候选基因策略在植物遗传学中的应用

随着遗传学研究技术的快速发展和后基因组学时代的来临,候选基因的概念和研究方法越来越多地被人们所应用,成 为功能克隆、图位克隆、表型克隆、插入突变等方法之外的又一重要基因克隆策略。候选基因策略的基本原理和主要步骤,以 及其在植物遗传学中具有重要的应用实例。     

A genome-wide survey of maize lipid-related genes: candidate genes mining,digital gene expression profiling and co-location with QTL for maize kernel oil

Lipids play an important role in plants due to their abundance and their extensive participation in many metabolic processes. Genes involved in lipid metabolism have been extensively studied in Arabidopsis and other plant species. In this study, a total of 1003 maize lipid-related genes were cloned and annotated, including 42 genes with experimental validation, 732 genes with full-length cDNA and protein sequences in public databases and 229 newly cloned genes. Ninety-seven maize lipid-related genes with tissue-preferential expression were discovered by in silico gene expression profiling based on 1984483 maize Expressed Sequence Tags collected from 182 cDNA libraries. Meanwhile, 70 QTL clusters for maize kernel oil were identified, covering 34.5% of the maize genome. Fifty-nine (84%) QTL clusters co-located with at least one lipid-related gene, and the total number of these genes amounted to 147. Interestingly, thirteen genes with kernel-preferential expression profiles fell within QTL clusters for maize kernel oil content. All the maize lipid-related genes identified here may provide good targets for maize kernel oil QTL cloning and thus help us to better understand the molecular mechanism of maize kernel oil accumulation.     

Genetic dissection of tocopherol content and composition in maize grain using quantitative trait loci analysis and the candidate gene approach

Tocopherols are essential micronutrients for humans and animals, with several beneficial effects in plants. Among cereals, only maize grains contain high concentrations of tocopherols. In this investigation we analyzed, during 2004 and 2005, by high-performance liquid chromatography (HPLC), a population of 233 recombinant inbred lines (RIL) which were derived from two diverse parents and had extremely variable tocopherol content and composition. A genetic map was constructed using 208 polymorphic molecular markers including gene-targeted markers based on six candidate genes of the tocopherol biosynthesis pathway (HPPD, VTE1, VTE3, VTE4, P3VTE5, and P4VTE5). Thirty-one quantitative trait loci (QTL) associated with quantitative variation of tocopherol content and composition were identified by composite interval mapping (CIM); these were located on sixteen genomic regions covering all the chromosomes except chromosome 4. Most (65%) QTL were co-located, suggesting that in some cases the same QTL predominantly affected the amounts of more than one tocopherol. Two candidate genes, HPPD and VTE4 showed co-localization with major QTL for tocopherol content and composition whereas only one interval (umc1075–umc1304) on chromosome eight exhibited a QTL for α, δ, γ, and total tocopherols with high LOD and PVE values. The candidate genes associated with tocopherol content and with composition, especially VTE4 and HPPD, could be precisely used for alteration of the tocopherol content and composition of maize grains by development of functional markers. Other identified major QTL especially those on chromosomes 8, 1, and 2 (near candidate gene VTE5) can also be used for improvement of maize grain quality by marker-assisted selection.     

Identification of functional candidate genes for drought tolerance in rice

Drought tolerance (DT) in rice is known to be controlled by many quantitative trait loci (QTLs) and involved differential expression of large numbers of genes, but linking QTLs with their underlying genes remains the most challenging issue in plant molecular biology. To shed some light on this issue, differential gene expression in response to PEG simulated drought in 3 unique genetic materials (a lowland rice, IR64 and its derived line, PD86 which has 11 introgressed DT QTLs, and a upland rice IRAT109) was investigated using a PCR-based subtractive hybridization strategy. More than 300 unique subtracted cDNA sequences, covering genes of diverse cellular activities and functions, were identified and confirmed by semi-quantitative and quantitative RT-PCR. Detailed bioinformatics analyses of the data revealed two interesting results. First, the levels and mechanisms of DT of the three rice lines were associated with the number and types of differentially expressed genes, suggesting different DT mechanisms in rice are controlled by different sets of genes and different metabolic pathways, and most differentially expressed genes under drought were able to contribute to DT. Second, there appeared a high correspondence in genomic location between DT QTLs and clusters of differentially expressed genes in rice, suggesting some DT QTLs may represent clusters of co-regulated and functionally related genes. Thus, differential gene expression analyses using genetically characterized materials can provide additional insights into the molecular basis of QTLs and convergent evidence to shortlist the candidate genes for target QTLs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Bin-Ying Fu and Jian-Hua Xiong are contributed to this work equally.     

Inheritance studies of low-phosphorus tolerance in maize (Zea mays L.), grown in a sand-alumina culture medium

Inbred lines of maize selected as tolerant and intolerant to low-P stress using a sand-alumina culture medium were used to obtain F₁ hybrids and advanced generations to be evaluated in diallel mating schemes and generation means analyses for the inheritance studies. Sand-alumina, a solid culture medium, which simulates a slow release, diffusion-limited P movement in soil solution was used in the inheritance studies. Tolerance to low-P stress conditions in maize seedlings is controlled largely by additive gene effects, but dominance is also important.     

A candidate gene approach identified phytoene synthase as the locus for mature fruit color in red pepper (Capsicum spp.)

Abstrat  The color of mature pepper fruit is determined by the composition of carotenoids. The fruit color of red pepper is genetically determined by three loci, y, c1, and c2. We have been developing a genetic map of hot pepper using RFLP and AFLP markers in the F₂ population of an interspecific cross between Capsicum annuum cv TF68 and Capsicum chinense cv Habanero. The color of the ripe fruit of TF68 is red and Habanero is orange. The red color is dominant over orange in the F₁ and the locus controlling this character has been marked in our SNU Linkage Group 7. To identify the gene or markers tightly linked to the red/orange locus, several candidate genes involved in the carotenoid biosynthesis pathway, namely FPS, GGPS, PSY, PDS, LCY and CCS, were examined. One of the candidate genes, phytoene synthase, cosegregated completely with fruit color in the F₂ population. QTL analysis of the pigment content of F₂ individuals quantified by HPLC also indicated that phytoene synthase is the locus responsible for the development of fruit color. The color, pigment content and genetic behavior of Habanero also suggest that phytoene synthase may be responsible for the c2 gene discriminating between red and orange cultivars. Received: 15 March 2000 / Accepted: 16 August 2000     

Positional cloning and characterization reveal the role of a miRNA precursor gene ZmLRT in the regulation of lateral root number and drought tolerance in maize

Lateral roots play essential roles in drought tolerance in maize(Zea mays L.). However, the genetic basis for the variation in the number of lateral roots in maize remains elusive. Here, we identified a major quantitative trait locus(QTL),q LRT5-1, controlling lateral root number using a recombinant inbred population from a cross between the maize lines Zong3(with many lateral roots) and 87-1(with few lateral roots).Fine-mapping and functional analysis determined that the candidate gene for qLRT...     

Candidate gene studies in the 21st century: meta-analysis, mediation, moderation

The results of a large body of candidate gene studies of behavioural and psychiatric phenotypes have been largely inconclusive, with most findings failing to replicate reliably. A variety of approaches that augment the 'traditional' candidate gene approach are discussed, including the use of meta-analysis to combine findings from existing published reports, the investigation of mediating variables (including the use of intermediate phenotypes or endophenotypes) and the awareness of possible moderating influences (such as sex or ethnicity) and gene–environment interactions on genetic associations, possibly via epigenetic mechanisms. Advances in genotyping technology will also allow the routine use of haplotype analysis and linkage disequilibrium mapping. Examples of how these approaches may improve our understanding of how genetic associations with behavioural and psychiatric phenotypes obtain are given.     

Ivr2, a candidate gene for a QTL of vacuolar invertase activity in maize leaves. Gene-specific expression under water stress

Water shortage produced an early and large stimulation of acid- soluble invertase activity in adult maize leaves whereas cell wall invertase activity remained constant. This response was closely related to the mRNA level for only one of the invertase gene (Ivr2), encoding a vacuolar isoform. In parallel, four quantitative trait loci (QTLs) were detected for invertase activity under control and nine under stressful conditions. One QTL in control and one in stressed plants was located near to the lvr2 gene on chromosome 5. Other QTLs for invertase activity were found close to carbohydrate QTLs; some of them formed stress clusters.     

Mapping of quantitative trait loci controlling adaptive traits in coastal Douglas-fir. IV. Cold-hardiness QTL verification and candidate gene mapping

Quantitative trait locus (QTL) analyses are used by geneticists to characterize the genetic architecture of quantitative traits, provide a foundation for marker-aided-selection (MAS), and provide a framework for positional selection of candidate genes. The most useful QTL for breeding applications are those that have been verified in time, space, and/or genetic background. In this study, spring cold-hardiness of Douglas-fir foliar tissues was evaluated in two clonally replicated (n=170 and 383 clones) full-sib cohorts derived from the same parental cross in two different years (made 5 years apart). The cohorts were established in widely separated forest test sites and tissues were artificially freeze tested using different cold injury assessment methods. Four of six unique QTL detected for spring cold-hardiness in needles of Cohort 1 were tentatively verified in the second cohort. Four additional QTL were detected in Cohort 2, two on linkage groups (LGs) not previously represented in the smaller cohort. In total, 10 unique QTL were identified across both cohorts. Seventeen of twenty-nine putative cold-hardiness candidate genes (Douglas-fir ESTs) placed on the Douglas-fir linkage map locate within the 95% confidence intervals of spring needle cold-hardiness QTL from the two cohorts and thus represent priority targets for initiating association mapping in Douglas-fir.     

Pollen-mediated gene flow in maize in real situations of coexistence

We present the first study on cross-fertilization between Bt and conventional maize in real situations of coexistence in two regions in which Bt and conventional maize were cultivated. A map was designed and the different crops were identified, as were the sowing and flowering dates, in Bt and conventional maize fields. These data were used to choose the non-transgenic fields for sampling and analysis by the real-time quantification system-polymerase chain reaction (RTQ-PCR) technique. In general, the rate of cross-fertilization was higher in the borders and, in most of the fields, decreased towards the centre of the field. Nine fields had values of genetically modified organism DNA to total DNA of much lower than 0.9%, whereas in three the rate was higher. Some differences were found when comparing our results with those of common field trials. In real conditions of coexistence and in cropping areas with smaller fields, the main factors that determined cross-pollination were the synchronicity of flowering and the distances between the donor and receptor fields. By establishing an index based on these two variables, the rate of the adventitious presence of genetically modified maize could be predicted, as well as the influence of other factors. By applying this index, and in the case of a fully synchronous flowering time, a security distance between transgenic and conventional fields of about 20 m should be sufficient to maintain the adventitious presence of genetically modified organisms as a result of pollen flow below the 0.9% threshold in the total yield of the field.     

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.     

A gene for resistance to the maize streak virus in the African CIMMYT maize inbred line CML202

Resistance to maize streak virus (MSV) is an essential trait of improved maize varieties in sub-Saharan Africa. We mapped quantitative trait loci (QTL) for resistance to MSV in a population of 196 F_2:3 lines derived from a cross between the maize inbred lines CML202 (resistant) from CIMMYT-Zimbabwe and Lo951 (susceptible) from Italy. Field tests were planted at two locations in Zimbabwe, inoculated with viruliferous leaf hoppers (Cicadulina mbila), and scored twice (21 and 83 days after infesting, DAI) on a 1–5 scale. The mean final streak intensity (score 2) of the parent lines was 2.2 (CML202) and 4.8 (Lo951). Genotype × location interaction was large for score 1 but negligible for score 2. Consequently, the heritability was higher for score 2 (0.93) than for score 1 (0.62). By composite interval mapping across locations, using a linkage map with 110 RFLP loci, four significant (LOD 3.0) QTL were identified for score 1 on chromosomes (C) 1, 2, 3, and 4, respectively. All four were contributed by CML202. For score 2, only the QTL on C 1 was significant (LOD =37), explaining 59% of the phenotypic and 64% of the genotypic variance. The QTL's partially dominant gene action was consistent with the nearly intermediate resistance of the F₁ generation (relative heterosis for resistance 12%). The presence of one major QTL is consistent with the bimodal frequency distribution of the mapping population showing a clear 3:1 segregation. This gene seems to be allelic or identical to Msv1, a major resistance gene which was previously identified in the same genomic region in Tzi4, an inbred line from IITA. Inbred CML202 had lower final disease ratings than Tzi4. The greater resistance of CML202 may be due to allelic differences at the msv1 locus or due to the minor QTL on C 2, 3, and 4 which were not detected in Tzi4.z y Trigo (International Maize and Wheat Improvement Center); IITA, International Institute of Tropical Agriculture; IRAT, Institute de Recherches Agronomiques Tropicales et des Cultures Vivrières; KARI, Kenya Agricultural Research Institute; MSV, maize streak virus; QTL, quantitative trait locus/loci     

农杆菌及基因枪在玉米遗传转化中的应用

目前外源基因导入玉米受体细胞的方法很多 ,最受瞩目的是农杆菌介导法和基因枪法。就农杆菌、基因枪转化玉米的基本原理、影响转化率的因素 ,以及近年来在玉米遗传转化中的最新动态进行了综述     

Sequential gene activation and gene imprinting during early embryo development in maize

Gene imprinting is a widely observed epigenetic phenomenon in maize endosperm; however, whether it also occurs in the maize embryo remains controversial. Here, we used high‐throughput RNA sequencing on laser capture microdissected and manually dissected maize embryos from reciprocal crosses between inbred lines B73 and Mo17 at six time points (3–13 days after pollination, DAP) to analyze allelic gene expression patterns. Co‐expression analysis revealed sequential gene activation during maize embryo development. Gene imprinting was observed in maize embryos, and a greater number of imprinted genes were identified at early embryo stages. Sixty‐four strongly imprinted genes were identified (at the threshold of 9:1) on manually dissected embryos 5–13 DAP (more imprinted genes at 5 DAP). Forty‐one strongly imprinted genes were identified from laser capture microdissected embryos at 3 and 5 DAP (more imprinted genes at 3 DAP). Furthermore, of the 56 genes that were completely imprinted (at the threshold of 99:1), 36 were not previously identified as imprinted genes in endosperm or embryos. In situ hybridization demonstrated that most of the imprinted genes were expressed abundantly in maize embryonic tissue. Our results shed lights on early maize embryo development and provide evidence to support that gene imprinting occurs in maize embryos.     

Genetic analysis of tolerance to low-phosphorus stress in maize using restriction fragment length polymorphisms

Summary An understanding of the genetic nature underlying tolerance to low-phosphorus (low-P) stress could aid in the efficient development of tolerant plant strains. The objective of this study was to identify the number of loci in a maize (Zea mays L.) population segregating for tolerance to low-P stress, their approximate location, and the magnitude of their effect.Seventy-seven restriction fragment length polymorphisms (RFLPs) were identified and scored in a maize F₂ population derived from a cross between line NY821 and line H99. The F₂ individuals were self-pollinated to produce F₃ families. Ninety F₃ families were grown in a sand-alumina system, which simulated diffusion-limited, low-P soil conditions. The F₃ families were evaluated for vegetative growth in a controlled-environment experiment. To identify quantitative trait loci (QTLs) underlying tolerance to low-P stress, the mean phenotypic performances of the F₃ families were contrasted based on genotypic classification at each of 77 RFLP marker loci.Six RFLP marker loci were significantly associated with performance under low-P stress (P<0.01). One marker locus accounted for 25% of the total phenotypic variation. Additive gene action was predominant for all of the QTLs identified. Significant marker loci were located on four separate chromosomes representing five unlinked genomic regions. Two marker loci were associated with an additive by additive epistatic interaction. A multiple regression model including three marker loci and the significant epistatic interaction accounted for 46% of the total phenotypic variation. Heterozygosity per se was not predictive of phenotypic performance.     

Two consensus quantitative trait loci clusters controlling anthesis–silking interval, ear setting and grain yield might be related with drought tolerance in maize

Unravelling the molecular basis of drought tolerance will provide novel opportunities for improving crop yield under water-limited conditions. The present study was conducted to identify quantitative trait loci (QTLs) controlling anthesis–silking interval (ASI), ear setting percentage (ESP) and grain yield (GY). The mapping population included 234 F₂ plants derived from the cross X178 (drought tolerant) × B73 (drought susceptible). The corresponding F_2:3 progenies, along with their parents, were evaluated for the above-mentioned traits under both well-watered and water-stressed field conditions in three different trials carried out in central and southern China. Interval mapping and composite interval mapping identified 45 and 65 QTLs for the investigated traits, respectively. Two QTL clusters influencing ASI and ESP on chromosomes 1 (bin 1.03) and 9 (bins 9.03–9.05) were identified in more than two environments, showing sizeable additive effects and contribution to phenotypic variance; these two QTL clusters influenced GY only in one environment. No significant interaction was detected between the two genomic regions. A comparative analysis of these two QTL clusters with the QTLs controlling maize drought tolerance previously described in three mapping populations confirmed and extended their relevance for marker-assisted breeding to improve maize production under water-limited conditions.     

Combined GWAS and QTL mapping revealed candidate genes and SNP network controlling recovery and tolerance traits associated with drought tolerance in seedling winter wheat

To date, very little research on drought tolerance has been conducted at the seedling stage in winter wheat. In this study, two types of traits, namely tolerance and recovery traits, associated with drought tolerance were scored in biparental mapping population (BPP) and association mapping population (A-set). The results of this study revealed no or weak significant correlation between the two types of traits. Based on GWAS and QTL mapping analyses, all QTLs associated with recovery traits were completely different from those associated with tolerance traits except one QTL in each population that was found to be associated with one tolerance trait and one recovery trait. The analysis of SNP and gene networks confirmed the results of combined GWAS and QTL mapping. One SNP marker located on the 2B chromosome (S2B_26494801) was found to be associated with recovery traits in both populations. The results of this study provided new information on understanding and improving drought tolerance in winter wheat.