers of each plant and analyzing the images (http://www.plant-image-analysis.org/). We will also collect other phenotypic data such as number of flowers, size of the plant and longevity of the flower. One plant per line will be genotyped using GBS. The GBS reads will be processed as was described in Objective 1. QTL analysis will be performed using R/QTL.Objective 4: Transcriptomic characterization of begonia accessionsTranscriptomics can be a useful tool to gain a better understanding of molecular mechanisms involved in any biological process. We will use RNA-seq to study the gene expression in several Sinningia accessions. We expect that in combination with the QTL results, RNA-seq will provide a complete framework in which to elucidate the most relevant genes involved in the traits that have been selected during the begonia breeding. We will use two different sets: (1) two wild and three or four cultivated accessions with different phenotypes with three biological replicates per sample and three plants per biological replicate, and (2) bulks of plants with similar phenotypes from different F2 mapping population lines (two bulks of 5-10 lines for each trait, three traits to analyze: flower color, single/double corolla, flower shape). Five organs/developmental stage will be analyzed: mature leaf, flower meristem, flower bud, flower petals, flower staments, flower pistils. Each of the samples will be subjected to Illumina sequencing (1x100 nucleotide reads, 5-10 million reads/sample). The reads will be processed as described previously in the first objective. Reads will be mapped with Tophat2 or HISAT. The differential expression analysis will be performed using Cufflinks or Stringtie. Some of the downstream analyses will involve cluster and Gene Set Enrichment Analysis (GSEA) using several R and Bioconductor software packages such as CummeRBund, Ballgown and TopGo.