Functional mapping of N deficiency‐induced response in wheat yield‐component traits by implementing high‐throughput phenotyping

As overfertilization leads to environmental concerns and the cost of N fertilizer increases, the issue of how to select crop cultivars that can produce high yields on N‐deficient soils has become crucially important. However, little information is known about the genetic mechanisms by which crops respond to environmental changes induced by N signaling. Here, we dissected the genetic architecture of N‐induced phenotypic plasticity in bread wheat (Triticum aestivum L.) by integrating functional mapping and semiautomatic high‐throughput phenotyping data of yield‐related canopy architecture. We identified a set of quantitative trait loci (QTLs) that determined the pattern and magnitude of how wheat cultivars responded to low N stress from normal N supply throughout the wheat life cycle. This analysis highlighted the phenological landscape of genetic effects exerted by individual QTLs, as well as their interactions with N‐induced signals and with canopy measurement angles. This information may shed light on our mechanistic understanding of plant adaptation and provide valuable information for the breeding of N‐deficiency tolerant wheat varieties.     

High‐density genotyping of the A.E. Watkins Collection of hexaploid landraces identifies a large molecular diversity compared to elite bread wheat

The importance of wheat as a food crop makes it a major target for agricultural improvements. As one of the most widely grown cereal grains, together with maize and rice, wheat is the leading provider of calories in the global diet, constituting 29% of global cereal production in 2015. In the last few decades, however, yields have plateaued, suggesting that the green revolution, at least for wheat, might have run its course and that new sources of genetic variation are urgently required. The overall aim of our work was to identify novel variation that may then be used to enable the breeding process. As landraces are a potential source of such diversity, here we have characterized the A.E. Watkins Collection alongside a collection of elite accessions using two complementary high‐density and high‐throughput genotyping platforms. While our results show the importance of using the appropriate SNP collection to compare diverse accessions, they also show that the Watkins Collection contains a substantial amount of novel genetic diversity which has either not been captured in current breeding programmes or which has been lost through previous selection pressures. As a consequence of our analysis, we have identified a number of accessions which carry an array of novel alleles along with a number of interesting chromosome rearrangements which confirm the variable nature of the wheat genome.     

A genome‐wide survey reveals abundant rice blast R genes in resistant cultivars

Plant resistance genes (R genes) harbor tremendous allelic diversity, constituting a robust immune system effective against microbial pathogens. Nevertheless, few functional R genes have been identified for even the best‐studied pathosystems. Does this limited repertoire reflect specificity, with most R genes having been defeated by former pests, or do plants harbor a rich diversity of functional R genes, the composite behavior of which is yet to be characterized? Here, we survey 332 NBS‐LRR genes cloned from five resistant Oryza sativa (rice) cultivars for their ability to confer recognition of 12 rice blast isolates when transformed into susceptible cultivars. Our survey reveals that 48.5% of the 132 NBS‐LRR loci tested contain functional rice blast R genes, with most R genes deriving from multi‐copy clades containing especially diversified loci. Each R gene recognized, on average, 2.42 of the 12 isolates screened. The abundant R genes identified in resistant genomes provide extraordinary redundancy in the ability of host genotypes to recognize particular isolates. If the same is true for other pathogens, many extant NBS‐LRR genes retain functionality. Our success at identifying rice blast R genes also validates a highly efficient cloning and screening strategy.     

High‐resolution melting analysis: a genotyping tool for population studies on Daphnia

Determining genetic variation at the DNA level within and between natural populations is important for understanding the role of natural selection on phenotypic traits, but many techniques of screening for genetic variation are either cost intensive, not sensitive enough or too labour‐ and time‐consuming. Here, we demonstrate high‐resolution melting analysis (HRMA) as a cost‐effective and powerful tool for screening variable target genes in natural populations. HRMA is based on monitoring the melting of PCR amplicons. Owing to saturating concentrations of a dye that binds at high concentrations to double‐stranded DNA, it is possible to genotype high numbers of samples rapidly and accurately. We analysed digestive trypsins of two Daphnia magna populations as an application example for HRMA. One population originated from a pond containing toxic cyanobacteria that possibly produce protease inhibitors and the other from a pond without such cyanobacteria. The hypothesis was that D. magna clones from ponds with cyanobacteria have undergone selection by these inhibitors, which has led to different trypsin alleles. We first sequenced pooled genomic PCR products of trypsins from both populations to identify variable DNA sequences of active trypsins. Second, we screened variable DNA sequences of each D. magna clone from both populations for single nucleotide polymorphisms via HRMA. The HRMA results revealed that both populations exhibited phenotypic differences in the analysed trypsins. Our results indicate that HRMA is a powerful genotyping tool for studying the variation of target genes in response to selection within and between natural Daphnia populations.     

High‐throughput fluorescence‐activated cell sorting for lipid hyperaccumulating Chlamydomonas reinhardtii mutants

The genetically tractable microalga Chlamydomonas reinhardtii has many advantages as a model for renewable bioproducts and/or biofuels production. However, one limitation of C. reinhardtii is its relatively low‐lipid content compared with some other algal species. To overcome this limitation, we combined ethane methyl sulfonate mutagenesis with fluorescence‐activated cell sorting (FACS) of cells stained with the lipophilic stain Nile Red to isolate lipid hyperaccumulating mutants of C. reinhardtii. By manipulating the FACS gates, we sorted mutagenized cells with extremely high Nile Red fluorescence signals that were rarely detected in nonmutagenized populations. This strategy successfully isolated several putative lipid hyperaccumulating mutants exhibiting 23% to 58% (dry weight basis) higher fatty acid contents than their progenitor strains. Significantly, for most mutants, nitrogen starvation was not required to attain high‐lipid content nor was there a requirement for a deficiency in starch accumulation. Microscopy of Nile Red stained cells revealed that some mutants exhibit an increase in the number of lipid bodies, which correlated with TLC analysis of triacyglycerol content. Increased lipid content could also arise through increased biomass production. Collectively, our findings highlight the ability to enhance intracellular lipid accumulation in algae using random mutagenesis in conjunction with a robust FACS and lipid yield verification regime. Our lipid hyperaccumulating mutants could serve as a genetic resource for stacking additional desirable traits to further increase lipid production and for identifying genes contributing to lipid hyperaccumulation, without lengthy lipid‐induction periods.     

A genome‐wide association study of heat stress‐associated SNPs in catfish

Heat tolerance is a complex and economically important trait for catfish genetic breeding programs. With global climate change, it is becoming an increasingly important trait. To better understand the molecular basis of heat stress, a genome‐wide association study (GWAS) was carried out using the 250 K catfish SNP array with interspecific backcross progenies, which derived from crossing female channel catfish with male F1 hybrid catfish (female channel catfish × male blue catfish). Three significant associated SNPs were detected by performing an EMMAX approach for GWAS. The SNP located on linkage group 14 explained 12.1% of phenotypical variation. The other two SNPs, located on linkage group 16, explained 11.3 and 11.5% of phenotypical variation respectively. A total of 14 genes with heat stress related functions were detected within the significant associated regions. Among them, five genes—TRAF2, FBXW5, ANAPC2, UBR1 and KLHL29— have known functions in the protein degradation process through the ubiquitination pathway. Other genes related to heat stress include genes involved in protein biosynthesis (PRPF4 and SYNCRIP), protein folding (DNAJC25), molecule and iron transport (SLC25A46 and CLIC5), cytoskeletal reorganization (COL12A1) and energy metabolism (COX7A2, PLCB1 and PLCB4) processes. The results provide fundamental information about genes and pathways that is useful for further investigation into the molecular mechanisms of heat stress. The associated SNPs could be promising candidates for selecting heat‐tolerant catfish lines after validating their effects on larger and various catfish populations.     

A genome‐wide association study for gestation length in swine

Selection for increased litter size in swine has potentially resulted in a correlated increase in preweaning mortality. Additional selection criteria should be considered when selecting for increased litter size to account for associated decreases in piglet quality, specifically piglet survival, initial weight and growth. Traits such as gestation length (GL), which have been associated with piglet performance, could be utilized to improve piglet development and survivability. The objective of this study was to conduct a genome‐wide association study to identify genomic regions associated with GL in differing parities in swine (n = 831) from the University of Nebraska–Lincoln reproductive longevity project. Gestation length was calculated as the number of days between last insemination administered and farrowing. Sows were genotyped with the Illumina SNP60 BeadArray, and the data were analyzed using Bayesian mixture models for GL at parity 1, 2, 3 and 4 (GL1, GL2, GL3 and GL4 respectively). Means (SD) for GL1–GL4 were 113 (1.4), 114 (1.2), 114 (1.3) and 115 (1.2) respectively. Posterior mean heritability estimates (PSD) for GL1, GL2, GL3 and GL4 were 0.33 (0.06), 0.34 (0.07), 0.32 (0.08) and 0.20 (0.08) respectively. Rank correlations between genomic estimated breeding values between GL1 and GL2, GL3 and GL4 respectively were moderate: 0.67, 0.65 and 0.60. The top SNP (ASGA0017859, SSC4, 7.8 Mb), located in the top common genomic region associated with GL1, GL2 and GL3, was associated with a difference of 1.1 days in GL1 between homozygote genotypes (P < 0.0001). The results of this study suggest that GL is a largely polygenic trait with relatively minor contributions from multiple genomic regions.     

High‐throughput sequencing and graph‐based cluster analysis facilitate microsatellite development from a highly complex genome

Despite recent advances in high‐throughput sequencing, difficulties are often encountered when developing microsatellites for species with large and complex genomes. This probably reflects the close association in many species of microsatellites with cryptic repetitive elements. We therefore developed a novel approach for isolating polymorphic microsatellites from the club‐legged grasshopper (Gomphocerus sibiricus), an emerging quantitative genetic and behavioral model system. Whole genome shotgun Illumina MiSeq sequencing was used to generate over three million 300 bp paired‐end reads, of which 67.75% were grouped into 40,548 clusters within RepeatExplorer. Annotations of the top 468 clusters, which represent 60.5% of the reads, revealed homology to satellite DNA and a variety of transposable elements. Evaluating 96 primer pairs in eight wild‐caught individuals, we found that primers mined from singleton reads were six times more likely to amplify a single polymorphic microsatellite locus than primers mined from clusters. Our study provides experimental evidence in support of the notion that microsatellites associated with repetitive elements are less likely to successfully amplify. It also reveals how advances in high‐throughput sequencing and graph‐based repetitive DNA analysis can be leveraged to isolate polymorphic microsatellites from complex genomes.     

High‐throughput SNP‐genotyping analysis of the relationships among Ponto‐Caspian sturgeon species

Legally certified sturgeon fisheries require population protection and conservation methods, including DNA tests to identify the source of valuable sturgeon roe. However, the available genetic data are insufficient to distinguish between different sturgeon populations, and are even unable to distinguish between some species. We performed high‐throughput single‐nucleotide polymorphism (SNP)‐genotyping analysis on different populations of Russian (Acipenser gueldenstaedtii), Persian (A. persicus), and Siberian (A. baerii) sturgeon species from the Caspian Sea region (Volga and Ural Rivers), the Azov Sea, and two Siberian rivers. We found that Russian sturgeons from the Volga and Ural Rivers were essentially indistinguishable, but they differed from Russian sturgeons in the Azov Sea, and from Persian and Siberian sturgeons. We identified eight SNPs that were sufficient to distinguish these sturgeon populations with 80% confidence, and allowed the development of markers to distinguish sturgeon species. Finally, on the basis of our SNP data, we propose that the A. baerii‐like mitochondrial DNA found in some Russian sturgeons from the Caspian Sea arose via an introgression event during the Pleistocene glaciation.     

Metabolome‐genome‐wide association study dissects genetic architecture for generating natural variation in rice secondary metabolism

Plants produce structurally diverse secondary (specialized) metabolites to increase their fitness for survival under adverse environments. Several bioactive compounds for new drugs have been identified through screening of plant extracts. In this study, genome‐wide association studies (GWAS) were conducted to investigate the genetic architecture behind the natural variation of rice secondary metabolites. GWAS using the metabolome data of 175 rice accessions successfully identified 323 associations among 143 single nucleotide polymorphisms (SNPs) and 89 metabolites. The data analysis highlighted that levels of many metabolites are tightly associated with a small number of strong quantitative trait loci (QTLs). The tight association may be a mechanism generating strains with distinct metabolic composition through the crossing of two different strains. The results indicate that one plant species produces more diverse phytochemicals than previously expected, and plants still contain many useful compounds for human applications.     

A genome‐wide search for local adaptation in a terrestrial‐breeding frog reveals vulnerability to climate change

Terrestrial‐breeding amphibians are likely to be vulnerable to warming and drying climates, as their embryos require consistent moisture for successful development. Adaptation to environmental change will depend on sufficient genetic variation existing within or between connected populations. Here, we use Single Nucleotide Polymorphism (SNP) data to investigate genome‐wide patterns in genetic diversity, gene flow and local adaptation in a terrestrial‐breeding frog (Pseudophryne guentheri) subject to a rapidly drying climate and recent habitat fragmentation. The species was sampled across 12 central and range‐edge populations (192 samples), and strong genetic structure was apparent, as were high inbreeding coefficients. Populations showed differences in genetic diversity, and one population lost significant genetic diversity in a decade. More than 500 SNP loci were putatively under directional selection, and 413 of these loci were correlated with environmental variables such as temperature, rainfall, evaporation and soil moisture. One locus showed homology to a gene involved in the activation of maturation in Xenopus oocytes, which may facilitate rapid development of embryos in drier climates. The low genetic diversity, strong population structuring and presence of local adaptation revealed in this study shows why management strategies such as targeted gene flow may be necessary to assist isolated populations to adapt to future climates.     

A genome‐wide significant association on chromosome 2 for footrot resistance/susceptibility in Swiss White Alpine sheep

Footrot is one of the most important causes of lameness in global sheep populations and is characterized by a bacterial infection of the interdigital skin. As a multifactorial disease, its clinical representation depends not only on pathogen factors and environmental components but also on the individual resistance/susceptibility of the host. A genetic component has been shown in previous studies; however, so far no causative genetic variant influencing the risk of developing footrot has been identified. In this study, we genotyped 373 Swiss White Alpine sheep, using the ovine high‐density 600k SNP chip, in order to run a DNA‐based comparison of individuals with known clinical footrot status. We performed a case–control genome‐wide association study, which revealed a genome‐wide significant association for SNP rs418747104 on ovine chromosome 2 at 81.2 Mb. The three best associated SNP markers were located at the MPDZ gene, which codes for the multiple PDZ domain crumbs cell polarity complex component protein, also known as multi‐PDZ domain protein 1 (MUPP1). This protein is possibly involved in maintaining the barrier function and integrity of tight junctions. Therefore, we speculate that individuals carrying MPDZ variants may differ in their footrot resistance/susceptibility due to modified horn and interdigital skin integrity. In conclusion, our study reveals that MPDZ might represent a functional candidate gene, and further research is needed to explore its role in footrot affected sheep.     

Genetic dissection of pre‐anthesis sub‐phase durations during the reproductive spike development of wheat

Flowering time is an important factor affecting grain yield in wheat. In this study, we divided reproductive spike development into eight sub‐phases. These sub‐phases have the potential to be delicately manipulated to increase grain yield. We measured 36 traits with regard to sub‐phase durations, determined three grain yield‐related traits in eight field environments and mapped 15 696 single nucleotide polymorphism (SNP, based on 90k Infinium chip and 35k Affymetrix chip) markers in 210 wheat genotypes. Phenotypic and genetic associations between grain yield traits and sub‐phase durations showed significant consistency (Mantel test; r = 0.5377, P < 0.001). The shared quantitative trait loci (QTLs) revealed by the genome‐wide association study suggested a close association between grain yield and sub‐phase duration, which may be attributed to effects on spikelet initiation/spikelet number (double ridge to terminal spikelet stage, DR‐TS) and assimilate accumulation (green anther to anthesis stage, GA‐AN). Moreover, we observed that the photoperiod‐sensitivity allele at the Ppd‐D1 locus on chromosome 2D markedly extended all sub‐phase durations, which may contribute to its positive effects on grain yield traits. The dwarfing allele at the Rht‐D1 (chromosome 4D) locus altered the sub‐phase duration and displayed positive effects on grain yield traits. Data for 30 selected genotypes (from among the original 210 genotypes) in the field displayed a close association with that from the greenhouse. Most importantly, this study demonstrated specific connections to grain yield in narrower time windows (i.e. the eight sub‐phases), rather than the entire stem elongation phase as a whole.     

Association between putative functional variants in the PSMB9 gene and risk of melanoma – re‐analysis of published melanoma genome‐wide association studies

To mine possibly hidden causal single‐nucleotide polymorphisms (SNPs) of melanoma, we investigated the association of SNPs in 76 M/G1 transition genes with melanoma risk using our published genome‐wide association study (GWAS) data set with 1804 melanoma cases and 1026 cancer‐free controls. We found multiple SNPs with P < 0.01 and performed validation studies for 18 putative functional SNPs in PSMB9 in two other GWAS data sets. Two SNPs (rs1351383 and rs2127675) were associated with melanoma risk in the GenoMEL data set (P = 0.013 and 0.004, respectively), but failed in validation using the Australian data set. Genotype–phenotype analysis revealed these two SNPs were significantly correlated with mRNA expression level of PSMB9. Further experiments revealed that SNP rs2071480, which is in high LD with rs1351383 and rs2127675, may have a weak effect on the promoter activity of PSMB9. Taken together, our data suggested that functional variants in PSMB9 may contribute to melanoma susceptibility.     

Population‐dependent reproducible deviation from natural bread wheat genome in synthetic hexaploid wheat

Sequence elimination is one of the main mechanisms that increases the divergence among homoeologous chromosomes after allopolyploidization to enhance the stability of recently established lineages, but it can cause a loss of some economically important genes. Synthetic hexaploid wheat (SHW) is an important source of genetic variation to the natural hexaploid wheat (NHW) genepool that has low genetic diversity. Here, we investigated the change between SHW and NHW genomes by utilizing a large germplasm set of primary synthetics and synthetic derivatives. Reproducible segment elimination (RSE) was declared if a large chromosomal chunk (>5 cM) produced no aligned reads in more than five SHWs. RSE in five genomic regions was the major source of variation between SHW and NHW. One RSE eliminated almost the complete short arm of chromosome 1B, which contains major genes for flour quality, disease resistance and different enzymes. The occurrence of RSE was highly dependent on the choice of diploid and tetraploid parental lines, their ancestral subpopulation and admixture, e.g. SHWs derived from Triticum dicoccon or from one of two Aegilops tauschii subpopulations were almost free of RSE, while highly admixed parents had higher RSE rates. The rate of RSE in synthetic derivatives was almost double that in primary synthetics. Genome‐wide association analysis detected four loci with minor effects on the occurrence of RSE, indicating that both parental lines and genetic factors were affecting the occurrence of RSE. Therefore, pre‐pre‐breeding strategies should be applied before introducing SHW into pre‐breeding programs to ensure genomic stability and avoid undesirable gene loss.     

A genome‐wide association study in Caucasian women suggests the involvement of HLA genes in the severity of facial solar lentigines

Solar lentigines are a common feature of sun‐induced skin ageing. Little is known, however, about the genetic factors contributing to their development. In this genome‐wide association study, we aimed to identify genetic loci associated with solar lentigines on the face in 502 middle‐aged French women. Nine SNPs, gathered in two independent blocks on chromosome 6, exhibited a false discovery rate below 25% when looking for associations with the facial lentigine score. The first block, in the 6p22 region, corresponded to intergenic SNPs and also exhibited a significant association with forehead lentigines (P = 1.37 × 10^?8). The second block, within the 6p21 HLA region, was associated with decreased HLA‐C expression according to several eQTL databases. Interestingly, these SNPs were also in high linkage disequilibrium with the HLA‐C*0701 allele (r² = 0.95). We replicated an association recently found by GWAS in the IRF4 gene. Finally, a complementary study on 44 selected candidate SNPs revealed novel associations in the MITF gene. Overall, our results point to several mechanisms involved in the severity of facial lentigines, including HLA/immunity and the melanogenesis pathway.