Walnut Breeding Programs Research Articles

Utilizing SSR markers to examine the population structure and molecular genetic diversity of walnut (Juglans regia L.) genotypes in the Northwestern Himalayan region of Jammu and Kashmir

By using 16 simple sequence repeat (SSR) markers, the genetic relatedness of 21 exceptional walnut genotypes was assessed. A significant degree of genetic diversity was observed within a given population, as indicated by the number of alleles per locus ranging from 2 to 4. WGA-1, WGA-4, and WGA-79 contained the greatest number of alleles (4), followed by WGA-118, WGA-202, and WGA-42. Conversely, WGA-27, WGA-69, and WGA-32 contained the fewest alleles. The range of the PIC value was 0.11 to 0.38. Using model-based cluster analysis, all genotypes were categorized into two primary clusters according to the UPGMA dendrogram, with varying degrees of sub-clustering. All the genotypes were categorized into six genetically distant subpopulations. The genotypes were genetically distinct but had variable degrees of admixture. The anticipated heterozygosity at a specific locus ranged from 0.563 to 0.741. Additionally, population differentiation (Fst) ranged between 0.176 and 0.261. These findings highlight the importance of considering germplasm diversity in walnut breeding programs and conservation efforts aimed at enhancing walnut cultivation in the region. Overall, this study contributes to our understanding of walnut genetic diversity in the Northwestern Himalayan region of Jammu and Kashmir and informs future breeding and conservation strategies.

Morphological and pomological assessments of seedling-originated walnut (Juglans regia L.) trees to select the promising late-leafing genotypes.

In many parts of the world, including Iran, walnut (Juglans regia L.) production is limited by late-spring frosts. Therefore, the use of late-leafing walnuts in areas with late-spring frost is the most important method to improve yield. In the present study, the phenotypic diversity of 141 seedling genotypes of walnut available in the Senejan area, Arak region, Markazi province, Iran was studied based on morphological traits to obtain superior late-leafing genotypes in the cropping seasons of 2022 and 2023. Based on the results of the analysis of variance, the studied genotypes showed a significant variation in terms of most of the studied morphological and pomological traits. Therefore, it is possible to choose genotypes for different values ​​of a trait. Kernel weight showed positive and significant correlations with leaf length (r = 0.32), leaf width (r = 0.33), petiole length (r = 0.26), terminal leaflet length (r = 0.34), terminal leaflet width (r = 0.21), nut length (r = 0.48), nut width (r = 0.73), nut weight (r = 0.83), kernel length (r = 0.64), and kernel width (r = 0.89). The 46 out of 141 studied genotypes were late-leafing and were analyzed separately. Among late-leafing genotypes, the length of the nut was in the range of 29.33-48.50mm, the width of the nut was in the range of 27.51-39.89mm, and nut weight was in the range of 8.18-16.06g. The thickness of shell was in the range of 1.11-2.60mm. Also, kernel length ranged from 21.97-34.84mm, kernel width ranged from 21.10-31.09mm, and kernel weight ranged from 3.10-7.97g. Based on important and commercial traits in walnut breeding programs, such as nut weight, kernel weight, kernel percentage, kernel color, and ease of kernel removal from nuts, 15 genotypes, including no.92, 91, 31, 38, 33, 18, 93, 3, 58, 108, 16, 70, 15, 82, and 32 were superior and could be used in walnut breeding programs in line with the introduction of new cultivars and the revival of traditional walnut orchards to commercialize them.

Unravelling the genetic diversity and population structure of common walnut in the Iranian Plateau

BackgroundCommon walnut (Juglans regia L.) has a long cultivation history, given its highly valuable wood and rich nutritious nuts. The Iranian Plateau has been considered as one of the last glaciation refugia and a centre of origin and domestication for the common walnut. However, a prerequisite to conserve or utilize the genetic resources of J. regia in the plateau is a comprehensive evaluation of the genetic diversity that is conspicuously lacking. In this regard, we used 31 polymorphic simple sequence repeat (SSR) markers to delineate the genetic variation and population structure of 508 J. regia individuals among 27 populations from the Iranian Plateau.ResultsThe SSR markers expressed a high level of genetic diversity (HO = 0.438, and HE = 0.437). Genetic differentiation among the populations was moderate (FST = 0.124), and genetic variation within the populations (79%) significantly surpassed among populations (21%). The gene flow (Nm = 1.840) may have remarkably influenced the population genetic structure of J. regia, which can be attributed to anthropological activities and wind dispersal of pollen. The STRUCTURE analysis divided the 27 populations into two main clusters. Comparing the neighbor-joining and principal coordinate analysis dendrograms and the Bayesian STRUCTURE analysis revealed the general agreement between the population subdivisions and the genetic relationships among the populations. However, a few geographically close populations dispersed into different clusters. Further, the low genetic diversity of the Sulaymaniyah (SMR) population of Iraq necessitates urgent conservation by propagation and seedling management or tissue culture methods; additionally, we recommend the indispensable preservation of the Gonabad (RGR) and Arak (AKR) populations in Iran.ConclusionsThese results reflected consistent high geographical affinity of the accession across the plateau. Our findings suggest that gene flow is a driving factor influencing the genetic structure of J. regia populations, whereas ecological and geological variables did not act as strong barriers. Moreover, the data reported herein provide new insights into the population structure of J. regia germplasm, which will help conserve genetic resources for the future, hence improving walnut breeding programs’ efficiency.

Tracing Superior Late-Leafing Genotypes of Persian Walnut for Managing Late-Spring Frost in Walnut Orchards

Evaluating genetic diversity in walnut (Juglans regia L.) populations is a rapid approach used by walnut breeding programs to distinguish superior genotypes. The present study was conducted on the walnut population of Hamedan Province, one of the richest and most genetically diverse regions in Iran, during 2018–2019. After the initial screening, 47 genotypes were selected for further evaluation of pomological and phenological traits based on International Plant Genetic Resources Institute (IPGRI) descriptors. Nut and kernel weights among the selected genotypes ranged from 7.15 to 21.05 g and 3.0 to 10.8 g, respectively. Principal component analysis (PCA) categorized the genotypes into three distinct groups. Whereas the cluster analysis (CA) revealed the similarities and dissimilarities among the genotypes by identifying four major clusters. Spearman correlation analysis showed a positive correlation (p < 0.01) between nut weight (NWT), nut size, and kernel weight (KW), while a negative correlation (p < 0.01) between shell thickness (STH) and packing tissue thickness (PTT) with kernel percentage (KP) was observed. Lastly, 10 of 47 genotypes (TAL8, TAL9, TAL10, TAL14, TAL19, TAL22, TB2, TB4, TB6, and RDGH5) were considered superior. Superior genotypes were late-leafing (25–40 days after the standard) and displayed a lateral bearing (LB) habit with heavy nuts (12.52–16.82 g) and kernels (6.53–8.15 g), thin shells (1.06–1.25 mm), and lightly colored kernels.

Morphometric traits and iPBS based molecular characterizations of walnut (Juglans regia L.) genotypes

In this study, walnut genotypes that were selected during two growing seasons among thousands of seedlings were analyzed in terms of detailed morphometric, phenological, and chemical traits. A multivariate analysis was conducted with valuable traits for breeding and selection such as morphometric traits, chemical composition, and phenological characteristics. Also, genotypes were characterized by a retrotransposon-based iPBS marker system. The correlation analysis showed significant positive and negative correlations between agro-morphological characters. The principal component analysis explained 71.44% of the total variance into five main components. Principal component and hierarchical cluster analysis divided genotypes into three groups and identified subgroups based on both agro-morphological characters and iPBS marker systems. A high level of polymorphism ratio was observed for tested markers. Mantel’s test demonstrated relatively low correlations between molecular and morphological treats (r = 0.04). The genetic similarities among all individuals ranged from 0.39 (between 018 and 015 or 045 genotypes) to 0.98 (between 090 and 094 genotypes) with a mean similarity of 0.67. Remarkable phenotypic and molecular variations were observed among the genotypes. The features of some investigated genotypes were above the acceptable thresholds for walnut selection in breeding programs, and our study indicated that iPBS markers can be beneficial in walnut breeding programs, allowing the evaluation of the genetic relationship between genotypes, helping to differentiate and select the best genotypes to improve agronomic properties.

Composite core set construction and diversity analysis of Iranian walnut germplasm using molecular markers and phenotypic traits.

Iran is a center of origin and diversity for walnuts (Juglans regia L.) with very good potential for breeding purposes. The rich germplasm available, creates an opportunity for study and selection of the diverse walnut genotypes. In this study, the population structure of 104 Persian walnut accessions was assessed using AFLP markers in combination with phenotypic variability of 17 and 18 qualitative and quantitative traits respetively. The primers E-TG/M-CAG, with high values of number of polymorphic bands, polymorphic information content, marker index and Shannon's diversity index, were the most effective in detecting genetic variation within the walnut germplasm. Multivariate analysis of variance indicated 93.98% of the genetic variability was between individuals, while 6.32% of variation was among populations. A relatively new technique, an advanced maximization strategy with a heuristic approach, was deployed to develop the core collection. Initially, three independent core collections (CC1-CC3) were created using phenotypic data and molecular markers. The three core collections (CC1-CC3) were then merged to generate a composite core collection (CC4). The mean difference percentage, variance difference percentage, variable rate of coefficient of variance percentage, coincidence rate of range percentage, Shannon's diversity index, and Nei's gene diversity were employed for comparative analysis. The CC4 with 46 accessions represented the complete range of phenotypic and genetic variability. This study is the first report describing development of a core collection in walnut using molecular marker data in combination with phenotypic values. The construction of core collection could facilitate the work for identification of genetic determinants of trait variability and aid effective utilization of diversity caused by outcrossing, in walnut breeding programs.

Eastern Black Walnut (Juglans nigra L.) Originating From Native Range Varies in Their Response to Inoculation With Geosmithia morbida

Thousand cankers disease (TCD) is caused by the walnut twig beetle (Pityophthorus juglandis) vectoring the fungal canker pathogen Geosmithia morbida, which can result in severe dieback and eventual death to species of walnut (Juglans spp.) and wingnut (Pterocarya spp.). This disease is most devastating to the highly valued species J. nigra (black walnut). This species is primarily grown and harvested for timber production in the Central Hardwood Region of the United States, which comprises part of its native range. Management options for TCD are limited; therefore, finding resistant genotypes is needed. Initial studies on black walnut susceptibility to G. morbida documented some genetic variation and suggested potential resistance. Furthermore, G. morbida is thought to be native to the United States, which may have allowed for co-evolution. To capture the representative genetic diversity and screen for resistance to G. morbida, J. nigra families were collected from across the native range. These wild trees, in conjunction with seedlings developed in a black walnut timber improvement program, were planted in a common garden in Fort Collins, Colorado and repeatedly inoculated with G. morbida over the course of four years and three growing seasons. Improved seedlings exhibited larger cankered areas than wild J. nigra of the same provenance. Cankers induced by G. morbida in wild germplasm were smaller on J. nigra collected from the western and central portions of the native range compared to those collected from the eastern portion. Although trees from the western and central part of the range still incurred cankers, our findings indicate that variation in genetic resistance to G. morbida is present in black walnut. This study was performed with G. morbida independent of the walnut twig beetle, but our results suggest the limited G. morbida resistance observed in J. nigra will prevent the full compromise of black walnut to TCD. Results from this study should be taken into consideration in future black walnut breeding programs.

Exploring low-chill genotypes of Persian walnut (Juglans regia L.) in west of Iran

Climate change may cause a significant decline in winter chill over the coming decades and is becoming an important issue for walnut (Juglans regia L.) breeding programs to address. The current study was conducted to identify superior walnut genotypes with low-chilling requirements in western Iran (Ilam province). In the first step, approximately 200 walnut trees of seed-origin were pre-selected based on a questionnaire. Based on this primary evaluation for phenotypic and horticultural traits, 55 of the 200 genotypes were selected for further study. Morphological and phenological traits of these 55 were evaluated for two consecutive years and 11 genotypes were selected as superior. These superior genotypes were moderate to late-leafing with high yield, lateral fruitfulness, thin shells, and light to extra light kernel color. Nut weight, kernel weight and kernel percentage of the 11 superior genotypes ranged between 12.6–15.9 g, 6.9–8.2 g, and 49.7–62.0%, respectively. These superior genotypes had high levels of protein (13.3–18.2%), oil (55.3–65.9%) and PUFAs content. Assessment of chilling requirement of these superior genotypes showed that eight (IrIlZg1, IrIlZg13, IrIlZg7, IrIlZg12, IrIlAa92, IrIlAa47, IrIlAa48 and IrIlAa27) had significantly lower chilling requirements than the cultivar ‘Chandler’. Although having low-chill requirements, these genotypes were moderate to late-leafing. This may be due to their high heat requirement, since chilling requirement together with heat accumulation determines the leafing and flowering date. In conclusion, the selected low chilling requirement genotypes having moderate to late leafing could be used in walnut breeding programs to release commercial cultivars which are suitable for areas with spring frost concerns and expected future loss of winter chill due to climate change.

Biochemical Properties of Superior Persian Walnut Genotypes Originated from Southwest of Iran

Evaluation of genetic diversity and identification of superior genotypes is a fundamental stepin walnut breeding programs. In addition, information on biochemical properties of superiorgenotypes can help walnut breeders to release commercial varieties with high kernel quality.To gain more information on superior genotypes, a walnut population located in southwest ofIran was morphologically evaluated from 2010 to 2016. Based on important walnut breedingtraits, nine superior walnut genotypes were selected from a total of 612 tested genotypes.These genotypes were characterized by high yield, moderate to late-leafing, lateral bearing,thin shell and large nuts with light and extra-light kernel color. Biochemical traits of theselected superior genotypes were evaluated for two consecutive years (2017 and 2018) and ahigh variation was observed among genotypes in respect of oil, protein and total phenolcontents. Oil, protein and phenol contents of walnut kernels ranged between 57.9 to 69.6%,13.0 to18.1% and 46.6 to 61.5 mg GAE g-1, respectively. Polyunsaturated fatty acids (PUFA),monounsaturated fatty acids (MUFA) and saturated fatty acids (SFA) constituted on average63.8%, 26.7% and 9.7% of fatty acid content, respectively. There was a negative correlationbetween some phenological traits and oil and protein contents. Lateral bearing genotypes haddarker kernels with higher amounts of saturated fatty acids. In general, the selected walnutgenotypes not only are superior in various aspects of phenotypic characteristics, but also havehigh kernel quality and nutritional value which can be used as a source of desirable genes forfuture walnut breeding programs.

Major QTL with pleiotropic effects controlling time of leaf budburst and flowering-related traits in walnut (Juglans regia L.)

Breeding studies in walnut (Juglans regia L.) are usually time consuming due to the long juvenile period and therefore, this study aimed to determine markers associated with time of leaf budburst and flowering-related traits by performing a genome-wide association study (GWAS). We investigated genotypic variation and its association with time of leaf budburst and flowering-related traits in 188 walnut accessions. Phenotypic data was obtained from 13 different traits during 3 consecutive years. We used DArT-seq for genotyping with a total of 33,519 (14,761 SNP and 18,758 DArT) markers for genome-wide associations to identify marker underlying these traits. Significant correlations were determined among the 13 different traits. Linkage disequilibrium decayed very quickly in walnut in comparison with other plants. Sixteen quantitative trait loci (QTL) with major effects (R2 between 0.08 and 0.23) were found to be associated with a minimum of two phenotypic traits each. Of these QTL, QTL05 had the maximum number of associated traits (seven). Our study is GWAS for time of leaf budburst and flowering-related traits in Juglans regia L. and has a strong potential to efficiently implement the identified QTL in walnut breeding programs.

Persian walnut breeding program at Naric Fruticulture Research Institute in Hungary

Persian walnut breeding program at Naric Fruticulture Research Institute in Hungary

Exploring of Walnut Genetic Resources in Kazakhstan and Evaluation of Promising Selections

Kazakhstan has very rich walnut genetic resources; however there is no ongoing walnut breeding program. Kazakhstan government has several projects in cooperation with Russia, USA, Bulgaria, Czech Republic and Poland for plant breeding purposes. In the present research walnut genetic resources originated from Jabagil, Tulkibas, Sayram, Lenger, and Botanical Garden of International Hodja Ahmet Yesevi Turkish-Kazakh University of Kazakhstan were evaluated during 2015-2018. In the pre-selection stage, 47 genotypes were selected according to their lateral bearing, disease and pest tolerance. In the next step, 10 genotypes with high nut quality and high yield were selected. These genotypes were grafted onto seedling walnut rootstocks in Turkey. All of the grafted genotypes had fruit at the first year. Among 47 genotypes, we recorded nut weight between 6.21-15.18 g, kernel weight 2.36-6.64 g, kernel percentage 33.55-70.96% and average nut length 2.61-4.19 cm and nut diameter between 2.65 to 3.39 cm. The selected genotypes have been found to have very low fruit quality compared to commercial walnut varieties in the world. However, these genotypes have been evaluated as a good genetic resource for lateral bearing which can be used in breeding programs.

Deploying Genome Editing Tools for Dissecting the Biology of Nut Trees

Walnuts are among the most important nut crops grown in temperate regions of the world. Commercial production in California, and increasingly worldwide, relies on only few clonally grafted scion genotypes, particularly ‘Chandler’, and more recently clonally propagated disease-resistant rootstocks. Diseases, nematodes, insect pests, abiotic stresses, and other nutritional and environmental factors, can reduce walnut productivity and quality, affecting grower profitability. The California Walnut Breeding Program at UC Davis has developed and released scion cultivars and rootstocks to help address some of these problems. Recent sequencing of the walnut genome is expected to speed walnut breeding by facilitating development of molecular markers that can be linked to phenotypic traits, and by reducing the time needed to screen new genotypes, but conventional breeding of tree crops is still a long-term proposition. Here we describe a toolkit that uses a CRISPR-Cas9 system to enable rapid and precise editing of the genomes of currently grown commercial clonal walnut genotypes. A computational tool was developed to aid selection of guide RNAs targeting specific genomic sites and to identify potential off-target sites. As a proof-of-concept, the gene encoding phytoene desaturase (PDS) of walnut was targeted, disrupting its expression. This resulted in albino leaves; a dominant visual phenotype associated with the successful editing of this gene. This toolkit can now be adapted to other important tree crop members of the order Fagales, which includes other valuable nuts and trees species such as pecans, butternuts, chestnuts, hickory nuts, hazelnut, wingnut and oaks.

Microsatellite Borders and Micro-sequence Conservation in Juglans

Walnuts (Juglans spp.) are economically important nut and timber species with a worldwide distribution. Using the published Persian walnut genome as a reference for the assembly of short reads from six Juglans species and several interspecific hybrids, we identified simple sequence repeats in 12 Juglans nuclear and organellar genomes. The genome-wide distribution and polymorphisms of nuclear and organellar microsatellites (SSRs) for most Juglans genomes have not been previously studied. We compared the frequency of nuclear SSR motifs and their lengths across Juglans, and identified section-specific chloroplast SSR motifs. Primer pairs were designed for more than 60,000 SSR-containing sequences based on alignment against assembled scaffold sequences. Of the >60,000 loci, 39,000 were validated by e-PCR using unique primer pairs. We identified primers containing 100% sequence identity in multiple species. Across species, sequence identity in the SSR-flanking regions was generally low. Although SSRs are common and highly dispersed in the genome, their flanking sequences are conserved at about 90 to 95% identity within Juglans and within species. In a few rare cases, flanking sequences are identical across species of Juglans. This comprehensive report of nuclear and organellar SSRs in Juglans and the generation of validated SSR primers will be a useful resource for future genetic analyses, walnut breeding programs, high-level taxonomic evaluations, and genomic studies in Juglandaceae.

First simple sequence repeat-based genetic linkage map reveals a major QTL for leafing time in walnut (Juglans regia L.)

Walnut (Juglans regia L.) is one of the most important nut tree species. Simple sequence repeat (SSR)-based genetic linkage maps are valuable and effective tools for studies on integrative and comparative mapping. The data available in literature on walnut is inadequate for linkage mapping. Therefore, in the present study, we aimed to construct the first SSR-based genetic linkage map using the ‘Chandler’ × ‘Kaplan-86’ F1 population and to identify QTL (Quantitative Trait Loci) for leafing time in walnut. A total of 386 SSRs were mapped to 16 linkage groups (LGs) after screening 1437 SSR primer pairs. Among the mapped markers, 166 SSRs (43.0%) were heterozygous in both the parents, whereas 113 (29.3%) and 107 (27.7%) markers were from one of the parents that were integrated into the female and male maps, respectively. The total length of the consensus map was 1568.2 cM, with an average length of 98.0 cM. It included an average of 24.1 markers per LG, and the mean distance between SSR markers was 4.3 cM. The Chandler maternal linkage map included 279 SSR markers of total map length 1285.8 cM, whereas the Kaplan-86 male genetic map contained 273 SSRs of total map length 1574.4 cM. The SSR-based linkage maps presented in this study are moderately dense and can be considered as a fundamental genetic linkage map for further genetic and molecular studies in walnut, as well as in other Juglans species. A major QTL was identified for leafing time on LG4 explaining 52.0–68.8% of the phenotypic variation at similar positions in parental and consensus maps. The identified QTL region with associate markers can be potential for marker-assisted selection in the future for walnut breeding programs.

Precocious genotypes and homozygous tendency generated by self-pollination in walnut

BackgroundObservations of precocious (early bearing) genotypes of walnut (Juglans regia L.) under natural conditions encouraged us to study the origin and genetic control of these fascinating traits.ResultsIn this study, the self-fertility, progeny performance, and simple sequence repeat (SSR) locus variation of iron walnut (Juglans sigillata Dode), an ecotype of J. regia, were investigated. The average self-pollinated fruit set rate of J. sigillata cv. ‘Dapao’ (DP) was 7.0% annually from 1979 to 1982. The average germination rate of self-pollinated seeds was 45.2% during the 4-year period. Most progeny had inbreeding depression. Nine representative self-pollinated progeny (SP1–SP9), with special or typical traits of DP, were selected. SP1–SP4 were precocious because they initiated flowers as early as 2 years after germination, compared to the 7–10-yr period that is typical of DP. SP9 had not flowered since 1980. Twelve SSR markers were used to analyze the SP and DP. The genome of SP had a tendency toward high levels of homozygosity. The high levels of homozygosity reported in 18 additional precocious walnut genotypes complemented the results of this study.ConclusionsThese results provide evidence of precocious phenotypes and genomes with high levels of homozygosity that might be generated from self-pollinating walnut. This suggests that self-pollination might facilitate the generation of unique homozygous parents for subsequent use in walnut-breeding programs. The results also indicate that more attention should be focused on adequate management of precocious walnut to avoid early depression in the production of nuts.

Analysis of genetic diversity and structure in a worldwide walnut (Juglans regia L.) germplasm using SSR markers.

Persian or English walnut (Juglans regia L.), the walnut species cultivated for nut production, is one of the oldest food sources known and is grown worldwide in temperate areas. France is the 7th leading producer as of 2016 with 39 kt. Deciphering walnut genetic diversity and structure is important for efficient management and use of genetic resources. In this work, 253 worldwide accessions from the INRA walnut germplasm collection, containing English walnut and several related species, were genotyped using 13 SSR (Single Sequence Repeat) markers selected from the literature to assess diversity and structure. Genetic diversity parameters showed a deficiency of heterozygotes and, for several SSRs, allele-specificities among the accessions tested. Principal Coordinate Analysis (PCoA) showed the 253 accessions clustered in largely in agreement with the existing botanical classification of the genus. Among the 217 J. regia accessions, two main clusters, accessions from Eastern Europe and Asia, and accessions from Western Europe and America, were identified using STRUCTURE software. This was confirmed by Principal Coordinate Analysis and supported by Neighbor-Joining tree construction using DARwin software. Moreover, a substructure was found within the two clusters, mainly according to geographical origin. A core collection containing 50 accessions was selected using the maximum length sub-tree method and prior knowledge about their phenotype. The present study constitutes a preliminary population genetics overview of INRA walnut genetic resources collection using SSR markers. The resulting estimations of genetic diversity and structure are useful for germplasm management and for future walnut breeding programs.

Genetic diversity of superior Persian walnut genotypes in Azadshahr, Iran.

Persian walnut (Juglans regia L.) is known to have originated in central and eastern Asia. Remnants of these wild populations can still be found in the Hyrcanian forest in north-eastern Iran. In this study, 102 individual walnut trees from four geographic populations in the Azadshahr province (Vamenan, Kashidar, Rudbar and Saidabad) were sampled. We characterized individual trees using 28 standard morphological traits. The range of traits varied widely for some economically important characteristics including nut weight (6.1-19.79g), kernel weight (2.9-9.4g), and kernel fill percentage (26.51-60.34%). After morphological evaluation, 39 superior individuals based on nut quality and kernel fill percentage were selected for further genetic analysis. Individual superior trees were genotyped using 10 simple sequence repeat markers (SSR) and genetic diversity. Number of alleles per locus ranged from 3 (WGA005) to 12 (WGA054). Clustering analysis of 10 SSR loci divided the genotypes into three main groups. PCoA analysis clearly sorted genotypes into one of four distinctive groups which aligned with the cluster analysis. All analyses showed that individuals from Saidabad were genetically distinct. Likewise, results indicated that the high level of genetic diversity in Azadshahr region walnuts may provide a diverse source for superior walnuts in walnut breeding programs.

Discrimination and assessment of black walnut (Juglans nigra L.) cultivars using phenology and microsatellite markers (SSRs)

Black walnut (Juglans nigra L.), a large tree native throughout the eastern United States, produces a high-quality edible nut. Our goal was to maintain the integrity of black walnut breeding programs by verifying the identity of accessions. We sampled 285 ramets of 78 cultivars from the black walnut nut breeding orchards and clonal repositories at the University of Missouri and Kansas State University. We employed both phenotypic and genotypic methods to identify and differentiate cultivars. Phenotypes were evaluated using seven phenological traits. Cultivars varied for all traits among each of the 4 yr, but the best morphological characteristics for evaluating cultivar identity were bud break date and date of first pistillate bloom. Samples (n = 285) were genotyped using 10 polymorphic microsatellite loci. The simple sequence repeats produced a total of 174 alleles and 17.2 alleles per locus. We detected 47 unique genotypes represented by more than one sample, including 128 instances of identical genotypes with different names (synonyms) and 106 instances of different genotypes with a shared name (homonyms). Our results indicated that multiple errors were committed during the propagation of these important cultivars. It may be difficult to determine which genotype is original to a cultivar name in the absence of a foundation plant materials collection or vouchered specimens. These results will assist black walnut breeders and producers by improving the integrity of breeding collections and by identifying the best phenological traits for rapid assessment of trueness to type.

Predicting breeding values and genetic components using generalized linear mixed models for categorical and continuous traits in walnut (Juglans regia)

Genetic components for economically important traits in walnut (Juglans regia) were estimated for the first time using historical pedigree and heirloom phenotypic data from the walnut breeding program at the University of California, Davis. The constructed pedigree is composed of ~ 15,000 individuals and is derived from current and historic phenotypic records dating back > 50 years and located across California. To predict the additive genetic values of individuals under selection, generalized linear mixed models (GLMM), implemented with MCMCglmm, were developed. Several repeatability models were established to obtain the best model and predict the genetic parameters for each trait. Repeatability for yield, harvest date, extra-light kernel color (ELKC), and lateral bearing were predicted at 0.82, 0.98, 0.63, and 0.96, respectively, and average narrow-sense heritabilities were 0.54, 0.77, 0.49, and 0.75, respectively. Each individual in the pedigree was ranked by its estimated breeding value (EBV). The genetic trend showed specific patterns for each trait, and real genetic improvement was found over time. The completed pedigree built here, the estimated breeding values, and the ranking of individuals according to their breeding values, can be used to guide future crossing designs in the walnut breeding program and future implementation of genomic selection methods in walnut.