Single Nucleotide Polymorphism Markers Research Articles

Mapping Single Nucleotide Polymorphism Markers Associated with the Pre-Flowering Morphological Performance of Fenugreek under Different Levels of Salt Stress

Salinity is a significant factor restricting plant growth and production. The effect of salinity stress on different growth parameters of 111 fenugreek genotypes was examined in an experiment with three salinity levels (0, 3000, 6000 mgL−1). A completely randomized block design with two replicated pots per treatment was used. Non-significant treatment effects were observed on fresh weight (FW); however, all traits showed significant genotype-by-treatment (GxT) interactions. This GxT was reflected in substantial SNP x environment interactions. Of 492 significant SNPs associated with the measured traits, 212 SNPs were linked to the correlated traits using an arbitrary threshold of three. Several SNPs were associated with FW and dry weight, measured under the same salinity treatment. The correlation between both traits was 0.98 under the three salinity treatments. In addition, 280 SNPs with conditional neutrality effects were mapped. The identified SNPs can be used in future marker-assisted breeding programs to select salt-tolerant genotypes. The results of this research shed light on the salt-tolerant properties of fenugreek.

Genetic linkage mapping and quantitative trait locus (QTL) analysis of sweet basil (Ocimum basilicum L.) to identify genomic regions associated with cold tolerance and major volatiles.

Chilling sensitivity is one of the greatest challenges affecting the marketability and profitability of sweet basil (Ocimum basilicum L.) in the US and worldwide. Currently, there are no sweet basils commercially available with significant chilling tolerance and traditional aroma profiles. This study was conducted to identify quantitative trait loci (QTLs) responsible for chilling tolerance and aroma compounds in a biparental mapping population, including the Rutgers advanced breeding line that served as a chilling tolerant parent, 'CB15', the chilling sensitive parent, 'Rutgers Obsession DMR' and 200 F2 individuals. Chilling tolerance was assessed by percent necrosis using machine learning and aroma profiling was evaluated using gas chromatography (GC) mass spectrometry (MS). Single nucleotide polymorphism (SNP) markers were generated from genomic sequences derived from double digestion restriction-site associated DNA sequencing (ddRADseq) and converted to genotype data using a reference genome alignment. A genetic linkage map was constructed and five statistically significant QTLs were identified in response to chilling temperatures with possible interactions between QTLs. The QTL on LG24 (qCH24) demonstrated the largest effect for chilling response and was significant in all three replicates. No QTLs were identified for linalool, as the population did not segregate sufficiently to detect this trait. Two significant QTLs were identified for estragole (also known as methyl chavicol) with only qEST1 on LG1 being significant in the multiple-QTL model (MQM). QEUC26 was identified as a significant QTL for eucalyptol (also known as 1,8-cineole) on LG26. These QTLs may represent key mechanisms for chilling tolerance and aroma in basil, providing critical knowledge for future investigation of these phenotypic traits and molecular breeding.

Genome‐wide association study of adult plant resistance to spot blotch in an elite Canadian two‐row barley germplasm collection

AbstractSpot blotch, caused by Bipolaris sorokiniana, is a growing concern for barley (Hordeum vulgare) production in most humid, temperate growing regions of the world. Under epidemic conditions, this disease can reduce yields significantly. Reduction of kernel plumpness is associated with lower malt extract and consequently grain quality. Enhanced resistance to this disease is a major goal of western Canadian two‐row barley breeding programmes; however, two‐row barley is considered to be more susceptible than six‐row barley. Plant–pathogen interactions observed for Canadian germplasm are polygenic, where quantitative breeding methods could be beneficial for the development of resistant germplasm. A germplasm panel consisting of 200 two‐row, spring barley genotypes with differential reaction to spot blotch was evaluated at Brandon, Manitoba (MB), and Melfort, Saskatchewan (SK), over eight site years. Genome‐wide association study was conducted using a 50k single‐nucleotide polymorphism (SNP) Illumina iSelect genotyping array. The most‐resistant germplasm was found to carry Midwest Six‐rowed Durable Resistant Haplotype (MSDRH) alleles at the loci located on chromosomes 1H (Rcs‐qtl‐1H‐11_10764), 3H (Rcs‐qtl‐3H‐11_10565) and 7H (Rcs‐qtl‐7H‐11_20162, Rcs5 locus), where the 3H locus was found to be the most under‐represented in Canadian germplasm. Additional marker‐trait associations within nurseries were identified on chromosomes 1H, 2H and 4H. Several of the SNP markers identified were found to be polymorphic within the Canadian two‐row germplasm panel and thus could be useful for enhancing spot blotch resistance in two‐row barley for development of resistant cultivars.

The use of SNP markers for cattle breed identification.

A potential application of single nucleotide polymorphisms (SNPs) in animal husbandry and production is identification of the animal breed. In this study, using chosen marker selection methods and genotypic data obtained with the use of Illumina Bovine SNP50 BeadChip for individuals belonging to ten cattle breeds, the reduced panels containing the most informative SNP markers were developed. The suitability of selected SNP panels for the effective and reliable assignment of the studied individuals to the breed of origin was checked by three allocation algorithms implemented in GeneClass 2. The studied breeds set included both Polish-native breeds under the genetic resources conservation programs and highly productive breeds with a global range. For all of the tested marker selection methods ("delta" and two FST-based variants), two separate methodological approaches of marker assortment were used and three marker panels were created with 96, 192, and 288 SNPs respectively, to determine the minimum number of markers required for effective differentiation of the studied breeds. Moreover, the usefulness of the most effective panels of markers to assess the population structure and genetic diversity of the analyzed breeds was examined. The conducted analyses showed the possibility of using SNP subsets from medium-density genotypic microarrays to distinguish breeds of cattle kept in Poland and to analyze their genetic structure.

Genotyping USA laboratory-maintained isolates and European clinical isolates of Dirofilaria immitis to assess macrocyclic lactone susceptibility or resistance at predictive SNP sites using droplet digital PCR

Dirofilaria immitis is a parasitic nematode that causes cardiovascular dirofilariosis ("heartworm disease”) primarily in canids. The principal approach for mitigating heartworm infection involves the use of macrocyclic lactone (ML) for prophylaxis. Recent research has substantiated the emergence of D. immitis displaying resistance to MLs in the USA. Numerous factors, such as the mobility of companion animals and competent vectors could impact the spread of drug resistance. Genomic analysis has unveiled that isolates resistant to ML exhibit unique genetic profiles when compared to their wild-type (susceptible) counterparts. Out of the ten single nucleotide polymorphism (SNP) markers validated in clinical samples of D. immitis from the USA, four have demonstrated their effectiveness in distinguishing between isolates with varying ML efficacy phenotypes. This study explores the potential of these confirmed SNPs for conducting surveillance studies. Genotypic analysis using SNP markers emerges as a valuable tool for carrying out surveys and evaluating individual clinical isolates. Two USA laboratory-maintained isolates (Berkeley, WildCat) and twenty-five random European clinical samples of either adult worms or microfilariae (mf) pools isolated from domestic dogs, were tested by droplet digital PCR (ddPCR)-based duplex assay. This approach elucidates genetic evidence pertaining to the development of drug resistance and provides baseline data on resistance related genotypes in Europe. The data on these clinical samples suggests genotypes consistent with the continued efficacy of ML treatment regimens in Europe. In addition, this assay can be significant in discriminating cases of drug-resistance from those possibly due to non-compliance to the recommended preventive protocols.

Quantitative trait locus mapping of hawthorn flavonoid content based on high-density linkage map

Hawthorn (Crataegus pinnatifida Bunge) belongs to the Rosaceae family and possess important value in food and medicinal product. Flavonoids in hawthorn leaves are major active ingredients and widely used for medicinal purposes. In our study, leaves from hybrid populations of 'DMQ' × 'QJX' and 'DMQ' × 'XBRZ' were used for flavonoid content quantitative trait locus (QTL) mapping in 2018 and 2019 based on our previously established high-density genetic map. In total, 10 and 27 QTL related to leaf flavonoid content in 'DMQ' × 'QJX' were discovered in 2018 and 2019, respectively; 10 QTL related to leaf flavonoid content in 'DMQ' × 'XBRZ' were discovered in 2019. Interestingly, three stable QTL were obtained in the 'DMQ' × 'QJX' population, which were related to the content of Vi rhamnoside and Hy in 2018 and 2019 and co-segregated with three single nucleotide polymorphism markers: m2172, m516, and f258.The results of this study provide important theoretical reference for molecular assisted breeding of flavonoid trait in hawthorn.

Identification of QTLs for symbiotic nitrogen fixation and related traits in a soybean recombinant inbred line population.

The genetic architecture of symbiotic N fixation and related traits was investigated in the field. QTLs were identified for percent N derived from the atmosphere, shoot [N] and C to N ratio. Soybean [Glycine max (L.) Merr.] is cultivated worldwide and is the most abundant source of plant-based protein. Symbiotic N2 fixation (SNF) in legumes such as soybean is of great importance; however, yields may still be limited by N in both high yielding and stressful environments. To better understand the genetic architecture of SNF and facilitate the development of high yielding cultivars and sustainable soybean production in stressful environments, a recombinant inbred line population consisting of 190 lines, developed from a cross between PI 442012A and PI 404199, was evaluated for N derived from the atmosphere (Ndfa), N concentration ([N]), and C to N ratio (C/N) in three environments. Significant genotype, environment and genotype × environment effects were observed for all three traits. A linkage map was constructed containing 3309 single nucleotide polymorphism (SNP) markers. QTL analysis was performed for additive effects of QTLs, QTL × environment interactions, and QTL × QTL interactions. Ten unique additive QTLs were identified across all traits and environments. Of these, two QTLs were detected for Ndfa and eight for C/N. Of the eight QTLs for C/N, four were also detected for [N]. Using QTL × environment analysis, six QTLs were detected, of which five were also identified in the additive QTL analysis. The QTL × QTL analysis identified four unique epistatic interactions. The results of this study may be used for genomic selection and introgression of favorable alleles for increased SNF, [N], and C/N via marker-assisted selection.

Mapping of a Major-Effect Quantitative Trait Locus for Seed Dormancy in Wheat.

The excavation and utilization of dormancy loci in breeding are effective endeavors for enhancing the resistance to pre-harvest sprouting (PHS) of wheat varieties. CH1539 is a wheat breeding line with high-level seed dormancy. To clarify the dormant loci carried by CH1539 and obtain linked molecular markers, in this study, a recombinant inbred line (RIL) population derived from the cross of weak dormant SY95-71 and strong dormant CH1539 was genotyped using the Wheat17K single-nucleotide polymorphism (SNP) array, and a high-density genetic map covering 21 chromosomes and consisting of 2437 SNP markers was constructed. Then, the germination percentage (GP) and germination index (GI) of the seeds from each RIL were estimated. Two QTLs for GP on chromosomes 5A and 6B, and four QTLs for GI on chromosomes 5A, 6B, 6D and 7A were identified. Among them, the QTL on chromosomes 6B controlling both GP and GI, temporarily named QGp/Gi.sxau-6B, is a major QTL for seed dormancy with the maximum phenotypic variance explained of 17.66~34.11%. One PCR-based diagnostic marker Ger6B-3 for QGp/Gi.sxau-6B was developed, and the genetic effect of QGp/Gi.sxau-6B on the RIL population and a set of wheat germplasm comprising 97 accessions was successfully confirmed. QGp/Gi.sxau-6B located in the 28.7~30.9 Mbp physical position is different from all the known dormancy loci on chromosomes 6B, and within the interval, there are 30 high-confidence annotated genes. Our results revealed a novel QTL QGp/Gi.sxau-6B whose CH1539 allele had a strong and broad effect on seed dormancy, which will be useful in further PHS-resistant wheat breeding.

Identification and Genetic Dissection of Resistance to Red Crown Rot Disease in a Diverse Soybean Germplasm Population.

Red crown rot (RCR) disease caused by Calonectria ilicicola negatively impacts soybean yield and quality. Unfortunately, the knowledge of the genetic architecture of RCR resistance in soybeans is limited. In this study, 299 diverse soybean accessions were used to explore their genetic diversity and resistance to RCR, and to mine for candidate genes via emergence rate (ER), survival rate (SR), and disease severity (DS) by a multi-locus random-SNP-effect mixed linear model of GWAS. All accessions had brown necrotic lesions on the primary root, with five genotypes identified as resistant. Nine single-nucleotide polymorphism (SNP) markers were detected to underlie RCR response (ER, SR, and DS). Two SNPs colocalized with at least two traits to form a haplotype block which possessed nine genes. Based on their annotation and the qRT-PCR, three genes, namely Glyma.08G074600, Glyma.08G074700, and Glyma.12G043600, are suggested to modulate soybean resistance to RCR. The findings from this study could serve as the foundation for breeding RCR-tolerant soybean varieties, and the candidate genes could be validated to deepen our understanding of soybean response to RCR.

Quantitative Trait Loci Mapping for Bacterial Wilt Resistance and Plant Height in Tomatoes.

Bacterial wilt (BW) of tomatoes, caused by Ralstonia solanacearum, is a devastating disease that results in large annual yield losses worldwide. Management of BW of tomatoes is difficult due to the soil-borne nature of the pathogen. One of the best ways to mitigate the losses is through breeding for disease resistance. Moreover, plant height (PH) is a crucial element related to plant architecture, which determines nutrient management and mechanical harvesting in tomatoes. An intraspecific F2 segregating population (NC 11212) of tomatoes was developed by crossing NC 84173 (tall, BW susceptible) × CLN1466EA (short, BW resistant). We performed quantitative trait loci (QTL) mapping using single nucleotide polymorphic (SNP) markers and the NC 11212 F2 segregating population. The QTL analysis for BW resistance revealed a total of three QTLs on chromosomes 1, 2, and 3, explaining phenotypic variation (R2) ranging from 3.6% to 14.9%, whereas the QTL analysis for PH also detected three QTLs on chromosomes 1, 8, and 11, explaining R2 ranging from 7.1% to 11%. This work thus provides information to improve BW resistance and plant architecture-related traits in tomatoes.

Dissecting the Genetic Diversity of USDA Cowpea Germplasm Collection Using Kompetitive Allele Specific PCR-Single Nucleotide Polymorphism Markers.

Cowpea (Vigna unguiculata L. Walp) is an important grain legume crop of the subtropics, particularly in West Africa, where it contributes to the livelihoods of small-scale farmers. Despite being a drought-resilient crop, cowpea production is hampered by insect pests, diseases, parasitic weeds, and various abiotic stresses. Genetic improvement can help overcome these limitations, and exploring diverse cowpea genetic resources is crucial for cowpea breeding. This study evaluated the genetic diversity of 361 cowpea accessions from the USDA core collection for the species using 102 Kompetitive Allele Specific PCR (KASP) single nucleotide polymorphism (SNP) markers. A total of 102 KASP-SNP was validated in the germplasm panel, and 72 showed polymorphism across the germplasm panel. The polymorphism information content (PIC) of all SNPs ranged from 0.1 to 0.37, with an average of 0.29, while the mean observed heterozygosity was 0.52. The population structure revealed three distinct populations that clustered into two major groups after phylogenetic analysis. Analysis of molecular variance (AMOVA) indicated greater genetic variation within populations than among populations. Although cowpea generally has a narrow genetic diversity, the accessions used in this study exhibited considerable variation across geographical regions, sub-species, and improvement status. These results indicated that the selected KASP genotyping assay can provide robust and accurate genotyping data for application in the selection and management of cowpea germplasm in breeding programs and genebanks.

Genome-wide scanning to identify and validate single nucleotide polymorphism markers associated with drought tolerance in spring wheat seedlings.

Unlike other growth stages of wheat, very few studies on drought tolerance have been done at the seedling stage, and this is due to the complexity and sensitivity of this stage to drought stress resulting from climate change. As a result, the drought tolerance of wheat seedlings is poorly understood and very few genes associated with drought tolerance at this stage were identified. To address this challenge, a set of 172 spring wheat genotypes representing 20 different countries was evaluated under drought stress at the seedling stage. Drought stress was applied on all tested genotypes by water withholding for 13 days. Two types of traits, namely morphological and physiological traits were scored on the leaves of all tested genotypes. Genome-wide association study (GWAS) is one of the effective genetic analysis methods that was used to identify target single nucleotide polymorphism (SNP) markers and candidate genes for later use in marker-assisted selection. The tested plant materials were genotyped using 25k Infinium iSelect array (25K) (herein after it will be identified as 25K) (for 172 genotypes) and genotyping-by-sequencing (GBS) (for 103 genotypes), respectively. The results of genotyping revealed 21,093 25K and 11,362 GBS-SNPs, which were used to perform GWAS analysis for all scored traits. The results of GWAS revealed that 131 and 55 significant SNPs were controlling morphological and physiological traits, respectively. Moreover, a total of eight and seven SNP markers were found to be associated with more than one morphological and physiological trait under drought stress, respectively. Remarkably, 10 significant SNPs found in this study were previously reported for their association with drought tolerance in wheat. Out of the 10 validated SNP markers, four SNPs were associated with drought at the seedling stage, while the remaining six SNPs were associated with drought stress at the reproductive stage. Moreover, the results of gene enrichment revealed 18 and six pathways as highly significant biological and molecular pathways, respectively. The selection based on drought-tolerant alleles revealed 15 genotypes with the highest number of different drought-tolerant alleles. These genotypes can be used as candidate parents in future breeding programs to produce highly drought-tolerant genotypes with high genetic diversity. Our findings in this study provide novel markers and useful information on the genetic basis of drought tolerance at early growth stages.

A genome-wide association study for resistance to Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) race 4 in diploid cotton (Gossypium arboreum)and resistance transfer to tetraploid Gossypium hirsutum.

Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum f. sp. vasinfectum (FOV), is a devastating disease affecting cotton (Gossypium spp.) worldwide. Understanding the genetic basis of resistance in diploid cotton and successfully transferring the resistance to tetraploid Upland cotton (G. hirsutum) are crucial for developing resistant cotton cultivars. Although numerous studies have been conducted to investigate the genetic basis of Fusarium wilt in tetraploid cotton, little research has been conducted on diploid species. In this study, an association mapping panel consisting of 246 accessions of G. arboreum, was used to identify chromosomal regions for FOV race 4 (FOV4) resistance based on foliar disease severity ratings in four greenhouse tests. Through a genome-wide association study (GWAS) based on 7,009 single nucleotide polymorphic (SNP) markers, 24 FOV4 resistance QTLs, including three major QTLs on chromosomes A04, A06, and A11, were detected. A validation panel consisting of 97 diploid cotton accessions was employed, confirming the presence of several QTLs. Evaluation of an introgressed BC2F7 population derived from G. hirsutum/G. aridum/G. arboreum showed significant differences in disease incidence and mortality rate, as compared to susceptible and resistant controls, suggesting that the resistance in G. arboreum and/or G. aridum was transferred into Upland cotton for the first time. The identification of novel major resistance QTLs, along with the transfer of resistance from the diploid species, expands our understanding of the genomic regions involved in conferring resistance to FOV4 and contributes to the development of resilient Upland cotton cultivars.

Unveiling the predominance of Saccharum spontaneum alleles for resistance to orange rust in sugarcane using genome-wide association.

Six QTLs of resistance to sugarcane orange rust were identified in modern interspecific hybrids by GWAS. For five of them, the resistance alleles originated from S. spontaneum. Altogether, they efficiently predict disease resistance. Sugarcane orange rust (SOR) is a threatening emerging disease in many sugarcane industries worldwide. Improving the genetic resistance of commercial cultivars remains the most promising solution to control this disease. In this study, an association panel of 568 modern interspecific sugarcane hybrids (Saccharum officinarum x S. spontaneum) from Réunion's breeding program was evaluated for its resistance to SOR under natural conditions of infection. Two genome-wide association studies (GWAS) were conducted between disease reactions and 183,842 single nucleotide polymorphism (SNP) markers obtained by targeted genotyping-by-sequencing. Five resistance quantitative trait loci (QTLs), named Oru1, Oru2, Oru3, Oru4 and Oru5, were identified using a single-locus GWAS (SL-GWAS). These five QTLs all originated from the species S. spontaneum. A multi-locus GWAS (ML-GWAS) uncovered an additional but less significant resistance QTL named Oru6, which originated from S. officinarum. All six QTLs had a moderate to major phenotypic effect on disease resistance. Prediction accuracy estimated with linear regression models based on each of the five QTLs identified by SL-GWAS was between 0.16-0.41. Altogether, these five QTLs provided a relatively high prediction accuracy of 0.60. In comparison, accuracies obtained with six genome-wide prediction models (i.e., GBLUP, Bayes-A, Bayes-B, Bayes-C, Bayesian Lasso and RKHS) reached only 0.65. The good prediction accuracy of disease resistance provided by the QTLs and the predominant S. spontaneum origin of their resistance alleles pave the way for effective marker-assisted breeding strategies.

Sequencing vs. amplification for the estimation of allele dosages in sugarcane (Saccharum spp.)

AbstractPremiseDetecting single‐nucleotide polymorphisms (SNPs) in a cost‐effective way is fundamental in any plant breeding pipeline. Here, we compare three genotyping techniques for their ability to reproduce the allele dosage of SNPs of interest in sugarcane (Saccharum spp.).MethodsTo identify a reproducible technique to estimate allele dosage for the validation of SNP markers, the correlation between Flex‐Seq, kompetitive allele‐specific PCR (KASP), and genotyping‐by‐sequencing and restriction site–associated DNA sequencing (GBS+RADseq) was determined for a set of 76 SNPs. To find alternative methodologies for allele dosage estimation, the KASP and Flex‐Seq techniques were compared for the same set of SNPs. For the three techniques, a population of 53 genotypes from the diverse sugarcane panel of the Centro de Investigación de la Caña de Azúcar (Cenicaña), Colombia, was selected.ResultsThe average Pearson correlation coefficients between GBS+RADseq and Flex‐Seq, GBS+RADseq and KASP, and Flex‐Seq and KASP were 0.62 ± 0.27, 0.38 ± 0.27, and 0.38 ± 0.30, respectively.DiscussionFlex‐Seq reproduced the allele dosages determined using GBS+RADseq with good levels of precision because of its depth of sequencing and ability to target specific positions in the genome. Additionally, Flex‐Seq outperformed KASP by allowing the conversion of a higher number of SNPs and a more accurate estimation of the allele dosage. Flex‐Seq has therefore become the genotyping methodology of choice for marker validation at Cenicaña.

Genomewide marker estimates of the level of dominance in maize heterosis

AbstractHeterosis is the superiority of an F1 hybrid over its parents, and overdominance and dominance are the two main hypotheses for the genetic basis of heterosis. Our objectives were to determine if genomewide marker effects accurately reflect the level of dominance at quantitative trait loci (QTL) and assess the distribution of per‐marker estimates of the level of dominance in maize (Zea mays L.). We simulated different levels of dominance at QTL that were in coupling, repulsion, and random linkage phases. Genomewide marker effects from ridge regression‐best linear unbiased prediction led to accurate estimates of the level of dominance when all linkages were in coupling phase, but repulsion and random linkage phases led to an upward bias. We then estimated levels of dominance from genomewide marker effects in a reciprocal testcross experiment with 1642 hybrids genotyped with 4805 single nucleotide polymorphism markers, and in a legacy Design III experiment with 351 backcross progenies (subjected to three generations of random mating to dissipate linkages) genotyped with 160 simple sequence repeats. In both experiments, the level of dominance was higher for yield than for moisture. For each trait, some loci showed little or no dominance, others showed partial to complete dominance, and others showed overdominance. A high level of dominance was typically due to a small difference between homozygote values. Overall, the results were consistent with previous QTL mapping results indicating that heterosis in maize is due to varying levels of dominance rather than a prevalent level of dominance.

FishSNP: a high quality cross-species SNP database of fishes

The progress of aquaculture heavily depends on the efficient utilization of diverse genetic resources to enhance production efficiency and maximize profitability. Single nucleotide polymorphisms (SNPs) have been widely used in the study of aquaculture genomics, genetics, and breeding research since they are the most prevalent molecular markers on the genome. Currently, a large number of SNP markers from cultured fish species are scattered in individual studies, making querying complicated and data reuse problematic. We compiled relevant SNP data from literature and public databases to create a fish SNP database, FishSNP (http://bioinfo.ihb.ac.cn/fishsnp), and also used a unified analysis pipeline to process raw data that the author of the literature did not perform SNP calling on to obtain SNPs with high reliability. This database presently contains 45,690,243 (45 million) nonredundant SNP data for 13 fish species, with 30,288,958 (30 million) of those being high-quality SNPs. The main function of FishSNP is to search, browse, annotate and download SNPs, which provide researchers various and comprehensive associated information.

Novel SNP markers for flowering and seed quality traits in faba bean (Vicia faba L.): characterization and GWAS of a diversity panel.

Faba bean (Vicia faba L.) is a legume crop grown in diverse climates worldwide. It has a high potential for increased cultivation to meet the need for more plant-based proteins in human diets, a prerequisite for a more sustainable food production system. Characterization of diversity panels of crops can identify variation in and genetic markers for target traits of interest for plant breeding. In this work, we collected a diversity panel of 220 accessions of faba bean from around the world consisting of gene bank material and commercially available cultivars. The aims of this study were to quantify the phenotypic diversity in target traits to analyze the impact of breeding on these traits, and to identify genetic markers associated with traits through a genome-wide association study (GWAS). Characterization under field conditions at Nordic latitude across two years revealed a large genotypic variation and high broad-sense heritability for eleven agronomic and seed quality traits. Pairwise correlations showed that seed yield was positively correlated to plant height, number of seeds per plant, and days to maturity. Further, susceptibility to bean weevil damage was significantly higher for early flowering accessions and accessions with larger seeds. In this study, no yield penalty was found for higher seed protein content, but protein content was negatively correlated to starch content. Our results showed that while breeding advances in faba bean germplasm have resulted in increased yields and number of seeds per plant, they have also led to a selection pressure towards delayed onset of flowering and maturity. DArTseq genotyping identified 6,606 single nucleotide polymorphisms (SNPs) by alignment to the faba bean reference genome. These SNPs were used in a GWAS, revealing 51 novel SNP markers significantly associated with ten of the assessed traits. Three markers for days to flowering were found in predicted genes encoding proteins for which homologs in other plant species regulate flowering. Altogether, this work enriches the growing pool of phenotypic and genotypic data on faba bean as a valuable resource for developing efficient breeding strategies to expand crop cultivation.

The genetic basis of apple shape and size unraveled by digital phenotyping.

Great diversity of shape, size, and skin color is observed among the fruits of different apple genotypes. These traits are critical for consumers and therefore interesting targets for breeding new apple varieties. However, they are difficult to phenotype and their genetic basis, especially for fruit shape and ground color, is largely unknown. We used the FruitPhenoBox to digitally phenotype 525 genotypes of the apple reference population (apple REFPOP) genotyped for 303,148 single nucleotide polymorphism (SNP) markers. From the apple images, 573 highly heritable features describing fruit shape and size as well as 17 highly heritable features for fruit skin color were extracted to explore genotype-phenotype relationships. Out of these features, seven principal components (PCs) and 16 features with the Pearson correlation r<0.75 (selected features) were chosen to carry out genome-wide association studies for fruit shape and size. Four PCs and eight selected features were used in genome-wide association studies for fruit skin color. In total, 69 SNPs scattered over all 17 apple chromosomes were significantly associated with round, conical, cylindrical, or symmetric fruit shapes and fruit size. Novel associations with major effect on round or conical fruit shapes and fruit size were identified on chromosomes 1 and 2. Additionally, 16 SNPs associated with PCs and selected features related to red overcolor as well as green and yellow ground color were found on eight chromosomes. The identified associations can be used to advance marker-assisted selection in apple fruit breeding to systematically select for desired fruit appearance.

Estimates of heterozygosity from single nucleotide polymorphism markers are context-dependent and oftenwrong.

Genetic diversity is frequently described using heterozygosity, particularly in a conservation context. Often, it is estimated using single nucleotide polymorphisms (SNPs); however, it has been shown that heterozygosity values calculated from SNPs can be biased by both study design and filtering parameters. Though solutions have been proposed to address these issues, our own work has found them to be inadequate in some circumstances. Here, we aimed to improve the reliability and comparability of heterozygosity estimates, specifically by investigating how sample size and missing data thresholds influenced the calculation of autosomal heterozygosity (heterozygosity calculated from across the genome, i.e. fixed and variable sites). We also explored how the standard practice of tri- and tetra-allelic site exclusion could bias heterozygosity estimates and influence eventual conclusions relating to genetic diversity. Across three distinct taxa (a frog, Litoria rubella; a tree, Eucalyptus microcarpa; and a grasshopper, Keyacris scurra), we found heterozygosity estimates to be meaningfully affected by sample size and missing data thresholds, partly due to the exclusion of tri- and tetra-allelic sites. These biases were inconsistent both between species and populations, with more diverse populations tending to have their estimates more severely affected, thus having potential to dramatically alter interpretations of genetic diversity. We propose a modified framework for calculating heterozygosity that reduces bias and improves the utility of heterozygosity as a measure of genetic diversity, whilst also highlighting the need for existing population genetic pipelines to be adjusted such that tri- and tetra-allelic sites be included in calculations.