To clone or not to clone plant QTLs: present and future challenges

Recent technical advancements and refinement of analytical methods have enabled the loci (quantitative trait loci, QTLs) responsible for the genetic control of quantitative traits to be dissected molecularly. To date, most plant QTLs have been cloned using a positional cloning approach following identification in experimental crosses. In some cases, an association between sequence variation at a candidate gene and a phenotype has been established by analysing existing genetic accessions. These strategies can be refined using appropriate genetic materials and the latest developments in genomics platforms. We foresee that although QTL analysis and cloning addressing naturally occurring genetic variation should shed light on mechanisms of plant adaptation, a greater emphasis on approaches relying on mutagenesis and candidate gene validation is likely to accelerate the pace of discovering the genes underlying QTLs.     

Advances in cereal genomics and applications in crop breeding

Recent advances in cereal genomics have made it possible to analyse the architecture of cereal genomes and their expressed components, leading to an increase in our knowledge of the genes that are linked to key agronomically important traits. These studies have used molecular genetic mapping of quantitative trait loci (QTL) of several complex traits that are important in breeding. The identification and molecular cloning of genes underlying QTLs offers the possibility to examine the naturally occurring allelic variation for respective complex traits. Novel alleles, identified by functional genomics or haplotype analysis, can enrich the genetic basis of cultivated crops to improve productivity. Advances made in cereal genomics research in recent years thus offer the opportunities to enhance the prediction of phenotypes from genotypes for cereal breeding.     

Development of a wide population of chromosome single-segment substitution lines in the genetic background of an elite cultivar of rice (Oryza sativa L.).

Naturally occurring allelic variations underlying complex traits are useful resources for the functional analysis of plant genes. To facilitate the genetic analysis of complex traits and the use of marker-assisted breeding in rice, we developed a wide population consisting of 217 chromosome single-segment substitution lines (SSSLs) using Oryza sativa L. 'Hua-Jing-Xian74' (HJX74), an elite Indica cultivar, as recipient, and 6 other accessions, including 2 Indica and 4 Japonica, as donors. Each SSSL contains a single substituted chromosome segment derived from 1 of the 6 donors in the genetic background of HJX74. The total size of the substituted segments in the SSSL population was 4695.0 cM, which was 3.1 times that of rice genome. To evaluate the potential application of these SSSLs for quantitative trait loci detection, phenotypic variations of the quantitative traits of days to heading and grain length in the population consisting of 210 SSSLs were observed under natural environmental conditions. The results demonstrated that there was a wide range of phenotypic variation in the traits in the SSSL population. These genetic materials will be powerful tools to dissect complex traits into a set of monogenic loci and to assign phenotypic values to different alleles at the locus of interest.     

植物数量性状变异的分子基础与QTL克隆研究进展

探讨数量性状变异规律以便对其进行遗传操纵一直是植物遗传学的一个重要领域。DNA分子标记和QTL作图技术的发展以及拟南芥和水稻全基因组测序的完成极大地促进了植物数量性状分子基础的研究。现已克隆了拟南芥ED1、水稻Hdl、玉米Tb1、番茄fw2．2和Brii9-2-5等控制目标数量性状的基因。数量性状表型变异不仅源于多个数量性状基因（QTL）的分离．而且还受到内外环境的修饰。QTL等位基因变异与孟德尔基因变异具有类似的分子基础,即基因表达或蛋白质功能发生改变。通过分析已克隆的植物QTL的变异特征及分子基础,讨论了植物QTL克隆技术策略,并对QTL研究所面临的挑战和应用前景进行了展望。     

High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.)

Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic polymorphism in plant genomes. SNP markers are valuable tools for genetic analysis of complex traits of agronomic importance, linkage and association mapping, genome-wide selection, map-based cloning, and marker-assisted selection. Current challenges for SNP genotyping in polyploid outcrossing species include multiple alleles per loci and lack of high-throughput methods suitable for variant detection. In this study, we report on a high-resolution melting (HRM) analysis system for SNP genotyping and mapping in outcrossing tetraploid genotypes. The sensitivity and utility of this technology is demonstrated by identification of the parental genotypes and segregating progeny in six alfalfa populations based on unique melting curve profiles due to differences in allelic composition at one or multiple loci. HRM using a 384-well format is a fast, consistent, and efficient approach for SNP discovery and genotyping, useful in polyploid species with uncharacterized genomes. Possible applications of this method include variation discovery, analysis of candidate genes, genotyping for comparative and association mapping, and integration of genome-wide selection in breeding programs.     

The candidate gene approach in plant genetics: a review

The candidate gene (CG) approach has been applied in plant genetics in the past decade for the characterisation and cloning of Mendelian and quantitative trait loci (QTLs). It constitutes a complementary strategy to map-based cloning and insertional mutagenesis. The goal of this paper is to present an overview of CG analyses in plant genetics. CG analysis is based on the hypothesis that known-function genes (the candidate genes) could correspond to loci controlling traits of interest. CGs refer either to cloned genes presumed to affect a given trait (`functional CGs') or to genes suggested by their close proximity on linkage maps to loci controlling the trait (`positional CGs'). In plant genetics, the most common way to identify a CG is to look for map co-segregation between CGs and loci affecting the trait. Statistical association analyses between molecular polymorphisms of the CG and variation in the trait of interest have also been carried out in a few studies. The final validation of a CG will be provided through physiological analyses, genetic transformation and/or sexual complementation. Theoretical and practical applications of validated CGs in plant genetics and breeding are discussed.     

Genetic and molecular dissection of quantitative traits in rice

Recent progress in the generation of a molecular genetic map and markers for rice has made possible a new phase of mapping individual genes associated with complex traits. This type of analysis is often referred to as quantitative trait locus (QTL) analysis. Increasing numbers of QTL analyses are providing enormous amounts of information about QTLs, such as the numbers of loci involved, their chromosomal locations and gene effects. Clarification of genetic bases of complex traits has a big impact not only on fundamental research on rice plant development, but it also has practical benefits for rice breeding. In this review, we summarize recent progress of QTL analysis of several complex traits in rice. A strategy for positional cloning of genes at QTLs is also discussed.     

A Multiparent Advanced Generation Inter-Cross to Fine-Map Quantitative Traits in Arabidopsis thaliana

Identifying natural allelic variation that underlies quantitative trait variation remains a fundamental problem in genetics. Most studies have employed either simple synthetic populations with restricted allelic variation or performed association mapping on a sample of naturally occurring haplotypes. Both of these approaches have some limitations, therefore alternative resources for the genetic dissection of complex traits continue to be sought. Here we describe one such alternative, the Multiparent Advanced Generation Inter-Cross (MAGIC). This approach is expected to improve the precision with which QTL can be mapped, improving the outlook for QTL cloning. Here, we present the first panel of MAGIC lines developed: a set of 527 recombinant inbred lines (RILs) descended from a heterogeneous stock of 19 intermated accessions of the plant Arabidopsis thaliana. These lines and the 19 founders were genotyped with 1,260 single nucleotide polymorphisms and phenotyped for development-related traits. Analytical methods were developed to fine-map quantitative trait loci (QTL) in the MAGIC lines by reconstructing the genome of each line as a mosaic of the founders. We show by simulation that QTL explaining 10% of the phenotypic variance will be detected in most situations with an average mapping error of about 300 kb, and that if the number of lines were doubled the mapping error would be under 200 kb. We also show how the power to detect a QTL and the mapping accuracy vary, depending on QTL location. We demonstrate the utility of this new mapping population by mapping several known QTL with high precision and by finding novel QTL for germination data and bolting time. Our results provide strong support for similar ongoing efforts to produce MAGIC lines in other organisms.     

Detection of epistatic interactions between exotic alleles introgressed from wild barley (H. vulgare ssp. spontaneum)

The expression of a quantitative phenotype can be controlled through genotype, environment and genotype by environment interaction effects. Further, genotype effects can be attributed to major genes, quantitative trait loci (QTL) and gene by gene interactions, which are also termed epistatic interactions. The present study demonstrates that two-way epistatic interactions can play an important role for the expression of domestication-related traits like heading date, plant height and yield. In the BC₂DH population S42, carrying wild barley introgressions in the genetic background of the spring barley cultivar Scarlett, 13, 8 and 12 marker by marker interaction effects could be detected for the traits heading date, plant height and yield, respectively. Significant allelic combinations at interacting loci coincided for heading date, plant height and yield suggesting the presence of pleiotropic effects rather than several linked QTL. The mode of epistasis observed was primarily characterised by either (1) compensatory effects, where allelic combinations from the same genotype buffered the phenotype, or (2) augmented effects, where only the combination of the exotic allele at both interacting loci caused an altered phenotype. The present study shows that estimates of main effects of QTL can be confounded by interactions with background loci, suggesting that the identification of epistatic effects is important for gene cloning and marker-assisted selection. Furthermore, interaction effects between loci and putative candidate genes detected in the present study reveal potential functional relationships, which can be used to further elucidate gene networks in barley.     

Identification, isolation and pyramiding of quantitative trait loci for rice breeding

Many agronomically important traits are governed by several genes known as quantitative trait loci (QTLs). The identification of important, QTL-controlled agricultural traits has been difficult because of their complex inheritance; however, completion of the rice genomic sequence has facilitated the cloning of QTLs and their pyramiding for breeding. Because QTLs are derived from natural variation, the use of a wider range of variations such as that found in wild species is important. In addition, Introgression Lines (ILs) developed from wild species in combination with Marker Assisted Selection should facilitate efficient gene identification. This review describes recent developments in rice QTL analysis including mapping, cloning and pyramiding QTLs.     

Epistasis,complex traits,and mapping genes

Using a three-locus model wherein two loci regulate a third, candidate locus, I examine physiological epistasis from the gene's eye view of the regulated locus. I show that, depending upon genetic background at the regulatory loci, an allele at the candidate locus can be dominant, additive, recessive, neutral, over-dominant, or under-dominant in its effects on fitness. This kind of variation in allelic effect caused by variation in genetic background from population to population, from time to time in the same population, or sample to sample makes finding and mapping the genes underlying a complex phenotype difficult. The rate of evolution of such genes can also be slowed, especially in genetically subdivided metapopulations with migration. Nevertheless, understanding how variation in genetic background causes variation in allelic effects permits the genetic architecture of such complex traits to be dissected into the interacting component genes. While some backgrounds diminish allelic effects and make finding and mapping genes difficult, other backgrounds enhance allelic effects and facilitate gene mapping.     

Genome-wide Characterization of Shared and Distinct Genetic Components that Influence Blood Lipid Levels in Ethnically Diverse Human Populations

Blood lipid concentrations are heritable risk factors associated with atherosclerosis and cardiovascular diseases. Lipid traits exhibit considerable variation among populations of distinct ancestral origin as well as between individuals within a population. We performed association analyses to identify genetic loci influencing lipid concentrations in African American and Hispanic American women in the Women’s Health Initiative SNP Health Association Resource. We validated one African-specific high-density lipoprotein cholesterol locus at CD36 as well as 14 known lipid loci that have been previously implicated in studies of European populations. Moreover, we demonstrate striking similarities in genetic architecture (loci influencing the trait, direction and magnitude of genetic effects, and proportions of phenotypic variation explained) of lipid traits across populations. In particular, we found that a disproportionate fraction of lipid variation in African Americans and Hispanic Americans can be attributed to genomic loci exhibiting statistical evidence of association in Europeans, even though the precise genes and variants remain unknown. At the same time, we found substantial allelic heterogeneity within shared loci, characterized both by population-specific rare variants and variants shared among multiple populations that occur at disparate frequencies. The allelic heterogeneity emphasizes the importance of including diverse populations in future genetic association studies of complex traits such as lipids; furthermore, the overlap in lipid loci across populations of diverse ancestral origin argues that additional knowledge can be gleaned from multiple populations.     

Evidence for a natural allelic series at the Maize domestication Locus teosinte branched1

Despite numerous quantitative trait loci and association mapping studies, our understanding of the extent to which natural allelic series contribute to the variation for complex traits is limited. In this study, we investigate the occurrence of a natural allelic series for complex traits at the teosinte branched1 (tb1) gene in natural populations of teosinte (Zea mays ssp. parviglumis, Z. mays ssp. mexicana, and Z. diploperennis). Previously, tb1 was shown to confer large effects on both plant architecture and ear morphology between domesticated maize and teosinte; however, the effect of tb1 on trait variation in natural populations of teosinte has not been investigated. We compare the effects of nine teosinte alleles of tb1 that were introgressed into an isogenic maize inbred background. Our results provide evidence for a natural allelic series at tb1 for several complex morphological traits. The teosinte introgressions separate into three distinct phenotypic classes, which correspond to the taxonomic origin of the alleles. The effects of the three allelic classes also correspond to known morphological differences between the teosinte taxa. Our results suggest that tb1 contributed to the morphological diversification of teosinte taxa as well as to the domestication of maize.     

TILLING技术在植物功能基因组及育种中的应用

随着拟南芥、水稻等模式植物基因组测序计划的全面完成,数据库中大量的DNA序列需要进行功能注释,而用传统的正向遗传学进行基因克隆和近年来发展的反向遗传学（如插入突变、反义RNA、RNAi等技术）方法已不能适应基因组学的发展需求,因此,研发大规模、高通量的基因功能分析方法成为当务之急。TILLING技术（Targeting induced local lesions in genomes）就是在基因组生物学大背景下出现的一种全新的反向遗传学技术。TILLING技术的基本步骤是通过化学诱变方法产生一系列点突变,经过PCR扩增放大和变性复性过程产生异源双链DNA分子,再通过特异性酶切和双色电泳分析识别异源双链中错配碱基,从而检测出突变发生的准确位置。由于具有高通量、大规模、高灵敏度和自动化等特点,能够适应植物功能基因组学研究的要求,TILLING技术已经和即将在功能基因组领域发挥越来越重要的作用。TILLING技术应用于已测序完成的拟南芥和水稻中的突变位点检测并取得了巨大成功;TILLING技术应用于农作物的品种改良,可以帮助实现快速、定向改良作物的品种,同时由于TILLING采用的化学诱变技术与传统诱变育种并无二致,因此在作物改良中采用TILLING技术不存在外源基因转入引发的转基因作物（GMO）争论;由TILLING技术发展来的EcoTILLING技术,具有通量高、成本低、定位准确等优点,可以很好地进行多态性检测和研究基因的功能,已成为开展物种DNA多态性检测和不同物种演替进化研究的有力工具。本文简要介绍了TILLING的原理及操作步骤,讨论了TILLING技术的特点和优点及TILLING技术的应用前景。     

Application of TILLING in Plant Improvement

TILLING (Targeting induced local lesions in genomes) is a general reverse-genetic strategy that is used to locate an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and cost-effective detection of induced point mutations in populations of chemically mutagenized individuals. The technique can be applied not only to model organisms but also to economically important organisms in plants. Owing to its full of advantages such as simple procedure, high sensitivity, and high efficiency, TILLING provides a powerful approach for gene discovery, DNA polymorphism assessment, and plant improvement. Coupled with other genomic resources, TILLING and EcoTILLING can be used immediately as a haplotyping tool in plant breeding for identifying allelic variation in genes exhibiting expression correlating with phenotypes and establishing an allelic series at genetic loci for the traits of interest in germplasm or induced mutants.     

Multiparent intercross populations in analysis of quantitative traits

Most traits of interest to medical, agricultural and animal scientists show continuous variation and complex mode of inheritance. DNA-based markers are being deployed to analyse such complex traits, that are known as quantitative trait loci (QTL). In conventional QTL analysis, F2, backcross populations, recombinant inbred lines, backcross inbred lines and double haploids from biparental crosses are commonly used. Introgression lines and near isogenic lines are also being used for QTL analysis. However, such populations have major limitations like predominantly relying on the recombination events taking place in the F1 generation and mapping of only the allelic pairs present in the two parents. The second generation mapping resources like association mapping, nested association mapping and multiparent intercross populations potentially address the major limitations of available mapping resources. The potential of multiparent intercross populations in gene mapping has been discussed here. In such populations both linkage and association analysis can be conductted without encountering the limitations of structured populations. In such populations, larger genetic variation in the germplasm is accessed and various allelic and cytoplasmic interactions are assessed. For all practical purposes, across crop species, use of eight founders and a fixed population of 1000 individuals are most appropriate. Limitations with multiparent intercross populations are that they require longer time and more resource to be generated and they are likely to show extensive segregation for developmental traits, limiting their use in the analysis of complex traits. However, multiparent intercross population resources are likely to bring a paradigm shift towards QTL analysis in plant species.     

Genome-wide Introgression Lines and their Use in Genetic and Molecular Dissection of Complex Phenotypes in Rice (Oryza sativa L.)

Tremendous efforts have been taken worldwide to develop genome-wide genetic stocks for rice functional genomic (FG) research since the rice genome was completely sequenced. To facilitate FG research of complex polygenic phenotypes in rice, we report the development of over 20 000 introgression lines (ILs) in three elite rice genetic backgrounds for a wide range of complex traits, including resistances/tolerances to many biotic and abiotic stresses, morpho-agronomic traits, physiological traits, etc., by selective introgression. ILs within each genetic background are phenotypically similar to their recurrent parent but each carries one or a few traits introgressed from a known donor. Together, these ILs contain a significant portion of loci affecting the selected complex phenotypes at which allelic diversity exists in the primary gene pool of rice. A forward genetics strategy was proposed and demonstrated with examples on how to use these ILs for large-scale FG research. Complementary to the genome-wide insertional mutants, these ILs opens a new way for highly efficient discovery, candidate gene identification and cloning of important QTLs for specific phenotypes based on convergent evidence from QTL position, expression profiling, functional and molecular diversity analyses of candidate genes, highlights the importance of genetic networks underlying complex phenotypes in rice that may ultimately lead to more complete understanding of the genetic and molecular bases of quantitative trait variation in rice. Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s11103-005-8519-3     

Heterogeneous selection at specific loci in natural environments in Arabidopsis thaliana

Genetic variation for quantitative traits is often greater than that expected to be maintained by mutation in the face of purifying natural selection. One possible explanation for this observed variation is the action of heterogeneous natural selection in the wild. Here we report that selection on quantitative trait loci (QTL) for fitness traits in the model plant species Arabidopsis thaliana differs among natural ecological settings and genetic backgrounds. At one QTL, the allele that enhanced the viability of fall-germinating seedlings in North Carolina reduced the fecundity of spring-germinating seedlings in Rhode Island. Several other QTL experienced strong directional selection, but only in one site and seasonal cohort. Thus, different loci were exposed to selection in different natural environments. Selection on allelic variation also depended upon the genetic background. The allelic fitness effects of two QTL reversed direction depending on the genotype at the other locus. Moreover, alternative alleles at each of these loci caused reversals in the allelic fitness effects of a QTL closely linked to TFL1, a candidate developmental gene displaying nucleotide sequence polymorphism consistent with balancing selection. Thus, both environmental heterogeneity and epistatic selection may maintain genetic variation for fitness in wild plant species.     

Quantitative trait loci analysis of nitrogen use efficiency in Arabidopsis

Improving plant nitrogen (N) use efficiency or controlling soil N requires a better knowledge of the regulation of plant N metabolism. This could be achieved using Arabidopsis as a model genetic system, taking advantage of the natural variation available among ecotypes. Here, we describe an extensive study of N metabolism variation in the Bay-0 x Shahdara recombinant inbred line population, using quantitative trait locus (QTL) mapping. We mapped QTL for traits such as shoot growth, total N, nitrate, and free-amino acid contents, measured in two contrasting N environments (contrasting nitrate availability in the soil), in controlled conditions. Genetic variation and transgression were observed for all traits, and most of the genetic variation was identified through QTL and QTL x QTL epistatic interactions. The 48 significant QTL represent at least 18 loci that are polymorphic between parents; some may correspond to known genes from the N metabolic pathway, but others represent new genes controlling or interacting with N physiology. The correlations between traits are dissected through QTL colocalizations: The identification of the individual factors contributing to the regulation of different traits sheds new light on the relations among these characters. We also point out that the regulation of our traits is mostly specific to the N environment (N availability). Finally, we describe four interesting loci at which positional cloning is feasible.