Association Genetics Approach Research Articles

HISS: Snakemake-based workflows for performing SMRT-RenSeq assembly, AgRenSeq and dRenSeq for the discovery of novel plant disease resistance genes

BackgroundIn the ten years since the initial publication of the RenSeq protocol, the method has proved to be a powerful tool for studying disease resistance in plants and providing target genes for breeding programmes. Since the initial publication of the methodology, it has continued to be developed as new technologies have become available and the increased availability of computing power has made new bioinformatic approaches possible. Most recently, this has included the development of a k-mer based association genetics approach, the use of PacBio HiFi data, and graphical genotyping with diagnostic RenSeq. However, there is not yet a unified workflow available and researchers must instead configure approaches from various sources themselves. This makes reproducibility and version control a challenge and limits the ability to perform these analyses to those with bioinformatics expertise.ResultsHere we present HISS, consisting of three workflows which take a user from raw RenSeq reads to the identification of candidates for disease resistance genes. These workflows conduct the assembly of enriched HiFi reads from an accession with the resistance phenotype of interest. A panel of accessions both possessing and lacking the resistance are then used in an association genetics approach (AgRenSeq) to identify contigs positively associated with the resistance phenotype. Candidate genes are then identified on these contigs and assessed for their presence or absence in the panel with a graphical genotyping approach that uses dRenSeq. These workflows are implemented via Snakemake, a python-based workflow manager. Software dependencies are either shipped with the release or handled with conda. All code is freely available and is distributed under the GNU GPL-3.0 license.ConclusionsHISS provides a user-friendly, portable, and easily customised approach for identifying novel disease resistance genes in plants. It is easily installed with all dependencies handled internally or shipped with the release and represents a significant improvement in the ease of use of these bioinformatics analyses.

Association genetics of carbon isotope discrimination and leaf morphology in a breeding population of Juglans regia L.

English walnut (Juglans regia L.) is an economically important crop with > 99% of US walnuts produced in California. Changes in climate and recent drought cycles have raised concerns regarding the future of nut production and responsible water use in California agriculture. Our study used an association genetics approach to characterize ecophysiological traits such as water use efficiency as estimated by carbon isotope discrimination (Δ13C), and photosynthetic capacity through foliar nitrogen composition, in important individuals of the Walnut Improvement Program, located at the University of California, Davis. Stable isotope and leaf measurements of 241 mature trees, representing 60 scion genotypes in established orchards were sampled in 2015 and 2016, followed by genotyping with the Walnut Axiom 700 k SNP Array. A mean Δ13C of 21.7‰ (σ: 0.9‰) was calculated for all individuals, as well as a mean nitrogen/leaf area (N/area) of 3.0 gN/m2 (σ: 0.5 gN/m2). A Bayesian analysis utilizing genomic relationships revealed rankings of the most water use efficient accessions as Solano (95-011-16), 67-013 (unreleased cross), and Tulare (67-011). 126,554 SNPs were used in a two-step association genetics approach identifying four loci associated with Δ13C after correction for multiple testing. Investigation of identified loci revealed an annotation on the J. regia genome of protein FAR1-related sequence 5-like, related to abiotic stress response. For uncharacterized markers, homologs were identified in Arabidopsis for two loci, similarly related to drought stress.

Association genetics of acetophenone defence against spruce budworm in mature white spruce

BackgroundOutbreaks of spruce budworm (SBW, Choristoneura fumiferana Clem.) cause major recurrent damage in boreal conifers such as white spruce (Picea glauca [Moench] Voss) and large losses of forest biomass in North America. Although defensive phenolic compounds have recently been linked to chemical resistance against SBW, their genetic basis remains poorly understood in forest trees, especially in conifers. Here, we used diverse association genetics approaches to discover genes and their variants that may control the accumulation of acetophenones, and dissect the genetic architecture of these defence compounds against SBW in white spruce mature trees.ResultsOut of 4747 single nucleotide polymorphisms (SNPs) from 2312 genes genotyped in a population of 211 unrelated individuals, genetic association analyses identified 35 SNPs in 33 different genes that were significantly associated with the defence traits by using single-locus, multi-locus and multi-trait approaches. The multi-locus approach was particularly effective at detecting SNP–trait associations that explained a large fraction of the phenotypic variance (from 20 to 43%). Significant genes were regulatory including the NAC transcription factor, or they were involved in carbohydrate metabolism, falling into the binding, catalytic or transporter activity functional classes. Most of them were highly expressed in foliage. Weak positive phenotypic correlations were observed between defence and growth traits, indicating little or no evidence of defence-growth trade-offs.ConclusionsThis study provides new insights on the genetic architecture of tree defence traits, contributing to our understanding of the physiology of resistance mechanisms to biotic factors and providing a basis for the genetic improvement of the constitutive defence of white spruce against SBW.

Association genetics studies on frost tolerance in wheat (Triticum aestivum L.) reveal new highly conserved amino acid substitutions in CBF-A3, CBF-A15, VRN3 and PPD1 genes

BackgroundUnderstanding the genetic basis of frost tolerance (FT) in wheat (Triticum aestivum L.) is essential for preventing yield losses caused by frost due to cellular damage, dehydration and reduced metabolism. FT is a complex trait regulated by a number of genes and several gene families. Availability of the wheat genomic sequence opens new opportunities for exploring candidate genes diversity for FT. Therefore, the objectives of this study were to identity SNPs and insertion-deletion (indels) in genes known to be involved in frost tolerance and to perform association genetics analysis of respective SNPs and indels on FT.ResultsHere we report on the sequence analysis of 19 candidate genes for FT in wheat assembled using the Chinese Spring IWGSC RefSeq v1.0. Out of these, the tandem duplicated C-repeat binding factors (CBF), i.e. CBF-A3, CBF-A5, CBF-A10, CBF-A13, CBF-A14, CBF-A15, CBF-A18, the vernalisation response gene VRN-A1, VRN-B3, the photoperiod response genes PPD-B1 and PPD-D1 revealed association to FT in 235 wheat cultivars. Within six genes (CBF-A3, CBF-A15, VRN-A1, VRN-B3, PPD-B1 and PPD-D1) amino acid (AA) substitutions in important protein domains were identified. The amino acid substitution effect in VRN-A1 on FT was confirmed and new AA substitutions in CBF-A3, CBF-A15, VRN-B3, PPD-B1 and PPD-D1 located at highly conserved sites were detected. Since these results rely on phenotypic data obtained at five locations in 2 years, detection of significant associations of FT to AA changes in CBF-A3, CBF-A15, VRN-A1, VRN-B3, PPD-B1 and PPD-D1 may be exploited in marker assisted breeding for frost tolerance in winter wheat.ConclusionsA set of 65 primer pairs for the genes mentioned above from a previous study was BLASTed against the IWGSC RefSeq resulting in the identification of 39 primer combinations covering the full length of 19 genes. This work demonstrates the usefulness of the IWGSC RefSeq in specific primer development for highly conserved gene families in hexaploid wheat and, that a candidate gene association genetics approach based on the sequence data is an efficient tool to identify new alleles of genes important for the response to abiotic stress in wheat.

A Novel Multivariate Approach to Phenotyping and Association Mapping of Multi-Locus Gametophytic Self-Incompatibility Reveals S, Z, and Other Loci in a Perennial Ryegrass (Poaceae) Population.

Self-incompatibility (SI) is a mechanism that many flowering plants employ to prevent fertilisation by self- and self-like pollen ensuring heterozygosity and hybrid vigour. Although a number of single locus mechanisms have been characterised in detail, no multi-locus systems have been fully elucidated. Historically, examples of the genetic analysis of multi-locus SI, to make analysis tractable, are either made on the progeny of bi-parental crosses, where the number of alleles at each locus is restricted, or on crosses prepared in such a way that only one of the SI loci segregates. Perennial ryegrass (Lolium perenne L.) possesses a well-documented two locus (S and Z) gametophytic incompatibility system. A more universal, realistic proof of principle study was conducted in a perennial ryegrass population in which allelic and non-allelic diversity was not artificially restricted. A complex pattern of pollinations from a diallel cross was revealed which could not possibly be interpreted easily per se, even with an already established genetic model. Instead, pollination scores were distilled into principal component scores described as Compatibility Components (CC1-CC3). These were then subjected to a conventional genome-wide association analysis. CC1 associated with markers on linkage groups (LGs) 1, 2, 3, and 6, CC2 exclusively with markers in a genomic region on LG 2, and CC3 with markers on LG 1. BLAST alignment with the Brachypodium physical map revealed highly significantly associated markers with peak associations with genes adjacent and four genes away from the chromosomal locations of candidate SI genes, S- and Z-DUF247, respectively. Further significant associations were found in a Brachypodium distachyon chromosome 3 region, having shared synteny with Lolium LG 1, suggesting further SI loci linked to S or extensive micro-re-arrangement of the genome between B. distachyon and L. perenne. Significant associations with gene sequences aligning with marker sequences on Lolium LGs 3 and 6 were also identified. We therefore demonstrate the power of a novel association genetics approach to identify the genes controlling multi-locus gametophytic SI systems and to identify novel loci potentially involved in already established SI systems.

Towards the Identification of Type III Effectors Associated with Ralstonia solanacearum Virulence on Tomato and Eggplant.

For the development of pathogen-informed breeding strategies, identifying the microbial genes involved in interactions with the plant is a critical step. To identify type III effector (T3E) repertoires associated with virulence of the bacterial wilt pathogen Ralstonia solanacearum on Solanaceous crops, we used an original association genetics approach combining DNA microarray data and pathogenicity data on resistant eggplant, pepper, and tomato accessions. From this first screen, 25 T3Es were further full-length polymerase chain reaction-amplified within a 35-strain field collection, to assess their distribution and allelic diversity. Six T3E repertoire groups were identified, within which 11 representative strains were chosen to challenge the bacterial wilt-resistant egg plants 'Dingras multiple Purple' and 'AG91-25', and tomato Hawaii 7996. The virulence or avirulence phenotypes could not be explained by specific T3E repertoires, but rather by individual T3E genes. We identified seven highly avirulence-associated genes, among which ripP2, primarily referenced as conferring avirulence to Arabidopsis thaliana. Interestingly, no T3E was associated with avirulence to both egg-plants. Highly virulence-associated genes were also identified: ripA5_2, ripU, and ripV2. This study should be regarded as a first step toward investigating both avirulence and virulence function of the highlighted genes, but also their evolutionary dynamics in natural R. solanacearum populations.

Genome-wide association links candidate genes to resistance to Plum Pox Virus in apricot (Prunus armeniaca).

In fruit tree species, many important traits have been characterized genetically by using single-family descent mapping in progenies segregating for the traits. However, most mapped loci have not been sufficiently resolved to the individual genes due to insufficient progeny sizes for high resolution mapping and the previous lack of whole-genome sequence resources of the study species. To address this problem for Plum Pox Virus (PPV) candidate resistance gene identification in Prunus species, we implemented a genome-wide association (GWA) approach in apricot. This study exploited the broad genetic diversity of the apricot (Prunus armeniaca) germplasm containing resistance to PPV, next-generation sequence-based genotyping, and the high-quality peach (Prunus persica) genome reference sequence for single nucleotide polymorphism (SNP) identification. The results of this GWA study validated previously reported PPV resistance quantitative trait loci (QTL) intervals, highlighted other potential resistance loci, and resolved each to a limited set of candidate genes for further study. This work substantiates the association genetics approach for resolution of QTL to candidate genes in apricot and suggests that this approach could simplify identification of other candidate genes for other marked trait intervals in this germplasm.

Genome-Tagged Amplification (GTA): a PCR-based method to prepare sample-tagged amplicons from hundreds of individuals for next generation sequencing

Sampling the sequence of a relatively small fraction of the genome in large numbers of individuals is an important objective for population genetics and association genetics approaches. However, currently available ‘sequence capture’ methods either require expensive instrumentation or have problems dealing with high sample numbers and relatively small target sizes. We have developed Genome-Tagged Amplification (GTA) as a flexible PCR-based method for preparing pools of hundreds of amplicons from hundreds of samples for next generation sequencing. The method involves tagging of genomic DNA with barcode adapters at restriction sites, followed by PCR amplification from flanking DNA. It is freely scalable for both sample number and amplicon number and has no specialized equipment requirement. An optimized protocol is presented which provides a matrix of 96 × 192 combinations of samples x amplicons, corresponding to a complete 454 Titanium run. Initially, we used 454 sequencing; however, GTA could easily be adapted to Illumina sequencing platforms as read lengths have significantly increased in this system.

Linkage Disequilibrium in a Diversity and Stress Adaptation Rice Panel for Association Mapping

Estimated of linkage disequilibrium (LD) are important as an indication of how useful LD-base association genetics approaches can be when compared with other available mapping methods. The success of association mapping efforts depends on the possibilities of separating LD due to linkage from LD due to other causes. In an attempt to associate markers with drought tolerance at vegetative stage, we examined the pattern of LD in a diversity and stress adaptation rice panel containing 184 rice germplasm accessions with 141 polymorphic SSR markers that were nearly evenly distributed at 3 mb bin on the 12 rice chromosomes. Significant LD was detected across the genome of the 184 rice genotypes and the extent of LD varied with different chromosomes, selfing in O. sativa species and existing of population structure in the rice panel might be the major factors of creating high LD. No genetic linkage was detected except for two pairs of SSR markers RM288 and RM464 (11.98758 Mb), RM215 and RM464 (14.61396 Mb), which were located on chromosome 9 at a relatively far distance but showed significantly higher correlation. The high degree of LD and fast decay of LD detected in this experiment indicating the 141 SSR markers used in the experiment could be feasible to carry out the whole-genome scan association studies with a relatively high resolution.

Association genetics of carbon isotope discrimination, height and foliar nitrogen in a natural population of Pinus taeda L

Loblolly pine, Pinus taeda L., is one of the most widely planted, commercially and ecologically important tree species in North America. We took an association genetics approach, using an unimproved population of 380 clonally replicated unrelated trees, to test 3,938 single nucleotide polymorphisms (SNPs) in as many genes for association with phenotypic variation in carbon isotope discrimination, foliar nitrogen concentration and total tree height after two growing seasons. Best linear unbiased prediction (BLUP) was used with a spatial adjustment to remove environmental variation from phenotypic data derived from a common garden experiment. After correction for multiple testing, a total of 14 SNPs were associated with the traits of carbon isotope discrimination (n = 7), height (n = 1) and foliar nitrogen concentration (n = 6) using 380 clones. Tails of the population phenotypic distribution were compared for allele frequency differences, revealing 10 SNPs with allele frequency in at least one tail significantly different from the overall population. Eight associated SNPs were in sequences similar to known genes, such as an AP2 transcription factor related to carbon isotope discrimination and glutamate decarboxylase associated with foliar nitrogen concentration, and others were from unknown genes without homologs in Arabidopsis.

Association genetics of traits controlling lignin and cellulose biosynthesis in black cottonwood (Populus trichocarpa, Salicaceae) secondary xylem

• An association genetics approach was used to examine individual genes and alleles at the loci responsible for complex traits controlling lignocellulosic biosynthesis in black cottonwood (Populus trichocarpa). Recent interest in poplars as a source of renewable energy, combined with the vast genomic resources available, has enabled further examination of their genetic diversity. • Forty candidate genes were resequenced in a panel of 15 unrelated individuals to identify single nucleotide polymorphisms (SNPs). Eight hundred and seventy-six SNPs were successfully genotyped in a clonally replicated population (448 clones). The association population (average of 2.4 ramets per clone) was phenotyped using pyrolysis molecular beam mass spectrometry. Both single-marker and haplotype-based association tests were implemented to identify associations for composite traits representing lignin content, syringyl : guaiacyl ratio and C6 sugars. • Twenty-seven highly significant, unique, single-marker associations (false discovery rate Q < 0.10) were identified across 40 candidate genes in three composite traits. Twenty-three significant haplotypes within 11 genes were discovered in two composite traits. • Given the rapid decay of within-gene linkage disequilibrium and the high coverage of amplicons across each gene, it is likely that the numerous polymorphisms identified are in close proximity to the causative SNPs and the haplotype associations reflect information present in the associations between markers.

Demographic processes shaping genetic variation

Demographic processes modulate genome-wide levels and patterns of genetic variation via impacting effective population size independently of natural selection. Such processes include the perturbation of population distributions from external events shaping habitat landscape and internal factors shaping the probability of contemporaneous alleles in a population (coalescence). Several patterns have recently emerged: spatial and temporal heterogeneity in population structure have different influences on the persistence of new mutations and genetic variation, multi-locus analyses indicate that gene flow continues to occur during speciation and the incorporation of demographic processes into models of molecular evolution and association genetics approaches has improved statistical power to detect deviations from neutral-equilibrium expectations and decreased false positive rates.

A Rapid Protocol for Isolating Genomic DNA from Tropical Grass Species Suitable for RAPD, ISSR and STS

Largely in apomictic tropical grasses molecular markers analyses including identifying genes through association genetics approach requires DNA from large numbers of samples in short span of time. A simple protocol for DNA isolation from 5 apomictic tropical grass species namely Dichanthium annulatum, Heteropogon concortus, Sehima nervosum, Chrysopogon fulvus and Cenchrus glaucus is described. The method allows DNA extraction from as little as 0.2 to 0.3 g of leaves ground in liquid nitrogen, followed by DNA isolation in 1.5 ml Eppendorf tubes involving modified cetyltrimethylammonium bromide (CTAB) procedure using 1% polyvinylpyrrolidone (PVP) to remove polysaccharides. By using the method, a DNA yield up to 345 μg/g leaf tissues was obtained. The quality of the DNA was quite suitable not only for PCR-based markers analyses but also for restriction enzyme digestion. No or insignificant DNA was obtained in plant samples extracted from the fixed solution (alcohol, alcohol and chloroform, alcohol and ethylene diamine tetra acetic acid (EDTA)), thus the use of liquid nitrogen was inevitable.