Trait-based analyses for the detection of linkage between marker loci and quantitative trait loci in crosses between inbred lines

Summary Methods are presented for determining linkage between a marker locus and a nearby locus affecting a quantitative trait (quantitative trait locus=QTL), based on changes in the marker allele frequencies in selection lines derived from the F-2 of a cross between inbred lines, or in the high and low phenotypic classes of an F-2 or BC population. The power of such trait-based (TB) analyses was evaluated and compared with that of methods for determining linkage based on the mean quantitative trait value of marker genotypes in F-2 or BC populations [marker-based (MB) analyses]. TB analyses can be utilized for marker-QTL linkage determination in situations where the MB analysis is not applicable, including analysis of polygenic resistance traits where only a part of the population survives exposure to the Stressor and analysis of marker-allele frequency changes in selection lines. TB analyses may be a useful alternative to MB analyses when interest is centered on a single quantitative trait only and costs of scoring for markers are high compared with costs of raising and obtaining quantitative trait information on F-2 or BC individuals. In this case, a TB analysis will enable equivalent power to be obtained with fewer individuals scored for the marker, but more individuals scored for the quantitative trait. MB analyses remain the method of choice when more than one quantitative trait is to be analyzed in a given population.Contribution from the ARO, Bet Dagan, Israel. No. 1698-E, 1986 series     

On the power of experimental designs for the detection of linkage between marker loci and quantitative loci in crosses between inbred lines

Summary The power of experiments aimed at detecting linkage between a quantitative locus and a marker locus, both segregating in the backross or F₂ generation of a cross between two inbred lines, is examined. Given that the two lines are close to fixation for alternative alleles of both marker locus and quantitative locus, it is concluded that experiments involving a few thousand offspring should be able to detect close linkages involving quantitative loci (or groups of loci) having rather modest effects (i.e., that contribute, say, 1% of the total phenotypic variance in the F₂).     

Optimum spacing of genetic markers for determining linkage between marker loci and quantitative trait loci

The cost of experiments aimed at determining linkage between marker loci and quantitative trait loci (QTL) was investigated as a function of marker spacing and number of individuals scored. It was found that for a variety of experimental designs, fairly wide marker spacings (ca. 50 cM) are optimum or close to optimum for initial studies of marker-QTL linkage, in the sense of minimizing overall cost of the experiment. Thus, even when large numbers of more or less evenly spaced markers are available, it will not always be cost effective to make full utilization of this capacity. This is particularly true when costs of rearing and trait evaluation per individual scored are low, as when marker data are obtained on individuals raised and evaluated for quantitative traits as part of existing programs. When costs of rearing and trait evaluation per individual scored are high, however, as in human family data collection carried out primarily for subsequent marker — QTL analyses, or when plants or animals are raised specifically for purposes of marker — QTL linkage experiments, optimum spacing may be rather narrow. It is noteworthy that when marginal costs of additional markers or individuals are constant, total resources allocated to a given experiment will determine total number of individuals sampled, but not the optimal marker spacing.     

Linkage analysis of quantitative traits in an interspecific cross of tomato (lycopersicon esculentum x lycopersicon pimpinellifolium) by means of genetic markers

Linkage relationships between loci affecting quantitative traits (QTL) and marker loci were examined in an interspecific cross between Lycopersicon esculentum and Lycopersicon pimpinellifolium. Parental lines differed for six morphological markers and for four electrophoretic markers. Almost 1700 F-2 plants were scored with respect to the genetic markers and also with respect to 18 quantitative traits. Major genes affecting the quantitative traits were not found, but out of 180 possible marker x trait combinations, 85 showed significant quantitative effects associated with the genetic markers. The average marker-associated main effect was on the order of 6% of the mean value of the trait. Most of the main effects were apparently due to linkage of QTL to the marker loci rather than to pleiotropy. Fourteen of the traits showed at least one highly significant effect of opposite sign to the overall difference between the parental lines, demonstrating the ability of this design to uncover cryptic genetic variation. Significant variance and skewness effects on the quantitative traits were found to be associated with the genetic markers, suggesting the possible presence of loci affecting the variance and shape of quantitative trait distribution in a population. Most marker-associated quantitative effects showed some degree of dominance, generally in the direction of the L. pimpinellifolium parent. When the significant marker-associated effects were examined in pairs, 12% showed significant interaction effects. The results of this study illustrate the potential usefulness of this type of analysis for the detailed genetic investigation of quantitative trait variation in suitably marked populations.     

Marker-based mapping of quantitative trait loci using replicated progenies

Summary When heritability of the trait under investigation is low, replicated progenies can bring about a major reduction in the number of individuals that need to be scored for marker genotype in determining linkage between marker loci and quantitative trait loci (QTL). Savings are greatest when heritability of the trait is low, but are much reduced when heritability of the quantitative trait is moderate to high. Required numbers for recombinant inbred lines will be greater than those required for a simple F₂ population when heritabilities are moderate to high and the proportion of recombination between marker locus and quantitative trait locus is substantial.Contribution No. 2613-E of the Agricultural Research Organization, 1989 series     

Mapping QTLs conferring salt tolerance during germination in tomato by selective genotyping

This study was conducted to identify genomic regions (quantitative trait loci, QTLs) affecting salt tolerance during germination in tomato. Germination response of an F2 population of a cross between UCT5 (Lycopersicon esculentum, salt-sensitive) and LA716 (L. pennellii, salt-tolerant) was evaluated at a salt-stress level of 175 mM NaCl + 17.5 mM CaCl2 (water potential ca. –950 kPa). Germination was scored visually as radicle protrusion at 6 h intervals for 30 consecutive days. Individuals at both extremes of the response distribution (i.e., salt-tolerant and salt-sensitive individuals) were selected. The selected individuals were genotyped at 84 genetic markers including 16 isozymes and 68 restriction fragment length polymorphisms (RFLPs). Trait-based marker analysis (TBA) which measures changes (differences) in marker allele frequencies in selected lines was used to identify marker-linked QTLs. Eight genomic regions were identified on seven tomato chromosomes bearing genes (QTLs) with significant effects on this trait. The results confirmed our previous suggestion that salt tolerance during germination in tomato is polygenically controlled. The salt-tolerant parent contributed favorable QTL alleles on chromosomes 1, 3, 9 and 12 whereas the salt sensitive parent contributed favorable QTL alleles on chromosomes 2, 7 and 8. The identification of favorable alleles in both parents suggests the likelihood of recovering transgressive segregants in progeny derived from these parental genotypes. The results can be used for marker-assisted selection and breeding of salt-tolerant tomatoes.     

Mapping quantitative trait loci in noninbred mosquito crosses

The identification of genes that affect quantitative traits has been of great interest to geneticists for many decades, and many statistical methods have been developed to map quantitative trait loci (QTL). Most QTL mapping studies in experimental organisms use purely inbred lines, where the two homologous chromosomes in each individual are identical. As a result, many existing QTL mapping methods developed for experimental organisms are applicable only to genetic crosses between inbred lines. However, it may be difficult to obtain inbred lines for certain organisms, e.g., mosquitoes. Although statistical methods for QTL mapping in outbred populations, e.g., humans, can be applied for such crosses, these methods may not fully take advantage of the uniqueness of these crosses. For example, we can generally assume that the two grandparental lines are homozygous at the QTL of interest, but such information is not be utilized through methods developed for outbred populations. In addition, mating types and phases can be relatively easy to establish through the analysis of adjacent markers due to the large number of offspring that can be collected, substantially simplifying the computational need. In this article, motivated by a mosquito intercross experiment involving two selected lines that are not genetically homozygous across the genome, we develop statistical methods for QTL mapping for genetic crosses involving noninbred lines. In our procedure, we first infer parental mating types and use likelihood-based methods to infer phases in each parent on the basis of genotypes of offspring and one parent. A hidden Markov model is then employed to estimate the number of high-risk alleles at marker positions and putative QTL positions between markers in each offspring, and QTL mapping is finally conducted through the inferred QTL configuration across all offspring in all crosses. The performance of the proposed methods is assessed through simulation studies, and the usefulness of this method is demonstrated through its application to a mosquito data set.     

Novel pleiotropic loci controlling panicle architecture across environments in japonica rice （Oryza sativa L.）

To identify quantitative trait loci (QTLs) controlling panicle architecture in japonica rice, a genetic map was constructed based on simple sequence repeat (SSR) markers and 254 recombinant inbred lines (RILs) derived from a cross between cultivars Xiushui 79 and C Bao. Seven panicle traits were investigated under three environments. Single marker analysis indicated that a total of 27 SSR markers were highly associated with panicle traits in all the three environments. Percentage of phenotypic variation explained by single locus varied from 2% to 35%. Based on the mixed linear model, a total of 40 additive QTLs for seven panicle traits were detected by composite interval mapping, explaining 1.2%-35% of phenotypic variation. Among the 9 QTLs with more than 10% of explained phenotypic variation, two QTLs were for the number of primary branches per panicle (NPB), two for panicle length (PL), two for spikelet density (SD), one for the number of secondary branches per panicle (NSB), one for secondary branch distribution density (SBD), and one for the number of spikelets per panicle (NS), respectively. qPLSD-9-1 and qPLSD-9-2 were novel pleiotropic loci, showing effects on PL and SD simultaneously. qPLSD-9-1 explained 34.7% of the phenotypic variation for PL and 25.4% of the phenotypic variation for SD, respec- tively. qPLSD-9-2 explained 34.9% and 24.4% of the phenotypic variation for PL and SD, respectively. The C Bao alleles at the both QTLs showed positive effects on PL, and the Xiushui 79 alleles at the both QTLs showed positive effects on SD. Genetic variation of panicle traits are mainly attributed to additive effects. QTL × environment interactions were not significant for additive QTLs and additive × additive QTL pairs.     

Theory for identification of marker locus-QTL associations in population by line crosses

The objective of this paper is to present genetic theory demonstrating the conditions under which it should be possible to identify molecular marker-quantitative trait locus (QTL) associations in crosses of random-mating populations to inbreds. Using as an example the cross of a corn (Zea mays L.) population to an inbred, the expected disequilibrium for testcross and per se performance of F₂, F₃, BC₁ (to the inbred) and recombinant inbred generations was derived for cases where a marker allele is linked to an unfavorable QTL allele in the inbred and where the marker allele is linked to a favorable QTL allele in the inbred. Disequilibrium in segregating generations was shown to be a function of disequilibrium in the parent population, the frequency of marker and QTL alleles in the parent population, and the recombination distance between the marker and the QTL. To maximize the opportunity to identify a favorable QTL the following procedures are suggested:

Quantitative trait locus analysis of a recombinant inbred line population derived from a Lycopersicon esculentum x Lycopersicon cheesmanii cross

Quantitative trait loci influencing fruit traits were identified by restriction fragment length polymorphism (RFLP) analysis in a population of recombinant inbred lines (RIL) derived from a cross of the cultivated tomato, Lycopersicon esculentum with a related wild species Lycopersicon cheesmanii. One hundred thirty-two polymorphic RFLP loci spaced throughout the tomato genome were scored for 97 F₈ RIL families. Fruit weight and soluble solids were measured in replicated trials during 1991 and 1992. Seed weight was measured in 1992. Significant (P<0.01 level) quantitative trait locus (QTL) associations of marker loci were identified for each trait. A total of 73 significant marker locus-trait associations were detected for the three traits measured. Fifty-three of these associations were for fruit weight and soluble solids, many of which involved marker loci signficantly associated with both traits. QTL with large effects on all three traits were detected on chromosome 6. Greater homozygosity at many loci in the RIL population as compared to F₂ populations and greater genomic coverage resulted in increased precision in the estimation of QTL effects, and large proportions of the total phenotypic variance were explained by marker class variation at significant marker loci for many traits. The RIL population was effective in detecting and discriminating among QTL for these traits previously identified in other investigations despite skewed segregation ratios at many marker loci. Large additive effects were measured at significant marker loci. Lower fruit weight, higher soluble solids, and lower seed weight were generally associated with RFLP alleles from theL. cheesmanii parent.     

New Arabidopsis recombinant inbred line populations genotyped using SNPWave and their use for mapping flowering-time quantitative trait loci

The SNPWave marker system, based on SNPs between the reference accessions Colombia-0 and Landsberg erecta (Ler), was used to distinguish a set of 92 Arabidopsis accessions from various parts of the world. In addition, we used these markers to genotype three new recombinant inbred line populations for Arabidopsis, having Ler as a common parent that was crossed with the accessions Antwerp-1, Kashmir-2, and Kondara. The benefit of using multiple populations that contain many similar markers and the fact that all markers are linked to the physical map of Arabidopsis facilitates the quantitative comparison of maps. Flowering-time variation was analyzed in the three recombinant inbred line populations. Per population, four to eight quantitative trait loci (QTL) were detected. The comparison of the QTL positions related to the physical map allowed the estimate of 12 different QTL segregating for flowering time for which Ler has an allele different from one, two, or three of the other accessions.     

Relationship between heterosis and heterozygosity at marker loci: a theoretical computation

Summary In this paper we have studied the linear correlation between a genetic distance index between two parent lines (based on marker loci information) and the heterosis observed in the F₁ hybrid from the two lines, for a quantitative character (determined by several loci, or QTL). Theoretical computations of the correlation coefficient () between the distance index and the heterosis were made, assuming the biallelic model (defined by Fisher). When the alleles at both marker loci and QTL are equally distributed among the whole population of considered lines, the coefficient is a function of the squares of linkage disequilibria between alleles at marker loci and alleles at QTL. The QTL that are not marked by marker loci and marker loci that do not mark any QTL play symmetrical roles and can decrease greatly. We conclude that the prediction of F₁ hybrid heterosis based on marker loci would be more efficient if these markers were selected for their relationship to the alleles implicated in the heterotic traits considered.     

Maximum likelihood techniques for the mapping and analysis of quantitative trait loci with the aid of genetic markers

A method is presented to estimate the biometric parameters of a quantitative trait locus linked to a genetic marker when both loci are segregating in the F-2 generation of a cross between two inbred lines. The method, which assumes underlying normal distributions, is a combination of maximum likelihood and moments methods and uses the statistics of the genetic marker genotype samples for the quantitative trait to estimate the recombination frequency between the two loci and the means and variances of the genotypes of the quantitative trait locus. With this method, the genetic parameters of a locus affecting plant height linked to an electrophoretic marker for esterase were accurately estimated from a sample of 1596 F-2 progeny of a cross between two species of Lycopersicon (tomato). Linkage distance between the two loci was 38 map units and the effect of the quantitative trait locus was 1.6 phenotypic standard deviation units. Accurate estimates of the genetic parameters and linkage distance for populations of 2000 individuals simulated with a segregating codominant locus with an effect of 1.63 standard deviations linked to a genetic marker with .2 recombination were also derived by this method. The method is not effective in distinguishing between complete and partial linkage in samples of only 500 individuals or for quantitative loci with effects less than a phenotypic standard deviation. The method is more effective for codominant than for dominant loci.     

Mapping quantitative trait loci with DNA microsatellites in a commercial dairy cattle population

Individual loci affecting economically important traits can be located using genetic linkage between quantitative trait loci and genetic markers. In the ‘granddaughter’ experimental design, heterozygous grandsires and their sons are genotyped for the genetic marker, while the quantitative trait records of the granddaughters are used for statistical analysis. Ten DNA microsatellite markers were used to look for associations with quantitative trait loci affecting milk production traits in seven Israeli Holstein grandsire families. At least 60% more grandsires were heterozygous, and 40% fewer individuals were discarded because of unknown paternal allele origin, as compared with diallelic markers. The effects of paternal alleles for locus D21S4 on kg milk and protein were significant (P < 0.025). The allele substitution effects for sire 783 were 283 kg milk and 5.7 kg protein. For both traits, progeny of sire 783 that inherited allele ‘18’ had higher evaluations than progeny that inherited allele ‘21’. These results were verified by genotyping 151 of his daughters. Thus, the rate of genetic gain for protein production can be increased by selecting progeny of sire 783 carrying allele ‘18’ at this locus.     

Bulk segregant analysis with molecular markers and its use for improving drought resistance in maize

The usual method to locate and compare loci regulating quantitative traits (QTLs) requires a segregating population of plants with each one genotyped with molecular markers. However, plants from such segregating populations can also be grouped according to phenotypic expression of a trait and tested for differences in allele frequency between the population bulks: bulk segregant analysis (BSA). The same probes used for making a genetic map (e.g. isozyme, RFLP, RAPD, etc) can be used for BSA. A molecular marker showing polymorphism between the parents of the population and which is closely-linked to a major QTL regulating a particular trait will mainly co-segregate with that QTL, i.e. segregate according to the phenotype if the QTL has a large effect. Thus, if plants are grouped according to expression of the trait and extreme groups tested with that polymorphic marker, the frequency of the two marker alleles present within each of the two bulks should deviate significantly from the ratio of 1 : 1 expected for most populations. As chromosomal locations of many molecular markers have now been determined in many species, the map location of closely-linked QTLs can therefore be deduced without having to genotype every individual in segregating populations. This has been used successfully with composite populations of maize to locate QTLs associated with yield under severe drought. An inbred line derived from one of the populations selected for higher drought yield has been crossed with a drought-susceptible inbred line to produce a mapping population for QTL analysis of physiological and developmental traits likely to regulate yield under drought. Future work to identify traits having QTLs with flanking markers showing significant allele frequency differences in the GSA studies will indicate those traits likely to be important in determining yield under drought.Key words: Bulk segregant analysis (BSA), drought resistance, genetic maps, maize, molecular markers, Zea mays (L.).      

The genetic basis of floral traits associated with mating system evolution in Leptosiphon (Polemoniaceae): an analysis of quantitative trait loci

Mapping of quantitative trait loci (QTL) was used to investigate the genetic architecture of divergence in floral characters associated with the mating system, an important adaptive trait in angiosperms. Two species of Leptosiphon (Polemoniaceae), one strongly self-fertilizing (L. bicolor) and the other partially outcrossing (L. jepsonii), were crossed to produce F2 and both backcross progenies. For each crossing population, a linkage map was created using amplified fragment length polymorphism markers, and QTL were identified for several dimensions of floral size. For each of the five traits examined, three to seven QTL were detected, with independent datasets yielding congruent results in some but not all cases. The phenotypic effect of individual QTL was generally moderate. We estimated that many of the QTL were additive or showed dominance toward L. bicolor, whereas comparison of mean trait values for parental and cross progenies showed apparent overall dominance of L. jepsonii traits. Colocalization of QTL for different dimensions of floral size was consistent with high phenotypic correlations between floral traits. Substantial segregation distortion was observed in marker loci, the majority favoring alleles from the large-flowered parent. A low frequency of male sterility in the F2 population is consistent with the Dobzhansky-Muller model for the evolution of reproductive isolation.     

Mapping quantitative trait loci associated with days to flowering and photoperiod sensitivity in rice (Oryza sativa L.)

This study was undertaken to identify putative quantitative trait loci (QTLs) associated with days to flowering (DTF) and photoperiod response in rice. A population of 143 recombinant inbred lines derived from a cross between CO39 and Moroberekan was grown under greenhouse conditions and exposed to two different photoperiod regimes. DTF of individual plants was evaluated under 10 h and 14 h day lengths, and loci associated with photoperiod sensitivity were identified based on the delay in flowering under the 14 h photoperiod (DTF at 14 h minus DTF at 10 h). An RFLP data set consisting of 127 markers provided the basis for the QTL analysis. Both single marker and interval analysis were used and interactions between putative QTLs were estimated based on two-way ANOVA. Out of 15 QTLs associated with DTF, only 4 were identified as influencing the response to photoperiod. Interactions between flowering QTLs indicated the complex nature of the control of flowering in rice. The effectiveness of using a single recombinant inbred population to study a variety of complex phenotypes is discussed in relation to practical plant breeding.     

Associations between quantitative traits and enzyme loci in the F2 population of a maize hybrid

Summary Univariate and multivariate analyses were used to identify associations between eight enzyme marker loci and 11 quantitative traits of maize (Zea mays L.). The material analyzed included inbred lines Wf9 and Pa405, single-cross hybrid Wf9 X Pa405, and the F2 generation of the selfed single-cross hybrid. Each enzyme locus assayed was associated with at least one quantitative trait, and all quantitative traits were associated with genotypes at particular enzyme loci. Significant associations also were found between the level of heterozygosity per individual and nine of 11 quantitative traits. The total contribution to heterosis, for seed yield per plant, of genes linked with the eight enzyme loci, was 27% of the F2 mean and 18% of the difference in mean between the F1 hybrid and the inbred parents. Genes linked with Glu1 accounted for nearly one third of the total dominance effect detected by the eight enzyme loci. The chromosome segments marked by loci with significant effects on seed yield were markedly overdominant. The large heterotic effects of chromosome segments marked by particular loci suggest that enzyme loci could be used to help transfer genes responsible for heterosis to inbred lines. We conclude that analyses of additional inbred lines, F1 hybrids, and F2 populations in more environments will halp identify specific associations between enzyme loci, or chromosome segments which they mark, and important agronomic traits.Cooperative investigations of the USDA, ARS and Dept. of Plant Sciences, South Dakota State Univ. (SDSU), Brookings, Journal Series No. 2039; and the Institute of Animal Resource Ecology, Univ. of British Columbia, Vancouver, B.C. V6T 1W5, Canada     

Identification of major quantitative trait loci qSBR11-1 for sheath blight resistance in rice

Sheath blight caused by Rhizoctonia solani Kühn is one of the important diseases of rice, resulting in heavy yield loss in rice every year. No rice line resistant to sheath blight has been identified till date. However, in some rice lines a high degree of resistance to R. solani has been observed. An indica rice line, Tetep, is a well documented source of durable and broad spectrum resistance to rice blast as well as quantitative resistance to sheath blight. The present study identified genetic loci for quantitative resistance to sheath blight in rice line Tetep. A mapping population consisting of 127 recombinant inbred lines derived from a cross between rice cultivars HP2216 (susceptible) and Tetep (resistant to sheath blight) was evaluated for sheath blight resistance and other agronomic traits for 4 years across three locations. Based on sheath blight phenotypes and genetic map with 126 evenly distributed molecular markers, a quantitative trait loci (QTLs) contributing to sheath blight resistance was identified on long arm of chromosome 11. Two QTL mapping approaches i.e., single marker analysis and composite interval mapping in multi environments were used to identify QTLs for sheath blight resistance and agronomical traits. The QTL qSBR11-1 for sheath blight resistance was identified between the marker interval RM1233 (26.45 Mb) to sbq33 (28.35 Mb) on chromosome 11. This region was further narrowed down to marker interval K39516 to sbq33 (~0.85 Mb) and a total of 154 genes were predicted including 11 tandem repeats of chitinase genes which may be responsible for sheath blight resistance in rice line Tetep. A set of 96 varieties and a F₂ population were used for validation of markers linked to the QTL region. The results indicate that there is very high genetic variation among varieties at this locus, which can serve as a starting point for allele mining of sheath blight resistance.     

Identification of quantitative trait loci (QTL) for fruit-quality traits and number of weeks of flowering in the cultivated strawberry

Fruit quality and repeat flowering are two major foci of several strawberry breeding programs. The identification of quantitative trait loci (QTL) and molecular markers linked to these traits could improve breeding efficiency. In this work, an F₁ population derived from the cross ‘Delmarvel’ × ‘Selva’ was used to develop a genetic linkage map for QTL analyses of fruit-quality traits and number of weeks of flowering. Some QTL for fruit-quality traits were identified on the same homoeologous groups found in previous studies, supporting trait association in multiple genetic backgrounds and utility in multiple breeding programs. None of the QTL for soluble solids colocated with a QTL for titratable acids, and, although the total soluble solid contents were significantly and positively correlated with titratable acids, the correlation coefficient value of 0.2452 and independence of QTL indicate that selection for high soluble solids can be practiced independently of selection for low acidity. One genomic region associated with the total number of weeks of flowering was identified quantitatively on LG IV-S-1. The most significant marker, FxaACAO2I8C-145S, explained 43.3 % of the phenotypic variation. The repeat-flowering trait, scored qualitatively, mapped to the same region as the QTL. Dominance of the repeat-flowering allele was demonstrated by the determination that the repeat-flowering parent was heterozygous. This genomic region appears to be the same region identified in multiple mapping populations and testing environments. Markers linked in multiple populations and testing environments to fruit-quality traits and repeat flowering should be tested widely for use in marker-assisted breeding.