PUTTING PHENOTYPIC AND GENOTYPIC TOOLS TO WORK FOR IMPROVING

WALNUT ROOTSTOCKS

Daniel Kluepfel, Ali McClean, Charles Leslie, Malli Aradhya, Ming-Cheng Luo, Pat Brown,

Ramesh Ramasamy, Jan Dvorak, Greg Browne, Janine Hasey, Wes Hackett, and Andreas

Westphal

ABSTRACT

Walnut production is a major contributor to the specialty crop economy of the USA. In 2016,

California produced 686,000 tons of in-shell walnuts on 315,000 acres with a farm gate value of

$1.24 billion. The continued success of this industry is threatened by multiple soil-borne plant

pathogens including Agrobacterium tumefaciens, (crown gall, CG), root-lesion (RLN), root-knot

nematode (RKN) (Pratylenchus vulnus, Meloidogyne incognita, respectively, NEM), and

Phytophthora crown and root rot (Phytophthora citricola, Phytophthora cinnamomi, PHY).

Remedial actions of pre-plant soil fumigation are costly, only partially effective, and pose potential

environmental and human risks. While methyl bromide has been phased out because of its’ ozone

depletion potential, alternative fumigants such as 1,3-D- containing materials (“Telone”) are

subject to township caps within a given year and their risk of generating hazardous volatile organic

compounds (VOCs) poses human health risks. The use of rootstocks that are resistant or tolerant

to soil borne pathogens have the greatest potential to minimize our dependence on soil fumigants

and maximize sustainability of the walnut industry.

INTRODUCTION

Currently, about 85% of the walnut industry uses either hybrid seedlings of J. hindsii x J. regia

(known as Paradox seedling rootstock), clonal selections of Juglans hindsii x J. regia (i.e., Vlach,

VX211) or a clonal selection of J. microcarpa x J. regia (RX1). In addition to the hybrid

rootstocks, about 10% of the industry uses seedlings of J. hindsii (Northern CA black walnut

seedling rootstock) while an estimated 5% rely on own rooted J. regia (English seedling rootstock)

(UC Farm Advisors-personal communications). Despite its popularity and relative advantages,

Paradox seedling rootstock is susceptible to all of the pathogens listed above. RX1 has been shown

to be resistant to P. cinnamomi, but it is not known to be sufficiently resistant to crown gall or

phytopathogenic nematodes. Combined, root system diseases cause an estimated 18% annual yield

loss worth an estimated $241 million to the California walnut industry. These high levels of

susceptibility are a strategic vulnerability. Consequently, the California Walnut Board has cited

these three soil-borne diseases as major threats to the industry in the PRAC priority documentation.

Because there are only limited pre- and post-plant management strategies for these biotic stresses,

rootstocks with elevated levels of genetic resistance/tolerance to these major pathogens offer the

most promising chemical-independent solution to soil-borne threats.

Despite pressing needs of the walnut industry for improved rootstocks, rootstock breeding is

challenging and slow. Breeding efficiency would greatly benefit from knowledge of the genetic

loci that mediate improved rootstock traits which could facilitate the development of molecular

markers for juvenile selection strategies.

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This project is designed to exploit the foundations of the walnut rootstock breeding technologies

and resources we developed over the last five years. We generated genetic and physical maps of

key Juglans species that have been used to identify genetic loci in mapping populations that appear

to be associated with disease resistance. Using this information we are in the process of developing

and validating potential genetic markers for use in marker assisted selection in our rootstock

breeding efforts. This will allow us to screen large populations to find candidates that contain not

only one but perhaps multiple disease resistance loci in a single genotype. Such a strategy will

produce superior elite material much quicker than a simple phenotyping approach, and will lead

to accelerated selection of improved walnut rootstocks in which we have stacked genes for

resistance. Finally, as in our previous project, we will continue to field test commercially viable

elite pathogen-and-stress-resistant rootstocks under a wide range of environmental conditions.

The central goal of this project is to identify, characterize and ultimately deploy crown gall,

Phytophthora and nematode resistant walnut rootstocks. Towards that end, we identified over 30

interspecific hybrid genotypes which exhibit an elevated level of tolerance/resistance to the

pathogens cited above. In addition, eleven candidates derived from crosses of J. microcarpa and

Juglans cathayensis with J. regia pollen, express higher levels of tolerance to P. vulnus and

Meloidogyne spp. in the field than the commercial Paradox clone VX211. All of these elite

putative disease resistant genotypes are being passed through out in vitro propagation pipeline in

preparation for rescreening and deployment in multiple field trials in the summer of 2020.

OBJECTIVES

Objective 1. Traditional and in vitro propagation of a genetically diverse collection of Juglans

species including creation and characterization of an enhanced mapping population from J.

microcarpa x J. regia crosses (Leslie, Hackett, and Brown).

Anticipated Outcome 1. Creation of a highly diverse collection of germplasm which is made

available for disease resistance screening and examination under field conditions.

Objective 2. Characterization of genetic loci which mediate disease resistance and

utilization of molecular markers for rapid screening/identification of resistant genotypes

(M.-C. Luo, Kluepfel, Brown, Browne, and Westphal).

Anticipated Outcome 2. a.) Development and validation of genetic markers useful for marker

assisted selection. b.) Identification of genotypes with resistance to two or more of our three target

pathogens, i.e. pyramiding resistance.

Objective 3. Outreach and development of new and existing field trials examining performance

of elite rootstock germplasm. Significant progress made last funding cycle on this objective

(Hasey, Westphal, Leslie, Brown, Browne, and Kluepfel).

Anticipated Outcome 3. Validation of the commercial potential/viability of candidate rootstock

genotypes under field conditions.

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SIGNIFICANT FINDINGS / ACCOMPLISHMENTS

• We continue to maintain 1600 unique walnut genotypes as micropropagated cultures; the

collection now includes Juglans sigillata and several Pterocarya (wingnut) accessions that

have shown grown gall resistance.

• Germinated an extensive collection of J. microcarpa (seeds) which were collected across

its natural range in the southwestern U.S. Using this material, we established a germplasm

orchard at the Armstrong field station (UC Davis) which contains 320 trees representing

160 wild J. microcarpa and J. major genotypes.

• Under field conditions, at least 14 Juglans genotypes have been identified which putatively

possess resistance or tolerance to root lesion nematode.

• Commercially generated clonal copies of over 25 “putative disease resistant” and

genetically diverse Juglans genotypes. Surviving genotypes from this group have been

planted out in liners at our cooperating nursery and will be used in future field trials.

• Generated clonal material for 5 additional disease resistant interspecific hybrids for

submission into the commercial in vitro propagation pipeline at our cooperating nursery.

• Four elite experimental genotypes continued to be examined in five large-scale field trials

across the state; yield was taken in three of the June bud trials.

• Harvested and propagated ~2,000 open pollinated nuts from two J. microcarpa mothers

trees (31.01 and 31.09). We then harvested tissue, extracted DNA, generated GBS data for

1,511 members of this population.

• Removed catkins from 7 J. microcarpa mother trees at the USDA-ARS national clonal

germplasm repository and then collected ~10,182 new open pollinated seeds which will be

germinated and screened for disease resistance in 2020.

• Produced approximately 1400 clonal plantlets from our J. microcarpa x J. regia breeding

population and open pollinated J. microcarpa genotypes, for distribution and disease

resistance testing (crown gall, Phytophthora and nematodes)

• Completed crown gall and Phytophthora screening of full-sib families from the J.

microcarpa x J. regia crosses.

• Hybrid saplings from 31.01 and 31.09 J. microcarpa mother trees (putative J. regia pollen

donor), were phenotyped for resistance to crown gall and P. cinnamomi. Phenotypes are

being used by the genotyping team for QTL / SNP validations.

• There were 248,720, 123,943 and 201,475 SNPs detected in J. microcarpa 31.01, and J.

microcarpa 31.09, respectively (see text for details).

• Generated high-quality assembled and annotated reference genomes for both mating

partners used to generate our breeding population (J. microcarpa x J. regia ‘Serr’).

• Identified 31,425 annotated genes in J. regia and 29,496 genes in J. microcarpa. For each

genome, 16 chromosomes were defined, 13 of which consist of a single super-scaffold.

One small gap was identified in each of 3 chromosomes in both parents.

• We genetically characterized all 600 interspecific progeny (Genotype By Sequencing),

which allowed us to map several thousand SNP markers on our genetic map.

• We confirmed/validated a major quantitative-trait-locus (QTL) in J. microcarpa that

mediate resistance to crown gall and Phytophthora root/crown rot.

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• With identified SNP markers associated with these QTLs, we are moving towards

development of marker assisted selection procedures for disease resistance in our

rootstock-breeding program.

PROCEDURES

Phytophthora resistance screening. In 2019, we completed phenotyping resistance to

Phytophthora cinnamomi and P. citricola among hybrid clones from controlled crosses of Juglans

microcarpa ’31.01’ x Juglans regia ‘Serr’ and controlled crosses of J. microcarpa ’31.09’ x J.

regia ‘Serr’. From 209 to 261 clones were phenotyped from each cross with both species of

Phytophthora. A greenhouse screening system with artificially infested soil and intermittent soil

flooding was used for the phenotyping; resistance to crown and root rot was phenotyped separately

for each species of Phytophthora. The phenotypes were summarized for the genotyping team,

which profiled the phenotypes against SNP markers to localize QTL.

In addition, we launched confirmatory phenotyping and genotyping efforts among seedlings from

the 31.01 and 31.09 mother trees of J. microcarpa; both hybrid (J. microcarpa x J. regia) and

maternal species (J. microcarpa) were represented among seedlings. There were 306 to 323

seedlings per mother tree. Resistance of each seedling was assessed according to: 1) an area under

the disease progress curve (largely based on plant survival over the 2-month phenotyping period)

and 2) average percentages of crown and root length rotted.

Nematode Resistance Screening. Field screening for resistance and tolerance to lesion (RLN)

and root-knot nematode (RKN) is being conducted at the Kearney Agricultural Research and

Extension Center. During the period 2015-2017, six phases of a total of >600 clonal plants of

interspecific Juglans hybrids from directed crosses and two phases of >1,600 seedlings of open-

pollinated crosses were established in the field.

Plants were planted in the spring, inoculated with RLN and RKN after establishment, and grown

for two seasons. At the end of the second growing season, plants were rated for vigor, growth

pattern and evaluated for nematode populations that developed in/on the roots. Selected Juglans

genotypes, with favorable horticultural and nematode resistance characteristics, were heavily

pruned and transplanted in a randomized complete block design with four to six replications in

RLN and RKN infested soil. Plants are grown for at least three additional years during which

growth response is monitored along with nematode population dynamics.

Agrobacterium tumefaciens resistance screening. Walnut clonal plants were grown and

maintained under greenhouse conditions. The plants were inoculated using a small wood chisel

blade dipped in a suspension (OD600 = 1.0) of Agrobacterium tumefaciens EC1. Each plant was

stabbed twice in the stem above the crown area and wrapped with parafilm. Negative controls were

prepared from each genotype by stabbing a single clonal plant with a sterile blade dipped in sterile

water. Three standard genotypes were included as plant controls: VX211, RX1, and Vlach. Plants

were evaluated for gall symptoms at 2 months post-inoculation then allowed to go dormant for the

winter. Plants were re-evaluated after emerging from dormancy 8 months post-inoculation. The

following rating system was used 1) = No symptoms, 2) = up to 25% of the stem surface girdled

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with gall tissue, 3) = 25-50% of the stem surface girdled with gall tissue, and 4) = >50 of the stem

girdled with gall tissue.

General Screening Comment. As mentioned previously, we generated ~50 clonal copies of each

of the 600 confirmed interspecific hybrids in our breeding population resulting from two J.

microcarpa x J. regia ‘Serr’ crosses. Nearly 75% of these progeny have been evaluated in our

disease resistance screening pipeline which is being conducted under greenhouse and/or field

conditions as described previously and above. The few remaining individuals not yet

screened/evaluated will be rated this coming spring. Final QTL analysis will then be conducted.

Plant Propagation. Plant propagation efforts are divided between maintenance of prior produced

crosses and newly produced orchard-derived crosses. We maintain tissue culture stocks of ca.

1,600 progeny from J. microcarpa with J. regia ‘Serr’ crosses, open pollinated genotypes, and

other assorted Juglans and Pterocarya species. This number includes genotypes that have not yet

been tested. Elite putative disease resistant genotypes were propagated and transferred to a

commercial nursery for increase to produce trees for field trials.

Seeds from crosses of MT31.01 and MT31.09 grafted into a J. regia orchard were stratified,

propagated in a greenhouse, and are currently being genotyped for evaluation with tentative

molecular markers for the presence of resistance loci to CG and PHY. Seedlings will be further

cultivated and marker-positives transferred to the nematode testing program at KARE, whereas

plants without the positive molecular signal will be culled with the exception of the same number

of plants that gave a positive signal. Both types of genotypes will be entered into phenotyping

assays for CG and PHY. The association of positive marker plus phenotypic carrier of the

resistance ascribed to that marker will be tested. This will be a proof of concept experiment

assessing marker validity. Only plants with a positive response to the marker for either CG or PHY

resistance will be transferred as seedlings to KARE for testing for nematode resistance. The in

vitro cultures of the selected plants will be maintained for the production of clonal plants.

Computational identification of conserved miRNAs and their putative target genes in J. regia

and J. microcarpa. High quality references genome assembly for J. regia and J. microcarpa were

used to identify miRNAs by homology-based approach and characterize the distribution of miRNA

on chromosomes of both species.

High quality internal SNP (SNPs between two haplotypes of genome) calling for Juglans

microcarpa accessions: J. microcarpa DJUG 31.01 (hereafter Jm31.01) genome sequence was

used as reference for variant calling. Illumina sequencing reads of J. microcarpa DJUG 31.05

(hereafter Jm31.05), DJUG 31.09 (hereafter Jm31.09), and Jm31.01 were download from NCBI.

Variant calling was performed and filtered with QC >=20. Only internal heterozygous sites

(polymorphism between two haplotypes) were kept. Due to the overall low sequencing depth

(mean depth<=7), the minimum depth of 10 was then used to call SNPs with high confidence.

Genotyping-By Sequencing (GBS) Analysis. Genomic DNAs were isolated from two

populations of open pollinated seedlings collected from J. microcarpa trees planted in a J. regia

breeding orchard. The first population consisted of OP seeds from Jm31.01and the second

population consisted of OP seeds from J. microcarpa 31.09. DNA from the hybrids and the parents

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were sequenced using GBS at U.C. Davis genome center. GBS involved genome complexity

reduction by digestion with restriction enzyme PstI followed by Next Generation Sequencing

(NGS) of DNA fragments (Elshire, R.J., et al., 2011). Each DNA fragment was labeled with a

unique ‘barcode’ and 96 samples were pooled for GBS. The pools were sequenced with the

Illumina HiSeq2000 NGS platform. The reads within each pool were deconvoluted using their

barcodes and mapped on J. microcarpa 31.01 and J. regia ‘Serr’ genome sequences using BWA-

mem (Li, H. and Durbin, R., 2009). SNPs were called using samtools (Li, H., et al., 2009).

VCFtools (Danecek, P., et al., 2011) and in-house python scripts were utilized to obtain high-

quality SNPs. Non-segregating SNPs, low read-depth calls, and SNPs with segregation distortion

were filtered out.

Field Trials. As mentioned in previous walnut research reports, five experimental Juglans

genotypes with putative resistance to one or more of the key pathogens targeted in this project,

were planted in replicated field trials in 2015 and 2016. These trials are ongoing. In 2016, Chandler

was budded onto these four rootstock genotypes and planted at four locations across California’s

walnut growing regions (Table 1). In addition to VX211, Vlach, and RX1, the June bud trials

included Paradox hybrid seedling rootstock for comparison. We established these trials in sites

with a known disease or soil problem and no artificial inoculations were used (Table 1). Baseline

caliper data were taken at planting and then again in November or December 2016 and reported in

2017 reports. 2017 rootstock and scion circumference data were taken in January 2018; 2018

growth data were taken winter 2018-19. Mortality percent over time was noted. Additionally,

rootstocks were surveyed for the presence of crown gall at the ground level, at the graft union, and

on the English scion. 2019 data will be taken in winter 2019-20.

In 2015, a trial was planted in Solano County (UC Davis-Armstrong Field Station) using potted

rootstock that was fall budded to Chandler. The experimental genotypes are being compared to the

standard clonal rootstocks VX211, RX1 and Vlach. If the first fall budding was unsuccessful, trees

were spring grafted or fall budded in 2016. This trial was artificially inoculated with Phytophthora

and A. tumefaciens in the spring of 2017. Trunk circumference was measured and tree mortality

was noted in April, 2018. 2018 growth measured as trunk circumference was taken on Feb. 22,

2019.

RESULTS AND DISCUSSION

Nematode Resistance Screening. Genotypes of the two breeding populations were evaluated two

years after inoculation. This increased the genotype number with determined host status to P.

vulnus and M. incognita to about half of both breeding populations (MT31.01: 134 genotypes;

MT31.09: 166 genotypes). Data are currently prepared for QTL analysis. The remaining genotypes

of the complete 300 genotypes of the two breeding populations are currently cultivated in field

plots at the Kearney center and are scheduled for the next dormant season (2019/2020) for

nematode evaluations. Additional promising open-pollinated seedlings from mother trees of J.

ailantifolia, J. major and J. microcarpa are continually monitored for nematode population

dynamics. These elites were grafted to the popular scion ‘Chandler’ in 2018 to concomitantly

assess their graft compatibility. The most promising genotypes had been transferred into tissue

culture, and replicate clonal plants were produced, and planted to nematode-infested field plots to

generate replicate data for nematode susceptibility of originally single seed-descent trees in spring

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2019. In a field trial with previously identified rootstock elites grafted to ‘Chandler’ and with

factorially applied water × nematode infestation, and differential water treatments and detail plant

measurements were initiated in cooperation with Dr. Andrew McElrone from USDA-ARS.

Phytophthora resistance screening; clones. In total, counting previous years’ efforts, replicated

phenotype data for resistance to P. cinnamomi and P. citricola were obtained for 231 and 233

clones, respectively, from crosses of J. microcarpa ’31.01’ x J. regia ‘Serr’ and for 209 and 261

clones, respectively, from crosses of J. microcarpa ’31.09’ x J. regia ‘Serr’. The updated

phenotype data are being used by the genotyping team to further resolve a QTL for resistance to

each Phytophthora sp. in linkage group 11 of Juglans microcarpa (no significant QTL was

detected in the parent, J. regia ‘Serr’). Clones with high levels of resistance to both pathogens

were transferred for nursery multiplication and confirmatory greenhouse and orchard testing.

Phytophthora resistance screening; seedlings. We also phenotyped resistance to P. cinnamomi

in seedlings from mother trees of J. microcarpa ‘31.01’ and J. microcarpa ‘31.09’ in 2019; hybrids

(J. microcarpa x J. regia) (306 plants) as well as maternal species (J. microcarpa) (323 plants)

were included. The hybrids and maternal seedlings from mother trees of J. microcarpa ’31.01’ and

J. microcarpa ’31.09’ varied widely in resistance as measured by disease progress curves and final

severity of crown rot and root rot (Figs. 1 and 2). The seedling phenotypes are being regressed on

their corresponding SNP genotypes to validate and further resolve the QTL found previously in

clonal genotypes.

Figure 1. Crown rot phenotype histogram, hybrid seedlings (J. microcarpa x J. regia) from 31.01 and 31.09

mother trees, soil infested with Phytophthora cinnamomi in 2019 greenhouse trial.

Nu

mb

er o

f se

edlin

gs

Severity classes (upper and lower limits) based on % crown rot

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Figure 2. Crown rot phenotype histogram, maternal seedlings (J. microcarpa) from 31.01 and 31.09 mother

trees, soil infested with Phytophthora cinnamomi in 2019 greenhouse trial.

Crown Gall Screening. Clonal walnut progeny from the AC cross 31.01 x ‘Serr’ (Fig. 3) and

the AD cross 31.09 x ‘Serr’ (Fig. 4) were inoculated with A. tumefaciens EC1 and evaluated 8

months post-inoculation after one dormancy period. Bars represent the average values from 3-5

replicates of each genotype. Ratings: 1) = No symptoms, 2) = up to 25% of stem surface girdled

with gall tissue, 3) = 25-50% of stem surface girdled with gall tissue, and 4) = >50 of stem girdled

with gall tissue. Control genotypes RX1, Vlach and VX211 are highlighted (indicated by black

bars). Three out of 34 AC genotypes and three out of 37 AD genotypes exhibited a high level of

resistance and remained symptomless 8 months post-inoculation. Several other AC and AD

progeny were more crown gall resistant than three control genotypes RX1, VX211, and Vlach.

These genotypes will be retested to assess the durability of the observed resistance.

Figure 3. Crown gall screen on clonal walnut genotypes.

Nu

mb

er o

f se

edlin

gs

Severity classes (upper and lower limits) based on % crown rot

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Figure 4. Crown gall screen on clonal walnut genotypes.

Screening “new” OP progeny; Open pollinated progeny from two J. microcarpa walnut mother

trees: 31.01 and 31.09 were inoculated with A. tumefaciens strain EC1. Two hundred eighty-

seven seedlings were hybrids and 334 were non-hybrids. Disease evaluations will be conducted

post-dormancy and the results compared to predictions based on QTL analysis.

Establishment of a new J. microcarpa germplasm orchard. Three hundred twenty seedlings

representing 160 wild J. microcarpa genotypes collected from throughout the southwestern United

States were planted in an orchard at the UC Davis Armstrong field station in Davis, CA.

Computational identification of conserved miRNAs and their putative target genes in J. regia

and J. microcarpa. MicroRNAs (miRNAs), small non-coding RNAs with 20-24nt, play important

roles in the post-transcriptional regulation of protein coding genes in plants. High quality

references genome assembly for J. regia and J. microcarpa make it possible to identify miRNAs

by homology-based approach and characterize the distribution of miRNA on chromosomes of both

species. We identified 39 and 40 conserved miRNAs belonging to 27 and 26 miRNA family in J.

regia and J. microcarpa. There is no significant difference in the quantity and category of miRNA

between the two species. An asymmetric distribution of miRNA precursors between the dominant

and subdominant chromosomes in both species was detected. We also predicted 325 and 316

potential target genes for these miRNAs in J. regia and J. microcarpa respectively. Of these target

genes, 12 and 18 resistance gene analogues and 56 and 54 transcript factors were found in J. regia

and J. microcarpa, respectively. Function annotation demonstrated, miRNA regulated similar

physiological activities in the two species.

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High quality internal SNP (SNPs between two haplotypes of genome) calling for Juglans

microcarpa accessions: J. microcarpa MT31.01 genome sequence was used as reference for

variant calling. The total length of MT31.01 assembly was 527,895,757bp with 16

pseudomolecules and 138 scaffolds. Illumina sequencing reads of J. microcarpa MT31.05,

MT31.09, and MT31.01 were download from NCBI. Sizes of raw sequencing reads are 4.800 Gb,

4.235 Gb and 4.416 Gb for MT31.01, MT31.05 and MT31.09, respectively. After quality

duplication filtration, there were 28,753,113, 25,518,667 and 27,031,847 reads of MT31.01,

MT31.05 and MT31.09, respectively, which were suitable for mapping with the mapping rates of

96.76%, 97.39% and 98.08% respectively. Variant calling was performed and filtered with QC

>=20. Only internal heterozygous sites (polymorphism between two haplotypes) were kept. Due

to the overall low sequencing depth (mean depth<=7), the minimum depth of 10 was then used to

call SNPs with high confidence. With the minimum depth of 10, there are 248,720, 123,943 and

201,475 SNPs detected in MT31.01, MT31.05, and MT31.09, respectively, which are summarized

in the Table 1. These SNPs can be used for fine mapping, gene tagging in genetic analysis and

breeding.

Table 1. Number of high quality internal SNPs identified in J. microcarpa accessions

Chrom. Length(bp) No. of SNP

(MT31.01)

No. of SNP

(MT31.05)

No. of SNP

(MT31.09)

Jm1D 49,856,174 23,652 7,459 20,196

Jm1S 30,169,828 13,314 8,076 10,464

Jm2D 43,614,719 19,625 10,784 14,199

Jm2S 34,707,362 14,590 7,528 13,834

Jm3D 39,900,371 16,092 11,189 14,384

Jm3S 27,832,763 15,458 7,685 11,718

Jm4D 35,629,462 16,591 9,197 12,164

Jm4S 27,577,207 15,829 7,630 11,636

Jm5D 35,798,223 20,684 3,876 14,109

Jm5S 32,113,326 16,484 9,613 11,350

Jm6D 38,364,907 18,328 7,825 15,971

Jm6S 22,373,256 10,531 5,914 7,836

Jm7D 26,765,518 9,799 7,071 9,054

Jm7S 21,889,424 10,556 6,058 8,568

Jm8D 36,225,362 20,740 9,214 16,693

Jm8S 19,962,766 4,352 3,517 8,004

JmUn 5,115,089 2,095 1,307 1,295

Total 527,895,757 248,720 123,943 201,475

Databases: Relational walnut genome databases were created to facilitate downloading, browsing,

blasting the genome resources of J. regia cv. Serr and J. microcarpa accessions. The URL of the

database is http://aegilops.wheat.ucdavis.edu/Walnut/data.php, and it is publicly accessible.

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Field Trials: 2016 June Bud Trials (Table 2). In 2018, there was very low tree mortality at all

four locations with the exception of JM8 at Glenn County site (36%), and 11-99 at the Tulare

County site (32%), and K3 at Sutter County site (16%). The standard seedling and clonal Paradox

rootstocks typically performed well across all sites. The new genotype clones performed

differently depending on the site. For the rootstock growth rate for all data combined across all

four field trials, VX211 grew significantly faster than RX1 among the standard rootstocks, while

K3 grew faster than the other new genotype clones as in 2016 and 2017 and was not significantly

different than RX1. The percent scion to rootstock ratio tends to be lowest for VX211 as in 2017

across the sites indicating that the trunk is less similar in size compared to the rootstock. Trees in

Glenn County were smaller than at other sites due to continuing severe deer browse damage and

the only site not harvested in 2019. Yield was taken in 2018 at the Sutter County site only. Yield

for the new genotypes was not significantly different from the standard clonal or seedling

rootstocks. All crown gall survey data was combined across the sites. Seedling Paradox rootstock

had the highest crown gall percentage at ground level, at the graft union, and on the English scion.

Crown gall was one percent or zero at ground level or on the graft union for all the clones.

Table 2. Clonal rootstock plots planted in 2016; Standards vs. experimental genotypes (June bud)

Location Advisor Putative Site

Problems

VX211,

RX1,

Vlach

Seedling

PDX

Experimental

Germplasm

Yield taken in

2019

Lindcove

REC

Tulare Co.

Fichtner Phytophthora,

lesion

nematode

Yes K3, JM4,

JM8,

11-99-1

Yes

Lake Co. Elkins Armillaria

root rot,

Nematodes,

Crown Gall

Yes K3, JM4,

JM8, CC

Yes

Glenn Co. Milliron Phytophthora,

lesion

nematode,

Crown Gall

Yes K3, JM4, JM8 No

Sutter Co. Hasey Marginal Soil Yes K3, JM4,

JM8, 11-99-1,

CC

Yes

(also 2018)

Field Trial: 2015 Potted Tree Trial (Solano County). Tree mortality was mostly limited to 3S17,

which generally did not grow. Thus, mortality is likely attributable to the general weakness of the

tree, not disease susceptibility.

When trunk circumference was analyzed for effect of rootstock or disease inoculation, it was found

that rootstock genotype significantly affected trunk circumference, but disease treatment had not

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caused a statistically detectable difference in trunk circumference. Two genotypes have

consistently small circumference, but all other genotypes show a wide range in circumference,

which may be making treatment difference hard to detect.

ACKNOWLEDGEMENTS

All of the work described here is supported, in part, by matching funds provided by the California

Walnut Board and a grant from the USDA Specialty Crop Research Initiative (SCRI). We would

also like to thank colleagues: Limin Chen and Ilene Battraw for in vitro and greenhouse

propagation, Natalia Ott, Holly Forbes, and Anna Lyn Robleza for Phytophthora resistance

screening and Tom Buzo Z.T.Z. Maung for nematode resistance screening,

REFERENCES

Danecek, P., Auton, A., Abecasis, G., Albers, C.A., Banks, E., DePristo, M.A., Handsaker, R.E.,

Lunter, G., Marth, G.T., Sherry, S.T. and McVean, G., 2011. The variant call format

and VCFtools. Bioinformatics, 27(15), pp.2156-2158.

Elshire, R.J., Glaubitz, J.C., Sun, Q., Poland, J.A., Kawamoto, K., Buckler, E.S. and Mitchell,

S.E., 2011. A robust, simple genotyping-by-sequencing (GBS) approach for high

diversity species. PloS one, 6(5), p.e19379.

Hishinuma, S.M. 2017. Quantifying the frequency, severity, and progression of thousand cankers

disease (TCD) in various walnut species and regions in California. Ph.D. dissertation,

University of California, Davis.

Li, H. and Durbin, R., 2009. Fast and accurate short read alignment with Burrows–Wheeler

transform. Bioinformatics, 25(14), pp.1754-1760.

Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G.

and Durbin, R., 2009. The sequence alignment/map format and SAMtools.

Bioinformatics, 25(16), pp.2078-2079.

Luo, M.C., You, F.M., Li, P., Wang, J.R., Zhu, T., Dandekar, A.M., Leslie, C.A., Aradhya, M.,

McGuire, P.E. and Dvorak, J., 2015. Synteny analysis in Rosids with a walnut physical

map reveals slow genome evolution in long-lived woody perennials. BMC genomics,

16(1), p.707.

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