Application of fluorescence-based semi-automated AFLP analysis in barley and wheat

Genetic mapping and the selection of closely linked molecular markers for important agronomic traits require efficient, large-scale genotyping methods. A semi-automated multifluorophore technique was applied for genotyping AFLP marker loci in barley and wheat. In comparison to conventional ³³P-based AFLP analysis the technique showed a higher resolution of amplicons, thus increasing the number of distinguishable fragments. Automated sizing of the same fragment in different lanes or different gels showed high conformity, allowing subsequent unambigous allele-typing. Simultaneous electrophoresis of different AFLP samples in one lane (multimixing), as well as simultaneous amplification of AFLP fragments with different primer combinations in one reaction (multiplexing), displayed consistent results with respect to fragment number, polymorphic peaks and correct size-calling. The accuracy of semi-automated co-dominant analysis for hemizygous AFLP markers in an F₂ population was too low, proposing the use of dominant allele-typing defaults. Nevertheless, the efficiency of genetic mapping, especially of complex plant genomes, will be accelerated by combining the presented genotyping procedures. Received: 10 April 1999 / Accepted: 11 May 1999     

High-throughput identification of genetic markers using representational oligonucleotide microarray analysis

We describe a novel approach for high-throughput development of genetic markers using representational oligonucleotide microarray analysis. We test the performance of the method in sugar beet (Beta vulgaris L.) as a model for crop plants with little sequence information available. Genomic representations of both parents of a mapping population were hybridized on microarrays containing in total 146,554 custom made oligonucleotides based on sugar beet bacterial artificial chromosome (BAC) end sequences and expressed sequence tags (ESTs). Oligonucleotides showing a signal with one parental line only, were selected as potential marker candidates and placed onto an array, designed for genotyping of 184 F₂ individuals from the mapping population. Utilizing known co-dominant anchor markers we obtained 511 new dominant markers (392 derived from BAC end sequences, and 119 from ESTs) distributed over all nine sugar beet linkage groups and calculated genetic maps. Further improvements for large-scale application of the approach are discussed and its feasibility for the cost-effective and flexible generation of genetic markers is presented.     

A simple note on how to save money in linkage analysis

Genetic linkage maps are often based on maximum-likelihood estimates of recombination fractions which are converted into map units by mapping functions. This paper presents a cost analysis of linkage analysis for a segregating F₂␣population with codominant or dominant molecular markers and a qualitative monogenic dominant–recessive trait. For illustration, a disease-resistance trait is considered, where the susceptible allele is recessive. Three sub-populations of the F₂ can be used for linkage analysis [susceptible (= recessive) individuals, resistant (= dominant) individuals, complete F₂]. While it is well-known that recessive individuals are more informative than dominant individuals, it is not obvious a priori, which of the three sub-populations should be preferred, when costs of phenotyping and genotyping are taken into consideration. A comparative economic analysis of alternative procedures of linkage detection based on these three sub-populations does exhibit a clear economic superiority of the sub-population of susceptible (= recessive) individuals, when costs of genotyping are high. This cost-effectiveness is due to the higher information content of this sub-population compared to the sub-population of dominant (= resistant) individuals and also compared to the complete F₂. Our final conclusion/recommendation is as follows: If the cost to genotype an individual is sufficiently large compared with the cost to phenotype an individual, then linkage analysis and genetic mapping should be only based on susceptible (= recessive) individuals. Conversely, if the cost of phenotyping exceeds that for genotyping, it may be preferable to genotype all plants. The exact conditions under which a strategy is preferable are described in the paper.     

Selective genotyping for determination of linkage between a marker locus and a quantitative trait locus

Summary Selective genotyping is the term used when the determination of linkage between marker loci and quantitative trait loci (QTL) affecting some particular trait is carried out by genotyping only individuals from the high and low phenotypic tails of the entire sample population. Selective genotyping can markedly decrease the number of individuals genotyped for a given power at the expense of an increase in the number of individuals phenotyped. The optimum proportion of individuals genotyped from the point of view of minimizing costs for a given experimental power depends strongly on the cost of completely genotyping an individual for all of the markers included in the experiment (including the costs of obtaining a DNA sample) relative to the cost of rearing and trait evaluation of an individual. However, in single trait studies, it will almost never be useful to genotype more than the upper and lower 25% of a population. It is shown that the observed difference in quantitative trait values associated with alternative marker genotypes in the selected population can be much greater than the actual gene effect at the quantitative trait locus when the entire population is considered. An expression and a figure is provided for converting observed differences under selective genotyping to actual gene effects.     

High-resolution mapping of quantitative trait loci by selective recombinant genotyping

Selective recombinant genotyping (SRG) is a three-stage procedure for high-resolution mapping of a QTL that has previously been mapped to a known confidence interval (target C.I.). In stage 1, a large mapping population is accessed and phenotyped, and a proportion, P, of the high and low tails is selected. In stage 2, the selected individuals are genotyped for a pair of markers flanking the target C.I., and a group of R individuals carrying recombinant chromosomes in the target interval are identified. In stage 3, the recombinant individuals are genotyped for a set of M markers spanning the target C.I. Extensive simulations showed that: (1) Standard error of QTL location (SEQTL) decreased when QTL effect (d) or population size (N) increased, but was constant for given "power factor" (PF = d(2)N); (2) increasing the proportion selected in the tails beyond 0.25 had only a negligible effect on SEQTL; and (3) marker spacing in the target interval had a remarkably powerful effect on SEQTL, yielding a reduction of up to 10-fold in going from highest (24 cM) to lowest (0.29 cM) spacing at given population size and QTL effect. At the densest marker spacing, SEQTL of 1.0-0.06 cM were obtained at PF = 500-16,000. Two new genotyping procedures, the half-section algorithm and the golden section/half-section algorithm, allow the equivalent of complete haplotyping of the target C.I. in the recombinant individuals to be achieved with many fewer data points than would be required by complete individual genotyping.     

Statistical recovering of incomplete genotypic information of DNA molecular markers

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Algorithms for selecting informative marker panels for population assignment.

Given a set of potential source populations, genotypes of an individual of unknown origin at a collection of markers can be used to predict the correct source population of the individual. For improved efficiency, informative markers can be chosen from a larger set of markers to maximize the accuracy of this prediction. However, selecting the loci that are individually most informative does not necessarily produce the optimal panel. Here, using genotypes from eight species--carp, cat, chicken, dog, fly, grayling, human, and maize--this univariate accumulation procedure is compared to new multivariate "greedy" and "maximin" algorithms for choosing marker panels. The procedures generally suggest similar panels, although the greedy method often recommends inclusion of loci that are not chosen by the other algorithms. In seven of the eight species, when applied to five or more markers, all methods achieve at least 94% assignment accuracy on simulated individuals, with one species--dog--producing this level of accuracy with only three markers, and the eighth species--human--requiring approximately 13-16 markers. The new algorithms produce substantial improvements over use of randomly selected markers; where differences among the methods are noticeable, the greedy algorithm leads to slightly higher probabilities of correct assignment. Although none of the approaches necessarily chooses the panel with optimal performance, the algorithms all likely select panels with performance near enough to the maximum that they all are suitable for practical use.     

Development of a simple functional marker for fragrance in rice and its validation in Indian Basmati and non-Basmati fragrant rice varieties

Fragrance development in rice has been reported due to a 8-bp deletion in the exon 7 of badh2 gene located on Chromosome 8S. Multiplex markers targeting the functional InDel polymorphism was earlier reported for genotyping fragrance trait, but the marker was observed to be inconsistent and difficult to use. We have developed a simple, co-dominant, functional marker for fragrance trait, which can be resolved in an agarose gel and validated in Basmati and non-Basmati aromatic rice varieties and in a mapping population segregated for fragrance trait. The marker targets the InDel polymorphism in badh2 gene and amplifies 95 and 103 bp fragments in fragrant and non-fragrant genotypes, respectively. The newly developed marker was highly efficient in discriminating all fragrant and non-fragrant genotypes and showed perfect co-segregation with the trait of fragrance in the mapping population. We recommend the use of this simple, low-cost marker in routine genotyping for fragrance trait in large scale breeding materials and germplasm.     

水杉人工林建植50年后的分化特征

通过种群统计学、格局分析和树干解析等对50 a生水杉人工林林分的分化特征进行了研究.结果表明:小个体林木占有相对多数(>50.4%);林木分化现象显著,特别是树高与材积的分化十分突出,林分已出现一定强度的自然稀疏;基于TSTRAT程式的林木分级结果表明,亚优势木占绝对优势(45.7%),其树冠构成该林分的主林冠;竞争指数依次为优势木级<亚优势木级<中庸木级<被压木级,各级的基尼系数基本上低于全林总计;优势木绝对生长率和相对生长率在林分成熟期以前均大于标准木,且胸径速生期持续时问较标准木长;林分高度生长受压抑程度低于胸径生长;林木分布格局总体呈均匀型,与造林初始基本一致;优势木和被压木为聚集分布,中庸木和枯死木为随机散布,亚优势木的分布型则介于随机和聚集之间.林木种群分化既表现在个体大小的不均等性上,也表现在空间分布的非匀质性上.目前林分结构特征的主要成因可以归结为密度压力下的种内竞争.     

Selective DNA Pooling for Determination of Linkage between a Molecular Marker and a Quantitative Trait Locus

Selective genotyping is a method to reduce costs in marker-quantitative trait locus (QTL) linkage determination by genotyping only those individuals with extreme, and hence most informative, quantitative trait values. The DNA pooling strategy (termed: ``selective DNA pooling') takes this one step further by pooling DNA from the selected individuals at each of the two phenotypic extremes, and basing the test for linkage on marker allele frequencies as estimated from the pooled samples only. This can reduce genotyping costs of marker-QTL linkage determination by up to two orders of magnitude. Theoretical analysis of selective DNA pooling shows that for experiments involving backcross, F(2) and half-sib designs, the power of selective DNA pooling for detecting genes with large effect, can be the same as that obtained by individual selective genotyping. Power for detecting genes with small effect, however, was found to decrease strongly with increase in the technical error of estimating allele frequencies in the pooled samples. The effect of technical error, however, can be markedly reduced by replication of technical procedures. It is also shown that a proportion selected of 0.1 at each tail will be appropriate for a wide range of experimental conditions.     

基于简化基因组数据开发岭南青冈微卫星引物

微卫星标记（simple sequence repeat，SSR）在基因组中普遍存在，具有共显性、高多态性等特点，在群体空间遗传研究中应用广泛。随着测序技术的发展，SSR的开发方法也更加多样化。岭南青冈（Quercus championii Benth）是中国南方常绿阔叶林的珍贵材用树种，对其分子标记的开发可促进岭南青冈的育种和种质保护。本研究基于4个岭南青冈个体的简化基因组序列进行SSR引物的开发。使用pyRAD软件分析显示：①每个个体中包含微卫星重复片段的序列在46 000~84 000条；②个体内聚类后得到的位点数在5 500~24 000；③个体间聚类后获得1 158个一致性位点。在1 158个位点中获得186个侧翼序列没有变异且重复碱基位于序列中间位置的位点。使用Primer Premier 5.0设计了25对引物，在来自3个群体的36个岭南青冈个体中进行验证，结果表明17对引物能成功扩增，共获得106个等位位点，每个引物的等位基因数在2~12，平均为6.2。引物的期望杂合度和观测杂合度分别为0.19~0.88和0.11~0.76。本研究的引物开发方法具有速度快、效率高、成本低的特点，可应用于群体遗传学分子标记的开发。     

Mapping quantitative trait loci with dominant markers in four-way crosses

 A common problem in mapping quantitative trait loci (QTLs) is that marker data are often incomplete. This includes missing data, dominant markers, and partially informative markers, arising in outbred populations. Here we briefly present an iteratively re-weighted least square method (IRWLS) to incorporate dominant and missing markers for mapping QTLs in four-way crosses under a heterogeneous variance model. The algorithm uses information from all markers in a linkage group to infer the QTL genotype. Monte Carlo simulations indicate that with half dominant markers, QTL detection is almost as efficient as with all co-dominant markers. However, the precision of the estimated QTL parameters generally decreases as more markers become missing or dominant. Notable differences are observed on the standard deviation of the estimated QTL position for varying levels of marker information content. The method is relatively simple so that more complex models including multiple QTLs or fixed effects can be fitted. Finally, the method can be readily extended to QTL mapping in full-sib families. Received: 16 June 1998 / Accepted: 29 September 1998     

Optimal designs for linkage disequilibrium mapping and candidate gene association tests in livestock populations

Simulation was used to evaluate the performance of different selective genotyping strategies when using linkage disequilibrium across large half-sib families to position a QTL within a previously defined genomic region. Strategies examined included standard selective genotyping and different approaches of discordant and concordant sib selection applied to arbitrary or selected families. Strategies were compared as a function of effect and frequency of QTL alleles, heritability, and phenotypic expression of the trait. Large half-sib families were simulated for 100 generations and 2% of the population was genotyped in the final generation. Simple ANOVA was applied and the marker with the greatest F-value was considered the most likely QTL position. For traits with continuous phenotypes, genotyping the most divergent pairs of half-sibs from all families was the best strategy in general, but standard selective genotyping was somewhat more precise when heritability was low. When the phenotype was distributed in ordered categories, discordant sib selection was the optimal approach for positioning QTL for traits with high heritability and concordant sib selection was the best approach when genetic effects were small. Genotyping of a few selected sibs from many families was generally more efficient than genotyping many individuals from a few highly selected sires.     

Selection for malathion-resistance in Drosophila melanogaster

Results from a long-term selection experiment for malathion-resistance in Drosophila melanogaster are described. A polled population of 40 locally-caught, iso-female lines was exposed to increasing concentrations of malathion in the food at both a high selection intensity (MH) and a lower intensity (ML). The response was consistent with a polygenic system. Both adult and larval resistance increased in parallel. Changes in the dose-response curve of adults could be approximately described by a dose-modification factor. Larval resistance was more complex; both selected populations showed a maternal effect which could not be explained by sex-linked genes. Larval resistance in the selected populations behaved as a co-dominant trait with respect to the susceptible controls. Adult resistance was dominant in the ML and co-dominant in the MH population, suggesting that different genes conferring resistance were selected. The selection procedure also produced a developmental delay in both populations, dependent on malathion concentration, but present even in its absence.     

Parentage and Sibship Inference From Multilocus Genotype Data Under Polygamy

Likelihood methods have been developed to partition individuals in a sample into sibling clusters using genetic marker data without parental information. Most of these methods assume either both sexes are monogamous to infer full sibships only or only one sex is polygamous to infer full sibships and paternal or maternal (but not both) half sibships. We extend our previous method to the more general case of both sexes being polygamous to infer full sibships, paternal half sibships, and maternal half sibships and to the case of a two-generation sample of individuals to infer parentage jointly with sibships. The extension not only expands enormously the scope of application of the method, but also increases its statistical power. The method is implemented for both diploid and haplodiploid species and for codominant and dominant markers, with mutations and genotyping errors accommodated. The performance and robustness of the method are evaluated by analyzing both simulated and empirical data sets. Our method is shown to be much more powerful than pairwise methods in both parentage and sibship assignments because of the more efficient use of marker information. It is little affected by inbreeding in parents and is moderately robust to nonrandom mating and linkage of markers. We also show that individually much less informative markers, such as SNPs or AFLPs, can reach the same power for parentage and sibship inferences as the highly informative marker simple sequence repeats (SSRs), as long as a sufficient number of loci are employed in the analysis.     

Comparing the distribution of RAPD and RFLP markers in a high density linkage map of sugar beet.

The distribution of RAPD markers was compared with that of RFLP markers in a high density linkage map of sugar beet. The same mapping population of 161 F2 individuals was used to generate all the marker data. The total map comprises 160 RAPD and 248 RFLP markers covering 508 cM. Both the RAPD and the RFLP markers show a high degree of clustering over the nine linkage groups. The pattern is compatible with a strong distal localization of recombination in the sugar beet. It leads generally to one major cluster of markers in the centre of each linkage group. In regions of high marker density, dominant RAPD markers present in either linkage phase and codominant RFLP markers are subclustered relative to each other. This phenomenon is shown to be attributable to: (i) effects of the mapping procedure when dominant and codominant data are combined, (ii) effects of the mapping procedure when dominant data in both linkage phases are combined, and (iii) genuine differences in the way RAPD and RFLP markers are recruited.     

Efficient fine mapping of the naked caryopsis gene (<Emphasis Type="Italic">nud</Emphasis>) by HEGS (High Efficiency Genome Scanning)/AFLP in barley

The hulled or naked caryopsis character of barley (Hordeum vulgare L.) is an important trait for edibility and to follow its domestication process. A single recessive gene, nud, controls the naked caryopsis character, and is located on the long arm of chromosome 7H. To develop a fine map around the nud locus efficiently, the HEGS (High Efficiency Genome Scanning) electrophoresis system was combined with amplified fragment length polymorphism (AFLP). From bulked segregant analysis of 1,894 primer combinations, 12 AFLP fragments were selected as linked markers. For mapping, an F₂ population of 151 individuals derived from a cross between Kobinkatagi (naked type) and Triumph (hulled type) was used. Seven AFLP markers were localized near the nud region. A fine map was developed with one-order higher resolution than before, along with the seven anchor markers. Among the seven linked AFLP markers (KT1–7), KT1, KT2 and KT6 were co-dominant, and the former two were detected for their single-nucleotide polymorphisms (SNPs) in the same length of fragments after electrophoresis with the non-denaturing gels of HEGS. The nud locus has co-segregated with KT3 and KT7, and was flanked by KT2 and KT4, at the 0.3-cM proximal and the 1.2-cM distal side, respectively. Four of these AFLP markers were converted into sequence-characterized amplified region (SCAR) markers, one of which was a dominant marker co-segregating with the nud gene.Communicated by G. Wenzel     

Development of a sequence-specific PCR marker linked to the gene “pauper” conferring low-alkaloids in white lupin (Lupinus albus L.) for marker assisted selection

Seeds and plants of wild type Lupinus albus are bitter and contain high level of alkaloids. During domestication, at least three genes conferring low-alkaloid content were identified and incorporated into commercial varieties. Australian lupin breeders exclusively utilize one of these sweetness genes, “pauper”, in all varieties to prevent possible bitterness contamination via out-crossing. A cross was made between a sweet variety Kiev Mutant (containing pauper gene) and a bitter type landrace P27174, and the population was advanced into F₈ recombinant inbred lines (RILs). Twenty-four plants representing sweetness and bitterness were subjected to DNA fingerprinting by the microsatellite-anchored fragment length polymorphism (MFLP) technique. A dominant polymorphism was discovered in an MFLP fingerprint. The MFLP marker was converted into a co-dominant, sequence-specific, simple PCR-based marker. Linkage analysis by the software program MapManager with marker score data and alkaloid phenotyping data from a segregating population containing 190 F₈ RILs indicated that the marker is linked to the pauper gene at the genetic distance of 1.4 centiMorgans (cM). This marker, which is designated as “PauperM1”, is capable of distinguishing the pauper gene from the other two low-alkaloid genes exiguus and nutricius. Validation on germplasm from the Australian lupin breeding program showed that the banding pattern of the marker PauperM1 is consistent with the alkaloid genotyping on a wide range of domesticated varieties and breeding lines. The PauperM1 marker is now being implemented for marker assisted selection in the Australian albus lupin breeding program.     

Linkage analysis between dominant and co-dominant makers in full-sib families of out-breeding species

As high-throughput genomic tools, such as the DNA microarray platform, have lead to the development of novel genotyping procedures, such as Diversity Arrays Technology (DArT) and Single Nucleotide Polymorphisms (SNPs), it is likely that, in the future, high density linkage maps will be constructed from both dominant and co-dominant markers. Recently, a strictly genetic approach was described for estimating recombination frequency (r) between co-dominant markers in full-sib families. The complete set of maximum likelihood estimators for r in full-sib families was almost obtained, but unfortunately, one particular configuration involving dominant markers, segregating in a 3:1 ratio and co-dominant markers, was not considered. Here we add nine further estimators to the previously published set, thereby making it possible to cover all combinations of molecular markers with two to four alleles (without epistasis) in a full-sib family. This includes segregation in one or both parents, dominance and all linkage phase configurations.     

利用分子标记分析遗传多样性时的玉米群体取样策略研究

利用分子标记技术对玉米种质资源进行遗传多样性分析对种质资源的保存和利用具有重要的指导意义。但是,在对地方品种和育种群体这些开放授粉群体进行大规模遗传多样性分析时,取样方法将会严重影响到研究结果和工作效率。本研究用2个育种群体和3个地方品种为试材,利用微卫星(SSR)标记对每个群体100个个体及其组成的不同随机混合样品进行了分子检测。结果表明,不同群体的群体内遗传变异大小存在差异;相同数目的个体随机混合的不同样品间的检测结果基本相同;不同数目的个体混合的样品间存在一定程度的差异,并且与材料本身的遗传变异大小有一定关系。考虑到结果的科学性和工作的可行性,建议在利用分子标记(如SSR)进行地方品种和育种群体的遗传多样性评估时,随机选取30个个体组成混合样(或用15个个体组成2个混合样)来代表1个地方品种或育种群体进行分子鉴定。