Integration of genetic and genomic methods for identification of genes and gene variants encoding QTLs in the nonhuman primate

We have developed an integrated approach, using genetic and genomic methods, in conjunction with resources from the Southwest National Primate Research Center (SNPRC) baboon colony, for the identification of genes and their functional variants that encode quantitative trait loci (QTL). In addition, we use comparative genomic methods to overcome the paucity of baboon specific reagents and to augment translation of our findings in a nonhuman primate (NHP) to the human population. We are using the baboon as a model to study the genetics of cardiovascular disease (CVD). A key step for understanding gene–environment interactions in cardiovascular disease is the identification of genes and gene variants that influence CVD phenotypes. We have developed a sequential methodology that takes advantage of the SNPRC pedigreed baboon colony, the annotated human genome, and current genomic and bioinformatic tools. The process of functional polymorphism identification for genes encoding QTLs involves comparison of expression profiles for genes and predicted genes in the genomic region of the QTL for individuals discordant for the phenotypic trait mapping to the QTL. After comparison, genes of interest are prioritized, and functional polymorphisms are identified in candidate genes by genotyping and quantitative trait nucleotide analysis. This approach reduces the time and labor necessary to prioritize and identify genes and their polymorphisms influencing variation in a quantitative trait compared with traditional positional cloning methods.     

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。     

From QTL to QTN identification in livestock--winning by points rather than knock-out: a review

Many quantitative trait loci (QTL) affecting economic traits in livestock have now been identified. However, the confidence interval (CI) of individual QTL as determined by linkage analysis often spans tens of map units, containing hundreds of genes. Linkage disequilibrium (LD) mapping can reduce the CI to individual map units, but this reduced interval will still contain tens of genes. Methods suitable for model animals to find and validate specific quantitative trait nucleotides (QTN) underlying the QTL cannot be easily applied to livestock species because of their long generation intervals, the cost of maintaining each animal and the difficulty of producing transgenics or 'knock-outs'. Considering these limitations, we review successful approaches for identifying QTN in livestock and outline a schematic strategy for QTN determination and verification. In addition to linkage and LD mapping, the methods include positional cloning, selection of candidate genes, DNA sequencing and statistical analyses. Concordance determination and functional assays are the critical tests for validation of a QTN; we provide a generalized formula for the probability of concordance by chance. Three genes that meet the burden of proof for QTN identification--DGAT1 in cattle, IGF2 in swine and GDF8 in sheep--are discussed in detail. The genetic and economic ramifications of identified QTN and the horizon for selection and introgression are also considered.     

Development and mapping of peach candidate genes involved in fruit quality and their transferability and potential use in other Rosaceae species

The goal of the present study was to identify candidate genes (CGs) involved in fruit quality in peach that can be transferred to other Rosaceae species. Two cDNA libraries from fruit of the “Fantasia” peach cultivar, constructed at two stages of development, were used to generate a set of expressed sequence tag sequences. A total of 1,730 peach unigenes were obtained after clustering. Sequences and corresponding annotations were stored in a relational database and are available through a web interface. Fifty-nine CGs involved in fruit growth and development or fruit quality at maturity, focusing on sweetness, acidity, and phenolic compound content, were selected according to their annotation. Fifty-five primer pairs, designed from peach CG sequences and giving PCR products in peach, were tested in strawberry and 36 gave amplified products. Eight CGs were mapped in peach, 14 in strawberry, four in both species and confirmed the pattern of synteny already proposed using comparative mapping. In peach, the CGs are located in three linkage groups (3, 5, 7), and in strawberry they are distributed in all seven Fragaria linkage groups. Colocalization between some of these CGs and quantitative trait loci for fruit quality traits were identified and are awaiting confirmation in further analyses.     

Recalculation of 23 mouse HDL QTL datasets improves accuracy and allows for better candidate gene analysis

In the past 15 years, the quantitative trait locus (QTL) mapping approach has been applied to crosses between different inbred mouse strains to identify genetic loci associated with plasma HDL cholesterol levels. Although successful, a disadvantage of this method is low mapping resolution, as often several hundred candidate genes fall within the confidence interval for each locus. Methods have been developed to narrow these loci by combining the data from the different crosses, but they rely on the accurate mapping of the QTL and the treatment of the data in a consistent manner. We collected 23 raw datasets used for the mapping of previously published HDL QTL and reanalyzed the data from each cross using a consistent method and the latest mouse genetic map. By utilizing this approach, we identified novel QTL and QTL that were mapped to the wrong part of chromosomes. Our new HDL QTL map allows for reliable combining of QTL data and candidate gene analysis, which we demonstrate by identifying Grin3a and Etv6, as candidate genes for QTL on chromosomes 4 and 6, respectively. In addition, we were able to narrow a QTL on Chr 19 to five candidates.     

Quantitative trait loci for glucosinolate accumulation in Brassica rapa leaves

Glucosinolates and their breakdown products have been recognized for their effects on plant defense, human health, flavor and taste of cruciferous vegetables. Despite this importance, little is known about the regulation of the biosynthesis and degradation in Brassica rapa. Here, the identification of quantitative trait loci (QTL) for glucosinolate accumulation in B. rapa leaves in two novel segregating double haploid (DH) populations is reported: DH38, derived from a cross between yellow sarson R500 and pak choi variety HK Naibaicai; and DH30, from a cross between yellow sarson R500 and Kairyou Hakata, a Japanese vegetable turnip variety. An integrated map of 1068 cM with 10 linkage groups, assigned to the international agreed nomenclature, is developed based on the two individual DH maps with the common parent using amplified fragment length polymorphism (AFLP) and single sequence repeat (SSR) markers. Eight different glucosinolate compounds were detected in parents and F(1)s of the DH populations and found to segregate quantitatively in the DH populations. QTL analysis identified 16 loci controlling aliphatic glucosinolate accumulation, three loci controlling total indolic glucosinolate concentration and three loci regulating aromatic glucosinolate concentrations. Both comparative genomic analyses based on Arabidopsis-Brassica rapa synteny and mapping of candidate orthologous genes in B. rapa allowed the selection of genes involved in the glucosinolate biosynthesis pathway that may account for the identified QTL.     

Development, genetic mapping of candidate gene-based markers and their significant association with the shoot fly resistance quantitative trait loci in sorghum [Sorghum bicolor (L.) Moench]

The associations of candidate genes with quantitative trait loci (QTL) for insect resistance provide primary insight into the molecular mechanisms of resistance. The objectives of the present study were to genetically map the candidate genes and identify their association with shoot fly resistance, and update the genetic map with new markers to locate additional QTL. In this study, 80 candidate gene (CG)-based markers were developed, targeting the seven most important shoot fly resistance genomic regions reported in our previous study. Of the 17 polymorphic CGs, the allelic polymorphisms of seven genes were significantly associated with 18 major QTL for component traits of resistance in multiple QTL mapping (MQM), and two genes in the single-marker analysis. MQM with an updated map revealed 20 new QTL with LOD and R ² (%) values ranging from 2.6 to 15.6 and 5.5 to 34.5?%, respectively. The susceptible parent 296B contributed resistance at 10 QTL. Interestingly, an orthologous insect resistance gene Cysteine protease-Mir1 (XnhsbmSFC34/SBI-10), previously presumed to be a CG based on synteny with maize, was significantly associated with major QTL for all traits (except seedling vigor) explaining 22.1?% of the phenotypic variation for deadhearts%, a direct measure of shoot fly resistance. Similarly, a NBS?CLRR gene (XnhsbmSFCILP2/SBI-10), involved in rice brown planthopper resistance, was associated with deadhearts% and number of eggs per plant. Beta-1,3-glucanase (XnhsbmSFC4/SBI-10), involved in aphid and brown planthopper resistance, was associated with deadhearts% and leaf glossiness. Comparative QTL analysis revealed the existence of common QTL for shoot fly and other important sorghum insect pests such as greenbug, head bug, and midge. Finally, the associated CGs should aid in elucidating the molecular basis of resistance, high-resolution mapping, and map-based cloning of major QTL, besides providing powerful gene tags for marker-assisted selection of shoot fly resistance.     

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.     

ezQTL: A Web Platform for Interactive Visualization and Colocalization of QTLs and GWAS Loci

Genome-wide association studies (GWAS) have identified thousands of genomic loci associated with complex diseases and traits, including cancer. The vast majority of common trait-associated variants identified via GWAS fall in non-coding regions of the genome, posing a challenge in elucidating the causal variants, genes, and mechanisms involved. Expression quantitative trait locus (eQTL) and other molecular QTL studies have been valuable resources in identifying candidate causal genes from GWAS loci through statistical colocalization methods. While QTL colocalization is becoming a standard analysis in post-GWAS investigation, an easy web tool for users to perform formal colocalization analyses with either user-provided or public GWAS and eQTL datasets has been lacking. Here, we present ezQTL, a web-based bioinformatic application to interactively visualize and analyze genetic association data such as GWAS loci and molecular QTLs under different linkage disequilibrium (LD) patterns (1000 Genomes Project, UK Biobank, or user-provided data). This application allows users to perform data quality control for variants matched between different datasets, LD visualization, and two-trait colocalization analyses using two state-of-the-art methodologies (eCAVIAR and HyPrColoc), including batch processing. ezQTL is a free and publicly available cross-platform web tool, which can be accessed online at https://analysistools.cancer.gov/ezqtl.     

Genetic mapping of the rice resistance-breaking gene of the brown planthopper Nilaparvata lugens

Host plant resistance has been widely used for controlling the major rice pest brown planthopper (BPH, Nilaparvata lugens). However, adaptation of the wild BPH population to resistance limits the effective use of resistant rice varieties. Quantitative trait locus (QTL) analysis was conducted to identify resistance-breaking genes against the anti-feeding mechanism mediated by the rice resistance gene Bph1. QTL analysis in iso-female BPH lines with single-nucleotide polymorphism (SNP) markers detected a single region on the 10th linkage group responsible for the virulence. The QTL explained from 57 to 84% of the total phenotypic variation. Bulked segregant analysis with next-generation sequencing in F₂ progenies identified five SNPs genetically linked to the virulence. These analyses showed that virulence to Bph1 was controlled by a single recessive gene. In contrast to previous studies, the gene-for-gene relationship between the major resistance gene Bph1 and virulence gene of BPH was confirmed. Identified markers are available for map-based cloning of the major gene controlling BPH virulence to rice resistance.     

猪的基因图谱及数量性状位点定位

在人类基因组计划的带动下,猪的遗传连锁图谱和细胞遗传学图谱有了较大的进步,利用目前猪基因组图谱的研究成果,通过基因组扫描法和候选基因法,可以对猪重要经济性状的主效基因位点进行区域定位,进而图位克隆,找到主效基因,为现代遗传育种奠定理论基础。     

Quantitative trait loci controlling agronomic and biochemical traits in Cannabis sativa

Understanding the genetic basis of complex traits is a fundamental goal of evolutionary genetics. Yet, the genetics controlling complex traits in many important species such as hemp (Cannabis sativa) remain poorly investigated. Because hemp’s change in legal status with the 2014 and 2018 U.S. Federal Farm Bills, interest in the genetics controlling its numerous agriculturally important traits has steadily increased. To better understand the genetics of agriculturally important traits in hemp, we developed an F₂ population by crossing two phenotypically distinct hemp cultivars (Carmagnola and USO31). Using whole-genome sequencing, we mapped quantitative trait loci (QTL) associated with variation in numerous agronomic and biochemical traits. A total of 69 loci associated with agronomic (34) and biochemical (35) trait variation were identified. We found that most QTL co-localized, suggesting that the phenotypic distinctions between Carmagnola and USO31 are largely controlled by a small number of loci. We identified TINY and olivetol synthase as candidate genes underlying co-localized QTL clusters for agronomic and biochemical traits, respectively. We functionally validated the olivetol synthase candidate by expressing the alleles in yeast. Gas chromatography-mass spectrometry assays of extracts from these yeast colonies suggest that the USO31 olivetol synthase is functionally less active and potentially explains why USO31 produces lower cannabinoids compared to Carmagnola. Overall, our results help modernize the genomic understanding of complex traits in hemp.     

Development of candidate gene markers associated to common bacterial blight resistance in common bean

Common bacterial blight (CBB), caused by Xanthomonas axonopodis pv. phaseoli (Xap), is a major yield-limiting factor of common bean (Phaseolus vulgaris L.) production around the world. Two major CBB-resistant quantitative trait loci (QTL), linked to the sequence characterized amplified region markers BC420 and SU91, are located at chromosomes 6 and 8, respectively. Using map-based cloning approach, four bacterial artificial chromosome (BAC) clones from the BC420-QTL locus and one BAC clone containing SU91 were sequenced by Roche 454 technique and subsequently assembled using merged assemblies from three different programs. Based on the quality of the assembly, only the sequences of BAC 32H6 and 4K7 were used for candidate gene marker (CGM) development and candidate gene (CG) selection. For the BC420-QTL locus, 21 novel genes were predicted in silico by FGENESH using Medicago gene model, whereas 16 genes were identified in the SU91-QTL locus. For each putative gene, one or more primer pairs were designed and tested in the contrasting near isogenic lines. Overall, six and nine polymorphic markers were found in the SU91- and BC420-QTL loci, respectively. Afterwards, association mapping was conducted in a breeding population of 395 dry bean lines to discover marker-trait associations. Two CGMs per each locus showed better association with CBB resistance than the BC420 and SU91 markers, which include BC420-CG10B and BC420-CG14 for BC420_QTL locus, and SU91-CG10 and SU91-CG11 for SU91_QTL locus. The strong associations between CBB resistance and the CGs 10 and 14 from BC420_QTL locus and the CGs 10 and 11 from SU91_QTL locus indicate that the genes 10 and 14 from the BC420 locus are potential CGs underlying the BC420_QTL locus, whereas the genes 10 and 11 from the SU91 locus are potential CGs underlying the SU91_QTL locus. The superiority of SU91-CG11 was further validated in a recombinant inbred line population Sanilac?×?OAC 09-3. Thus, co-dominant CGMs, BC420-CG14 and SU91-CG11, are recommended to replace BC420 and SU91 for marker-assisted selection of common bean with resistance to CBB.     

番茄果重数量性状基因的研究进展及在遗传学教学中的应用

遗传学发展史上一系列经典的研究案例对学科的发展起了巨大的推动作用,将这些经典案例与教学内容相结合应用到遗传学课程教学中,对学生的科学思维和遗传分析能力是一个很好的训练。番茄果重基因的定位与克隆在数量性状基因座研究中是开创性的工作,完整的体现了植物数量性状基因的研究历程。将其作为一个综合案例应用于遗传学教学,可以生动直观地给学生展示一个精彩的科学发现过程,展现遗传学研究的魅力,激发学生的学习兴趣,收到了很好的教学效果。     

Quantitative trait loci and candidate genes associated with starch pasting viscosity characteristics in cassava (Manihot esculenta Crantz)

Starch pasting viscosity is an important quality trait in cassava (Manihot esculenta Crantz) cultivars. The aim here was to identify loci and candidate genes associated with the starch pasting viscosity. Quantitative trait loci (QTL) mapping for seven pasting viscosity parameters was carried out using 100 lines of an F₁ mapping population from a cross between two cassava cultivars Huay Bong 60 and Hanatee. Starch samples were obtained from roots of cassava grown in 2008 and 2009 at Rayong, and in 2009 at Lop Buri province, Thailand. The traits showed continuous distribution among the F₁ progeny with transgressive variation. Fifteen QTL were identified from mean trait data, with Logarithm of Odds (LOD) values from 2.77–13.01 and phenotype variations explained (PVE) from10.0–48.4%. In addition, 48 QTL were identified in separate environments. The LOD values ranged from 2.55–8.68 and explained 6.6–43.7% of phenotype variation. The loci were located on 19 linkage groups. The most important QTL for pasting temperature (PT) (qPT.1LG1) from mean trait values showed largest effect with highest LOD value (13.01) and PVE (48.4%). The QTL co‐localised with PT and pasting time (PTi) loci that were identified in separate environments. Candidate genes were identified within the QTL peak regions. However, the major genes of interest, encoding the family of glycosyl or glucosyl transferases and hydrolases, were located at the periphery of QTL peaks. The loci identified could be effectively applied in breeding programmes to improve cassava starch quality. Alleles of candidate genes should be further studied in order to better understand their effects on starch quality traits.     

质量性状和数量性状含义的辨析

植物或动物的性状一般分为质量性状和数量性状,而实际上,许多性状并不是绝对的质量性状或数量性状,而是同时受到一个或少数几个主基因和或数量性状多基因的控制.因此,在遗传学教学中,有必要对此类性状进行分析.为加深学生对此类性状的遗传及这两个概念的理解,通过性状次数分布图分析,结合最新的遗传学研究成果,对之进行了分析和讨论.     

植物数量性状基因定位研究概述

植物重要的性状多为数量性状。长期以来,人类一直寻求解释植物数量性状的遗传规律以便对其进行遗传操纵。现代分子生物技术的发展为植物数量性状基因的定位、分离等研究提供了条件。本文从数量性状基因座(QTL)作图群体类型及其特点,QTL定位方法,植物QTL研究现状,以及QTL精细定位、克隆、利用等方面进行了综述,并对今后植物QTL研究进行了展望。     

Construction of a genetic linkage map of Thlaspi caerulescens and quantitative trait loci analysis of zinc accumulation

Zinc (Zn) hyperaccumulation seems to be a constitutive species-level trait in Thlaspi caerulescens. When compared under conditions of equal Zn availability, considerable variation in the degree of hyperaccumulation is observed among accessions originating from different soil types. This variation offers an excellent opportunity for further dissection of the genetics of this trait. A T. caerulescens intraspecific cross was made between a plant from a nonmetallicolous accession [Lellingen (LE)], characterized by relatively high Zn accumulation, and a plant from a calamine accession [La Calamine (LC)], characterized by relatively low Zn accumulation. Zinc accumulation in roots and shoots segregated in the F3 population. This population was used to construct an LE/LC amplified fragment length polymorphism (AFLP)-based genetic linkage map and to map quantitative trait loci (QTL) for Zn accumulation. Two QTL were identified for root Zn accumulation, with the trait-enhancing alleles being derived from each of the parents, and explaining 21.7 and 16.6% of the phenotypic variation observed in the mapping population. Future development of more markers, based on Arabidopsis orthologous genes localized in the QTL regions, will allow fine-mapping and map-based cloning of the genes underlying the QTL.