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Uncovering the genetic basis of photosynthetic trait variation under drought stress is essential for breeding climate-resilient walnut cultivars. To this end, we examined photosynthetic capacity in a diverse panel of 150 walnut families (1500 seedlings) from various agro-climatic zones in their habitats and grown in a common garden experiment. Photosynthetic traits were measured under well-watered (WW), water-stressed (WS) and recovery (WR) conditions. We performed genome-wide association studies (GWAS) using three genomic datasets: genotyping by sequencing data (∼43 K SNPs) on both mother trees (MGBS) and progeny (PGBS) and the Axiom™ Juglans regia 700 K SNP array data (∼295 K SNPs) on mother trees (MArray). We identified 578 unique genomic regions linked with at least one trait in a specific treatment, 874 predicted genes that fell within 20 kb of a significant or suggestive SNP in at least two of the three GWAS datasets (MArray, MGBS, and PGBS), and 67 genes that fell within 20 kb of a significant SNP in all three GWAS datasets. Functional annotation identified several candidate pathways and genes that play crucial roles in photosynthesis, amino acid and carbohydrate metabolism, and signal transduction. Further network analysis identified 15 hub genes under WW, WS and WR conditions including GAPB, PSAN, CRR1, NTRC, DGD1, CYP38, and PETC which are involved in the photosynthetic responses. These findings shed light on possible strategies for improving walnut productivity under drought stress.

Walnut pellicle color is a key quality attribute that drives consumer preference and walnut sales. For the first time a high-throughput, computer vision-based phenotyping platform using a custom algorithm to quantitatively score each walnut pellicle in L* a* b* color space was deployed at large-scale. This was compared to traditional qualitative scoring by eye and was used to dissect the genetics of pellicle pigmentation. Progeny from both a bi-parental population of 168 trees ('Chandler' × 'Idaho') and a genome-wide association (GWAS) with 528 trees of the UC Davis Walnut Improvement Program were analyzed. Color phenotypes were found to have overlapping regions in the 'Chandler' genetic map on Chr01 suggesting complex genetic control. In the GWAS population, multiple, small effect QTL across Chr01, Chr07, Chr08, Chr09, Chr10, Chr12 and Chr13 were discovered. Marker trait associations were co-localized with QTL mapping on Chr01, Chr10, Chr14, and Chr16. Putative candidate genes controlling walnut pellicle pigmentation were postulated.

BACKGROUND:Unravelling the genetic architecture of agronomic traits in walnut such as budbreak date and bearing habit, is crucial for climate change adaptation and yield improvement. A Genome-Wide Association Study (GWAS) using multi-locus models was conducted in a panel of 170 walnut accessions genotyped using the Axiom™ J. regia 700 K SNP array, with phenological data from 2018, 2019 and legacy data. These accessions come from the INRAE walnut germplasm collection which is the result of important prospecting work performed in many countries around the world. In parallel, an F1 progeny of 78 individuals segregating for phenology-related traits, was genotyped with the same array and phenotyped for the same traits, to construct linkage maps and perform Quantitative Trait Loci (QTLs) detection. RESULTS:Using GWAS, we found strong associations of SNPs located at the beginning of chromosome 1 with both budbreak and female flowering dates. These findings were supported by QTLs detected in the same genomic region. Highly significant associated SNPs were also detected using GWAS for heterodichogamy and lateral bearing habit, both on chromosome 11. We developed a Kompetitive Allele Specific PCR (KASP) marker for budbreak date in walnut, and validated it using plant material from the Walnut Improvement Program of the University of California, Davis, demonstrating its effectiveness for marker-assisted selection in Persian walnut. We found several candidate genes involved in flowering events in walnut, including a gene related to heterodichogamy encoding a sugar catabolism enzyme and a cell division related gene linked to female flowering date. CONCLUSIONS:This study enhances knowledge of the genetic architecture of important agronomic traits related to male and female flowering processes and lateral bearing in walnut. The new marker available for budbreak date, one of the most important traits for good fruiting, will facilitate the selection and development of new walnut cultivars suitable for specific climates.

The release of the first reference genome of walnut (Juglans regia L.) enabled many achievements in the characterization of walnut genetic and functional variation. However, it is highly fragmented, preventing the integration of genetic, transcriptomic, and proteomic information to fully elucidate walnut biological processes. Here, we report the new chromosome-scale assembly of the walnut reference genome (Chandler v2.0) obtained by combining Oxford Nanopore long-read sequencing with chromosome conformation capture (Hi-C) technology. Relative to the previous reference genome, the new assembly features an 84.4-fold increase in N50 size, with the 16 chromosomal pseudomolecules assembled and representing 95% of its total length. Using full-length transcripts from single-molecule real-time sequencing, we predicted 37,554 gene models, with a mean gene length higher than the previous gene annotations. Most of the new protein-coding genes (90%) present both start and stop codons, which represents a significant improvement compared with Chandler v1.0 (only 48%). We then tested the potential impact of the new chromosome-level genome on different areas of walnut research. By studying the proteome changes occurring during male flower development, we observed that the virtual proteome obtained from Chandler v2.0 presents fewer artifacts than the previous reference genome, enabling the identification of a new potential pollen allergen in walnut. Also, the new chromosome-scale genome facilitates in-depth studies of intraspecies genetic diversity by revealing previously undetected autozygous regions in Chandler, likely resulting from inbreeding, and 195 genomic regions highly differentiated between Western and Eastern walnut cultivars. Overall, Chandler v2.0 will serve as a valuable resource to better understand and explore walnut biology.

Walnut shell suture strength directly impacts the ability to maintain shell integrity during harvest and processing, susceptibility to insect damage and other contamination, and the proportion of kernel halves recovered during cracking. Suture strength is therefore an important breeding objective. Here, two methods of phenotyping this trait were investigated: 1) traditional, qualitative and rather subjective scoring on an interval scale by human observers, and; 2) quantitative and continuous measurements captured by a texturometer. The aim of this work was to increase the accuracy of suture strength phenotyping and to then apply two mapping approaches, quantitative trait loci (QTL) mapping and genome wide association (GWAS) models, in order to dissect the genetic basis of the walnut suture trait. Using data collected on trees within the UC Davis Walnut Improvement Program (n = 464), the genetic correlation between the texturometer method and qualitatively scored method was high (0.826). Narrow sense heritability calculated using quantitative measurements was 0.82. A major QTL for suture strength was detected on LG05, explaining 34% of the phenotypic variation; additionally, two minor QTLs were identified on LG01 and LG11. All three QTLs were confirmed with GWAS on corresponding chromosomes. The findings reported in this study are relevant for application towards a molecular breeding program in walnut.

Over the last 20 years, global production of Persian walnut (Juglans regia L.) has grown enormously, likely reflecting increased consumption due to its numerous benefits to human health. However, advances in genome-wide association (GWA) studies and genomic selection (GS) for agronomically important traits in walnut remain limited due to the lack of powerful genomic tools. Here, we present the development and validation of a high-density 700K single nucleotide polymorphism (SNP) array in Persian walnut. Over 609K high-quality SNPs have been thoroughly selected from a set of 9.6 m genome-wide variants, previously identified from the high-depth re-sequencing of 27 founders of the Walnut Improvement Program (WIP) of University of California, Davis. To validate the effectiveness of the array, we genotyped a collection of 1284 walnut trees, including 1167 progeny of 48 WIP families and 26 walnut cultivars. More than half of the SNPs (55.7%) fell in the highest quality class of 'Poly High Resolution' (PHR) polymorphisms, which were used to assess the WIP pedigree integrity. We identified 151 new parent-offspring relationships, all confirmed with the Mendelian inheritance test. In addition, we explored the genetic variability among cultivars of different origin, revealing how the varieties from Europe and California were differentiated from Asian accessions. Both the reconstruction of the WIP pedigree and population structure analysis confirmed the effectiveness of the Applied Biosystems™ Axiom™ J. regia 700K SNP array, which initiates a novel genomic and advanced phase in walnut genetics and breeding.

Yield, nut quality, and ability to adapt to specific climate conditions, are all important factors to consider in the development and selection of new Persian walnut (Juglans regia L.) varieties. The genetic control of these traits is still unknown in walnut, limiting the accuracy and rapidity of releasing new cultivars for commercial use. We studied the genetic architecture of five traits crucial for either marketing (i.e., yield, lateral fruit-bearing, and pellicle color) or selection of individuals with specific phenology (i.e., leafing and harvest date). By combining over 30 years of phenotypic data with genetic profiles generated using the latest Axiom™ J. regia 700K SNP array, we were able to identify and confirm major loci for all these traits. In particular, we revealed that a genomic region at the beginning of Chr1 controls both leafing and harvest date in walnut, consistent with the observed strong phenotypical correlation between these traits, and including candidate genes involved in plant development, leaf formation, and cell division. In addition, a large genomic region on Chr11 that includes genes with a central role in flowering control and shoot meristem growth underlies both lateral fruit-bearing and yield in walnut. We observed a more complex genetic architecture for pellicle color, strongly influenced by the environment (h 2 = 0.43). We identified two marker-trait associations on Chr6 and 7 for pellicle color, where genes encoding a UDP-glycosyltransferase or involved in the response to oxidation were found. In conclusion, by combining classical quantitative trait loci (QTL) mapping and genome-wide association mapping, we deciphered, for the first time, the molecular pathways controlling walnut phenology, yield, lateral fruitfulness, and pellicle color. Our findings represent a further milestone in the transition from conventional to genome-assisted breeding in Persian walnut.

Walnut production is challenged by climate change and abiotic stresses. Elucidating the genomic basis of adaptation to climate is essential to breeding drought-tolerant cultivars for enhanced productivity in arid and semi-arid regions. Here, we aimed to identify loci potentially involved in water use efficiency (WUE) and adaptation to drought in Persian walnut using a diverse panel of 95 walnut families (950 seedlings) from Iran, which show contrasting levels of water availability in their native habitats. We analyzed associations between phenotypic, genotypic, and environmental variables from data sets of 609 000 high-quality single nucleotide polymorphisms (SNPs), three categories of phenotypic traits [WUE-related traits under drought, their drought stress index, and principal components (PCs)], and 21 climate variables and their combination (first three PCs). Our genotype–phenotype analysis identified 22 significant and 266 suggestive associations, some of which were for multiple traits, suggesting their correlation and a possible common genetic control. Also, genotype–environment association analysis found 115 significant and 265 suggestive SNP loci that displayed potential signals of local adaptation. Several sets of stress-responsive genes were found in the genomic regions significantly associated with the aforementioned traits. Most of the candidate genes identified are involved in abscisic acid signaling, stomatal regulation, transduction of environmental signals, antioxidant defense system, osmotic adjustment, and leaf growth and development. Upon validation, the marker–trait associations identified for drought tolerance-related traits would allow the selection and development of new walnut rootstocks or scion cultivars with superior WUE. [ABSTRACT FROM AUTHOR]