Performing Department
Plant Biology
Non Technical Summary
Potatofaces special challenges in terms of genetic improvement due to the nature of its genome. Potato possesses four copies of each chromosome, making it more difficult than usual to incorporate desirable agronomic traits, such as disease resistance and yield,during the breeding process. This trait of possessing more than two copies of each chromosome, called polyploidy, also makes it difficult to identify the genes and genetic variants underlying traits of interest. To simplify the system and make genetic studies more feasible, one strategy that can be utilized is the production of potatoes that possess only two copies of each chromosome, called dihaploids. Potato dihaploids are made by fertilizing the target plant with a special group of potatoes known as in vitro pollinators. Once the pollination is performed, the eggs from the target plant develop with only half the number of chromosomes as a usual potato plant. This makes them far easier to use in genetic studies, and they can also be used to incorporate genetic material from wild potato that only possess two copies of each chromosome and are otherwise incompatible with cultivated potato. During the formation of dihaploids, some genetic material is sometimes incorporated from the pollinator plant into the mother plant. This outcome is undesirable, and the proposed projects seek to identify the prevalence of this phenomenon in two dihaploid populations generated from the common potato cultivars Atlantic and Superior. The project will also utilize the dihaploid populations from these plants to study, on a genome-wide scale, how changes at the genetic and epigenetic level influence agronomic traits in the field and expression of genes at the molecular level. The studies, altogether, will contribute to the community's knowledge of how dihaploid production works andprovide the potato breeding community withmore fine-scaled knowledge on how variation in the potato genome contributes to observable trait differences between different plants.
Animal Health Component
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Research Effort Categories
Basic
100%
Applied
0%
Developmental
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Goals / Objectives
This project will investigate the molecular mechanism of dihaploid induction in potato, a common technique to reduce ploidy level for genetic studies and breeding. Previous work demonstrates that DNA from the dihaploid inducer is transferred to progeny, an undesirable result. The first goal of this project seeks to characterize the extend of this phenomenon. The objectives to meet his goal are as follows:Characterize single nucleotide polymorphism in potato dihaploid populations and the dihaploid inducer parent plant. This will be achieved through the analysis of next-generation sequencing data.Discover instances of incidental introgression from the dihaploid inducerDevelop bioinformatics approaches to explain cases of introgressionDihaploid populations will then be used to characterize genes and genomic variation influencing field-measured agronomic traits, including yield, vine vigor, and height. This will be accomplished with the following objectives:Identify and characterize genome-wide copy number variation in dihaploid populationsIn QTL regions associated with traits, determine differential expression regulated by copy number variation of genesDetermine differential gene regulation regulated by histone marks contributing to extreme phenotypes
Project Methods
Aim 1. Determine extent and mechanisms of incidental IVP introgressionHypothesis and experimental approaches: For the following studies, dihaploid (2x) populations were generated by the fertilization of two tetraploid (4x) varieties, Atlantic and Superior, using a haploid-inducing S. phureja in vitro pollinator, IVP101. IVP101 has been demonstrated to show no evidence of somatic translocation during haploid induction using AFLP markers (Straadt and Rasmussen, 2003). However, another in vitro pollinator, IVP48, has been demonstrated to cause introgressions in the maternal genome (Clulow and Rousselle-Bourgeois, 1997; Straadt and Rasmussen, 2003; Wilkinson et al., 1995). A whole-genome assessment has not yet been performed to search for incorporation chromosomal segments from IVP101 during dihaploid induction. To assess this possibility, the Atlantic and Superior dihaploid populations will be analyzed at the whole-genome level to determine if chromosomal translocation has occurred, which would result in the invasion of Atlantic and Superior haplotypes by IVP101 genetic material.Atlantic and Superior tetraploids have already been sequenced and genotyped in previous work focused on intragenome heterogeneity of six tetraploid cultivars (Pham et al. in preparation). Using this information, and newly generated sequence data from IVP101, IVP101-specific SNPs will be identified for use as markers for chromosomal translocation in the dihaploid populations (Figure 1). At this time, the Atlantic and Superior dihaploid populations consisting of 158 and 95 individuals, respectively, have been sequenced using a whole-shotgun approach at a depth of approximately 8X genome coverage per line (Figure 1). The Atlantic and Superior dihaploids, as well as S. phureja IVP101, will be genotyped using the Genome Analysis Toolkit workflow. Occurrences of chromosomal translocation will be inferred by locating IVP101-specific SNPs in the dihaploid individuals. Bioinformatics methods will be developed to determine the possible molecular mode of introgression, including non-homologous end joining, non-crossover double-stranded break repair, and crossover double-stranded break repair (Table 1).Potential pitfalls and alternative strategies: Low-coverage sequencing, depending on filtering stringency, may result in spurious genotype calls. To address this issue, genotypes called in the dihaploid populations will only be considered if they are monomorphic in the entire population, effectively using the combined read depth in multiple samples to ensure that homozygous sites are properly identified. These sites can then be used to locate IVP101-specific alleles. Additionally, it will be difficult to distinguish between non-homologous end-joining and aneuploidy because it is not possible to tell if chromosomal segments are fused or separate. To distinguish these two, it may be necessary to implement use of a breakpoint determining software.Aim 2. Study differential gene expression and epigenetic regulation of QTL regions underlying agronomic traitsHypothesis and experimental approaches: The Atlantic and Superior dihaploid populations have been grown at two field locations in Michigan and data for various agronomic traits, including emergence, plant height, total tuber yield, and other traits, were collected in 2014 and 2015. Using SNP genotyping data generated by whole-genome sequencing, a genetic map will be constructed and QTLs for the measured traits will be identified. A subset of three lines each has been selected to represent high, medium, and low vigor individuals. For these individuals, RNA sequencing will be performed on tubers and leaves, and ChIP sequencing will be performed surveying H3K9me3, a mark of repression, and H3K4me3, a mark of activation. The RNA-seq data will be analyzed to find differential expression of genes between high, medium, and low vigor individuals, and differential histone methylation will be examined to determine if gene expression differences are epigenetically regulated. Additionally, these data can be used on a genome-wide scale to find other genes that may be transcriptionally regulated by epigenetic changes that are responsible for phenotypic extremes in potato. Because DNA sequence data will also be available for these lines, intragenome variation can be characterized to determine the role of structural variation and SNPs in phenotypic variation. For example, the inheritance of different gene copy numbers within QTLs may be a key driver of plant vigor. The inheritance of recessive deleterious alleles in a homozygous state may also result in deleterious phenotypes.Potential pitfalls and alternative strategies: Focusing on QTL regions is a strategy to limit the focus of genomic analyses on pre-defined regions of the genome. This narrow focus may dilute the importance of genome-wide changes in epigenetic status, for example. To address this, global epigenetic changes will be compared between lines if comparisons between high and low vigor lines in QTL regions indicates that there may be larger genome-wide changes affecting plant vigor. Additionally, if there have been gross structural genomic aberrations during dihaploid induction, this may impact QTL analysis results. To address this, the results from Aim 1 will be considered during analysis of data in Aim 2 to account for the possibility of large-scale genomic alterations.