Adam D. Henk

Genetic diversity and population structure in Malus sieversii, a wild progenitor species of domesticated apple

Malus sieversii (Lebed.) M. Roem. is a wild progenitor species of the domesticated apple. It is found across a mountainous region of central Asia and has been the focus of several collection expeditions by the USDA-ARS-National Plant Germplasm System. This study used microsatellite variation at seven loci to estimate diversity and differentiation within M. sieversii using several complimentary approaches. Multilocus genotypes were amplified from 949 individuals representing seedling trees from 88 half-sib families from eight M. sieversii populations collected in Kazakhstan. Apportioning of genetic variation was estimated at both the family and site level. Analyses using a hierarchical model to estimate Fst showed that differentiation among individual families is more than three times greater than differentiation among sites. In addition, average gene diversity and allelic richness varied significantly among sites. A rendering of a genetic network among all sites showed that differentiation is largely congruent with geographical location. In addition, nonhierarchical Bayesian assignment methods were used to infer genetic clusters across the collection area. We detected four genetic clusters in the data set. The quality of these assignments was evaluated over multiple Markov Chain Monte Carlo runs using both posterior likelihood and stability of the assignments. The spatial pattern of genetic assignments among the eight collection sites shows two broadly distributed and two narrowly distributed clusters. These data indicate that the southwestern collection sites are more admixed and more diverse than the northern sites.

Genetic diversity in Malus ×domestica (Rosaceae) through time in response to domestication

UNLABELLED • PREMISE OF THE STUDY Patterns of genetic diversity in domesticated plants are affected by geographic region of origin and cultivation, intentional artificial selection, and unintentional genetic bottlenecks. While bottlenecks are mainly associated with the initial domestication process, they can also affect diversity during crop improvement. Here, we investigate the impact of the improvement process on the genetic diversity of domesticated apple in comparison with other perennial and annual fruit crops.• METHODS Apple cultivars that were developed at various times (ranging from the 13th through the 20th century) and 11 of the 15 apple cultivars that are used for 90% of the apple production in the United States were surveyed for genetic diversity based on either 9 or 19 simple sequence repeats (SSRs). Diversity was compared using standard metrics and model-based approaches based on expected heterozygosity (He) at equilibrium. Improvement bottleneck data for fruit crops were also collected from the literature.• KEY RESULTS Domesticated apples showed no significant reduction in genetic diversity through time across the last eight centuries. Diversity was generally high, with an average He > 0.7 for apples from all centuries. However, diversity of the apples currently used for the bulk of commercial production was lower.• CONCLUSIONS The improvement bottleneck in domesticated apples appears to be mild or nonexistent, in contrast to improvement bottlenecks in many annual and perennial fruit crops, as documented from the literature survey. The low diversity of the subset of cultivars used for commercial production, however, indicates that an improvement bottleneck may be in progress for this perennial crop.

Chloroplast heterogeneity and historical admixture within the genus Malus

UNLABELLED • PREMISE OF THE STUDY The genus Malus represents a unique and complex evolutionary context in which to study domestication. Several Malus species have provided novel alleles and traits to the cultivars. The extent of admixture among wild Malus species has not been well described, due in part to limited sampling of individuals within a taxon.• METHODS Four chloroplast regions (1681 bp total) were sequenced and aligned for 412 Malus individuals from 30 species. Phylogenetic relationships were reconstructed using maximum parsimony. The distribution of chloroplast haplotypes among species was examined using statistical parsimony, phylogenetic trees, and a median-joining network.• KEY RESULTS Chloroplast haplotypes are shared among species within Malus. Three major haplotype-sharing networks were identified. One includes species native to China, Western North America, as well as Malus domestica Borkh, and its four primary progenitor species: M. sieversii (Ledeb.) M. Roem., M. orientalis Uglitzk., M. sylvestris (L.) Mill., and M. prunifolia (Willd.) Borkh; another includes five Chinese Malus species, and a third includes the three Malus species native to Eastern North America.• CONCLUSIONS Chloroplast haplotypes found in M. domestica belong to a single, highly admixed network. Haplotypes shared between the domesticated apple and its progenitors may reflect historical introgression or the retention of ancestral polymorphisms. Multiple individuals should be sampled within Malus species to reveal haplotype heterogeneity, if complex maternal contributions to named species are to be recognized.

Probabilistic viability calculations for cryopreserving vegetatively propagated collections in genebanks

Vegetatively propagated plant collections within genebanks are vulnerable to threats when they are maintained in field or greenhouse conditions. International genebanking standards recommend that these collections be securely backed-up at either secondary locations or by using in vitro culture or cryopreservation. A series of tools and tables were developed to assist genebank managers in determining the predicted number of viable explants after cryopreservation procedures have been employed based on the number of samples processed and the viability for a range of confidence levels (0.5, 0.75, 0.90, 0.95, and 0.99). In addition, critical viabilities were estimated which represent the minimum viability required to have at least one viable explant for a given number of explants stored per cryovial or tube. The plant cryobanking literature was surveyed to summarize the sample processing practices that have been implemented within genebanks. The minimum number of explants predicted to be viable for accessions within these cryobanked collections was calculated based on the published minimum viability levels and numbers of explants processed. Implementation of the proposed tools and tables will allow genebank managers to determine if shifts in their current methods could result in more efficient back-up procedures.

Seeds capture the diversity of genetic resource collections of Malus sieversii maintained in an orchard

Many species in genebank collections are maintained vegetatively either as in vitro cultures or as plants in the field or greenhouse. In these cases, individual genotypes (cultivars) are the focus of conservation. In crop wild relatives, where the value of an accession is in the heterogeneity of segregating genotypes, seeds may be the most representative form of conservation. We used two sets of field-planted Malus sieversii apple trees that originated from seeds collected from two wild populations in Kazakhstan as a model system to determine if seeds from hand-pollinated crosses could be used to represent the allelic diversity of the parent trees in a genebank orchard. A crossing design was developed that used each of the selected trees from the two Kazakhstan collection sites as mothers and also as fathers in the form of bulked pollen pools. The sets of offspring from these crosses were genotyped using microsatellite markers. Paternity analyses revealed the percentage of offspring that was derived from each of the parent trees. We also determined the contribution of each parent to the fertilization events from the pollen pools. Although there were some unintended pollination events (fertilization by trees in unexpected pollen pools) and unequal contribution of pollen parents within pollen pools, we demonstrated that the seedlings effectively captured the allelic frequency and diversity of the parents. A seed collection strategy is the most efficient way to conserve the diversity of crop wild relatives even in collections typically conserved as individual clones.

Chloroplast sequence data differentiate Maleae, and specifically Pyrus, species in the USDA-ARS National Plant Germplasm System

The USDA-ARS National Plant Germplasm System (NPGS) maintains a large collection of Maleae, including 49 Malus taxa, 36 Pyrus taxa, and 24 additional genera with ornamental and plant breeding value. These plant genetic resources are primarily maintained clonally as trees or shrubs in field conditions, and seeds are also conserved for some species. We used NPGS Maleae taxa to assess the genetic diversity across the tribe Maleae and placed Pyrus taxa within this broader context using analytical methods that displayed the genetic relationships as a network, rather than as a traditional dendrogram. Sequence variation from four conserved chloroplast regions unraveled the complex and often reticulate genetic relationships among and within 109 economically important Maleae taxa. In a broad sense, chloroplast haplotypes differentiated Pyrus species within Sections Pyrus and Pashia. The genetic relationships amongst Pyrus species were found to be complex, likely resulting from multiple hybridization and expansion/contraction events during the speciation process. Knowledge of the genetic relationships among Maleae genera and/or species may aid in the selection of materials for use as rootstocks and or breeding (hybridization) programs. Future collection efforts to augment the NPGS accessions within the tribe Maleae will improve the coverage and representation and assure conservation of important Rosaceae genetic resources in the NPGS.