Family-based association mapping in crop species

Identification of allelic variants associated with complex traits provides molecular genetic information associated with variability upon which both artificial and natural selections are based. Family-based association mapping (FBAM) takes advantage of linkage disequilibrium among segregating progeny within crosses and among parents to provide greater power than association mapping and greater resolution than linkage mapping. Herein, we discuss the potential adaption of human family-based association tests and quantitative transmission disequilibrium tests for use in crop species. The rapid technological advancement of next generation sequencing will enable sequencing of all parents in a planned crossing design, with subsequent imputation of genotypes for all segregating progeny. These technical advancements are easily adapted to mating designs routinely used by plant breeders. Thus, FBAM has the potential to be widely adopted for discovering alleles, common and rare, underlying complex traits in crop species.     

Future tools for association mapping in crop plants

Association mapping currently relies on the identification of genetic markers. Several technologies have been adopted for genetic marker analysis, with single nucleotide polymorphisms (SNPs) being the most popular where a reasonable quantity of genome sequence data are available. We describe several tools we have developed for the discovery, annotation, and visualization of molecular markers for association mapping. These include autoSNPdb for SNP discovery from assembled sequence data; TAGdb for the identification of gene specific paired read Illumina GAII data; CMap3D for the comparison of mapped genetic and physical markers; and BAC and Gene Annotator for the online annotation of genes and genomic sequences.     

Whole-genome association mapping in elite inbred crop varieties

We have previously shown that linkage disequilibrium (LD) in the elite cultivated barley (Hordeum vulgare) gene pool extends, on average, for <1-5 cM. Based on this information, we have developed a platform for whole genome association studies that comprises a collection of elite lines that we have characterized at 3060 genome-wide single nucleotide polymorphism (SNP) marker loci. Interrogating this data set shows that significant population substructure is present within the elite gene pool and that diversity and LD vary considerably across each of the seven barley chromosomes. However, we also show that a subpopulation comprised of only the two-rowed spring germplasm is less structured and well suited to whole genome association studies without the need for extensive statistical intervention to account for structure. At the current marker density, the two-rowed spring population is suited for fine mapping simple traits that are located outside of the genetic centromeres with a resolution that is sufficient for candidate gene identification by exploiting conservation of synteny with fully sequenced model genomes and the emerging barley physical map.     

Epistatic association and linkage analysis in human families

Summary A new method is given to test for phenotypic association using related individuals in pedigree analysis. It is also shown how an extension of this method allows analyses of genetic linkage in the presence of epistatic associations. A published pedigree with strong evidence for linkage between Lp and ESD is reanalyzed, resulting in a considerable drop of the lod score for linkage.Dr. Falk is supported by a grant from the National Institutes of Health (GM 29177)     

Genome-wide association mapping of agronomic and morphologic traits in highly structured populations of barley cultivars

Genome-wide association study (GWAS) has become an obvious general approach for studying traits of agricultural importance in higher plants, especially crops. Here, we present a GWAS of 32 morphologic and 10 agronomic traits in a collection of 615 barley cultivars genotyped by genome-wide polymorphisms from a recently developed barley oligonucleotide pool assay. Strong population structure effect related to mixed sampling based on seasonal growth habit and ear row number is present in this barley collection. Comparison of seven statistical approaches in a genome-wide scan for significant associations with or without correction for confounding by population structure, revealed that in reducing false positive rates while maintaining statistical power, a mixed linear model solution outperforms genomic control, structured association, stepwise regression control and principal components adjustment. The present study reports significant associations for sixteen morphologic and nine agronomic traits and demonstrates the power and feasibility of applying GWAS to explore complex traits in highly structured plant samples.     

Linkage disequilibrium and population-structure analysis among Capsicum annuum L. cultivars for use in association mapping

Knowledge of population structure and linkage disequilibrium among the worldwide collections of peppers currently classified as hot, mild, sweet and ornamental types is indispensable for applying association mapping and genomic selection to improve pepper. The current study aimed to resolve the genetic diversity and relatedness of Capsicum annuum germplasm by use of simple sequence repeat (SSR) loci across all chromosomes in samples collected in 2011 and 2012. The physical distance covered by the entire set of SSRs used was 2,265.9 Mb from the 3.48-Gb hot-pepper genome size. The model-based program STRUCTURE was used to infer five clusters, which was further confirmed by classical molecular-genetic diversity analysis. Mean heterozygosity of various loci was estimated to be 0.15. Linkage disequilibrium (LD) was used to identify 17 LD blocks across various chromosomes with sizes from 0.154 Kb to 126.28 Mb. CAMS-142 of chromosome 1 was significantly associated with both capsaicin (CA) and dihydrocapsaicin (DCA) levels. Further, CAMS-142 was located in an LD block of 98.18 Mb. CAMS-142 amplified bands of 244, 268, 283 and 326 bp. Alleles 268 and 283 bp had positive effects on both CA and DCA levels, with an average R ² of 12.15 % (CA) and 12.3 % (DCA). Eight markers from seven different chromosomes were significantly associated with fruit weight, contributing an average effect of 15 %. CAMS-199, HpmsE082 and CAMS-190 are the three major quantitative trait loci located on chromosomes 8, 9, and 10, respectively, and were associated with fruit weight in samples from both years of the study. This research demonstrates the effectiveness of using genome-wide SSR-based markers to assess features of LD and genetic diversity within C. annuum.     

Molecular mapping and tagging of genes in crop plants

In India, molecular mapping and tagging of agronomically important genes using RFLP and RAPD markers have been carried out in three different crops: rice, mustard and chickpea. In rice, tagging of genes for resistance to gall midge and blast has been accomplished. Molecular mapping of cooking quality traits in rice is in progress. For fingerpringting rice cultivars, suitable probe enzyme combinations have been identified. In mustard, a partial RFLP linkage map has been constructed and one of the yellow seed-coat colour loci has been mapped. Significant associations of RFLP markers with quantitative traits have also been established. Potential use of RAPD markers to identify heterotic groups among mustard accessions has been demonstrated. In chickpea, the occurrence of considerable interspecific DNA polymorphism as revealed by RAPD analysis has facilitated construction of a partial linkage map.     

Genome-wide association mapping of fruit-quality traits using genotyping-by-sequencing approach in citrus landraces,modern cultivars,and breeding lines in Japan

Association mapping is an attractive method to identify QTLs in perennial horticultural crops such as citrus, as it does not need a designed cross between parental genotypes and can save time and labor to construct a segregating population. It usually requires more genetic markers than linkage-based QTL mapping owing to a lower degree of linkage disequilibrium (LD). However, recent advances in next-generation sequencing offer high-throughput, cost-effective methods, including genotyping-by-sequencing (GBS), for genotyping massive amounts of single nucleotide polymorphisms (SNPs). In this study, we performed a genome-wide association study (GWAS) of fruit-quality traits in citrus using SNPs obtained by GBS. We evaluated 110 citrus accessions, including landraces, modern cultivars, and breeding lines, for eight fruit-quality traits (fruit weight, fruit skin color, fruit surface texture, peelability, pulp firmness, segment firmness, sugar content, and acid content) during 2005 to 2012 (except 2007). GBS found 2309 SNPs, which we anchored to the clementine reference genome. We evaluated LD in the 110 accessions and confirmed that GBS gave enough SNPs to conduct GWAS. We identified seven QTLs, including four novel ones, comprising four significant QTLs for fruit weight and one QTL each for fruit skin color, pulp firmness, and segment firmness. These QTLs offer promise for use in citrus crossbreeding.     

Responses of various crop species and cultivars to fertilizer application

Summary Crop response to fertilizer application depends not only on the level of available plant nutrients in the soil but is also related to crop physiology and morphology. For a well balanced nutrition the rate of nutrient supply to the roots must correspond with the rate of nutrient required for growth. Species or cultivars with a high growth rate generally respond more favourably to fertilizer application than those with low growth rates. An analogous relationship holds for the biomass produced per unit soil surface. Thus modern rice and wheat cultivars tolerate a more dense spacing than older ones. Due to the dense stand the yield and particularly the grain yield of the modern varieties may be several times higher than those of older cultivars, and therefore also the nutrient requirement, especially the demand for N and P, is higher for the modern cultivars.Modern cereal cultivars are characterized by a high crop index which means that after flowering a high proportion of grain filling material must be produced by photosynthesis. Assimilation and translocation of photosynthates are favoured by K⁺. Thus in particular modern cultivars require a high K⁺ content for optimum grain filling.Nutrient exploitation of soils by plant roots depends on root morphology and root physiology. Grasses generally have much longer roots than dicots. Thus the rate of K⁺ and phosphate uptake per unit root length is lower for grasses than for dicots. It is for this reason that dicots respond earlier to a K⁺ and phosphate dressing than grasses.Species living symbiotically with Rhizobium may depress the rhizosphere pH considerably and thus promote the dissolution of phosphate rock.     

QTL mapping under truncation selection in homozygous lines derived from biparental crosses

In plant breeding, a large number of progenies that will be discarded later in the breeding process must be phenotyped and marker genotyped for conducting QTL analysis. In many cases, phenotypic preselection of lines could be useful. However, in QTL analyses even moderate preselection can have a significant effect on the power of QTL detection and estimation of effects of the target traits. In this study, we provide exact formulas for quantifying the change of allele frequencies within marker classes, expectations of marker contrasts and the variance of the marker contrasts under truncation selection, for the general case of two QTL affecting the target trait and a correlated trait. We focused on homozygous lines derived at random from biparental crosses. The effects of linkage between the marker and the QTL under selection as well as the effect of selection on a correlated trait can be quantified with the given formulas. Theoretical results clearly show that depending on the magnitude of QTL effects, high selection intensities can lead to a dramatic reduction in power of QTL detection and that approximations based on the infinitesimal model deviate substantially from exact solutions. The presented formulas are valuable for choosing appropriate selection intensity when performing QTL mapping experiments on the data on phenotypically preselected traits and enable the calculation and bias correction of the effects of QTL under selection. Application of our theory to experimental data revealed that selection-induced bias of QTL effects can be successfully corrected.     

Association mapping of yield and its components in rice cultivars

To make advances in rice breeding it is important to understand the relatedness and ancestry of introduced rice accessions, and identify SSR markers associated with agronomically important phenotypic traits, for example yield. Ninety-two rice germplasm accessions recently introduced from seven geographic regions of Africa, Asia, and Latin America, and eleven US cultivars, included as checks, were evaluated for yield and kernel characteristics, and genotyped with 123 SSR markers. The SSR markers were highly polymorphic across all accessions. Population structure analysis identified eight main clusters for the accessions which corresponded to the major geographic regions, indicating agreement between genetic and predefined populations. Linkage disequilibrium (LD) patterns and distributions are of fundamental importance for genome-wide mapping association. LD between linked markers decreased with distance and with a substantial drop in LD decay values between 20 and 30 cM, suggesting it should be possible to achieve resolution down to the 25 cM level. For the 103 cultivars, the complex traits yield, kernel width, kernel length, kernel width/length ratio, and 1000-kernel weight, were estimated by analysis of variety trial data. The mixed linear model method was used to disclose marker-trait associations. Many of the associated markers were located in regions where QTL had previously been identified. In conclusion, association mapping in rice is a viable alternative to QTL mapping based on crosses between different lines.     

Genetic diversity trends in twentieth century crop cultivars: a meta analysis

In recent years, an increasing number of papers has been published on the genetic diversity trends in crop cultivars released in the last century using a variety of molecular techniques. No clear general trends in diversity have emerged from these studies. Meta analytical techniques, using a study weight adapted for use with diversity indices, were applied to analyze these studies. In the meta analysis, 44 published papers were used, addressing diversity trends in released crop varieties in the twentieth century for eight different field crops, wheat being the most represented. The meta analysis demonstrated that overall in the long run no substantial reduction in the regional diversity of crop varieties released by plant breeders has taken place. A significant reduction of 6% in diversity in the 1960s as compared with the diversity in the 1950s was observed. Indications are that after the 1960s and 1970s breeders have been able to again increase the diversity in released varieties. Thus, a gradual narrowing of the genetic base of the varieties released by breeders could not be observed. Separate analyses for wheat and the group of other field crops and separate analyses on the basis of regions all showed similar trends in diversity.     

作物抗旱相关性状的QTLs定位研究进展

提高作物品种的抗旱性、获得高产稳产是抗旱育种的最终目标。作物分子连锁图谱的构建及其它分子遗传学研究进展为改良作物抗旱性提供了新机遇。Ｔａｎｋｓｌｅｙ等[1]指出,通过ＲＦＬＰ或其他分子标记进行标记辅助选择,为难以测量的性状提供了一条重要的选择途径。通过对抗旱相关性状的数量性状位点(ＱＴＬｓ)的标记,就可以把复杂的性状作为一套单基因性状进行分析和选择。一旦找到紧密连锁的标记(1-5ｃＭ),就可以在育种工作中进行标记辅助选择[2]。分子标记使育种家无需测定表型就能够追踪调控抗旱性状的遗传位点,可免去多年多点的大量田间…     

Coalescent-based association mapping and fine mapping of complex trait loci

We outline a general coalescent framework for using genotype data in linkage disequilibrium-based mapping studies. Our approach unifies two main goals of gene mapping that have generally been treated separately in the past: detecting association (i.e., significance testing) and estimating the location of the causative variation. To tackle the problem, we separate the inference into two stages. First, we use Markov chain Monte Carlo to sample from the posterior distribution of coalescent genealogies of all the sampled chromosomes without regard to phenotype. Then, averaging across genealogies, we estimate the likelihood of the phenotype data under various models for mutation and penetrance at an unobserved disease locus. The essential signal that these models look for is that in the presence of disease susceptibility variants in a region, there is nonrandom clustering of the chromosomes on the tree according to phenotype. The extent of nonrandom clustering is captured by the likelihood and can be used to construct significance tests or Bayesian posterior distributions for location. A novelty of our framework is that it can naturally accommodate quantitative data. We describe applications of the method to simulated data and to data from a Mendelian locus (CFTR, responsible for cystic fibrosis) and from a proposed complex trait locus (calpain-10, implicated in type 2 diabetes).     

Association mapping for seed size and shape traits in soybean cultivars

Seed size and shape traits are closely related to seed yield and appearance quality in soybean (Glycine max L.). Previous studies were mainly derived from bi-parental segregating populations and relatively little is known about the results in soybean cultivars. In this study, 257 soybean cultivars obtained by stratified random sampling from six geographic ecotypes in China were used to carry out association mapping for these traits using information from 135 simple sequence repeat markers and an epistatic association mapping approach implemented using an empirical Bayes algorithm. In this analysis, seed size was measured by seed length (SL), width (SW) and thickness (ST), and seed shape was evaluated by seed length-to-width (SLW), length-to-thickness (SLT) and width-to-thickness (SWT) ratios, in 2008–2010. A total of 59 main-effect quantitative trait loci (QTL) and 31 QTL-by-environment interactions were identified. Among them, 25 QTL were associated simultaneously with at least two traits; 80 QTL (90 %) could be confirmed by enriched compression mixed linear model analysis; and the size for a large number of detected QTL was minor, except for qSL-5, qSW-7e, qST-5-2 and qSLW-2. According to the estimates for the allelic effects of the detected QTL, elite alleles could be mined: for example, the 307-bp allele of QTL linked to satt453 was the best for seed length. These elite alleles could be used to design parental combinations; e.g., the cross of Zhenghezhibanzi and Nannongdahuangdou might improve seed length, and the combination of cultivars Lindou10, Deqingxiangzhudou, Ninghaixiazhidou, Zhenghezhibanzi, 0803, Shangqiu7605 and 0831 might pyramid 42 elite alleles.     

Allelic association and disease mapping.

The application of allelic association to map genes for complex traits, particularly using high-density maps of single nucleotide polymorphisms in candidate regions, is an area of very active research. Here we present some aspects of the methodology and applications to both major gene mapping, which illustrates the effectiveness of the method, and oligogenes, where methods are still in flux and for which there have been relatively few successes to date. Several important considerations emerge, including the selection of the optimal metric for measuring association and the importance of modelling the decline in association with distance given the variability in association in a candidate region. The Malecot model of association with distance is shown to have a resolution of greater than 50 kilobases but the available evidence suggests that considerably higher resolution might be achieved with dense single nucleotide polymorphism (SNP) maps.     

Single-nucleotide-polymorphism-based association mapping of dog stereotypes

Phenotypic stereotypes are traits, often polygenic, that have been stringently selected to conform to specific criteria. In dogs, Canis familiaris, stereotypes result from breed standards set for conformation, performance (behaviors), etc. As a consequence, phenotypic values measured on a few individuals are representative of the breed stereotype. We used DNA samples isolated from 148 dog breeds to associate SNP markers with breed stereotypes. Using size as a trait to test the method, we identified six significant quantitative trait loci (QTL) on five chromosomes that include candidate genes appropriate to regulation of size (e.g., IGF1, IGF2BP2 SMAD2, etc.). Analysis of other morphological stereotypes, also under extreme selection, identified many additional significant loci. Less well-documented data for behavioral stereotypes tentatively identified loci for herding, pointing, boldness, and trainability. Four significant loci were identified for longevity, a breed characteristic not under direct selection, but inversely correlated with breed size. The strengths and limitations of the approach are discussed as well as its potential to identify loci regulating the within-breed incidence of specific polygenic diseases.     

Fine mapping versus replication in whole-genome association studies

Association replication studies have a poor track record and, even when successful, often claim association with different markers, alleles, and phenotypes than those reported in the primary study. It is unknown whether these outcomes reflect genuine associations or false-positive results. A greater understanding of these observations is essential for genomewide association (GWA) studies, since they have the potential to identify multiple new associations that that will require external validation. Theoretically, a repeat association with precisely the same variant in an independent sample is the gold standard for replication, but testing additional variants is commonplace in replication studies. Finding different associated SNPs within the same gene or region as that originally identified is often reported as confirmatory evidence. Here, we compare the probability of replicating a gene or region under two commonly used marker-selection strategies: an "exact" approach that involves only the originally significant markers and a "local" approach that involves both the originally significant markers and others in the same region. When a region of high intermarker linkage disequilibrium is tested to replicate an initial finding that is only weak association with disease, the local approach is a good strategy. Otherwise, the most powerful and efficient strategy for replication involves testing only the initially identified variants. Association with a marker other than that originally identified can occur frequently, even in the presence of real effects in a low-powered replication study, and instances of such association increase as the number of included variants increases. Our results provide a basis for the design and interpretation of GWA replication studies and point to the importance of a clear distinction between fine mapping and replication after GWA.