Integrative analysis of physiology and genomics provides insights into freeze tolerance adaptations of Acacia koa along an elevational cline.

Natural selection for plant species in heterogeneous environments creates genetic variation for traits such as cold tolerance. While physiological or molecular analyses have been used to evaluate stress tolerance adaptations, combining these approaches may provide deeper insight. Acacia koa (koa) occurs from sea level to 2300 m in Hawai'i, USA. At high elevations, natural koa populations have declined due to deforestation, and freeze tolerance is a limiting factor for tree regeneration. We used physiology and molecular analyses to evaluate cold tolerance of koa populations from low (300-750 m), middle (750-1500 m), and high elevations (1500-2100 m). Half of the seedlings were cold acclimated by exposure to progressively lowered air temperatures for eight weeks (from 25.6/22.2°C to 8/4°C, day/night). Using the whole plant physiology-freezing test and koa C-repeat Binding Factor CBF genes, our results indicated that koa can be cold-acclimated when exposed to low, non-freezing temperatures. Seedlings from high elevations had consistently higher expression of Koa CBF genes associated with cold tolerance, helping to explain variation in cold-hardy phenotypes. Evaluation of the genetic background of 22 koa families across the elevations with low coverage RNA sequencing indicated that high elevation koa had relatively low values of heterozygosity, suggesting that adaptation is more likely to arise in the middle and low elevation sources. This physiology and molecular data for cold tolerance of koa across the elevation gradient of the Hawaiian Islands provides insights into natural selection processes and may help to support guidelines for conservation and seed transfer in forest restoration efforts.

Domestication and selection footprints in Persian walnuts (Juglans regia).

Walnut (Juglans) species are economically important hardwood trees cultivated worldwide for both edible nuts and high-quality wood. Broad-scale assessments of species diversity, evolutionary history, and domestication are needed to improve walnut breeding. In this study, we sequenced 309 walnut accessions from around the world, including 55 Juglans relatives, 98 wild Persian walnuts (J. regia), 70 J. regia landraces, and 86 J. regia cultivars. The phylogenetic tree indicated that J. regia samples (section Dioscaryon) were monophyletic within Juglans. The core areas of genetic diversity of J. regia germplasm were southwestern China and southern Asia near the Qinghai-Tibet Plateau and the Himalayas, and the uplift of the Himalayas was speculated to be the main factor leading to the current population dynamics of Persian walnut. The pattern of genomic variation in terms of nucleotide diversity, linkage disequilibrium, single nucleotide polymorphisms, and insertions/deletions revealed the domestication and selection footprints in Persian walnut. Selective sweep analysis, GWAS, and expression analysis further identified two transcription factors, JrbHLH and JrMYB6, that influence the thickness of the nut diaphragm as loci under selection during domestication. Our results elucidate the domestication and selection footprints in Persian walnuts and provide a valuable resource for the genomics-assisted breeding of this important crop.

Whole genome based insights into the phylogeny and evolution of the Juglandaceae.

BACKGROUND: The walnut family (Juglandaceae) contains commercially important woody trees commonly called walnut, wingnut, pecan and hickory. Phylogenetic relationships and diversification within the Juglandaceae are classic and hot scientific topics that have been elucidated by recent fossil, morphological, molecular, and (paleo) environmental data. Further resolution of relationships among and within genera is still needed and can be achieved by analysis of the variation of chloroplast, mtDNA, and nuclear genomes. RESULTS: We reconstructed the backbone phylogenetic relationships of Juglandaceae using organelle and nuclear genome data from 27 species. The divergence time of Juglandaceae was estimated to be 78.7 Mya. The major lineages diversified in warm and dry habitats during the mid-Paleocene and early Eocene. The plastid, mitochondrial, and nuclear phylogenetic analyses all revealed three subfamilies, i.e., Juglandoideae, Engelhardioideae, Rhoipteleoideae. Five genera of Juglandoideae were strongly supported. Juglandaceae were estimated to have originated during the late Cretaceous, while Juglandoideae were estimated to have originated during the Paleocene, with evidence for rapid diversification events during several glacial and geological periods. The phylogenetic analyses of organelle sequences and nuclear genome yielded highly supported incongruence positions for J. cinerea, J. hopeiensis, and Platycarya strobilacea. Winged fruit were the ancestral condition in the Juglandoideae, but adaptation to novel dispersal and regeneration regimes after the Cretaceous-Paleogene boundary led to the independent evolution of zoochory among several genera of the Juglandaceae. CONCLUSIONS: A fully resolved, strongly supported, time-calibrated phylogenetic tree of Juglandaceae can provide an important framework for studying classification, diversification, biogeography, and comparative genomics of plant lineages. Our addition of new, annotated whole chloroplast genomic sequences and identification of their variability informs the study of their evolution in walnuts (Juglandaceae).

A comparative assessment of morphological and molecular characterization among three Ziziphus species.

Genetic variability of 84 accessions of three Ziziphus species including Z. spina-christi, Z. nummularia and Z. mauritiana were analyzed using a combination of morphological traits and translation initiation codon (ATG) polymorphism. Both morphological and molecular data revealed a high level of inter and intra specific variations among the accessions. Accordingly, 90.49% of amplified fragments were polymorphic among the accessions with the mean values of 0.37 for polymorphic information content (PIC), 3.31 for resolving power (RP), and 1.95 for marker index (MI). The phylogenetic clustering clearly delineated the entire germplasm into three well supported distinct clusters according to the species sources. According to the Nei's genetic identity, Z. spina-christi and Z. nummularia were the most similar species and had high differentiation with Z. mauritiana. Moreover, the highest values for Shannon's information index (I = 0.505) and gene diversity (h = 0.347) were recorded in Z. spina-christi indicating there is higher genetic diversity compared with two other species. Four private alleles were identified in two species which could be beneficial for accessions authentication in argumentative situations. Moreover, results of the Mantel test showed there were moderate correlation between molecular and morphological matrices. In addition, estimation of bivariate correlations revealed there were significant positive and negative correlations between different variables, which offer a practical application of this information during phenotype based selection in ber improvement programs. The results of this investigation highlight the efficiency of translation initiation codon polymorphism for genetic characterization and accurate authentication of Ziziphus accessions as well as detecting and tagging morphologically important traits in this genus that would be helpful for implementation of effective conservation strategies and even broaden current genetic diversity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01000-7.

Morpho-Physiological and Genomic Evaluation of Juglans Species Reveals Regional Maladaptation to Cold Stress.

Climate change may have unpredictable effects on the cold hardiness of woody species planted outside of their range of origin. Extreme undulations in temperatures may exacerbate susceptibility to cold stress, thereby interfering with productivity and ecosystem functioning. Juglans L. and their naturally occurring interspecific F1 hybrids, are distributed natively across many temperate regions, and J. regia has been extensively introduced. Cold hardiness, an environmental and genetic factor yet to be evaluated in many native and introduced Juglans species, may be a limiting factor under future climate change and following species introductions. We evaluated cold hardiness of native North American and Eastern Asian Juglans along with J. regia genotypes using field data from the Midwestern United States (Indiana), controlled freezing tests, and genome sequencing with close assessment of Juglans cold hardy genes. Many Juglans species previously screened for cold-hardiness were genotypes derived from the Midwest, California, and Europe. In 2014, despite general climate adaptation, Midwestern winter temperatures of -30°C killed J. regia originating from California; however, naturalized Midwestern J. regia survived and displayed low damage. Hybridization of J. regia with black walnut (J. nigra) and butternut (J. cinerea) produced F1s displaying greater cold tolerance than pure J. regia. Cold hardiness and growth are variable in Midwestern J. regia compared to native Juglans, East Asian Juglans, and F1 hybrids. Phylogeny analyses revealed that J. cinerea sorted with East Asian species using the nuclear genome but with North American species using the organellar genome. Investigation of selected cold hardy genes revealed that J. regia was distinct from other species and exhibited less genetic diversity than native Juglans species Average whole genome heterozygosity and Tajima's D for cold hardy genes was low within J. regia samples and significantly higher for hybrid as well as J. nigra. We confirmed that molecular and morpho-physiological data were highly correlated and thus can be used effectively to characterize cold hardiness in Juglans species. We conclude that the genetic diversity within local J. regia populations is low and additional germplasm is needed for development of more regionally adapted J. regia varieties.

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.

Pollen flow and paternity in an isolated and non-isolated black walnut (Juglans nigra L.) timber seed orchard.

Artificial pollination of black walnut (Juglans nigra L.) is not practical and timber breeders have historically utilized only open-pollinated half-sib families. An alternate approach called "breeding without breeding," consists of genotyping open-pollinated progeny using DNA markers to identify paternal parents and then constructing full-sib families. In 2014, we used 12 SSR markers to genotype 884 open-pollinated half-sib progeny harvested from two clonal orchards containing 206 trees, comprised of 52 elite timber selections. Seed was harvested in 2011 from each of two ramets of 23 clones, one upwind and one downwind, based on prevailing wind direction from the west-southwest. One orchard was isolated from wild black walnut and composed of forward selections while the other orchard was adjacent to a natural forest containing mature black walnut composed of backward selections. Isolation significantly increased within-orchard pollination (85%) of the progeny from the isolated orchard compared to 42% from the non-isolated orchard. Neither prevailing wind direction nor seed tree position in the orchard affected paternity patterns or wild pollen contamination. Genetic diversity indices revealed that progeny from both orchards were in Hardy-Weinberg equilibrium with very little inbreeding and no selfing. A significant level of inbreeding was present among the forward selected parents, but not the first generation (backward selected) parents. Some orchard clones failed to sire any progeny while other clones pollinated upwards of 20% of progeny.

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 (6.1-19.79 g), kernel weight (2.9-9.4 g), 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.

Evaluation of genetic variability among "Early Mature" Juglans regia using microsatellite markers and morphological traits.

Limiting the juvenile phase and reducing tree size are the two main challenges for breeders to improve most fruit crops. Early maturation and dwarf cultivars have been reported for many fruit species. "Early mature" and low vigor walnut genotypes were found among seedlings of Persian walnut. Nine microsatellite markers were used to evaluate genetic diversity among "Early Mature" Persian walnut accessions and provide a comparison with "normal growth" accessions. Six maturation related characteristics were also measured in "Early Mature" samples. Phenotypic traits and diversity indices showed relatively high levels of genetic diversity in "Early Mature" seedlings and indicated high differentiation between individuals. Seedling height, the most diverse phenotypic trait, has an important role in the clustering of "Early Mature" accessions. The "Early Mature" type had higher number of alleles, number of effective allele, and Shannon index compared to the "Normal Growth" group. The two types of studied walnuts had different alleles, with more than half of produced alleles specific to a specific group. "Early Mature" and "Normal Growth" walnuts had 27 and 17 private alleles, respectively. Grouping with different methods separated "Early Mature" and "Normal Growth" samples entirely. The presence of moderate to high genetic diversity in "Early Mature" walnuts and high genetic differentiation with "Normal Growth" walnuts, indicated that "Early Mature" walnuts were more diverse and distinct from "Normal Growth" samples. Moreover, our results showed SSR markers were useful for differentiating between "Early Mature" and "Normal Growth" walnuts. A number of identified loci have potential in breeding programs for identification of "Early Mature" walnuts at the germination phase.