The genetic diversity and evolution of field pea (<Emphasis Type="Italic">Pisum</Emphasis>) studied by high throughput retrotransposon based insertion polymorphism (RBIP) marker analysis

Background  

The genetic diversity of crop species is the result of natural selection on the wild progenitor and human intervention by ancient and modern farmers and breeders. The genomes of modern cultivars, old cultivated landraces, ecotypes and wild relatives reflect the effects of these forces and provide insights into germplasm structural diversity, the geographical dimension to species diversity and the process of domestication of wild organisms. This issue is also of great practical importance for crop improvement because wild germplasm represents a rich potential source of useful under-exploited alleles or allele combinations. The aim of the present study was to analyse a major Pisum germplasm collection to gain a broad understanding of the diversity and evolution of Pisum and provide a new rational framework for designing germplasm core collections of the genus.     

The use of RAPD analysis to classify Triticum accessions

 Crop germplasm collections contain a considerable percentage of misclassified accessions which may affect the use of germplasm for agricultural crop improvement. The objective of this study was to determine if random amplified polymorphic DNA (RAPD) analysis could be used to reclassify misclassified Triticum accessions. Twelve accessions suspected to be misclassified, based on morphological characters, as either macha or vavilovii wheat were studied using RAPD and cytological analyses. In the RAPD analysis, a dendrogram, based on Jaccard genetic similarity coefficients, grouped 5 dicoccum-like, 1 timopheevii-like, and 6 monococcum-like accessions with Triticum dicoccum, T. timopheevii, and T. monococcum accessions, respectively. These results were confirmed by the cytological analysis. A RAPD marker specific to the D genome was also detected. This study suggests that RAPD analysis can be used to classify germplasm and to distinguish some species in Triticum. Received: 12 June 1998 / Accepted: 18 August 1998     

Identification of interspecific hybrids among domesticated apple and its wild relatives

Potential interspecific hybrids are usually identified in natural populations by their proximity to interbreeding species or their intermediate phenotypes; hybridization can then be confirmed by comparing the genetic makeup of putative hybrids to pure species. In contrast, detecting interspecific hybridization and misclassifications in ex situ collections can be difficult because fine-scale geographic locations and species-specific phenotypic data are generally unavailable. Thus, there is little a priori information available to suggest which individuals might be hybrids. Instead, hybrids or misclassified individuals must be identified based on molecular data via population assignment and admixture detection programs. We have applied a variety of population assignment and admixture detection programs to over 400 samples of four closely related Malus species held in the US Department of Agriculture?CAgricultural Research Service National Plant Germplasm System that were genotyped at 19 simple sequence repeat loci. Our findings indicate that over 10?% of the samples of the wild species Malus sieversii and Malus orientalis and nearly 20?% of the samples of the wild species Malus sylvestris may be admixed or misclassified. The percentage of admixed or misclassified samples of the domesticated species, Malus × domestica, was much lower, at <5?%. These findings provide an illustration of how to detect hybridization and misclassification in ex situ collections using molecular data and, ultimately, should help to maximize the utility of the collections. In addition, the presence of wild-collected samples that show admixture with domesticated apple suggests that gene flow may be occurring from the crop into natural populations of the wild species.     

Temperature- and predator-induced phenotypic plasticity in Bosmina cornuta and B. pellucida (Crustacea: Cladocera)

SUMMARY 1. Clones of Bosmina cornuta and B. pellucida (B. longirostris species complex) were derived from samples collected from Scheuermühlenteich and Lake Windsborn(westernGermany). Experimental temperature change (to 10 °C and 20 °C) and exposure to Acanthocyclops vernalis copepods (12 L^?1) significantly altered external morphology in laboratory cultures of the two species. Morphological traits were derived from eight log₁₀‐transformed and standardised morphometric distances by factorial analysis: factor 1 represented body size, factor 2, size of appendages and factor 3, the head size. 2. Acclimation of clones to cold water (10 °C, >14 days) led to an increase in body, antennule and mucrone size in B. cornuta and B. pellucida. Moreover, at 10 °C, B. cornuta cultures usually collapsed within a few weeks. Compared to the trials at 10 °C, acclimation to 20 °C (the two species) and to 15 °C (B. pellucida only) left the size of body appendages unchanged. Individuals were unequivocally assigned to each species by discriminant functions. Conspecific individuals that were acclimated to different temperatures between 10 and 20 °C also differed in external morphology, but discriminant analysis yielded misclassification rates of 5.3–23.3%. 3. Morphological response to the presence of copepod predators was weaker than that caused by temperature change. Long‐term exposure of clones to copepod predators induced a significant increase in size of appendages in the two species but left body size unaffected. Again, species identification by discriminant functions could be made without any error, whereas conspecific controls and experimentals were misclassified at rates between 19.4 and 29.5%. 4. It is suggested that temperature is the main proximal cue for Bosmina cyclomorphosis. The distinct response to temperature of B. pellucida and B. cornuta may also account for seasonal differences in abundance observed in field.     

Molecular identification of cryptic bumblebee species from degraded samples using PCR–RFLP approach

The worldwide decline and local extinctions of bumblebees have raised a need for fast and accurate tools for species identification. Morphological characters are often not sufficient, and molecular methods have been increasingly used for reliable identification of bumblebee species. Molecular methods often require high‐quality DNA which makes them less suitable for analysis of low‐quality or older samples. We modified the PCR–RFLP protocol for an efficient and cost‐effective identification of four bumblebee species in the subgenus Bombus s. str. (B. lucorum, B. terrestris, B. magnus and B. cryptarum). We used a short partial mitochondrial COI fragment (446 bp) and three diagnostic restriction enzymes (Hinf I, Hinc II and Hae III) to identify species from degraded DNA material. This approach allowed us to efficiently determine the correct species from all degraded DNA samples, while only a subset of samples 64.6% (31 of 48) resulted in successful amplification of a longer COI fragment (1064 bp) using the previously described method. This protocol can be applied for conservation and management of bumblebees within this subgenus and is especially useful for fast species identification from degraded samples.     

Comparative analysis of core collection sampling methods for mandarin germplasm based on molecular and phenotypic data

Gene banks have been established to conserve the genetic diversity of crop species. Large germplasm collections lead to management problems (space, maintenance costs, etc.), especially in collections involving species with recalcitrant seeds that must be maintained as growing plants. Core collections (CCs) are thus developed to reduce the size of large germplasm collections while keeping the maximum variability. This also facilitates fine phenotypic evaluation. In this study, several software packages (DARwin, PowerMarker and MSTRAT) and methods (Max length subtree, M strategy, simulated annealing and MinSD) were compared to define a mandarin (Citrus reticulata) CC. One hundred and sixty‐seven accessions were sampled from two germplasm collections, which were genotyped with 50 SSR, 24 InDel and 68 single nucleotide polymorphism markers. All the CC obtained were tested for the maintenance of the genetic variability parameters (Ho and He) of the initial collection, the level of linkage disequilibrium (LD) and the phenotypic diversity retention. The Max length subtree function from DARWin seemed to be the most appropriate method for establishing a CC in C. reticulata. It maintained 96.82% of the allelic richness and 17.96% of the size of the initial collection with only 30 accessions. Besides it did not increase the LD (r² value) of the initial collection and retained the vast majority of the phenotypic variability. However, a CC with 70 accessions would be more helpful for genetic association studies.     

Peanut Resistance Gene Expression in Response to Aspergillus flavus Infection During Seed Germination

Aspergillus flavus produces potent mutagenic and carcinogenic polyketide‐derived secondary metabolites known as aflatoxins. Development of host plant resistance in peanut and other crops is the most environmentally friendly and cost‐effective method to eliminate the serious problem of aflatoxin contamination in grains. To confirm that putative peanut genes identified in a previous microarray study were involved in peanut resistance to A. flavus infection, 14 genes were selected for further investigation through real‐time PCR. The results revealed diverse patterns of gene expression during seed germination after A. flavus inoculation. Based on the expression levels and the relative‐expression patterns over a 7‐day period, the 14 host genes could be classified into six different groups belonging to three main biochemical and genetic defence processes of lipid metabolism, oxidative signalling and cell‐wall synthesis during counter‐attack. A network of gene expression patterns was activated in sequential order in response to A. flavus invasion in both resistant and susceptible peanut lines during seed germination. Understanding gene expression patterns in peanut will be useful to breeders and other scientists interested in incorporating genetic resources of resistance against A. flavus into peanut germplasm and/or commercial cultivars via conventional and/or molecular methods.     

Germplasm collections and breeding needs in Europe

Finding new ways to increase usage of germplasm from national genebanks for crop improvement remains an international challenge. Collections still appear deficient both in the quality and quantity of the data that describe the samples and this is clearly limiting the flow of material. Based on a survey of European breeders for two crops, it appears that breeders in developed countries often maintain substantial working collections and furthermore that material from these collections is commonly exchanged among the breeders. While the variability in toto in these working collections generally appears to be more than adequate for most present needs, there is a strong and continuing interest by breeders in gaining access to novel variability. It is suggested that to maximize the utility of collections, increased emphasis should be placed on studying and assembling small sub-collections that contain representative samples of the available diversity.     

Accuracy of nest fate classification and predator identification from evidence at nests of Least Terns and Piping Plovers

For federally listed species such as Least Tern Sternula antillarum and Piping Plover Charadrius melodus, correct determination of nest fates and causes of nest failure is crucial for understanding population dynamics and improving monitoring programmes. We used video cameras to evaluate nest fate misclassification rate and to identify factors that may cause researchers monitoring nests at different intervals to classify Least Tern and Piping Plover nest fates incorrectly. During the 2013–2015 breeding seasons, we installed miniature surveillance cameras at 65 of 294 Least Tern and 89 of 551 Piping Plover nests under observation on the Missouri River in North Dakota. Nest fates were assigned in the field from remains found at the nest‐site and then again by an independent researcher who reviewed camera footage. We used ordinal logistic regressions to examine whether monitoring interval, clutch age or temporal factors influenced a correct, partially misclassified (probable successful in the field vs. successful by camera) or misclassified nest fate classification. During a 7‐day monitoring interval between visits, 45% of nests were partially and 27.5% were fully misclassified. The percentage of partially (20%) and fully (8.0%) misclassified nests decreased with a more intensive (3‐day) monitoring schedule. Researchers were also less likely to correctly classify nest fates for Least Terns than for Piping Plovers, and as clutch age and monitoring interval increased for both species. Furthermore, causes of failure (e.g. predators, weather) as determined from field evidence vs. video disagreed for 53.5% of nests. The ability to identify accurately nest fate and cause of nest failure will facilitate a better understanding of factors that limit productivity and will lead to better informed management decisions for improving nest survival.     

Early phenology and growth trait variation in closely related European pine species

Closely related taxa occupying different environments are valuable systems for studying evolution. In this study, we examined differences in early phenology (bud set, bud burst) and early growth in a common garden trial of closely related pine species: Pinus sylvestris, P. mugo, and P. uncinata. Seeds for the trial were sourced from populations across the ranges of each species in Europe. Over first 4 years of development, clear differences were observed between species, while the most significant intraspecific differentiation was observed among plants from P. sylvestris populations from continental European locations. Trait differences within P. sylvestris were highly correlated with altitude and latitude of the site of origin. Meanwhile, P. mugo populations from the Carpathians had the earliest bud set and bud flush compared to other populations of the species. Overall, populations from the P. mugo complex from heterogeneous mountain environments and P. sylvestris from the Scottish Highlands showed the highest within‐population variation for the focal traits. Although the three species have been shown to be genetically highly similar, this study reveals large differences in key adaptive traits both among and within species.     

Molecular characterisation and interpretation of genetic diversity within globally distributed germplasm collections of tall fescue (Festuca arundinacea Schreb.) and meadow fescue (F. pratensis Huds.)

Allohexaploid tall fescue (Festuca arundinacea Schreb. syn. Lolium arundinaceum [Schreb.] Darbysh.) is an agriculturally important grass cultivated for pasture and turf world-wide. Genetic improvement of tall fescue could benefit from the use of non-domesticated germplasm to diversify breeding populations through the incorporation of novel and superior allele content. However, such potential germplasm must first be characterised, as three major morphotypes (Continental, Mediterranean and rhizomatous) with varying degrees of hybrid interfertility are commonly described within this species. As hexaploid tall fescue is also a member of a polyploid species complex that contains tetraploid, octoploid and decaploid taxa, it is also possible that germplasm collections may have inadvertently sampled some of these sub-species. In this study, 1,040 accessions from the publicly available United States Department of Agriculture tall fescue and meadow fescue germplasm collections were investigated. Sequence of the chloroplast genome-located matK gene and the nuclear ribosomal DNA internal transcribed spacer (rDNA ITS) permitted attribution of accessions to the three previously known morphotypes and also revealed the presence of tall fescue sub-species of varying ploidy levels, as well as other closely related species. The majority of accessions were, however, identified as Continental hexaploid tall fescue. Analysis using 34 simple sequence repeat markers was able to further investigate the level of genetic diversity within each hexaploid tall fescue morphotype group. At least two genetically distinct sub-groups of Continental hexaploid tall fescue were identified which are probably associated with palaeogeographic range expansion of this morphotype. This work has comprehensively characterised a large and complex germplasm collection and has identified genetically diverse accessions which may potentially contribute valuable alleles at agronomic loci for tall fescue cultivar improvement programs.     

Genomic prediction of seedling root length in maize (Zea mays L.)

Genotypes with extreme phenotypes are valuable for studying ‘difficult’ quantitative traits. Genomic prediction (GP) might allow the identification of such extremes by phenotyping a training population of limited size and predicting genotypes with extreme phenotypes in large sequences of germplasm collections. We tested this approach employing seedling root traits in maize and the extensively genotyped Ames Panel. A training population made up of 384 inbred lines from the Ames Panel was phenotyped by extracting root traits from images using the software program aria . A ridge regression best linear unbiased prediction strategy was used to train a GP model. Genomic estimated breeding values for the trait ‘total root length’ (TRL) were predicted for 2431 inbred lines, which had previously been genotyped by sequencing. Selections were made for 100 extreme TRL lines and those with the predicted longest or shortest TRL were validated for TRL and other root traits. The two predicted extreme groups with regard to TRL were significantly different (P = 0.0001). The difference in predicted means for TRL between groups was 145.1 cm and 118.7 cm for observed means, which were significantly different (P = 0.001). The accuracy of predicting the rank between 1 and 200 of the validation population based on TRL (longest to shortest) was determined using a Spearman correlation to be ρ = 0.55. Taken together, our results support the idea that GP may be a useful approach for identifying the most informative genotypes in sequenced germplasm collections to facilitate experiments for quantitative inherited traits.     

Development of a Real‐time Fluorescence Loop‐mediated Isothermal Amplification Assay for Detection of Burkholderia gladioli pv. alliicola

Burkholderia gladioli pv. alliicola is a causal agent of rot on a wide range of hosts including onion and tulip. It is one of quarantine phytopathogenic bacteria in China. To reduce the economic losses associated with this pathogen, simple and rapid detection methods are needed. In this study, an efficient loop‐mediated isothermal amplification (LAMP) assay with a real‐time fluorometer was developed. The analysis of 16S‐23S rRNA intergenic transcribed spacer (ITS) sequences showed considerable variability between different Burkholderia species and B. gradioli pathovars. A set of LAMP primers was designed based on the ITS region. The sensitivity and specificity of the developed assay were evaluated at the optimal temperature of 65°C. The primers were specific for B. gladioli pv. alliicola and did not react to strains of others species and other pathovars in the species B. gladioli. The sensitivity of the real‐time LAMP assay was 1 fg DNA which was 100 times higher than that of conventional PCR. The method was verified by testing natural samples and inoculated onion seeds, and it showed effectiveness. The real‐time LAMP assay established in this study is an effective method for detection of B. gladioli pv. alliicola.     

Last exit before the brink: Conservation genomics of the Cambodian population of the critically endangered southern river terrapin

The southern river terrapin, Batagur affinis is one of the world's 25 most endangered freshwater turtle species. The major portion of the global population is currently found in peninsular Malaysia, with the only remnant Indochinese population in southern Cambodia. For more than a decade, wild nests in this remnant Cambodian population have been fenced and hatchlings reared in captivity. Here we amplified 10 microsatellite markers from all 136 captive individuals, obtained 2,658 presumably unlinked and neutral single nucleotide polymorphisms from 72 samples with ddRAD‐seq, and amplified 784 bp of mtDNA from 50 samples. Our results reveal that the last Indochinese population comprised only four kinship groups as of 2012, with all offspring sired from <10 individuals in the wild. We demonstrate an obvious decrease in genetic contributions of breeders in the wild from 2006–2012 and identify high‐value breeders instrumental for ex‐situ management of the contemporary genetic stock of the species.     

Development,validation and genetic analysis of a large soybean SNP genotyping array

Cultivated soybean (Glycine max) suffers from a narrow germplasm relative to other crop species, probably because of under‐use of wild soybean (Glycine soja) as a breeding resource. Use of a single nucleotide polymorphism (SNP) genotyping array is a promising method for dissecting cultivated and wild germplasms to identify important adaptive genes through high‐density genetic mapping and genome‐wide association studies. Here we describe a large soybean SNP array for use in diversity analyses, linkage mapping and genome‐wide association analyses. More than four million high‐quality SNPs identified from high‐depth genome re‐sequencing of 16 soybean accessions and low‐depth genome re‐sequencing of 31 soybean accessions were used to select 180 961 SNPs for creation of the Axiom^® SoyaSNP array. Validation analysis for a set of 222 diverse soybean lines showed that 170 223 markers were of good quality for genotyping. Phylogenetic and allele frequency analyses of the validation set data indicated that accessions showing an intermediate morphology between cultivated and wild soybeans collected in Korea were natural hybrids. More than 90 unanchored scaffolds in the current soybean reference sequence were assigned to chromosomes using this array. Finally, dense average spacing and preferential distribution of the SNPs in gene‐rich chromosomal regions suggest that this array may be suitable for genome‐wide association studies of soybean germplasm. Taken together, these results suggest that use of this array may be a powerful method for soybean genetic analyses relating to many aspects of soybean breeding.     

Mature male parr contribution to the effective size of an anadromous Atlantic salmon (Salmo salar) population over 30 years

We describe temporal changes in the genetic composition of a small anadromous Atlantic salmon (Salmo salar) population from South Newfoundland, an area where salmon populations are considered threatened (COSEWIC 2010). We examined the genetic variability (13 microsatellite loci) in 869 out‐migrating smolt and post‐spawning kelt samples, collected from 1985 to 2011 for a total of 22 annual collections and a 30 year span of assigned cohorts. We estimated the annual effective number of breeders (N_b) and the generational effective population size (N_e) through genetic methods and demographically using the adult sex ratio. Comparisons between genetic and demographic estimates show that the adult spawners inadequately explain the observed N_e estimates, suggesting that mature male parr are significantly increasing N_b and N_e over the study period. Spawning as parr appears to be a viable and important strategy in the near absence of adult males.     

Population genetics of the American eel (Anguilla rostrata): FST = 0 and North Atlantic Oscillation effects on demographic fluctuations of a panmictic species

We performed population genetic analyses on the American eel (Anguilla rostrata) with three main objectives. First, we conducted the most comprehensive analysis of neutral genetic population structure to date to revisit the null hypothesis of panmixia in this species. Second, we used this data to provide the first estimates of contemporary effective population size (N_e) and to document temporal variation in effective number of breeders (N_b) in American eel. Third, we tested for statistical associations between temporal variation in the North Atlantic Oscillation (NAO), the effective number of breeders and two indices of recruit abundance. A total of 2142 eels from 32 sampling locations were genotyped with 18 microsatellite loci. All measures of differentiation were essentially zero, and no evidence for significant spatial or temporal genetic differentiation was found. The panmixia hypothesis should thus be accepted for this species. N_b estimates varied by a factor of 23 among 12 cohorts, from 473 to 10 999. The effective population size N_e was estimated at 10 532 (95% CI, 9312–11 752). This study also showed that genetically based demographic indices, namely N_b and allelic richness (Ar), can be used as surrogates for the abundance of breeders and recruits, which were both shown to be positively influenced by variation during high (positive) NAO phases. Thus, long‐term genetic monitoring of American glass eels at several sites along the North American Atlantic coast would represent a powerful and efficient complement to census monitoring to track demographic fluctuations and better understand their causes.     

Population structure and genetic diversity of Bromus tectorum within the small grain production region of the Pacific Northwest

Bromus tectorum L. is an invasive winter annual grass naturalized across the United States. Numerous studies have investigated B. tectorum population structure and genetics in the context of B. tectorum as an ecological invader of natural areas and rangeland. Despite the wealth of information regarding B. tectorum, previous studies have not focused on, or made comparisons to, B. tectorum as it persists in individual agroecosystems. The objectives of this study were to assess the genetic diversity and structure, the occurrence of generalist and specialist genotypes, and the influence of climate on distribution of B. tectorum sourced exclusively from within small grain production regions of the Pacific Northwest. Genetic diversity of B. tectorum sourced from agronomic fields was found to be similar to what has been observed from other land use histories. Six distinct genetic clusters of B. tectorum were identified, with no evidence to indicate that any of the genetic clusters were better adapted to a particular geographical area or climate within the region. Given the apparent random spatial distribution of B. tectorum genetic clusters at the spatial scale of this analysis, unique genotypes may be well mixed within region, similar to what was reported for other inbreeding weedy grass species.     

Cryopreservation of fruit germplasm

Most temperate fruit species are genetically heterozygous and vegetatively propagated. Active collections of fruit genetic resources in Germany are generally maintained in the field, e.g., as potted plants for Fragaria and as trees for pome and stone fruit species. The plant material in active collections should be kept in duplicate to ensure security in case of disease or environmental disaster. The aim of this study was to develop an efficient complementary conservation strategy for fruit genetic resources. Although costly, fruit tree cultivars can be duplicated as field collections at a second site within the framework of the German Fruit Genebank, which is a decentralized fruit-specific network. Wild species accessions, particularly those of the genera Malus spp. (apple) and Fragaria spp. (strawberry) as well as strawberry cultivars, can also be duplicated by means of cryopreservation. In the current study, long-term cryopreservation was initiated for 194 Fragaria genotypes. A protocol combining vitrification with cold acclimation was effective and highly reproducible, with an average regrowth rate of 86%. In Malus, a general cryopreservation protocol based on dormant winter buds was adopted. Based on the results provided in this study, a combination of traditional ex situ conservation and cryopreservation can greatly improve the stability and security of fruit germplasm conservation.     

Assemblage structure and dynamics of terrestrial birds in the southwest Amazon: a camera‐trap case study

Habitat spatial distribution, seasonal variation, and activity patterns influence changes in vertebrate assemblages over time. Terrestrial birds play major roles in the dynamics of tropical forests, but there are few effective methods to study these species due to their cryptic coloration and elusive behavior. We used camera‐trap data collected during 16 mo (February 2017–June 2018) to describe the terrestrial avifauna in southeastern Peru, assess to what extent the composition of terrestrial avifauna changes among seasons and across two major habitats (terra firme and floodplain forests), and determine daily activity patterns of terrestrial birds. We used overlap analyses to examine temporal co‐occurrence between ecologically similar and sympatric species. Camera traps recorded 16 species, including eight species in the family Tinamidae. Capture rates were highest for Pale‐winged Trumpeters (Psophia leucoptera; Psophiidae) and Gray‐fronted Doves (Leptolila rufaxilla; Columbidae). Species composition did not differ between habitats or seasons, and capture rates between habitats only differed for White‐throated Tinamous (Tinamus guttatus). Overlaps of activity patterns were high between ecologically similar species and species found in terra firme habitats (White‐throated Tinamous and Cinereous Tinamous, Crypturellus cinereus) and in both habitat types (Pale‐winged Trumpeters and Gray‐fronted Doves). Low numbers of captures of possibly locally rare or less abundant species hindered a complete analysis of spatial and seasonal patterns of terrestrial bird assemblages. We suggest a greater sampling effort and greater spatial replication to better understand the spatial and seasonal dynamics of the terrestrial avifauna. Further studies that assess the mechanisms that allow the coexistence of sympatric tinamous would be valuable, both in our study area and elsewhere. The use of camera traps in long‐term monitoring projects proved to be an effective tool for monitoring terrestrial birds, identifying cryptic and often rare animals to species level, and providing valuable ecological information at species and community levels.