Comparative analysis of QTLs affecting plant height and flowering among closely-related diploid and polyploid genomes.

Quantitative trait loci (QTLs) affecting plant height and flowering were studied in the two Saccharum species from which modern sugarcane cultivars are derived. Two segregating populations derived from interspecific crosses between Saccharum officinarum and Saccharum spontaneum were genotyped with 735 DNA markers. Among the 65 significant associations found between these two traits and DNA markers, 35 of the loci were linked to sugarcane genetic maps and 30 were unlinked DNA markers. Twenty-one of the 35 mapped QTLs were clustered in eight genomic regions of six sugarcane homologous groups. Some of these could be divergent alleles at homologous loci, making the actual number of genes implicated in these traits much less than 35. Four QTL clusters controlling plant height in sugarcane corresponded closely to four of the six plant-height QTLs previously mapped in sorghum. One QTL controlling flowering in sugarcane corresponded to one of three flowering QTLs mapped in sorghum. The correspondence in locations of QTLs affecting plant height and flowering in sugarcane and sorghum reinforce the notion that the simple sorghum genome is a valuable "template" for molecular dissection of the much more complex sugarcane genome.     

Mapping of quantitative trait loci and development of allele-specific markers for seed weight in Brassica napus

Seed weight is an important component of grain yield in oilseed rape (Brassica napus L.), but the genetic basis for the important quantitative trait is still not clear. In order to identify the genes for seed weight in oilseed rape, QTL mapping for thousand seed weight (TSW) was conducted with a doubled haploid (DH) population and an F₂ population. A complete linkage map of the DH population was constructed using 297 simple sequence repeat (SSR) markers. Among nine TSW QTLs detected, two major QTLs, TSWA7a and TSWA7b, were stably identified across years and collectively explained 27.6–37.9% of the trait variation in the DH population. No significant epistatic interactions for TSW detected in the DH population indicate that the seed weight variation may be primarily attributed to additive effects. The stability and significance of TSWA7a and TSWA7b were further validated in the F₂ population with different genetic backgrounds. By cloning BnMINI3a and BnTTG2a, two B. napus homologous genes to Arabidopsis thaliana, allele-specific markers were developed for TSWA5b and TSWA5c, two TSW QTLs on A5, respectively. The importance of the major and minor QTLs identified was further demonstrated by analysis of the allelic effects on TSW in the DH population.     

Identification and validation of QTLs for green plant percentage in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) anther culture

In cereals, albinism is a major obstacle to produce doubled haploids (DH) for breeding programs. In order to identify QTLs for green plant percentage in barley anther culture, a specific population was developed. This population, consisting of 100 DH lines, was generated by crossing the model cultivar for anther culture “Igri” with an albino-producing DH line (DH46) selected from Igri × Dobla, in search of a maximum segregation for the trait and minimum for the other anther culture variables. A combination of bulked segregant analysis and AFLP methodology was used to identify markers linked to the trait. A linkage map was constructed using these AFLPs, together with RAPD, STS and SSR markers. This study identified a new QTL for green plant percentage on chromosome 3H and confirmed the previously reported one on chromosome 5H. Up to 65.2% of the phenotypic variance for this trait was explained by the additive effects of these two QTLs. Thirty elite cultivars of barley from different origin, row type, growth habit and end use, were selected to validate these QTLs. Since two of the markers linked to the QTLs were AFLPs, we successfully converted them into simple PCR-based SCAR markers. Only the SSR HVM60, on chromosome 3H, was significantly associated with the trait, explaining near 20% of the phenotypic variance. Among the allelic variants identified for this marker, HVM60-120bp was associated with the highest values of green plant percentage.     

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.).      

A high-density consensus map of barley to compare the distribution of QTLs for partial resistance to Puccinia hordei and of defence gene homologues

A consensus map of barley was constructed based on three reference doubled haploid (DH) populations and three recombinant inbred line (RIL) populations. Several sets of microsatellites were used as bridge markers in the integration of those populations previously genotyped with RFLP or with AFLP markers. Another set of 61 genic microsatellites was mapped for the first time using a newly developed fluorescent labelling strategy, referred to as A/T labelling. The final map contains 3,258 markers spanning 1,081 centiMorgans (cM) with an average distance between two adjacent loci of 0.33 cM. This is the highest density of markers reported for a barley genetic map to date. The consensus map was divided into 210 BINs of about 5 cM each in which were placed 19 quantitative trait loci (QTL) contributing to the partial resistance to barley leaf rust (Puccinia hordei Otth) in five of the integrated populations. Each parental barley combination segregated for different sets of QTLs, with only few QTLs shared by any pair of cultivars. Defence gene homologues (DGH) were identified by tBlastx homology to known genes involved in the defence of plants against microbial pathogens. Sixty-three DGHs were located into the 210 BINs in order to identify candidate genes responsible for the QTL effects. Eight BINs were co-occupied by a QTL and DGH(s). The positional candidates identified are receptor-like kinase, WIR1 homologues and several defence response genes like peroxidases, superoxide dismutase and thaumatin. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Fine Mapping QTL for Drought Resistance Traits in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) Using Bulk Segregant Analysis

Drought stress is a major limitation to rice (Oryza sativa L.) yields and its stability, especially in rainfed conditions. Developing rice cultivars with inherent capacity to withstand drought stress would improve rainfed rice production. Mapping quantitative trait loci (QTLs) linked to drought resistance traits will help to develop rice cultivars suitable for water-limited environments through molecular marker-assisted selection (MAS) strategy. However, QTL mapping is usually carried out by genotyping large number of progenies, which is labour-intensive, time-consuming and cost-ineffective. Bulk segregant analysis (BSA) serves as an affordable strategy for mapping large effect QTLs by genotyping only the extreme phenotypes instead of the entire mapping population. We have previously mapped a QTL linked to leaf rolling and leaf drying in recombinant inbred (RI) lines derived from two locally adapted indica rice ecotypes viz., IR20/Nootripathu using BSA. Fine mapping the QTL will facilitate its application in MAS. BSA was done by bulking DNA of 10 drought-resistant and 12 drought-sensitive RI lines. Out of 343 rice microsatellites markers genotyped, RM8085 co-segregated among the RI lines constituting the respective bulks. RM8085 was mapped in the middle of the QTL region on chromosome 1 previously identified in these RI lines thus reducing the QTL interval from 7.9 to 3.8 cM. Further, the study showed that the region, RM212–RM302–RM8085–RM3825 on chromosome 1, harbours large effect QTLs for drought-resistance traits across several genetic backgrounds in rice. Thus, the QTL may be useful for drought resistance improvement in rice through MAS and map-based cloning.     

Novel linkage mapping approach using DNA pooling in human and animal genetics. II. Detection of quantitative traits loci in dairy cattle

Selective DNA pooling is an advanced methodology for linkage mapping of quantitative trait loci (QTL) in farm animals. The principle is based on densitometric estimates of marker allele frequency in pooled DNA samples of phenotypically extreme individuals from half-sib, backcross and F(2) experimental designs in farm animals. This methodology provides a rapid and efficient analysis of a large number of individuals with short tandem repeat markers that are essential to detect QTL through the genome - wide searching approach. Several strategies involving whole genome scanning with a high statistical power have been developed for systematic search to detect the quantitative traits loci and linked loci of complex traits. In recent studies, greater success has been achieved in mapping several QTLs in Israel-Holstein cattle using selective DNA pooling. This paper outlines the currently emerged novel strategies of linkage mapping to identify QTL based on selective DNA pooling with more emphasis on its theoretical pre-requisite to detect linked QTLs, applications, a general theory for experimental half-sib designs, the power of statistics and its feasibility to identify genetic markers linked QTL in dairy cattle. The study reveals that the application of selective DNA pooling in dairy cattle can be best exploited in the genome-wide detection of linked loci with small and large QTL effects and applied to a moderately sized half-sib family of about 500 animals.     

The effect of semi-dwarf genes on root system size in field-grown barley

Root system size (RSS) was measured in 12 diverse barley genotypes and 157 double-haploid lines (DHs), using electric capacitance. The parents of the DHs, Derkado and B83-12/21/5, carry different semi-dwarfing genes, sdw1 and ari-e.GP, respectively. Estimates of RSS were taken in the field thrice during plant development: stem elongation (RSS1), heading (RSS2) and grain filling (RSS3). The 12 barley genotypes were assessed over 3 years and at two or three locations each year; the DH mapping population was assessed at two locations in 2002. Among the 12 barley genotypes, those with the semi-dwarf genes had greater RSS values in all 3 years (28.9, 24.6 and 15.0% in years 1, 2 and 3, respectively) compared to non-semi-dwarf controls. The DH population showed transgressive segregation on both sides of the parent means, indicating polygenic control of RSS. Quantitative trait loci (QTLs) for RSS were found on five of the seven chromosomes: 1H, 3H, 4H, 5H and 7H and these were compared with previously mapped agronomic traits. The TotalRSS QTL on 3H was associated with sdw1 and QTLs for height, plant yield and plant weight. The RSS3 QTL on 5H was associated with ari-e.GP and QTLs for height, plant yield, plant weight, harvest index and tiller number. The RSS3 QTL on 7H was also associated with a TotalRSS QTL and QTLs for plant weight and harvest index. Other RSS QTLs were not associated with any other trait studied. RSS is considered to be a polygenic trait linked to important traits, in particular to yield. The study highlights the effects of semi-dwarfing genes and discusses the potential for breeding for root traits.     

Quantitative trait loci for aluminum resistance in wheat

Quantitative trait loci (QTL) for wheat resistance to aluminum (Al) toxicity were analyzed using simple sequence repeats (SSRs) in a population of 192 F₆ recombinant inbred lines (RILs) derived from a cross between an Al-resistant cultivar, Atlas 66 and an Al-sensitive cultivar, Chisholm. Wheat reaction to Al was measured by relative root growth and root response to hematoxylin stain in nutrient-solution culture. After screening 1,028 SSR markers for polymorphisms between the parents and bulks, we identified two QTLs for Al resistance in Atlas 66. One major QTL was mapped on chromosome 4D that co-segregated with the Al-activated malate transporter gene (ALMT1). Another minor QTL was located on chromosome 3BL. Together, these two QTLs accounted for about 57% of the phenotypic variation in hematoxylin staining score and 50% of the variation in net root growth (NRG). Expression of the minor QTL on 3BL was suppressed by the major QTL on 4DL. The two QTLs for Al resistance in Atlas 66 were also verified in an additional RIL population derived from Atlas 66/Century. Several SSR markers closely linked to the QTLs were identified and have potential to be used for marker-assisted selection (MAS) to improve Al-resistance of wheat cultivars in breeding programs.     

QTL mapping and molecular marker analysis for the resistance of rice to ozone

The resistance of rice to ozone (O3) is a quantitative trait controlled by nuclear genes. The identification of quantitative trait loci (QTL) and analysis of molecular markers of O3 resistance is important for increasing the resistance of rice to O3 stress. QTL associated with the O3 resistance of rice were mapped on chromosomes 1, 7 and 11 using 164 recombinant inbred (RI) lines from a cross between 'Milyang 23' and 'Gihobyeo'. The quantitative trait loci were tightly linked to the markers RG109, C507 and RG1094 and were detected in each of three replications. The association between these markers and O3 resistance in 26 rice cultivars and doubled haploid (DH) populations was analysed. The markers permit the screening of rice germplasm for O3 resistance and the introduction of resistance into elite lines in breeding programs.     

Verification of QTL linked markers for propagation traits in <Emphasis Type="Italic">Eucalyptus</Emphasis>

In a previous study, several quantitative trait loci (QTLs) affecting vegetative propagation traits were detected in a hybrid cross between Eucalyptus tereticornis and Eucalyptus globulus. The objective of this work was to confirm stable QTL linked markers (detected in different years) for propagation traits in an independent set of the same segregating population and in two related crosses involving the original E. globulus parent. Phenotypic averages of groups of individuals carrying alternative allelic forms of the stable QTL linked markers were statistically tested for significant differences. Adventitious rooting and petrification marker–trait associations, detected previously in the E. tereticornis parent, were verified in an independent sample of the original progeny. In the E. globulus parent, the QTL linked marker was only verified in one related genetic background. Verification was possible only for high-effect QTL linked markers. This study highlights the importance of sample size in QTL detection for low-heritability traits.     

Merino sheep: a further look at quantitative trait loci for wool production

A quantitative trait loci (QTL) analysis of wool traits from experimental half-sib data of Merino sheep is presented. A total of 617 animals distributed in 10 families were genotyped for 36 microsatellite markers on four ovine chromosomes OAR1, OAR3, OAR4 and OAR11. The markers covering OAR3 and OAR11 were densely spaced, at an average distance of 2.8 and 1.2 cM, respectively. Body weight and wool traits were measured at first and second shearing. Analyses were conducted under three hypotheses: (i) a single QTL controlling a single trait (for multimarker regression models); (ii) two linked QTLs controlling a single trait (using maximum likelihood techniques) and (iii) a single QTL controlling more than one trait (also using maximum likelihood techniques). One QTL was identified for several wool traits on OAR1 (average curvature of fibre at first and second shearing, and clean wool yield measured at second shearing) and on OAR11 (weight and staple strength at first shearing, and coefficient of variation of fibre diameter at second shearing). In addition, one QTL was detected on OAR4 affecting weight measured at second shearing. The results of the single trait method and the two-QTL hypotheses showed an additional QTL segregating on OAR11 (for greasy fleece weight at first shearing and clean wool yield trait at second shearing). Pleiotropic QTLs (controlling more than one trait) were found on OAR1 (clean wool yield, average curvature of fibre, clean and greasy fleece weightand staple length, all measured at second shearing).     

Mapping of quantitative trait loci (QTLs) for oil yield using SSRs and gene-based markers in African oil palm (<Emphasis Type="Italic">Elaeis guineensis</Emphasis> Jacq.)

The identification of quantitative trait loci (QTLs) based on anchor markers, especially candidate genes that control a trait of interest, has been noted to increase the power of QTL detection. Since these markers can be scored as co-dominant data, they are also valuable for comparing and integrating the QTL linkage maps from diverse mapping populations. To estimate the position and effects of QTLs linked to oil yield traits in African oil palm, co-dominant microsatellites (SSR) and candidate gene-based sequence polymorphisms were applied to construct a linkage map for a progeny showing large differences in oil yield components. The progeny was genotyped for 97 SSR markers, 93 gene-linked markers, and 12 non-gene-linked SNP markers. From these, 190 segregating loci could be arranged into 31 linkage groups while 12 markers remained unmapped. Using the single marker linkage, interval mapping and multiple QTL methods, 16 putative QTLs on seven linkage groups affecting important oil yield related traits such as fresh fruit bunch yield (FFB), ratio of oil per fruit (OF), oil per bunch (OB), fruit per bunch (FB) and wet mesocarp per fruit (WMF) could be identified in the segregating population with estimated values for explained variance ranging from 12.4 % to 54.5 %. Markers designed from some candidate genes involved in lipid biosynthesis were found to be mapped near significant QTLs for various economic yield traits. Associations between QTLs and potential candidate genes are discussed.     

Genetic dissection of chlorophyll content at different growth stages in common wheat

Quantitative trait loci (QTLs) for chlorophyll content were studied using a doubled haploid (DH) population with 168 progeny lines, derived from a cross between two elite Chinese wheat cultivars Huapei 3 × Yumai 57. Chlorophyll content was evaluated at the maximum tillering stage (MS), the heading stage (HS), and the grain filling stage (GS), at three different environments in 2005 and 2006 cropping seasons. QTL analyses were performed using a mixed linear model approach. A total of 17 additive QTLs and nine pairs of epistatic QTLs were detected. Ten of 17 additive QTLs for chlorophyll content were persistently expressed at more than two growth stages, which suggest developmentally regulated loci controlling genetics for chlorophyll content in different growth stages in wheat. One novel major QTL for chlorophyll content was closely linked with the PCR marker Xwmc215 and was persistently expressed at three growth stages.     

Linkage of RAPD markers to NESTUR, a stem growth index in radiata pine seedlings

 Needle-to-stem unit rate (NESTUR) is a stem growth index of conifer seedlings that measures the efficiency of stemwood production per unit of foliage growth. The random amplified polymorphic DNA (RAPD) technique was applied to haploid DNA from the megagametophytes of a full-sib radiata pine cross to find markers linked to factors controlling the NESTUR trait. Using the bulked segregant analysis approach, 23 of 933 primers displayed putative linkage to factors controlling NESTUR. Based on the genotypic analysis of 174 individuals, two quantitative trait loci (QTLs) controlling NESTUR were identified at ANOVA P-levels of 0.01–0.001. The QTLs were identified by RAPD markers OPE-06₄₅₀ and OPA-10₁₂₀₀, which were linked to each other (r=7%), and UBC-333₅₅₀, which was not linked to the other two. Linkage to components of NESTUR (increments in stem diameter and stem volume) was demonstrated for UBC-333₅₅₀, while the others were not linked to NESTUR components. Received: 18 December 1996/Accepted: 24 January 1997     

Assessment of DNA pooling strategies for mapping of QTLs

The synthesis of DNA pools from segregating populations is an efficient strategy for identifying DNA markers closely linked to genes or genomic regions of interest. To-date, DNA pooling based solely upon phenotypic information, or bulked segregant analysis, has been employed only in the analysis of simply-inherited traits. We have assessed the utility of phenotype-based DNA pools for tagging (e.g., identifying DNA markers closely-linked to) quantitative trait loci (QTLs), segregating in the presence of other such loci, and expressing phenotypes which are influenced by the environment. Theoretical estimates suggest that QTL alleles with phenotypic effects of 0.75–1.0 standard deviations (SD), or larger, should be detectable in back-cross (BC), F₂ and recombinant inbred (RI) or doubled haploid (DH) populations of manageable size (100–200 plants/lines). However, post hoc analysis of three data sets, used in QTL mapping of tomato and rice, indicate that the majority of QTLs identified had allele effects of less than 0.75 SD, and thus could not be easily tagged in DNA pools. Segregation distortion can have a large effect on the allelic composition of DNA pools, necessitating the use of more individuals in the pools to minimize false positive and false negative results. In general, we suggest that use of phenotype-based DNA pools might be successful in tagging QTLs of very large effect, but is unlikely to permit comprehensive identification of the majority of QTLs affecting a complex trait. DNA pools constructed from a priori information should, however, be useful in identifying new DNA markers for regions of the genome known to contain QTLs.     

QTLs for a component of partial resistance to cucumber mosaic virus in pepper: restriction of virus installation in host-cells

 Ninety four doubled-haploid (DH) lines obtained from the F₁ between Perennial, a cucumber mosaic virus (CMV)-partially resistant Capsicum annuum line, and Yolo Wonder, a CMV-susceptible C. annuum line, were analysed with 138 markers including mostly RFLPs and RAPDs. Clustering of RAPD markers was observed on five linkage groups of the intraspecific linkage map. These clusters could correspond to the centromeric regions of pepper chromosomes. The same progenies were evaluated for restriction of CMV installation in pepper cells in order to map quantitative trait loci (QTLs) controlling CMV resistance. This component of partial resistance to CMV was quantitatively assessed using a CMV strain that induced necrotic local lesions on the inoculated leaves. The number of local lesions gave an estimation of the density of the virus-infection sites. Genotypic variance among the DH lines was highly significant for the number of local lesions, and heritability was estimated to be 0.94. Using both analysis of variance and non-parametric tests, three genomic regions significantly affecting CMV resistance were detected on chromosomes Noir, Pourpre and linkage group 3, together explaining 57% of the phenotypic variation. A digenic epistasis between one locus that controlled significant trait variation and a second locus that by itself had no demonstrable effect on the trait was found to have an effect on CMV resistance. For each QTL, the allele from Perennial was associated with an increased resistance. Implications of QTL mapping in marker-based breeding for CMV resistance are discussed. Received: 16 September 1996     

Quantitative Trait Loci for Light Sensitivity,Body Weight,Body Size,and Morphological Eye Parameters in the Bumblebee,Bombus terrestris

Bumblebees such as Bombus terrestris are essential pollinators in natural and managed ecosystems. In addition, this species is intensively used in agriculture for its pollination services, for instance in tomato and pepper greenhouses. Here we performed a quantitative trait loci (QTL) analysis on B. terrestris using 136 microsatellite DNA markers to identify genes linked with 20 traits including light sensitivity, body size and mass, and eye and hind leg measures. By composite interval mapping (IM), we found 83 and 34 suggestive QTLs for 19 of the 20 traits at the linkage group wide significance levels of p = 0.05 and 0.01, respectively. Furthermore, we also found five significant QTLs at the genome wide significant level of p = 0.05. Individual QTLs accounted for 7.5-53.3% of the phenotypic variation. For 15 traits, at least one QTL was confirmed with multiple QTL model mapping. Multivariate principal components analysis confirmed 11 univariate suggestive QTLs but revealed three suggestive QTLs not identified by the individual traits. We also identified several candidate genes linked with light sensitivity, in particular the Phosrestin-1-like gene is a primary candidate for its phototransduction function. In conclusion, we believe that the suggestive and significant QTLs, and markers identified here, can be of use in marker-assisted breeding to improve selection towards light sensitive bumblebees, and thus also the pollination service of bumblebees.     

Targeted identification of markers linked to malaria and filarioid nematode parasite resistance genes in the mosquito Aedes aegypti.

Quantitative trait loci (QTL) have been identified for competence of the mosquito Aedes aegypti to transmit the avian malaria parasite Plasmodium gallinaceum and the human filarial parasite Brugia malayi. Efforts towards the map-based cloning of the associated genes are limited by the availability of genetic markers for fine-scale mapping of the QTL positions. Two F2 mosquito populations were subjected to bulked segregant analysis to identify random amplified polymorphic DNA (RAPD)-PCR fragments linked with the major QTL determining susceptibility to both parasites. Individual mosquitoes for the bulks were selected on the basis of their genotypes at restriction fragment length polymorphism (RFLP) loci tightly linked with the QTL. Pool-positive RAPD fragments were cloned and evaluated as RFLP markers. Of the 62 RAPD/RFLP fragments examined, 10 represented low-copy number sequences. Five of these clones were linked with the major QTL for P. gallinaceum susceptibility (pgs1), of which one clone mapped within the flanking markers that define the QTL interval. The remaining five clones were linked with the major QTL for B. malayi susceptibility (fsb1), and again one clone mapped within the flanking markers that define the QTL interval. In addition, nine RAPD/RFLP fragments were isolated that seem to be of non-mosquito origin.