Developing core collections to optimize the management and the exploitation of diversity of the coffee Coffea canephora

The management of diversity for conservation and breeding is of great importance for all plant species and is particularly true in perennial species, such as the coffee Coffea canephora. This species exhibits a large genetic and phenotypic diversity with six different diversity groups. Large field collections are available in the Ivory Coast, Uganda and other Asian, American and African countries but are very expensive and time consuming to establish and maintain in large areas. We propose to improve coffee germplasm management through the construction of genetic core collections derived from a set of 565 accessions that are characterized with 13 microsatellite markers. Core collections of 12, 24 and 48 accessions were defined using two methods aimed to maximize the allelic diversity (Maximization strategy) or genetic distance (Maximum-Length Sub-Tree method). A composite core collection of 77 accessions is proposed for both objectives of an optimal management of diversity and breeding. This core collection presents a gene diversity value of 0.8 and exhibits the totality of the major alleles (i.e., 184) that are present in the initial set. The seven proposed core collections constitute a valuable tool for diversity management and a foundation for breeding programs. The use of these collections for collection management in research centers and breeding perspectives for coffee improvement are discussed.     

Ex situ conservation of underutilised fruit tree species: establishment of a core collection for Ficus carica L. using microsatellite markers (SSRs)

Ex situ germ plasm collections of woody crops are necessary to ensure the optimal use of plant genetic resources. The fig tree (Ficus carica L.) germ plasm bank, consisting of 229 accessions, is located in Centro de Investigación ‘La Orden’. Despite great progress in conservation, ex situ collections face size and organization problems. Core collections obtained from structured samples of bigger collections are a useful tool to improve germ plasm management. In this work, we used simple sequence repeat (SSR) markers to establish a core collection in this underutilised Mediterranean fruit tree species. Four approaches have been carried out (random sampling, maximization, simulated annealing and stepwise clustering) to determine the best method to develop a core collection in this woody plant. The genetic diversity obtained with each subset was compared with that of the complete collection. It was found that the most efficient way to achieve the maximum diversity was the maximization strategy, which, with 30 accessions, recovers all the SSR alleles and does not show significant differences in allele frequency distribution in any of the loci or in the variability parameters (H _O, H _E) between the whole and core collections. Thus, this core collection, a representative of most fig diversity conserved in the germ plasm bank, could be used as a basis for plant material exchange among researchers and breeders.     

Design of a Brassica rapa core collection for association mapping studies

A Brassica rapa collection of 239 accessions, based on two core collections representing different morphotypes from different geographical origins, is presented and its use for association mapping is illustrated for flowering time. We analyzed phenotypic variation of leaf and seed pod traits, plant architecture, and flowering time using data collected from three field experiments and evaluated the genetic diversity with a set of SSR markers. The Wageningen University and Research Centre (WUR) and the Vavilov Research Institute of Plant Industry (VIR) core collections had similar representations of most morphotypes, as illustrated by the phenotypic and genetic variation within these groups. The analysis of population structure revealed five subgroups in the collection, whereas previous studies of the WUR core collection indicated four subgroups; the fifth group identified consisted mainly of oil accessions from the VIR core collection, winter oils from Pakistan, and a number of other types. A very small group of summer oils is described, that is not related to other oil accessions. A candidate gene approach was chosen for association mapping of flowering time with a BrFLC1 biallelic CAPS marker and a BrFLC2 multiallelic SSR marker. The two markers were significantly associated with flowering time, but their effects were confined to certain morphotypes and (or) alleles. Based on these results, we discuss the optimal design for an association mapping population and the need to fix the heterogeneous accessions to facilitate phenotyping and genotyping.     

长江春大豆核心种质构建及分析

利用长江春大豆初选核心种质SSR(simple sequence repeat）标记和农艺性状表型等基础数据,对用不同个体取样方法以及不同数据类型建立的核心种质进行评价,目的是确定中国大豆（Glycine max）核心种质的最佳取样策略提供依据,结果表明,根据SSR分子数据聚类,采用类内随机取样,类内以遗传相似性系数取样以及仅依据遗传相似性系数取样都可用于大豆核心种质构建,但是综合不同评价参数发现,以类内随机取样最佳,类内按遗传相似性系数取样次之,单独以遗传相似性系数取样较差。分析不同SSR等位变异保留比例的遗传多样性指数发现,当保留90%和80%的SSR等位变异时,核心种质具有更高的遗传多样性,由于与SSR分子数据种质遗传关系评价的不一致性,农艺性状等基础数据虽然可用来构建核心种质,但其SSR分子水平代表性相对较低,本研究结果还表明,用不同方法或同一方法不同重复次数取样建立的核心种质具有异质性,且这种异质性随核心种质取样比例的降低而增大,因此,虽然可依据不同数据类型确定相应的方法建立核心种质,但综合表型和分子数据建立的核心种质更具有代表性。     

Assessing genetic potential in germplasm collections of crop plants by marker-trait association: a case study for potatoes with quantitative variation of resistance to late blight and maturity type

Genetic diversity of crop plants resulting from breeding and selection is preserved in gene banks. Utilization of such materials for further crop improvement depends on knowledge of agronomic performance and useful traits, which is usually obtained by phenotypic evaluation. Associations between DNA markers and agronomic characters in collections of crop plants would (i) allow assessment of the genetic potential of specific genotypes prior to phenotypic evaluation, (ii) identify superior trait alleles in germplasm collections, (iii) facilitate high resolution QTL mapping and (iv) validate candidate genes responsible for quantitative agronomic characters. The feasibility of association mapping was tested in a gene bank collection of 600 potato cultivars bred between 1850 and 1990 in different countries. The cultivars were genotyped with five DNA markers linked to previously mapped QTL for resistance to late blight and plant maturity. Specific DNA fragments were tested for association with these quantitative characters based on passport evaluation data. Highly significant association with QTL for resistance to late blight and plant maturity was detected with PCR markers specific for R1, a major gene for resistance to late blight, and anonymous PCR markers flanking the R1 locus at 0.2 Centimorgan genetic distance. The marker alleles associated with increased resistance and later plant maturity were traced to an introgression from the wild species S. demissum. These DNA markers are the first marker that are diagnostic for quantitative agronomic characters in a large collection of cultivars.     

The power to detect linkage disequilibrium with quantitative traits in selected samples

Results from power studies for linkage detection have led to many ongoing and planned collections of phenotypically extreme nuclear families. Given the great expense of collecting these families and the imminent availability of a dense diallelic marker map, the families are likely to be used in allelic-association as well as linkage studies. However, optimal selection strategies for linkage may not be equally powerful for association. We examine the power to detect linkage disequilibrium for quantitative traits after phenotypic selection. The results encompass six selection strategies that are in widespread use, including single selection (two designs), affected sib pairs, concordant and discordant pairs, and the extreme-concordant and -discordant design. Selection of sibships on the basis of one extreme proband with high or low trait scores provides as much power as discordant sib pairs but requires the screening and phenotyping of substantially fewer initial families from which to select. Analysis of the role of allele frequencies within each selection design indicates that common trait alleles generally offer the most power, but similarities between the marker- and trait-allele frequencies are much more important than the trait-locus frequency alone. Some of the most widespread selection designs, such as single selection, yield power gains only when both the marker and quantitative trait loci (QTL) are relatively rare in the population. In contrast, discordant pairs and the extreme-proband design provide power for the broadest range of QTL-marker-allele frequency differences. Overall, proband selection from either tail provides the best balance of power, robustness, and simplicity of ascertainment for family-based association analysis.     

Comparison of different methods to construct a core germplasm collection in woody perennial species with simple sequence repeat markers. A case study in cherimoya (Annona cherimola, Annonaceae), an underutilised subtropical fruit tree species

Although molecular markers are becoming the tool of choice to develop core collections in plants, the examples of their use in woody perennial species are very scarce. In this work, we used simple sequence repeat (SSR) marker data to develop a core collection in an underutilised subtropical fruit tree species, cherimoya ( Annona cherimola , Annonaceae), from an initial collection of 279 genotypes from different countries. We compared six alternative allocation methods to construct the core collection, four not based upon the similarity dendrogram [random sampling, maximisation strategy (M strategy) and simulated annealing algorithm maximising both genetic diversity and number of SSR alleles] and two based on dendrogram data (logarithmic strategy and stepwise clustering). The diversity maintained in each subset was compared with that present in the entire collection. The results obtained indicate that the use of SSRs together with the M strategy is the most efficient method to develop a core collection in cherimoya. In the best subset, with 40 accessions, all the SSR alleles present in the whole collection were recovered and no significant differences in frequency distribution of alleles for any of the loci studied or in variability parameters ( H _O, H _E) were recorded between the core and the whole collection.     

Targeted association analysis identified japonica rice varieties achieving Na+/K+ homeostasis without the allelic make-up of the salt tolerant indica variety Nona Bokra

During the last decade, a large number of QTLs and candidate genes for rice tolerance to salinity have been reported. Using 124 SNP and 52 SSR markers, we targeted 14 QTLs and 65 candidate genes for association mapping within the European Rice Core collection (ERCC) comprising 180 japonica accessions. Significant differences in phenotypic response to salinity were observed. Nineteen distinct loci significantly associated with one or more phenotypic response traits were detected. Linkage disequilibrium between these loci was extremely low, indicating a random distribution of favourable alleles in the ERCC. Analysis of the function of these loci indicated that all major tolerance mechanisms were present in the ERCC although the useful level of expression of the different mechanisms was scattered among different accessions. Under moderate salinity stress some accessions achieved the same level of control of Na(+) concentration and Na(+)/K(+) equilibrium as the indica reference variety for salinity tolerance Nona Bokra, although without sharing the same alleles at several loci associated with Na(+) concentration. This suggests (a) differences between indica and japonica subspecies in the effect of QTLs and genes involved in salinity tolerance and (b) further potential for the improvement of tolerance to salinity above the tolerance level of Nona Bokra, provided the underlying mechanisms are complementary at the whole plant level. No accession carried all favourable alleles, or showed the best phenotypic responses for all traits measured. At least nine accessions were needed to assemble the favourable alleles and all the best phenotypic responses. An effective strategy for the accumulation of the favourable alleles would be marker-assisted population improvement.     

Associative overdominance: Evaluating the effects of inbreeding and linkage disequilibrium

Expressions are obtained for the expected phenotypic values of homozygous and heterozygous genotypes for a neutral marker locus linked to a locus segregating for a recessive deleterious gene. The phenotypic values are functions of the allele frequencies at the marker locus, the inbreeding coefficient and the degree of association of the deleterious gene with the marker alleles. The analysis is extended to more than two alleles at the marker locus. Either linkage disequilibrium or inbreeding alone can produce an apparent superiority of heterozygotes for the marker locus (unless specified otherwise, the terms ‘homozygote’ and ‘heterozygote’ will refer to the marker locus). The effect of linkage disequilibrium on the difference between the heterozygote and homozygote values can be positive (associative overdominance) or negative (associative underdominance), depending on the frequencies of the marker alleles and the degree of their association with the deleterious gene. Inbreeding has always a positive effect. In general, the expected value of a homozygote is a positive function of its allele frequency. When the various homozygous genotypes are combined into one class and the various heterozygous genotypes into another, the phenotypic difference of the two classes is a function of the evenness of the allelic frequency distribution. Inbreeding is a more likely explanation of associative overdominance if the frequency of the deleterious gene is low, but its effect on the character high. Conversely, linkage disequilibrium is more likely if the frequency is high and the effect low. The degrees of association between marker alleles and the deleterious gene can, in principle, be estimated from the observed phenotypic scores and used to calculate expected multi-locus genotype scores. This could provide the basis for statistical tests of the associative overdominance hypothesis as an explanation of observed correlations between multi-locus heterozygosity and phenotypic traits.     

PowerCore: a program applying the advanced M strategy with a heuristic search for establishing core sets

MOTIVATION: Core sets are necessary to ensure that access to useful alleles or characteristics retained in genebanks is guaranteed. We have successfully developed a computational tool named 'PowerCore' that aims to support the development of core sets by reducing the redundancy of useful alleles and thus enhancing their richness. RESULTS: The program, using a new approach completely different from any other previous methodologies, selects entries of core sets by the advanced M (maximization) strategy implemented through a modified heuristic algorithm. The developed core set has been validated to retain all characteristics for qualitative traits and all classes for quantitative ones. PowerCore effectively selected the accessions with higher diversity representing the entire coverage of variables and gave a 100% reproducible list of entries whenever repeated. AVAILABILITY: PowerCore software uses the .NET Framework Version 1.1 environment which is freely available for the MS Windows platform. The files can be downloaded from http://genebank.rda.go.kr/powercore/. The distribution of the package includes executable programs, sample data and a user manual.     

Autosegregation of enzyme loci in agamospermous progenies of triploid plants of sugar beet (Beta vulgaris L.)

The ratios of the phenotypic classes of glucosephosphate isomerase (GP12) and malate dehydrogenase (MDH1 and MDH2) were studied in agamospermous progenies of triploid sugar beet plants. The ratio of the phenotypic classes of these enzymes corresponds to the calculations based on the assumption of polyteny of chromosomes carrying alleles of the enzyme loci accompanied by the loss of extra copies of the alleles in the first division of a cell entering embryogenesis. An increase in the gene dosage due to polyteny leads to the appearance in the progeny with a definite frequency of alleles that were absent in the original parental plant. The notions of meiotic autosegregation and mitotic autosegregation characteristic of meiotic and mitotic agamospermy are introduced, as well as the term locus polygenotype characterizing not only the allelic composition and the number of chromosomes, but also the number of chromatids carrying alleles of the marker locus in the cell before its entry into embryogenesis.     

Generating novel allelic variation through Activator insertional mutagenesis in maize

The maize transposable element Activator (Ac) has been exploited as an insertional mutagen to disrupt, clone, and characterize genes in a number of plant species. To develop an Ac-based mutagenesis platform for maize, a large-scale mutagenesis was conducted targeting the pink scutellum1 locus. We selected 1092 Ac transposition events from a closely linked donor Ac, resulting in the recovery of 17 novel ps1 alleles. Multiple phenotypic classes were identified corresponding to Ac insertions in the 5'-UTR and coding region of the predicted Ps1 gene. To generate a stable allelic series, we employed genetic screens and identified 83 germinally heritable ps1 excision alleles. Molecular characterization of these excision alleles revealed a position-dependent bias in excision allele frequencies and the predominance of 7- and 8-bp footprint products. In total, 19 unique ps1 excision alleles were generated in this study, including several that resulted in weak mutant phenotypes. The analysis of footprint alleles suggests a model of Ac excision in maize that is consistent with recent in vitro studies of hAT element excision. Importantly, the genetic and molecular methods developed in this study can be extended to generate novel allelic variation at any Ac-tagged gene in the genome.     

Power analysis for case–control association studies of samples with known family histories

Genome-wide case–control studies have been widely used to identify genetic variants that predispose to human diseases. Such studies are powerful in detecting common genetic variants with moderate effects, but quickly lose power as allele frequency and genotype relative risk decrease. Because patients with one or more affected relatives are more likely to inherit disease-predisposing alleles of a genetic disease than patients without family histories of the disease, sampling patients with affected relatives almost always increases the frequency of disease predisposing alleles in cases and improves the power of case–control association studies. This paper evaluates the power of case–control studies that select cases and/or controls according to their family histories of disease. Our results showed that this study design can dramatically increase the power of a case–control association study for a wide range of disease types. Because each additional affected relative of a patient reduces the required sample size roughly by a pair of case and control, inclusion of cases with affected relatives can dramatically decrease the required sample size and thus the cost of such studies.     

Methods of developing core collections based on the predicted genotypic value of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The selection of an appropriate sampling strategy and a clustering method is important in the construction of core collections based on predicted genotypic values in order to retain the greatest degree of genetic diversity of the initial collection. In this study, methods of developing rice core collections were evaluated based on the predicted genotypic values for 992 rice varieties with 13 quantitative traits. The genotypic values of the traits were predicted by the adjusted unbiased prediction (AUP) method. Based on the predicted genotypic values, Mahalanobis distances were calculated and employed to measure the genetic similarities among the rice varieties. Six hierarchical clustering methods, including the single linkage, median linkage, centroid, unweighted pair-group average, weighted pair-group average and flexible-beta methods, were combined with random, preferred and deviation sampling to develop 18 core collections of rice germplasm. The results show that the deviation sampling strategy in combination with the unweighted pair-group average method of hierarchical clustering retains the greatest degree of genetic diversities of the initial collection. The core collections sampled using predicted genotypic values had more genetic diversity than those based on phenotypic values.Communicated by D.J. Mackill     

Autosegregation of enzyme loci in agamospermous progenies of triploid plants of sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

The ratios of the phenotypic classes of glucosephosphate isomerase (GPI2) and malate dehydrogenase (MDH1 and MDH2) were studied in agamospermous progenies of triploid sugar beet plants. The ratio of the phenotypic classes of these enzymes corresponds to the calculations based on the assumption of polyteny of chromosomes carrying alleles of the enzyme loci accompanied by the loss of extra copies of the alleles in the first division of a cell entering embryogenesis. An increase in the gene dosage due to polyteny leads to the appearance in the progeny with a definite frequency of alleles that were absent in the original parental plant. The notions of “meiotic autosegregation” and “mitotic autosegregation” characteristic of meiotic and mitotic agamospermy are introduced, as well as the term locus polygenotype characterizing not only the allelic composition and the number of chromosomes, but also the number of chromatids carrying alleles of the marker locus in the cell before its entry into embryogenesis.     

Construction of prediction models for growth traits of soybean cultivars based on phenotyping in diverse genotype and environment combinations

As soybean cultivars are adapted to a relatively narrow range of latitude, the effects of climate changes are estimated to be severe. To address this issue, it is important to improve our understanding of the effects of climate change by applying the simulation model including both genetic and environmental factors with their interactions (G×E). To achieve this goal, we conducted the field experiments for soybean core collections using multiple sowing times in multi-latitudinal fields. Sowing time shifts altered the flowering time (FT) and growth phenotypes, and resulted in increasing the combinations of genotypes and environments. Genome-wide association studies for the obtained phenotypes revealed the effects of field and sowing time to the significance of detected alleles, indicating the presence of G×E. By using accumulated phenotypic and environmental data in 2018 and 2019, we constructed multiple regression models for FT and growth pattern. Applicability of the constructed models was evaluated by the field experiments in 2020 including a novel field, and high correlation between the predicted and measured values was observed, suggesting the robustness of the models. The models presented here would allow us to predict the phenotype of the core collections in a given environment.     

Statistical analysis strategies for association studies involving rare variants

The limitations of genome-wide association (GWA) studies that focus on the phenotypic influence of common genetic variants have motivated human geneticists to consider the contribution of rare variants to phenotypic expression. The increasing availability of high-throughput sequencing technologies has enabled studies of rare variants but these methods will not be sufficient for their success as appropriate analytical methods are also needed. We consider data analysis approaches to testing associations between a phenotype and collections of rare variants in a defined genomic region or set of regions. Ultimately, although a wide variety of analytical approaches exist, more work is needed to refine them and determine their properties and power in different contexts.     

Evidence for a natural allelic series at the Maize domestication Locus teosinte branched1

Despite numerous quantitative trait loci and association mapping studies, our understanding of the extent to which natural allelic series contribute to the variation for complex traits is limited. In this study, we investigate the occurrence of a natural allelic series for complex traits at the teosinte branched1 (tb1) gene in natural populations of teosinte (Zea mays ssp. parviglumis, Z. mays ssp. mexicana, and Z. diploperennis). Previously, tb1 was shown to confer large effects on both plant architecture and ear morphology between domesticated maize and teosinte; however, the effect of tb1 on trait variation in natural populations of teosinte has not been investigated. We compare the effects of nine teosinte alleles of tb1 that were introgressed into an isogenic maize inbred background. Our results provide evidence for a natural allelic series at tb1 for several complex morphological traits. The teosinte introgressions separate into three distinct phenotypic classes, which correspond to the taxonomic origin of the alleles. The effects of the three allelic classes also correspond to known morphological differences between the teosinte taxa. Our results suggest that tb1 contributed to the morphological diversification of teosinte taxa as well as to the domestication of maize.     

Genetic architecture of flag leaf length and width in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) revealed by association mapping

Rice flag leaf is the main photosynthetic organ and plays a key role in grain yield. In this study, 106 loci associated with flag leaf length and width were identified using association mapping in a rice mini core collection. Analyzing the phenotypic effects of each allele represented 156 positive and 167 negative alleles, with a few alleles showing inconsistent effect in different environments. Among the 106 loci, 69 were environment-specific loci, 16 were environmentally stable and 21 were environmentally sensitive. Fifteen associated markers were shared by two traits and were designated as pleiotropic markers. According to the frequency distribution of alleles in different variety types, 343 alleles were categorized into three types based on geographic source and usage in breeding. Among these, 156 were used alleles, 138 were unused and 49 were foreign alleles. The results further our understanding of the genetic mechanisms of flag leaf length and width.