Recipient Organization
UNIVERSITY OF NEVADA
(N/A)
RENO,NV 89557
Performing Department
(N/A)
Non Technical Summary
The overarching goal of this PARTNERSHIP proposal is to use new CRISPR/Cas9 mutants of sorghum [Sorghum bicolor (L.) Moench] to identify which gene at the INHIBITION OF ALIEN POLLEN (IAP) locus controls the ability of alien pollen tubes to reach the ovary during interspecific cross-pollination. IAP is the only known reproductive barrier locus in sorghum. We will characterize general plant morphology, reproductive biology, off-target copy number, and target gene expression in three mutant lines having lesions in three candidate genes at IAP (one putatively knocked-out gene per line). The specific iap allele (or combination thereof) that reduces/knocks out the expression of the inhibited alien pollen phenotype will be identified. Our specific aims are to 1) Identify which iap allele (or combination of alleles) enables alien pollen tubes to reach the sorghum ovary, 2) Differentiate tissue-specific gene expression profiles during cross-pollination events, and 3) Identify key genes involved in a compatible versus incompatible cross-pollination event using a co-expression analysis across different timepoints. Our long-term vision is to use information gained from the new CRISPR mutants to develop methods to either prevent or encourage outcrossing among sorghum and its wild relatives, depending on context. For example, outcrossing between the crop and closely-related weeds is undesirable, but enhancing the abiotic stress tolerance of the crop through breeding with more distant relatives is desirable.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The long-term goal of this research is to develop two types of new sorghum cultivars with different outcrossing abilities: those having a reduced capacity to accept the pollen of close wild relatives (by strengthening the reproductive barrier), and those having an enhanced capacity to accept the "alien" pollen of distant wild relatives (by removing the barrier).With this in mind, the specific goal of this project is to identify the causal genes contained within the INHIBITION OF ALIEN POLLEN (IAP) locus, which is currently the only known reproductive barrier locus (pre- or post-zygotic) in the Sorghum genus. Our previous efforts have identified three candidate genes involved in pistil-expressed cross-incompatibility at the IAP locus, but the strongest candidate (Sobic.002g023300) is not represented in any publicly available mutant libraries. Therefore, we have used CRISPR/Cas9 to generate three iap mutant lines (each having a lesion in one candidate gene) in a common genetic background (Tx430), and we propose to characterize the effects of each mutation on gene expression, plant morphology, and reproductive biology in order to pinpoint the causal allele(s) and ultimately create a set of publicly available plant materials and resources that will enable long-term studies of reproductive biology in the Sorghum crop-wild species complex.
Project Methods
Our efforts will be used to:Identify which iap allele (or combination of alleles) enables alien pollen tubes to reach the sorghum ovary in an interspecific cross. The winning mutant plants (having one or more iap alleles) will be evaluated for general plant morphology, reproductive biology, off-target copy number, and target gene expression. The causal allele will be validated by crossing to untransformed wild-type sorghum and observing restoration of the IAP phenotype, then backcrossing to the iap donor and observing restoration of the iap phenotype. The outputs of this aim will include confirmation of the causative IAP allele and a set of backcrossed lines with the iap knockout that can be used in further studies of CI in the Sorghum genus.Differentiate tissue-specific gene expression profiles for pollen and pistils during cross-pollination events. RNA-Seq data from the pistils and pollen of S. bicolor and S. plumosum will first be evaluated separately to identify variants that can be used to distinguish the two species, and then mixed samples of pollinated pistils will be genotyped based on these variants. The outputs of this aim will include tissue-specific expression profiles that can be used to distinguish cross-talk between male and female factors involved in pollination, and a bioinformatics pipeline that can be used to efficiently analyze pollinated pistils in future studies.Identify key genes involved in a compatible versus incompatible cross-pollination event using a co-expression analysis across different timepoints. RNA-Seq data from pollinated pistils will be collected at multiple timepoints following the initial pollination event and evaluated in networks of correlated genes that will be constructed based on similarities in their expression profiles over time. The outputs of this aim will include a list of genes possibly interacting with the IAP locus, and results will also be used to validate phenotypes observed in Aim 1 to confirm the expression or knockout of causal genes. Data will be analyzed to preliminarily identify any additional, as-yet-unidentified mechanisms of CI in Sorghum species by comparing it to other grasses.