Effect of population size and unbalanced data sets on QTL detection using genome-wide association mapping in barley breeding germplasm

Over the past two decades many quantitative trait loci (QTL) have been detected; however, very few have been incorporated into breeding programs. The recent development of genome-wide association studies (GWAS) in plants provides the opportunity to detect QTL in germplasm collections such as unstructured populations from breeding programs. The overall goal of the barley Coordinated Agricultural Project was to conduct GWAS with the intent to couple QTL detection and breeding. The basic idea is that breeding programs generate a vast amount of phenotypic data and combined with cheap genotyping it should be possible to use GWAS to detect QTL that would be immediately accessible and used by breeding programs. There are several constraints to using breeding program-derived phenotype data for conducting GWAS namely: limited population size and unbalanced data sets. We chose the highly heritable trait heading date to study these two variables. We examined 766 spring barley breeding lines (panel #1) grown in balanced trials and a subset of 384 spring barley breeding lines (panel #2) grown in balanced and unbalanced trials. In panel #1, we detected three major QTL for heading date that have been detected in previous bi-parental mapping studies. Simulation studies showed that population sizes greater than 384 individuals are required to consistently detect QTL. We also showed that unbalanced data sets from panel #2 can be used to detect the three major QTL. However, unbalanced data sets resulted in an increase in the false-positive rate. Interestingly, one-step analysis performed better than two-step analysis in reducing the false-positive rate. The results of this work show that it is possible to use phenotypic data from breeding programs to detect QTL, but that careful consideration of population size and experimental design are required.     

Multi-trait QTL mapping in barley using multivariate regression

Many studies of QTL locations record several different traits on the same population, but most analyses look at this information on a trait-by-trait basis. In this paper we show how the regression approach to QTL mapping of Haley & Knott (1992) may be extended to a multi-trait analysis via multivariate regression, easily programmed in statistical packages. A procedure for identifying QTL locations using forward selection and bootstrapping is proposed. The method is applied to examine the locations for QTLs for six yield characters (the number of fertile stems, the grain number of the main stem, the main stem grain weight, the single plant yield, the plot yield and the thousand grain weight) in a doubled haploid population of spring barley. Several chromosomal locations with effects on more than one trait are found. The method is also suitable for examining a single trait measured in different years or environments, and is used here to examine data on heading date, a highly heritable trait, and plot yield, a trait with moderate heritability and showing QTL-environment interactions.     

Genes mapping to boron tolerance QTL in barley identified by suppression subtractive hybridization

Boron tolerance is a quantitative trait controlled by multiple genes. Suppression subtractive hybridization was carried out on root cDNA from bulked boron tolerant and intolerant doubled haploid barley lines grown under moderate boron stress to identify genes associated with boron tolerance. One hundred and eleven clones representing known proteins were found to be up‐regulated in the tolerant bulk upon boron stress. Nine clones were genetically mapped to previously reported boron tolerance QTL. These include a clone identical to the boron transporter gene Bot1 and a clone coding for a bromo‐adjacent homology domain‐containing protein, mapping to the 6H boron tolerance locus and co‐segregating with reduced boron intake in a Clipper × Sahara‐3771 mapping population. A third clone mapping to the 2H QTL region encoding an S‐adenosylmethionine decarboxylase precursor was found to provide tolerance to high boron by heterologous expression. Yeast cells expressing Sahara SAMDC were able to grow on 15 mm boron solid media and maintained cellular boron concentrations at 13% lower than control cells expressing empty vector. The data suggest that an antioxidative response mechanism involving polyamines and the ascorbate–glutathione pathway in Sahara barley may provide an advantage in tolerating high soil concentrations of boron.     

Fine-scale mapping in Neurospora crassa by using genome-wide knockout strains

Fine-scale genetic mapping is often hindered by the lack of adequate markers surrounding the locus of interest. In the filamentous ascomycete Neurospora crassa, the genome has been sequenced and an effort has been made to generate genome-wide deletion strains for the entire gene set. Accordingly, the hygromycin-resistant marker in each deletion strain can be used as a mapping locus in a classical three-point cross, along with the mapping target and a standard marker. We have demonstrated the feasibility of this fine-scale mapping approach in N. crassa by refining the location of r(Sk-2).     

A new semi-dwarfing gene identified by molecular mapping of quantitative trait loci in barley

Semi-dwarfing genes have been widely used in spring barley (Hordeum vulgare L.) breeding programs in many parts of the world, but the success in developing barley cultivars with semi-dwarfing genes has been limited in North America. Exploiting new semi-dwarfing genes may help in solving this dilemma. A recombinant inbred line population was developed by crossing ZAU 7, a semi-dwarf cultivar from China, to ND16092, a tall breeding line from North Dakota. To identify quantitative trait loci (QTL) controlling plant height, a linkage map comprised of 111 molecular markers was constructed. Simple interval mapping was performed for each of the eight environments. A consistent QTL for plant height was found on chromosome 7HL. This QTL is not associated with maturity and rachis internode length. We suggest the provisional name Qph-7H for this QTL. Qph-7H from ZAU 7 reduced plant height to about 3/4 of normal; thus, Qph-7H is considered a semi-dwarfing gene. Other QTLs for plant height were found, but their expression was variable across the eight environments tested.     

Identification of marker-trait associations in the German winter barley breeding gene pool (Hordeum vulgare L.)

A genome-wide association mapping approach for grain yield and traits of high agronomic relevance was carried out on basis of a set of 61 six-rowed and 48 two-rowed German winter barley (Hordeum vulgare L.) cultivars representing breeding progress in the period 1959?C2003. Extensive phenotyping was conducted in field trials carried out at 12 locations in 3?years. Heritability was estimated at between 0.45 for grain yield and 0.94 for grains per spike. By using the Illumina Golden Gate Bead Array technology, 833 single nucleotide polymorphisms with an allele frequency higher than 5% were obtained. Linkage disequilibrium on the whole genome extends to 7.35?cM. Based on a mixed linear model approach taking into account the population structure estimated on the basis of 72 simple sequence repeat markers covering the whole barley genome, 91 significant marker-trait associations were detected, corresponding to 48 different genomic regions.     

Multiple phenotypes in genome-wide genetic mapping studies

For many psychiatric and other traits, diagnoses are based on a number of different criteria or phenotypes. Rather than carrying out genetic analyses on the final diagnosis, it has been suggested that relevant phenotypes should be analyzed directly. We provide an overview of statistical methods for the joint analysis of multiple phenotypes in case-control association studies.     

Identification of SSR markers associated with height using pool-based genome-wide association mapping in sorghum

Sorghum has been proposed as a potential energy crop. However, it has been traditionally bred for grain yield and forage quality, not traits related to bioenergy production. To develop tools for genetic improvement of bioenergy-related traits such as height, genetic markers associated with these traits have first to be identified. Association mapping has been extensively used in humans and in some crop plants for this purpose. However, genome-wide association mapping using the whole association panel is costly and time-consuming. A variation of this method called pool-based genome-wide association mapping has been extensively used in humans. In this variation, pools of individuals with contrasting phenotypes, instead of the whole panel, are screened with genetic markers and polymorphic markers are confirmed by screening the individuals in the pools. Here, we identified several new simple sequence repeats (SSR) markers associated with height using this pool-based genome-wide association mapping in sorghum. After screening the tall and short pools of sorghum accessions from the sorghum Mini Core collection developed at the International Crops Research Institute for the Semi-Arid Tropics with 703 SSR markers, we have identified four markers that are closely associated with sorghum height on chromosomes 2, 6, and 9. Comparison with published maps indicates that all four markers are clustered with markers previously mapped to height or height-related traits and with candidate genes involved in regulating plant height such as FtsZ, Ugt, and GA 2-oxidase. The mapping method can be applied to other crop plants for which a high-throughput genome-wide association mapping platform is not yet available.     

Prospects for using multimarker dihaploid lines for mapping the barley genome

Model population of spring barley Oregon Wolfe Pack dihaploid lines, derived from F1 of cross between dominant marker stock and recessive line, was studied on presence of morphological markers. For detection of DNA variability RAPD-analysis of these genotypes was carried out. Polymorphic fragment correlating with morphological trait was detected using primer P89.     

Identification of Ug99 stem rust resistance loci in winter wheat germplasm using genome-wide association analysis

The evolution of a new race of stem rust, generally referred to as Ug99, threatens global wheat production because it can overcome widely deployed resistance genes that had been effective for many years. To identify loci conferring resistance to Ug99 in wheat, a genome-wide association study was conducted using 232 winter wheat breeding lines from the International Winter Wheat Improvement Program. Breeding lines were genotyped with diversity array technology, simple sequence repeat and sequence-tagged site markers, and phenotyped at the adult plant stage for resistance to stem rust in the stem rust resistance screening nursery at Njoro, Kenya during 2009-2011. A mixed linear model was used for detecting marker-trait associations. Twelve loci associated with Ug99 resistance were identified including markers linked to known genes Sr2 and Lr34. Other markers were located in the chromosome regions where no Sr genes have been previously reported, including one each on chromosomes 1A, 2B, 4A and 7B, two on chromosome 5B and four on chromosome 6B. The same data were used for investigating epistatic interactions between markers with or without main effects. The marker csSr2 linked to Sr2 interacted with wPt4930 on 6BS and wPt729773 in an unknown location. Another marker, csLV34 linked to Lr34, also interacted with wPt4930 on 6BS and wPt4916 on 2BS. The frequent involvement of wPt4916 on 2BS and wPt4930 on 6BS in interactions with other significant loci on the same or different chromosomes suggested complex genetic control for adult plant resistance to Ug99 in winter wheat germplasm.     

QTL linkage mapping of wing length in zebra finch using genome-wide single nucleotide polymorphisms markers

Avian wing length is an important trait that covaries with the ecology and migratory behaviour of a species and tends to change rapidly when the conditions are altered. Long-distance migrants typically have longer wings than short-distance migrants and sedentary species, and long-winged species also tend to be more dispersive. Although the substantial heritability of avian wing length is well established, the identification of causal genes has remained elusive. Based on large-scale genotyping of 1404 informative single nucleotide polymorphisms (SNP) in a captive population of 1067 zebra finches, we here show that the within-population variation of relative wing length (h(2) = 0.74 ± 0.05) is associated with standing genetic variation in at least six genomic regions (one genome-wide significant and five suggestive). The variance explained by these six quantitative trait loci (QTL) sums to 36.8% of the phenotypic variance (half of the additive genetic variance), although this likely is an overestimate attributable to the Beavis effect. As avian wing length is primarily determined by the length of the primary feathers, we then searched for candidate genes that are related to feather growth. Interestingly, all of the QTL signals co-locate with Wnt growth factors and closely interacting genes (Wnt3a, Wnt5a, Wnt6, Wnt7a, Wnt9a, RhoU and RhoV). Our findings therefore suggest that standing genetic variation in the Wnt genes might be linked to avian wing morphology, although there are many other genes that also fall within the confidence regions.     

Phenotype/genotype associations for yield and salt tolerance in a barley mapping population segregating for two dwarfing genes

Barley traits related to salt tolerance are mapped in a population segregating for a dwarfing gene associated with salt tolerance. Twelve quantitative trait loci (QTLs) were detected for seven seedling traits in doubled haploids from the spring barley cross Derkado x B83-12/21/5 when given saline treatment in hydroponics. The location of QTLs for seedling growth stage (leaf appearance rate), stem weight prior to elongation, and tiller number are reported for the first time. In addition, four QTLs were found for the mature plant traits grain nitrogen and plot yield. In total, seven QTLs are co-located with the dwarfing genes sdw1, on chromosome 3H, and ari-e.GP, on chromosome 5H, including seedling leaf response (SGa) to gibberellic acid (GA(3)). QTLs controlling the growth of leaves (GS2) on chromosomes 2H and 3H and emergence of tillers (TN2) and grain yield were independent of the dwarfing genes. Field trials were grown in eastern Scotland and England to estimate yield and grain composition. A genetic map was used to compare the positions of QTLs for seedling traits with the location of QTLs for the mature plant traits. The results are discussed in relation to the study of barley physiology and the location of genes for dwarf habit and responses to GA.     

Identification of SSR markers associated with saccharification yield using pool-based genome-wide association mapping in sorghum

Saccharification describes the conversion of plant biomass by cellulase into glucose. Because plants have never been selected for high saccharification yield, cellulosic ethanol production faces a significant bottleneck. To improve saccharification yield, it is critical to identify the genes that affect this process. In this study, we used pool-based genome-wide association mapping to identify simple sequence repeat (SSR) markers associated with saccharification yield. Screening of 703 SSR markers against the low and high saccharification pools identified two markers on the sorghum chromosomes 2 (23-1062) and 4 (74-508c) associated with saccharification yield. The association was significant at 1% using either general or mixed linear models. Localization of these markers based on the whole genome sequence indicates that 23-1062 is 223 kb from a β-glucanase (Bg) gene and 74-508c is 81 kb from a steroid-binding protein (Sbp) gene. Bg is critical for cell wall assembly and degradation, but Sbp can suppress the expression of Bg as demonstrated in Arabidopsis (Yang et al. 2005). These markers are found physically close to genes encoding plant cell wall synthesis enzymes such as xyloglucan fucosyltransferase (149 kb from 74-508c) and UDP-D-glucose 4-epimerase (46 kb from 23-1062). Genetic transformation of selected candidate genes is in progress to examine their effect on saccharification yield in plants.     

RFLP mapping of manganese efficiency in barley

In many cropping regions of the world, yield is limited by the availability of micronutrients, and micronutrient-efficient cultivars provide a yield advantage. Traditional methods of testing cultivars for micronutrient efficiency are time-consuming and laborious. Molecular markers linked to loci controlling micronutrient efficiency will allow more rapid and efficient selection and introgression of these traits than is currently possible. Using a pot-based bioassay and bulked segregant analysis of an F₂ population, we have identified several RFLPs (grouped distally on chromosome 4HS) linked to a locus for manganese efficiency in barley. This manganese efficiency locus has been designated Mel1. Pot bioassay analysis of intercrosses suggests that three useful sources of manganese efficiency are likely to be allelic at the Mel1 locus. Field evaluation of marker selected F₄ progeny supports the major role of Mel1 in the genetic control of manganese efficiency. Adoption of marker assisted selection for this trait in the Southern Australian barley breeding program has occurred. This has been facilitated by the demonstration that the Mel1 allele of Amagi Nijo can be distinguished from 95 other locally useful varieties and breeder’s lines on the basis of RFLPs identified by just two molecular markers. Received: 20 October 1999 / Accepted: 18 February 2000     

Physical mapping of barley genes using an ultrasensitive fluorescence in situ hybridization technique.

The primary objective of this study was to elucidate gene organization and to integrate the genetic linkage map for barley (Hordeum vulgare L.) with a physical map using ultrasensitive fluorescence in situ hybridization (FISH) techniques for detecting signals from restriction fragment length polymorphism (RFLP) clones. In the process, a single landmark plasmid, p18S5Shor, was constructed that identified and oriented all seven of the chromosome pairs. Plasmid p18S5Shor was used in all hybridizations. Fourteen cDNA probes selected from the linkage map for barley H. vulgare 'Steptoe' x H. vulgare 'Morex' (Kleinhofs et al. 1993) were mapped using an indirect tyramide signal amplification technique and assigned to a physical location on one or more chromosomes. The haploid barley genome is large and a complete physical map of the genome is not yet available; however, it was possible to integrate the linkage map and the physical locations of these cDNAs. An estimate of the ratio of base pairs to centimorgans was an average of 1.5 Mb/cM in the distal portions of the chromosome arms and 89 Mb/cM near the centromere. Furthermore, while it appears that the current linkage maps are well covered with markers along the length of each arm, the physical map showed that there are large areas of the genome that have yet to be mapped.