Milan Lstibůrek

Breeding without breeding: is a complete pedigree necessary for efficient breeding?

Complete pedigree information is a prerequisite for modern breeding and the ranking of parents and offspring for selection and deployment decisions. DNA fingerprinting and pedigree reconstruction can substitute for artificial matings, by allowing parentage delineation of naturally produced offspring. Here, we report on the efficacy of a breeding concept called “Breeding without Breeding” (BwB) that circumvents artificial matings, focusing instead on a subset of randomly sampled, maternally known but paternally unknown offspring to delineate their paternal parentage. We then generate the information needed to rank those offspring and their paternal parents, using a combination of complete (full-sib: FS) and incomplete (half-sib: HS) analyses of the constructed pedigrees. Using a random sample of wind-pollinated offspring from 15 females (seed donors), growing in a 41-parent western larch population, BwB is evaluated and compared to two commonly used testing methods that rely on either incomplete (maternal half-sib, open-pollinated: OP) or complete (FS) pedigree designs. BwB produced results superior to those from the incomplete design and virtually identical to those from the complete pedigree methods. The combined use of complete and incomplete pedigree information permitted evaluating all parents, both maternal and paternal, as well as all offspring, a result that could not have been accomplished with either the OP or FS methods alone. We also discuss the optimum experimental setting, in terms of the proportion of fingerprinted offspring, the size of the assembled maternal and paternal half-sib families, the role of external gene flow, and selfing, as well as the number of parents that could be realistically tested with BwB.

Comparison of genetic parameters from marker-based relationship, sibship, and combined models in Scots pine multi-site open-pollinated tests

Nine microsatellite DNA markers (simple sequence repeats, SSRs) were used to estimate pairwise relationships among 597 Scots pine (Pinus sylvestris) trees as well as to generate a sibship structure for quantitative genetic parameters’ estimation comparison. The studied trees were part of an open-pollinated progeny test of 102 first-generation parents. Three methods were used to estimate variance components and heritabilities, namely, structured pedigree (half- and full-sib), marker-based pairwise relationships (four pairwise estimators), and a combined pedigree and marker-based relationship. In each of the three methods, the same animal model was used to compute variances except when marker-based relationship was used wherein we substituted the average numerator relationship matrix (i.e., pedigree-based matrix) with that computed based on markers’ pairwise relationships. Our results showed a high correlation in estimated breeding values between the pedigree (full-sib) and the combined marker-pedigree estimates. The marker-based relationship method produced high correlations when individual site data were analyzed. In contrast, the marker-based relationship method resulted in a significant decrease in both variance estimation and their standard errors which were in concordance with earlier published results; however, no estimates were produced when across-site analyses were attempted. We concluded that the combined pedigree method is the best approach as it represents the historical (pairwise) and contemporary (pedigree) relationships among the tested individuals, a situation that cannot be attained by any of the used methods individually. This method is dependent on the number and informativeness of the markers used.

Breeding without Breeding

We developed a deterministic model to optimize DNA fingerprinting effort in the presence of gene flow during the application of Breeding without Breeding. The method considers trait’s heritability, level of gene flow, selection differential, and the proportion of progeny test subjected to fingerprinting (truncation). All the model’s variables individually or in concert proved important; however, the trait’s heritability magnitude played the most important role in minimizing the selection of contaminated individuals and it could be used as a surrogate to traits with low heritability.

Breeding without breeding: minimum fingerprinting effort with respect to the effective population size

We present a probabilistic model to minimize the fingerprinting effort associated with the implementation of the “breeding without breeding” scheme under partial pedigree reconstruction. Our approach is directed at achieving a declared target population’s minimum effective population size (Ne), following the pedigree reconstruction and genotypic selection and is based on the graph theory algorithm. The primary advantage of the proposed method is to reduce the cost associated with fingerprinting before the implementation of the pedigree reconstruction for seed parent–offspring derived from breeding arboreta and production or natural populations. Stochastic simulation was conducted to test the method’s efficiency assuming a simple polygenic model and a single trait. Hypothetical population consisted of 30 parental trees that were paired at random (selfing excluded), resulting in 600 individuals (potential candidates for forwards selection). The male parentage was assumed initially unknown. The model was used to estimate the minimum genotyping sample size needed to reaching the prescribed Ne. Results were compared with the known pedigree data. The model was successful in revealing the true relationship pattern over the whole range of Ne. Two to three offspring entered genotyping to meet the Ne = 2 while 41 to 43 were required to satisfy the Ne = 14. Importantly, genetic gain was affected at the lower limits of the genotyping effort. Doubling the number of parents resulted in considerable reduction of the genotyping effort at higher Ne values.

Expansion of the minimum-inbreeding seed orchard design to operational scale

The minimum-inbreeding (MI) seed orchard layout, formulated originally as a global quadratic assignment problem, was expanded into realistic problem sizes that are often encountered in operational forestry, where two modifications were tested: (1) the merging algorithm of independent MI’s solutions (i.e., smaller blocks) and (2) the extended global (genetic-tabu) algorithm. Extending the global heuristic algorithm of the quadratic assignment problem seems to be the most efficient strategy. The reported minimum-inbreeding distance of the extended MI scheme was the lowest in comparison to the completely randomized and the randomized, replicated, staggered clonal-row (R2SCR) seed orchard design schemes. These conclusions also hold for more complex scenarios when added relatedness among orchard’s parents or unequal deployment was considered. This improved MI scheme is suitable to large and complex advanced-generation seed orchards, where many practical constraints have to be jointly considered.

Randomized, replicated, staggered clonal-row (R2SCR) seed orchard design

Spatial randomization of clones across a seed orchard’s grid is commonly applied to promote cross-fertilization and minimize selfing. The high selection differential attained from advanced-generation breeding programs sets high premier on the genetic gain and diversity delivery from seed orchards, thus clonal allocation is important and even more challenging when clones share common ancestry. Evidences of low selfing in many conifers’ seed orchards, as a result of their high genetic load, inbreeding depression, and polyembryony are abundant and call for orchards’ design re-evaluation, specifically when randomization is associated with added managerial burden. Clonal-rows represent a viable option for simplifying orchards management; however, they are often associated with elevated correlated matings between adjacent clones. Here, we propose a modified clonal-row design that replicates, staggers, and randomizes the rows, thus doubling the number of adjacent clones and providing different set of neighboring clones at each replication, thus allowing accommodating related parents more readily than any single-tree arrangement. We present a novel algorithm packaged in user-friendly software for executing various seed orchards’ designs. The developed program is interactive and suitable for any orchard size and configuration, accommodates any number of clones that are allocated to rows with variable length (ranging from a single tree to any even number) and pre-set separation zone between ramets of the same clone. The program offers three deployment modes (equal, linear, and custom) each with multiple layouts determined by the number of iterations requested. The resulting layouts are ranked based on four criteria including: (1) the number of empty positions, (2) deviation between expected and observed clone size, (3) minimum inbreeding, and (4) a neighborhood index that expresses the efficiency of clonal distribution.

Dynamic Gene-Resource Landscape Management of Norway Spruce: Combining Utilization and Conservation.

Traditional gene-resource management programs for forest trees are long-term endeavors requiring sustained organizational commitment covering extensive landscapes. While successful in maintaining adaptation, genetic diversity and capturing traditional growth attributes gains, these programs are dependent on rigid methods requiring elaborate mating schemes, thus making them slow in coping with climate change challenges. Here, we review the significance of Norway spruce in the boreal region and its current management practices. Next, we discuss opportunities offered by novel technologies and, with the use of computer simulations, we propose and evaluate a dynamic landscape gene-resource management in Norway. Our suggested long-term management approach capitalizes on: (1) existing afforestation activities, natural crosses, and DNA-based pedigree assembly to create structured pedigree for evaluation, thus traditional laborious control crosses are avoided and (2) landscape level genetic evaluation, rather than localized traditional progeny trials, allowing for screening of adapted individuals across multiple environmental gradients under changing climate. These advantages lead to greater genetic response to selection in adaptive traits without the traditional breeding and testing scheme, facilitating conservation of genetic resources within the breeding population of the most important forest tree species in Norway. The use of in situ selection from proven material exposed to realistic conditions over vast territories has not been conducted in forestry before. Our proposed approach is in contrast to worldwide current programs, where genetic evaluation is constrained by the range of environments where testing is conducted, which may be insufficient to capture the broad environmental variation necessary to tackle adaptation under changing climate.

Optimum neighborhood seed orchard design

Original seed orchard algorithm “Optimum Neighborhood Seed Orchard Design” was developed as local heuristics to facilitate the establishment of advanced-generation seed orchards with complex configurations. The scheme leads to uniform spatial distribution among adjacent genetic entries, promoting panmixia. The resulting scheme is thus suitable to establishing both productive seed orchards as well as clonal archives of threatened species, etc. Further, it can accommodate variable clonal sizes, uneven grids, relatedness, assortative mating. It may supplement existing layouts (clonal rows, etc.). Layouts outperformed existing advanced-generation alternative schemes in most parameters and thus could be recommended to operational forestry and gene conservation management.

Congruence between theory and practice: reduced contamination rate following phenotypic pre-selection within the Breeding without Breeding framework

Wind-pollinated seed orchards are often subjected to pollen intrusion from outside pollen sources. When wind-pollinated seed orchards seed are used to establish progeny trials, the offspring is expected to harbour varying proportion sired by outside sources. Theoretically the magnitude of selection differential between the orchards population and the contaminant pollen sources will affect the proportion of offspring resulting from outside pollen sources matings. If phenotypic pre-selection is implemented through selecting the top phenotypically ranked individuals from these progeny trials, then it is expected that the proportion of individuals sired by outside pollen sources will be lower within the pre-selected vs. unselected individuals. Here, we present empirical data from two Scots pine progeny trials supporting these theoretical expectations. The observed reduced contamination rate among fingerprinted elite offspring was 3.9 and 4.2%, suggesting a significant reduction in comparison to reported contamination rates between 21 and 70% in Scots pine. Results provide support to the proposed phenotypic pre-selection during the implementation of Breeding without Breeding scheme.

Innovative multiplex and its evaluation for effective genotyping of wild cherry

Trees from the family Rosaceae play an important role in forest and agricultural ecosystems. Therefore, they are often an object of interest for both forest and horticultural tree breeders. Here, we present the utilization of an effective microsatellite (SSRs) genotyping method for wild cherry (Prunus avium L.) and verified the discriminatory power of the presented multiplex by genotyping 48 genetically distinctive individuals (plus-trees). Concerned loci were previously proven to be cross-compatible among various cultivars of cherry, hence, the method could have a broader utilization beyond to the field of forestry. Our technique is based on post-PCR processing of 15 polymorphic SSRs loci amplified in three multiplex reactions with fluorescently labeled primers (6-FAM, VIC, PET and NED). All PCR products could be pooled and analyzed simultaneously (pseudo 15-plex). In order to make this approach feasible, we redefined sequences of several primers. Thus, utilizing modified primers provides non-overlapping amplicons of each fluorescent dye.