The overall goal of the genetics research of the rootstock working group is to discover the underlying genetic basis of resistance in walnut to crown gall, Phytophthora, lesion nematode, and Armillaria through pathology screening and development of molecular markers to utilize in the development of improved walnut rootstock. Mapping resistance genes and quantitative trait loci (QTL) with molecular markers will allow us to identify markers linked to resistance genes/QTL thus accelerating the development of improved rootstocks at reduced cost.

The group has previously developed, or currently has in development, multiple genetic and genomic resources for J. regia. Resources currently available are single nucleotide polymorphisms (SNPs) located in gene coding regions, a 6K Illumina Infinium SNP assay, a SNP-based genetic map and a physical map for J. regia, bacterial artificial chromosome (BAC) end sequences (BES), gene tags mapped on the physical map, and RNA expression profiles of 20 tissues and/or developmental stages. Currently a whole genome shotgun (WGS) sequence of J. regia ‘Chandler’ is being assembled and WGS sequencing is in preparation for wild species J. microcarpa and J. cathayensis, a separate project funded by the California Walnut Board.

Our specific goals are to 1) complete development of a high resolution English walnut (J. regia) reference genome sequence, which will facilitate marker development and mapping resistance genes, 2) map genes controlling resistance to soil-borne pathogens in wild Juglans mother trees, and 3) develop molecular markers for efficient development of resistant rootstocks.


Though technically this phase of the research is just beginning, the group already has numerous resources developed or in development (listed previously). One foundation for future work is to produce a completed assembly of the J. regia ‘Chandler’ genome sequence. To facilitate completion of the genome assembly we will nanomap the walnut genome, which will aid ordering and orienting the scaffolds onto the physical map and close any gaps between them. Nanomapping of the walnut genome will aid in transforming the currently assembled scaffolds into pseudomoleules, representing the 16 walnut chromosomes, and resulting in a reference genome for J. regia. To better annotate the genome for genes expressed in walnut roots, we will also add four root transcriptomes from rootstocks showing susceptibility or tolerance in response to soil-borne pathogens to the 20 scion tissue and/or developmental stage transcriptomes available from prior work.

Preliminary data supports that resistance is expressed in hybrids of wild Juglans spp. and J. regia, such as the commercially available rootstock RX1 (J. microcarpa x J. regia) that exhibits elevated resistance to Phytopthora spp. (PHY). Our preliminary crown gall (CG) screening demonstrates the ability of wild Juglans spp. and J. regia hybrids to express CG resistance. To utilize disease resistance found in wild species we must first identify mother trees with resistance from half-sib families, which are more efficient to produce in terms of cost, time, and labor. The half-sib family mean resistance of J. microcarpa accessions DJUG31.01 and DJUG31.07 to PHY and CG was significantly higher than the population mean. Likewise, half-sib progeny of J. cathayensis (accession DJUG 11.03) showed a greater degree of resistance to lesion nematode (NEM) than progeny of other mother trees tested. The segregation of resistant progeny in these and several other mother trees where the half-sib family means suggest resistance will be validated in a second round of half-sib evaluations. Mother trees showing significantly higher half-sib family means for resistance than the population mean will be used for gene/QTL mapping of resistance genes using populations of full-sib F1 interspecific hybrids.

We are working with resistance from several different Juglans species in this project, but SNPs are largely species-specific. Therefore our SNP discovery and genotyping strategy must be applicable to all species. Genotyping by sequencing (GBS) is a highly parallel SNP discovery and genotyping approach utilizing next generation sequencing (NGS) technology that can be used for genotyping polymorphisms and genetic map construction. The GBS informatic pipeline will utilize the walnut reference genome by aligning NGS reads to it and generating sequence tags to a file containing the genomic position of each. Partial WGS sequences will also be produced for J. microcarpa and J. cathayensis in a separate project by this group and funded by the California Walnut Board. The reference genome sequences of the three species will facilitate efficient use of GBS and discovery of genes controlling resistance to targeted diseases.

DNA from each tree in the full-sib populations will be submitted to the Institute for Genomic Diversity at Cornell University for GBS, resulting in thousands to tens of thousands of SNPs, for mapping segregating SNPs and disease resistance. We anticipate that each map will contain several thousand gene loci aligned against the reference genome sequences. Since we do not know the genetic architecture of resistance in the MTs, we a priori assume quantitative inheritance of resistance and will use a QTL mapping approach.

Nucleotide sequences surrounding gene-based SNPs linked to a resistance gene on the genetic map will be used in the development of molecular markers for application in rootstock breeding. Though a number of commercial platforms for high throughput SNP genotyping exist, the Walnut Improvement Program uses the competitive allele specific PCR (KASP) genotyping services offered through LGC Genomics (Boston, Mass., USA). The advantage of this system is its ability to genotype thousands of individuals using a small number of markers at low cost and high accuracy matching the needs of a breeding program. Genomic DNA sequences for J. regia and wild walnut species will play an important role in the design of robust, allele-specific KASP markers for deployment in walnut rootstock breeding.

•    Walnut reference genome sequence 
•    Comparative genetic maps densely populated with molecular markers for the discovery and mapping of disease resistance genes 
•    Discovery and mapping of genes conferring resistance to soil-borne pathogens 
•    Development of markers for selection of resistance genes in segregating populations 
•    Knowledge-base and genomic tools for gene discovery and the deployment of marker assisted selection in walnut rootstock breeding
•    Genomic resources for wild relatives of English walnut, which will accelerate biological, agronomic and forestry research