Detection of quantitative trait Loci influencing recombination using recombinant inbred lines

The genetic basis of variation in recombination in higher plants is polygenic and poorly understood, despite its theoretical and practical importance. Here a method of detecting quantitative trait loci (QTL) influencing recombination in recombinant inbred lines (RILs) is proposed that relies upon the fact that genotype data within RILs carry the signature of past recombination. Behavior of the segregational genetic variance in numbers of chromosomal crossovers (recombination) over generations is described for self-, full-sib-, and half-sib-generated RILs with no dominance in true crossovers. This genetic variance, which as a fraction of the total phenotypic variance contributes to the statistical power of the method, was asymptotically greatest with half sibbing, less with sibbing, and least with selfing. The statistical power to detect a recombination QTL declined with diminishing QTL effect, genome target size, and marker density. For reasonably tight marker linkage power was greater with less intense inbreeding for later generations and vice versa for early generations. Generational optima for segregation variance and statistical power were found, whose onset and narrowness varied with marker density and mating design, being more pronounced for looser marker linkage. Application of this method to a maize RIL population derived from inbred lines Mo17 and B73 and developed by selfing suggested two putative QTL (LOD > 2.4) affecting certain chromosomes, and using a canonical transformation another putative QTL was detected. However, permutation tests failed to support their presence (experimentwise alpha = 0.05). Other populations with more statistical power and chosen specifically for recombination QTL segregation would be more effective.     

A Set of Lotus japonicus Gifu x Lotus burttii Recombinant Inbred Lines Facilitates Map-based Cloning and QTL Mapping

Model legumes such as Lotus japonicus have contributed significantly to the understanding of symbiotic nitrogen fixation. This insight is mainly a result of forward genetic screens followed by map-based cloning to identify causal alleles. The L. japonicus ecotype 'Gifu' was used as a common parent for inter-accession crosses to produce F2 mapping populations either with other L. japonicus ecotypes, MG-20 and Funakura, or with the related species L. filicaulis. These populations have all been used for genetic studies but segregation distortion, suppression of recombination, low polymorphism levels, and poor viability have also been observed. More recently, the diploid species L. burttii has been identified as a fertile crossing partner of L. japonicus. To assess its qualities in genetic linkage analysis and to enable quantitative trait locus (QTL) mapping for a wider range of traits in Lotus species, we have generated and genotyped a set of 163 Gifu × L. burttii recombinant inbred lines (RILs). By direct comparisons of RIL and F2 population data, we show that L. burttii is a valid alternative to MG-20 as a Gifu mapping partner. In addition, we demonstrate the utility of the Gifu × L. burttii RILs in QTL mapping by identifying an Nfr1-linked QTL for Sinorhizobium fredii nodulation.     

Quantitative trait loci mapping in five new large recombinant inbred line populations of Arabidopsis thaliana genotyped with consensus single-nucleotide polymorphism markers

Quantitative approaches conducted in a single mapping population are limited by the extent of genetic variation distinguishing the parental genotypes. To overcome this limitation and allow a more complete dissection of the genetic architecture of complex traits, we built an integrated set of 15 new large Arabidopsis thaliana recombinant inbred line (RIL) populations optimized for quantitative trait loci (QTL) mapping, having Columbia as a common parent crossed to distant accessions. Here we present 5 of these populations that were validated by investigating three traits: flowering time, rosette size, and seed production as an estimate of fitness. The large number of RILs in each population (between 319 and 377 lines) and the high density of evenly spaced genetic markers scored ensure high power and precision in QTL mapping even under a minimal phenotyping framework. Moreover, the use of common markers across the different maps allows a direct comparison of the QTL detected within the different RIL sets. In addition, we show that following a selective phenotyping strategy by performing QTL analyses on genotypically chosen subsets of 164 RILs (core populations) does not impair the power of detection of QTL with phenotypic contributions >7%.     

Is genotypic variation of H⁺ efflux under P deficiency linked with nodulated-root respiration of N₂ fixing common-bean (Phaseolus vulgaris L.)?

To examine genotypic variation of common bean in growth, phosphorus uptake, nodulated-root proton release, and nodule gas permeability, seven common bean recombinant inbred lines (RIL) from the cross of BAT477×DOR364 were inoculated by Rhizobium tropici CIAT 899 and grown in hydroaeroponic culture under glasshouse conditions. A positive correlation was observed between shoot and nodule biomass for most of the studied RILs. Under P deficiency, the tolerant common bean RILs acidified more of their rhizosphere than the sensitive ones. The proton release of the RILs 147, 124, 104, 75 and RIL34 was positively correlated with nodule O? permeability. We conclude that nodulated common bean plants release a substantial amount of H? into the rhizosphere that is linked to the symbiotic N? fixation. It depends upon the nodule permeability to O? diffusion, and varies with genotype.     

Genetic and physical mapping of<Emphasis Type="Italic"> Pi5(t)</Emphasis>, a locus associated with broad-spectrum resistance to rice blast

To gain an understanding of the molecular basis for resistance to rice blast (Magnaporthe grisea), we have initiated a project to clone Pi5(t), a locus associated with broad-spectrum resistance to diverse blast isolates. AFLP-derived markers linked to Pi5(t)-mediated resistance were isolated using bulked segregant analysis of F(2) populations generated by crossing three recombinant inbred lines (RILs), RIL125, RIL249, and RIL260 with the susceptible line CO39. The most tightly linked AFLP marker, S04G03, was positioned on chromosome 9 of the fingerprint-based physical map of Nipponbare, a well-characterized rice genotype. Flanking BAC-based Nipponbare markers were generated for saturation mapping using four populations, the three initial RILs and an additional one derived from a cross between M202 and RIL260. A BIBAC (binary BAC) library was constructed from RIL260. Using these resources Pi5(t) was mapped to a 170-kb interval, and a contiguous set of BIBAC clones spanning this region was constructed. It had previously been suggested that Pi3(t) and Pi5(t) might be allelic, due to their identical resistance spectrum and tight linkage. We therefore compared genomic regions for lines containing Pi3(t) using the Pi5(t)-linked markers. DNA gel-blot analyses indicated that the region around Pi3(t) is identical to that of Pi5(t), suggesting that Pi3(t) and Pi5(t) are the same resistance gene.     

Fine mapping of the Bsr1 barley stripe mosaic virus resistance gene in the model grass Brachypodium distachyon

The ND18 strain of Barley stripe mosaic virus (BSMV) infects several lines of Brachypodium distachyon, a recently developed model system for genomics research in cereals. Among the inbred lines tested, Bd3-1 is highly resistant at 20 to 25 °C, whereas Bd21 is susceptible and infection results in an intense mosaic phenotype accompanied by high levels of replicating virus. We generated an F(6:7) recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21 and used the RILs, and an F(2) population of a second Bd21 × Bd3-1 cross to evaluate the inheritance of resistance. The results indicate that resistance segregates as expected for a single dominant gene, which we have designated Barley stripe mosaic virus resistance 1 (Bsr1). We constructed a genetic linkage map of the RIL population using SNP markers to map this gene to within 705 Kb of the distal end of the top of chromosome 3. Additional CAPS and Indel markers were used to fine map Bsr1 to a 23 Kb interval containing five putative genes. Our study demonstrates the power of using RILs to rapidly map the genetic determinants of BSMV resistance in Brachypodium. Moreover, the RILs and their associated genetic map, when combined with the complete genomic sequence of Brachypodium, provide new resources for genetic analyses of many other traits.     

Power to detect higher-order epistatic interactions in a metabolic pathway using a new mapping strategy

Epistatic interactions among quantitative trait loci (QTL) contribute substantially to the variation in complex traits. The main objectives of this study were to (i) compare three- vs. four-step genome scans to identify three-way epistatic interactions among QTL belonging to a metabolic pathway, (ii) investigate by computer simulations the power and proportion of false positives (PFP) for detecting three-way interactions among QTL in recombinant inbred line (RIL) populations derived from a nested mating design, and (iii) compare these estimates to those obtained for detecting three-way interactions among QTL in RIL populations derived from diallel and different partial diallel mating designs. The single-nucleotide polymorphism haplotype data of B73 and 25 diverse maize inbreds were used to simulate the production of various RIL populations. Compared to the three-step genome scan, the power to detect three-way interactions was higher with the four-step genome scan. Higher power to detect three-way interactions was observed for RILs derived from optimally allocated distance-based designs than from nested designs or diallel designs. The power and PFP to detect three-way interactions using a nested design with 5000 RILs were for both the 4-QTL and the 12-QTL scenario of a magnitude that seems promising for their identification.     

Using parthenogenesis‐inducing Wolbachia for the selection of optimal lines of the egg parasitoid Trichogramma pretiosum for use in biocontrol

Trichogramma wasps (Hymenoptera: Trichogrammatidae) are egg parasitoids commonly employed in augmentative biological control releases against a variety of mainly lepidopteran pests. By exploiting the mechanism by which the endosymbiotic bacterium Wolbachia induces parthenogenesis in this genus, we created a set of completely homozygous Wolbachia‐infected recombinant isofemale lines (RILs), each consisting of a different combination of the genome of two well‐characterized lines of Trichogramma pretiosum Riley. We subsequently use 16 of these RILs to investigate the effect of genetic variation on various measures associated with offspring production under laboratory conditions. Unsurprisingly, substantial differences were found between the RILs in their propensity to parasitize hosts, the number of hosts they parasitize, and the levels of mortality in their offspring. Such measures can be used to choose an optimal line for biological control purposes. A method was also developed to characterize the 16 RILs using their allelic state at five loci. Essentially, this binary system uses high‐resolution melt analysis to resolve identity at each locus, with alleles originating from either the grandmaternal or grandpaternal line, and is such that each RIL can be distinguished from each other RIL by their allelic state at one or more loci. The method facilitates the easy diagnosis of line origin when two or more lines are competing with each other in competition assays, allowing for the design of more complicated tests of parasitoid quality. Future field experiments will determine which genetic line performs best under more realistic biological control conditions. The fact that these lines are infected with parthenogenesis‐inducing Wolbachia will allow for prolonged rearing without appreciable change in their genetic makeup, which should lead to a predictable biological control performance.     

The Power of QTL Mapping with RILs

QTL (quantitative trait loci) mapping is commonly used to identify genetic regions responsible to important phenotype variation. A common strategy of QTL mapping is to use recombinant inbred lines (RILs), which are usually established by several generations of inbreeding of an F1 population (usually up to F6 or F7 populations). As this inbreeding process involves a large amount of labor, we are particularly interested in the effect of the number of inbreeding generations on the power of QTL mapping; a part of the labor could be saved if a smaller number of inbreeding provides sufficient power. By using simulations, we investigated the performance of QTL mapping with recombinant inbred lines (RILs). As expected, we found that the power of F4 population could be almost comparable to that of F6 and F7 populations. A potential problem in using F4 population is that a large proportion of RILs are heterozygotes. We here introduced a new method to partly relax this problem. The performance of this method was verified by simulations with a wide range of parameters including the size of the segregation population, recombination rate, genome size and the density of markers. We found our method works better than the commonly used standard method especially when there are a number of heterozygous markers. Our results imply that in most cases, QTL mapping does not necessarily require RILs at F6 or F7 generations; rather, F4 (or even F3) populations would be almost as useful as F6 or F7 populations. Because the cost to establish a number of RILs for many generations is enormous, this finding will cause a reduction in the cost of QTL mapping, thereby accelerating gene mapping in many species.     

QTL mapping for combining ability in different population-based NCII designs: a simulation study

The NCII design (North Carolina mating design II) has been widely applied in studies of combining ability and heterosis. The objective of our research was to estimate how different base populations, sample sizes, testcross numbers and heritability influence QTL analyses of combining ability and heterosis. A series of Monte Carlo simulation experiments with QTL mapping were then conducted for the base population performance, testcross population phenotypic values and the general combining ability (GCA), specific combining ability (SCA) and Hmp (midparental heterosis) datasets. The results indicated that: (i) increasing the number of testers did not necessarily enhance the QTL detection power for GCA, but it was significantly related to the QTL effect. (ii) The QTLs identified in the base population may be different from those from GCA dataset. Similar phenomena can be seen from QTL detected in SCA and Hmp datasets. (iii) The QTL detection power for GCA ranked in the order of DH(RIL) based > F₂ based > BC based NCII design, when the heritability was low. The recombinant inbred lines (RILs) (or DHs) allows more recombination and offers higher mapping resolution than other populations. Further, their testcross progeny can be repeatedly generated and phenotyped. Thus, RIL based (or DH based) NCII design was highly recommend for combining ability QTL analysis. Our results expect to facilitate selecting elite parental lines with high combining ability and for geneticists to research the genetic basis of combining ability.     

Haldane,Waddington and recombinant inbred lines: extension of their work to any number of genes

In the early 1930s, J. B. S. Haldane and C. H. Waddington collaborated on the consequences of genetic linkage and inbreeding. One elegant mathematical genetics problem solved by them concerns recombinant inbred lines (RILs) produced via repeated self or brother–sister mating. In this classic contribution, Haldane and Waddington derived an analytical formula for the probabilities of 2-locus and 3-locus RIL genotypes. Specifically, the Haldane–Waddington formula gives the recombination rate R in such lines as a simple function of the per generation recombination rate r. Interestingly, for more than 80 years, an extension of this result to four or more loci remained elusive. In 2015, we generalized the Haldane–Waddington self-mating result to any number of loci. Our solution used self-consistent equations of the multi-locus probabilities ‘for an infinite number of generations’ and solved these by simple algebraic operations. In practice, our approach provides a quantum leap in the systems that can be handled: the cases of up to six loci can be solved by hand while a computer program implementing our mathematical formalism tackles up to 20 loci on standard desktop computers.     

Properties and power of the Drosophila Synthetic Population Resource for the routine dissection of complex traits

The Drosophila Synthetic Population Resource (DSPR) is a newly developed multifounder advanced intercross panel consisting of >1600 recombinant inbred lines (RILs) designed for the genetic dissection of complex traits. Here, we describe the inference of the underlying mosaic founder structure for the full set of RILs from a dense set of semicodominant restriction-site-associated DNA (RAD) markers and use simulations to explore how variation in marker density and sequencing coverage affects inference. For a given sequencing effort, marker density is more important than sequence coverage per marker in terms of the amount of genetic information we can infer. We also assessed the power of the DSPR by assigning genotypes at a hidden QTL to each RIL on the basis of the inferred founder state and simulating phenotypes for different experimental designs, different genetic architectures, different sample sizes, and QTL of varying effect sizes. We found the DSPR has both high power (e.g., 84% power to detect a 5% QTL) and high mapping resolution (e.g., ～1.5 cM for a 5% QTL).     

ECOLOGICAL NOVELTY BY HYBRIDIZATION: EXPERIMENTAL EVIDENCE FOR INCREASED THERMAL TOLERANCE BY TRANSGRESSIVE SEGREGATION IN TIGRIOPUS CALIFORNICUS

Early generations of hybrids can express both genetic incompatibilities and phenotypic novelty. Insights into whether these conflicting interactions between intrinsic and extrinsic selection persist after a few generations of recombination require experimental studies. To address this question, we use interpopulation crosses and recombinant inbred lines (RILs) of the copepod Tigriopus californicus, and focus on two traits that are relevant for the diversification of this species: survivorship during development and tolerance to thermal stress. Experimental crosses between two population pairs show that most RILs between two heat‐tolerant populations show enhanced tolerance to temperatures that are lethal to the respective parentals, whereas RILs between a heat‐tolerant and a heat‐sensitive population are intermediate. Although interpopulation crosses are affected by intrinsic selection at early generational hybrids, most of the sampled F₉ RILs have recovered fitness to the level of their parentals. Together, these results suggest that a few generations of recombination allows for an independent segregation of the genes underlying thermal tolerance and cytonuclear incompatibilities, permitting certain recombinant lineages to survive in niches previously unused by parental taxa (i.e., warmer thermal environments) without incurring intrinsic selection.     

Identification of quantitative trait loci across recombinant inbred lines and testcross populations for traits of agronomic importance in rice

This study was conducted to determine whether quantitative trait loci (QTL) controlling traits of agronomic importance detected in recombinant inbred lines (RILs) are also expressed in testcross (TC) hybrids of rice. A genetic map was constructed using an RIL population derived from a cross between B5 and Minghui 63, a parent of the most widely grown hybrid rice cultivar in China. Four TC hybrid populations were produced by crossing the RILs with three maintaining lines for the widely used cytoplasmic male-sterile (CMS) lines and the genic male-sterile line Peiai64s. The mean values of the RILs for the seven traits investigated were significantly correlated to those of the F1 hybrids in the four TC populations. Twenty-seven main-effect QTL were identified in the RILs. Of these, the QTL that had the strongest effect on each of the seven traits in the RILs was detected in two or more of the TC populations, and six other QTL were detected in one TC population. Epistatic analysis revealed that the effect of epistatic QTL was relatively weak and cross combination specific. Searching publicly available QTL data in rice revealed the positional convergence of the QTL with the strongest effect in a wide range of populations and under different environments. Since the main-effect QTL is expressed across different testers, and in different genetic backgrounds and environments, it is a valuable target for gene manipulation and for further application in rice breeding. When a restorer line that expresses main-effect QTL is bred, it could be used in a number of cross combinations.     

Genetic Control of the Leaf Angle and Leaf Orientation Value as Revealed by Ultra-High Density Maps in Three Connected Maize Populations

Plant architecture is a key factor for high productivity maize because ideal plant architecture with an erect leaf angle and optimum leaf orientation value allow for more efficient light capture during photosynthesis and better wind circulation under dense planting conditions. To extend our understanding of the genetic mechanisms involved in leaf-related traits, three connected recombination inbred line (RIL) populations including 538 RILs were genotyped by genotyping-by-sequencing (GBS) method and phenotyped for the leaf angle and related traits in six environments. We conducted single population quantitative trait locus (QTL) mapping and joint linkage analysis based on high-density recombination bin maps constructed from GBS genotype data. A total of 45 QTLs with phenotypic effects ranging from 1.2% to 29.2% were detected for four leaf architecture traits by using joint linkage mapping across the three populations. All the QTLs identified for each trait could explain approximately 60% of the phenotypic variance. Four QTLs were located on small genomic regions where candidate genes were found. Genomic predictions from a genomic best linear unbiased prediction (GBLUP) model explained 45±9% to 68±8% of the variation in the remaining RILs for the four traits. These results extend our understanding of the genetics of leaf traits and can be used in genomic prediction to accelerate plant architecture improvement.     

Development of a near-isogenic line population of Arabidopsis thaliana and comparison of mapping power with a recombinant inbred line population

In Arabidopsis recombinant inbred line (RIL) populations are widely used for quantitative trait locus (QTL) analyses. However, mapping analyses with this type of population can be limited because of the masking effects of major QTL and epistatic interactions of multiple QTL. An alternative type of immortal experimental population commonly used in plant species are sets of introgression lines. Here we introduce the development of a genomewide coverage near-isogenic line (NIL) population of Arabidopsis thaliana, by introgressing genomic regions from the Cape Verde Islands (Cvi) accession into the Landsberg erecta (Ler) genetic background. We have empirically compared the QTL mapping power of this new population with an already existing RIL population derived from the same parents. For that, we analyzed and mapped QTL affecting six developmental traits with different heritability. Overall, in the NIL population smaller-effect QTL than in the RIL population could be detected although the localization resolution was lower. Furthermore, we estimated the effect of population size and of the number of replicates on the detection power of QTL affecting the developmental traits. In general, population size is more important than the number of replicates to increase the mapping power of RILs, whereas for NILs several replicates are absolutely required. These analyses are expected to facilitate experimental design for QTL mapping using these two common types of segregating populations.     

A SNP-based genetic dissection of versatile traits in bread wheat (Triticum aestivum L.)

The continuous increase in global population prompts increased wheat production. Future wheat (Triticum aestivum L.) breeding will heavily rely on dissecting molecular and genetic bases of wheat yield and related traits which is possible through the discovery of quantitative trait loci (QTLs) in constructed populations, such as recombinant inbred lines (RILs). Here, we present an evaluation of 92 RILs in a bi-parental RIL mapping population (the International Triticeae Mapping Initiative Mapping Population [ITMI/MP]) using newly generated phenotypic data in 3-year experiments (2015), older phenotypic data (1997–2009), and newly created single nucleotide polymorphism (SNP) marker data based on 92 of the original RILs to search for novel and stable QTLs. Our analyses of more than 15 unique traits observed in multiple experiments included analyses of 46 traits in three environments in the USA, 69 traits in eight environments in Germany, 149 traits in 10 environments in Russia, and 28 traits in four environments in India (292 traits in 25 environments) with 7584 SNPs (292 × 7584 = 2 214 528 data points). A total of 874 QTLs were detected with limit of detection (LOD) scores of 2.01–3.0 and 432 QTLs were detected with LOD > 3.0. Moreover, 769 QTLs could be assigned to 183 clusters based on the common markers and relative proximity of related QTLs, indicating gene-rich regions throughout the A, B, and D genomes of common wheat. This upgraded genotype–phenotype information of ITMI/MP can assist breeders and geneticists who can make crosses with suitable RILs to improve or investigate traits of interest.