Location: Nat'l Clonal Germplasm Rep - Tree Fruit & Nut Crops & Grapes
Project Number: 2032-30100-001-006-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2024
End Date: Jul 31, 2025
Objective:
Objective 1. Characterize the field performance, nut quality, and transcriptional profiles of Juglans trees from the Himalayan center of origin: So far, the ancestral allele at the selective sweep is perfectly correlated with the presence of very thick shells. However, data are sparse, and other phenotypes potentially associated with either the selective sweep or wild walnuts in general have not been characterized. For example, it remains unknown whether wild walnuts show differences in hull dehiscence compared to domesticated J. regia.
Objective 2. Phenotype and genotype all trees in the Juglans D block: This is the only Juglans block that was not included in previous global diversity survey of J. regia (Arab et al. in prep).
Objective 3. Assemble two long-read genome assemblies for wild walnuts: Currently no truly wild walnuts have been sequenced, and the sequence of the ancestral allele at the selective sweep is not known. All existing J. regia assemblies are from individuals homozygous for the derived allele, this haplotype contains numerous structural rearrangements relative to haplotypes in other Juglans species, and it is not clear which of these rearrangements may have occurred during the domestication of J. regia.
Approach:
Objective 1: All trees from the Kashmir region of northern Pakistan and India, including the 35 individual trees from the 10 accessions that carry the ancestral allele, will be phenotyped for the same set of field and quality traits that Cooperator used for breeding evaluation previously. In addition, eight trees will be selected for a transcriptome experiment: developing nuts will be sampled for RNA extraction and RNAseq at two time points in four trees heterozygous for the ancestral allele and four trees homozygous for the wild-type allele. Individuals homozygous for the ancestral allele are few in number and are not currently bearing sufficient nuts to support both this objective and the nut quality phenotyping. STAR, FeatureCounts, and limma will be used for alignment and identification of differential gene expression between genotypes.
Objective 2: 175 additional trees, mostly J. regia from Kyrgyzstan, China, and Azerbaijan, that could add to the understanding of J. regia diversity and domestication, will be phenotyped for the same set of field and quality traits used for breeding evaluation and included in Objective 1.
Objective 3: Cooperator will use the PacBio Revio platform to generate phased long-read assemblies for two individuals, one homozygous for the ancestral allele, and one heterozygote used for transcriptome work in Objective 1. The resulting haplotypes will be used to identify SNPs and structural variants that differentiate the derived and ancestral J. regia haplotypes in this region. The resulting dataset will be used to catalog all functional mutations, including non-synonymous SNPs in predicted coding regions, for potential use in allele mining.
