Mapping of markers related to self-incompatibility, disease resistance, and quality traits in Lolium perenne L

Several linkage maps, mainly based on anonymous markers, are now available for Lolium perenne. The saturation of these maps with markers derived from expressed sequences would provide information useful for QTL mapping and map alignment. Therefore, we initiated a study to develop and map DNA markers in genes related to self-incompatibility, disease resistance, and quality traits such as digestibility and sugar content in two L. perenne families. In total, 483 and 504 primer pairs were designed and used to screen the ILGI and CLO-DvP mapping populations, respectively, for length polymorphisms. Finally, we were able to map 67 EST markers in at least one mapping population. Several of these markers coincide with previously reported QTL regions for the traits considered or are located in the neighbourhood of the self-incompatibility loci, S and Z. The markers developed expand the set of gene-derived markers available for genetic mapping in ryegrasses.     

Transmission ratio distortion in intraspecific hybrids of Mimulus guttatus: implications for genomic divergence

We constructed a genetic linkage map between two divergent populations of Mimulus guttatus. We genotyped an F(2) mapping population (N = 539) at 154 AFLP, microsatellite, and gene-based markers. A framework map was constructed consisting of 112 marker loci on 14 linkage groups with a total map length of 1518 cM Kosambi. Nearly half of all markers (48%) exhibited significant transmission ratio distortion (alpha = 0.05). By using a Bayesian multipoint mapping method and visual inspection of significantly distorted markers, we detected 12 transmission ratio distorting loci (TRDL) throughout the genome. The high degree of segregation distortion detected in this intraspecific map indicates substantial genomic divergence that perhaps suggests genomic incompatibilities between these two populations. We compare the pattern of transmission ratio distortion in this map to an interspecific map constructed between M. guttatus and M. nasutus. A similar level of segregation distortion is detected in both maps. Collinear regions between maps are compared to determine if there are shared genetic patterns of non-Mendelian segregation distortion within and among Mimulus species.     

Identification of perennial ryegrass (Lolium perenne (L.)) and meadow fescue (Festuca pratensis (Huds.)) candidate orthologous sequences to the rice Hd1(Se1) and barley HvCO1 CONSTANS-like genes through comparative mapping and microsynteny

Microsynteny with rice and comparative genetic mapping were used to identify candidate orthologous sequences to the rice Hd1(Se1) gene in Lolium perenne and Festuca pratensis. A F. pratensis bacterial artificial chromosome (BAC) library was screened with a marker (S2539) physically close to Hd1 in rice to identify the equivalent genomic region in F. pratensis. The BAC sequence was used to identify and map the same region in L. perenne. Predicted protein sequences for L. perenne and F. pratensis Hd1 candidates (LpHd1 and FpHd1) indicated they were CONSTANS-like zinc finger proteins with 61-62% sequence identity with rice Hd1 and 72% identity with barley HvCO1. LpHd1 and FpHd1 were physically linked in their respective genomes (< 4 kb) to marker S2539, which was mapped to L. perenne chromosome 7. The identified candidate orthologues of rice Hd1 and barley HvCO1 in L. perenne and F. pratensis map to chromosome 7, a region of the L. perenne genome which has a degree of conserved genetic synteny both with rice chromosome 6, which contains Hd1, and barley chromosome 7H, which contains HvCO1.     

Use of amplified fragment length polymorphism markers to assess genetic diversity of Lolium species from Portugal

We evaluated the use of amplified fragment length polymorphism (AFLP) markers to distinguish genotypes, populations and species of Lolium. Accessions of two species Lolium perenne and Lolium multiflorum and their hybrid Lolium x hybridum, collected by the Institute of Grass and Environmental Research in 1995 from locations across Portugal, were used. The genetic variation within and between populations from the extremes of latitude and altitude was determined and assessed. Three primer pair combinations generated 765 polymorphic bands. Principal coordinate analysis of similarities between 127 plants showed high dimensionality in the data. Axes 1-3 were associated primarily with species differences, axes 4-14 with population differences within species and axis 15 onwards with within population differences. UPGMA analysis confirmed the groupings. The three populations of L. perenne formed a discrete cluster widely separated from all other populations. There were two distinct groups of L. x hybridum, of which one was similar to and overlapped with L. multiflorum and the second formed a distinct cluster. Analyses of individual bands showed that every inter- and intraspecific contrast involved a different sets of bands, again confirming the high dimensionality of the data. No single band was strictly diagnostic of any population or species. Nevertheless, the UPGMA analysis showed little or no overlap between populations. Thus, despite the high ratio of within-to-between population genetic variance, the full AFLP banding pattern of each genotype is a relatively reliable fingerprint diagnostic of its parent population. The high dimensionality implies that many different factors contribute to the differences observed. This adds to the potential value of the methodology, since it implies that there is a reasonably high likelihood of finding bands relevant to a given environmental gradient or other factor influencing the distribution of genetic diversity.     

Prevalence of segregation distortion in diploid alfalfa and its implications for genetics and breeding applications

Segregation distortion (SD) is often observed in plant populations; its presence can affect mapping and breeding applications. To investigate the prevalence of SD in diploid alfalfa (Medicago sativa L.), we developed two unrelated segregating F₁ populations and one F₂ population. We genotyped all populations with SSR markers and assessed SD at each locus in each population. The three maps were syntenic and largely colinear with the Medicago truncatula genome sequence. We found genotypic SD for 24 and 34% of markers in the F₁ populations and 68% of markers in the F₂ population; distorted markers were identified on every linkage group. The smaller percentage of genotypic SD in the F₁ populations could be because they were non-inbred and/or due to non-fully informative markers. For the F₂ population, 60 of 90 mapped markers were distorted, and they clustered into eight segregation distortion regions (SDR). Most SDR identified in the F₁ populations were also identified in the F₂ population. Genotypic SD was primarily due to zygotic rather than allelic distortion, suggesting zygotic not gametic selection is the main cause of SD. On the F₂ linkage map, distorted markers in all SDR except two showed heterozygote excess. The severe SD in the F₂ population likely biased genetic distances among markers and possibly also marker ordering and could affect QTL mapping of agronomic traits. To reduce the effects of SD and non-fully informative markers, we suggest constructing linkage maps and conducting QTL mapping in advanced generation populations.     

Excess heterozygosity contributes to genetic map expansion in pea recombinant inbred populations

Several plant genetic maps presented in the literature are longer than expected from cytogenetic data. Here we compare F(2) and RI maps derived from a cross between the same two parental lines and show that excess heterozygosity contributes to map inflation. These maps have been constructed using a common set of dominant markers. Although not generally regarded as informative for F(2) mapping, these allowed rapid map construction, and the resulting data analysis has provided information not otherwise obvious when examining a population from only one generation. Segregation distortion, a common feature of most populations and marker systems, found in the F(2) but not the RI, has identified excess heterozygosity. A few markers with a deficiency of heterozygotes were found to map to linkage group V (chromosome 3), which is known to form rod bivalents in this cross. Although the final map length was longer for the F(2) population, the mapped order of markers was generally the same in the F(2) and RI maps. The data presented in this analysis reconcile much of the inconsistency between map length estimates from chiasma counts and genetic data.     

不同作图群体对显隐性分子标记位点的作图效率

根据位点组合和位点的有效性,发展了一种对使用３种不同的作用图群体作图显隐性分子标记的作图效率评价方法,应用该方法所评价的结果是,双单倍体（ＤＨ）群体的作图效率最高,自交群体（Ｆ２群体）与回交群体的作图效率相同,因此使用双单倍体群体作图不仅所用费用低,而且作图速度快,但只有在极少数植物中能获得双单倍体群体,对于那些不能获得双单倍体的动植物物种而言,可使用自交群体或回交群体作图。如果作高密度的分子标记     

A highly recombined,high‐density,eight‐founder wheat MAGIC map reveals extensive segregation distortion and genomic locations of introgression segments

Multiparent Advanced Generation Intercross (MAGIC) mapping populations offer unique opportunities and challenges for marker and QTL mapping in crop species. We have constructed the first eight‐parent MAGIC genetic map for wheat, comprising 18 601 SNP markers. We validated the accuracy of our map against the wheat genome sequence and found an improvement in accuracy compared to published genetic maps. Our map shows a notable increase in precision resulting from the three generations of intercrossing required to create the population. This is most pronounced in the pericentromeric regions of the chromosomes. Sixteen percent of mapped markers exhibited segregation distortion (SD) with many occurring in long (>20 cM) blocks. Some of the longest and most distorted blocks were collinear with noncentromeric high‐marker‐density regions of the genome, suggesting they were candidates for introgression fragments introduced into the bread wheat gene pool from other grass species. We investigated two of these linkage blocks in detail and found strong evidence that one on chromosome 4AL, showing SD against the founder Robigus, is an interspecific introgression fragment. The completed map is available from http://www.niab.com/pages/id/326/Resources .     

Genetic mapping of local adaptation along the altitudinal gradient in <Emphasis Type="Italic">Abies sachalinensis</Emphasis>

Understanding the genetic bases of local adaptation in dominant conifer species is critical in predicting the impacts of rapid climate change on forest ecosystems. However, the genetic basis of adaptation is not yet fully understood due to the huge and complex genomes of conifers and the unavailability to date of suitable crossing material. In this study, we constructed a linkage map for Abies sachalinensis (2n = 24) and investigated quantitative trait loci (QTLs) associated with local adaptation along an altitudinal gradient. A segregating population of 239 seedlings was produced from a cross between two F₁ hybrids (high-altitude × low-altitude genotypes). QTL mapping of phenological and growth traits was performed using a pseudo-testcross strategy with linkage maps based on 1251 single-nucleotide polymorphism (SNP) and three simple sequence repeat (SSR) markers. Two maps consisting of 12 linkage groups with an average marker interval of ca. 3 cM were constructed for each parent. The total lengths of the maps were 1861 and 1949 cM. A permutation test identified four significant QTLs and 11 additional suggestive QTLs, with high logarithm of odds (LOD) scores (> 3.0). This is the first highly saturated linkage map produced for Abies taxa. Our results suggest that spring bud phenology is controlled by several QTLs with moderate effects. The use of the mapping population created by crossing two hybrids (high × low altitude genotypes) and numerous SNP markers enabled us to investigate the genetic basis of adaptive traits in conifer species.     

Construction of an integrated map of rose with AFLP, SSR, PK, RGA, RFLP, SCAR and morphological markers

A high-density genetic map with a number of anchor markers has been created to be used as a tool to dissect genetic variation in rose. Linkage maps for the diploid 94/1 population consisting of 88 individuals were constructed using a total of 520 molecular markers including AFLP, SSR, PK, RGA, RFLP, SCAR and morphological markers. Seven linkage groups, putatively corresponding to the seven haploid rose chromosomes, were identified for each parent, spanning 487 cM and 490 cM, respectively. The average length of 70 cM may cover more than 90% of the rose genome. An integrated map was constructed by incorporating the homologous parental linkage groups, resulting in seven linkage groups with a total length of 545 cM. The present linkage map is currently the most advanced map in rose with regard to marker density, genome coverage and with robust markers, giving good perspectives for QTL mapping and marker-assisted breeding in rose. The SSR markers, together with RFLP markers, provide good anchor points for future map alignment studies in rose and related species. Codominantly scored AFLP markers were helpful in the integration of the parental maps.     

Genetic mapping of biomass yield in three interconnected Miscanthus populations

Improving biomass yield is a major goal of Miscanthus breeding. We conducted a study on one interspecific Miscanthus sinensis × Miscanthus sacchariflorus F₁ population and two intraspecific M. sinensis F₁ populations, each of which shared a common parent. A field trial was established at Urbana, IL during spring 2011, and phenotypic data were collected in 2012 and 2013 for fourteen yield traits. Six high‐density parental genetic maps, as well as a consensus genetic map integrating M. sinensis and M. sacchariflorus, were developed via the pseudotestcross strategy for noninbred parents with ≥1214 single‐nucleotide polymorphism markers generated from restriction site‐associated DNA sequencing. We confirmed for the first time a whole‐genome duplication in M. sacchariflorus relative to Sorghum bicolor, similar to that observed previously for M. sinensis. Four quantitative trait locus (QTL) analysis methods for detecting marker‐trait associations were compared: (1) individual parental map composite interval mapping analysis, (2) individual parental map stepwise analysis, (3) consensus map single‐population stepwise analysis and (4) consensus map joint‐population stepwise analysis. These four methods detected 288, 264, 133 and 109 total QTLs, which resolved into 157, 136, 106 and 86 meta‐QTLs based on QTL congruency, respectively, including a set of 59 meta‐QTLs common to all four analysis methods. Composite interval mapping and stepwise analysis co‐identified 118 meta‐QTLs across six parental maps, suggesting high reliability of stepwise regression in QTL detection. Joint‐population stepwise analysis yielded the highest resolution of QTLs compared to the other three methods across all meta‐QTLs. Strong, frequently advantageous transgressive segregation in the three populations indicated a promising future for breeding new higher‐yielding cultivars of Miscanthus.     

A Multi-Population Consensus Genetic Map Reveals Inconsistent Marker Order among Maps Likely Attributed to Structural Variations in the Apple Genome

Genetic maps serve as frameworks for determining the genetic architecture of quantitative traits, assessing structure of a genome, as well as aid in pursuing association mapping and comparative genetic studies. In this study, a dense genetic map was constructed using a high-throughput 1,536 EST-derived SNP GoldenGate genotyping platform and a global consensus map established by combining the new genetic map with four existing reliable genetic maps of apple. The consensus map identified markers with both major and minor conflicts in positioning across all five maps. These major inconsistencies among marker positions were attributed either to structural variations within the apple genome, or among mapping populations, or genotyping technical errors. These also highlighted problems in assembly and anchorage of the reference draft apple genome sequence in regions with known segmental duplications. Markers common across all five apple genetic maps resulted in successful positioning of 2875 markers, consisting of 2033 SNPs and 843 SSRs as well as other specific markers, on the global consensus map. These markers were distributed across all 17 linkage groups, with an average of 169±33 marker per linkage group and with an average distance of 0.70±0.14 cM between markers. The total length of the consensus map was 1991.38 cM with an average length of 117.14±24.43 cM per linkage group. A total of 569 SNPs were mapped onto the genetic map, consisting of 140 recombinant individuals, from our recently developed apple Oligonucleotide pool assays (OPA). The new functional SNPs, along with the dense consensus genetic map, will be useful for high resolution QTL mapping of important traits in apple and for pursuing comparative genetic studies in Rosaceae.     

<Emphasis Type="Italic">In silico</Emphasis> genotyping of the maize nested association mapping population

Nested Association Mapping (NAM) has been proposed as a means to combine the power of linkage mapping with the resolution of association mapping. It is enabled through sequencing or array genotyping of parental inbred lines while using low-cost, low-density genotyping technologies for their segregating progenies. For purposes of data analyses of NAM populations, parental genotypes at a large number of Single Nucleotide Polymorphic (SNP) loci need to be projected to their segregating progeny. Herein we demonstrate how approximately 0.5 million SNPs that have been genotyped in 26 parental lines of the publicly available maize NAM population can be projected onto their segregating progeny using only 1,106 SNP loci that have been genotyped in both the parents and their 5,000 progeny. The challenge is to estimate both the genotype and genetic location of the parental SNP genotypes in segregating progeny. Both challenges were met by estimating their expected genotypic values conditional on observed flanking markers through the use of both physical and linkage maps. About 90%, of 500,000 genotyped SNPs from the maize HapMap project, were assigned linkage map positions using linear interpolation between the maize Accessioned Gold Path (AGP) and NAM linkage maps. Of these, almost 70% provided high probability estimates of genotypes in almost 5,000 recombinant inbred lines.     

Guidelines for genotyping in genomewide linkage studies: single-nucleotide-polymorphism maps versus microsatellite maps

Genomewide linkage scans have traditionally employed panels of microsatellite markers spaced at intervals of approximately 10 cM across the genome. However, there is a growing realization that a map of closely spaced single-nucleotide polymorphisms (SNPs) may offer equal or superior power to detect linkage, compared with low-density microsatellite maps. We performed a series of simulations to calculate the information content associated with microsatellite and SNP maps across a range of different marker densities and heterozygosities for sib pairs (with and without parental genotypes), sib trios, and sib quads. In the case of microsatellite markers, we varied density across 11 levels (1 marker every 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cM) and marker heterozygosity across 6 levels (2, 3, 4, 5, 10, or 20 equally frequent alleles), whereas, in the case of SNPs, we varied marker density across 4 levels (1 marker every 0.1, 0.2, 0.5, or 1 cM) and minor-allele frequency across 7 levels (0.5, 0.4, 0.3, 0.2, 0.1, 0.05, and 0.01). When parental genotypes were available, a map consisting of microsatellites spaced every 2 cM or a relatively sparse map of SNPs (i.e., at least 1 SNP/cM) was sufficient to extract most of the inheritance information from the map (>95% in most cases). However, when parental genotypes were unavailable, it was important to use as dense a map of markers as possible to extract the greatest amount of inheritance information. It is important to note that the information content associated with a traditional map of microsatellite markers (i.e., 1 marker every ~10 cM) was significantly lower than the information content associated with a dense map of SNPs or microsatellites. These results strongly suggest that previous linkage studies that employed sparse microsatellite maps could benefit substantially from reanalysis by use of a denser map of markers.     

Genetic analysis and genome mapping in Raphanus.

The first genetic map of the Raphanus genome was developed based on meiosis in a hybrid between Raphanus sativus (cultivated radish) and Raphanus raphanistrum (wild radish). This hybrid was used to produce a BC1 population of 54 individuals and an F2 population of 85 individuals. A total of 236 marker loci were assayed in these populations using a set of 144 informative Brassica RFLP probes previously used for genetic mapping in other crucifer species. The genetic maps derived from the BC1 and F2 populations were perfectly collinear and were integrated to produce a robust Raphanus map. Cytological observations demonstrated strict bivalent pairing in the R. sativus x R. raphanistrum hybrids. Productive pairing along the length of each chromosome was confirmed by the identification of nine extensive linkage groups and the lack of clustering of marker loci. Indeed, the distributions of both marker loci and crossovers was more random than those reported for other crop species. The genetic markers and the reference map of Raphanus will be of considerable value for future trait mapping and marker-assisted breeding in this crop, as well as in the intergenomic transfer of Raphanus genes into Brassica crops. The future benefits of comparative mapping with Arabidopsis and Brassica species are also discussed.     

Creating a saturated reference map for the apple (Malus × domestica Borkh.) genome

The availability of a high quality linkage map is essential for the detection and the analysis of quantitative traits. Such a map should cover a significant part of the genome, should be densely populated with markers, and in order to gain the maximum advantage should be transferable to populations or cultivars other than the ones on which it has been constructed. An apple genetic linkage map has been constructed on the basis of a segregating population of the cross between the cultivars Fiesta and Discovery. A total of 840 molecular markers, 475 AFLPs, 235 RAPDs, 129 SSRs and 1 SCAR, were used for the two parental maps constructed with JoinMap and spanning 1,140 cM and 1,450 cM, respectively. Large numbers of codominant markers, like SSRs, enable a rapid transfer of the map to other populations or cultivars, allowing the investigation of any chosen trait in another genetic background. This map is currently the most advanced linkage map in apple with regard to genome coverage and marker density. It represents an ideal starting point for future mapping projects in Malus since the stable and transferable SSR frame of the map can be saturated quickly with dominant AFLP markers.     

Molecular genecology of temperature response in Lolium perenne: 2. association of AFLP markers with ecogeography

Improved winter hardiness is an important breeding objective in the forage grass Lolium perenne. This is a complex trait with several components, including the ability to survive and grow at low temperature, to acclimate to cold, tolerate wind, snow cover and ice encasement. Marker-assisted selection has the potential to increase the efficiency of breeding for improved cold tolerance. Here we describe a genecological approach to identifying molecular markers that are associated with adaptation to low winter temperatures. AFLP was used to assess the genetic diversity in 29 wild populations of ryegrass (Lolium perenne) representing a pan-European temperature cline in terms of their geographical origin. A further 18 populations from a temperature cline in Bulgaria were also analysed. In addition, two varieties and five populations representing parents of mapping families currently in use at IGER were included in the analysis. Principal coordinate (PCoA) and cluster analyses of the molecular marker data showed that the Bulgarian altitude cline populations could be distinguished clearly from the other populations. Two regression analyses were carried out; one to identify AFLP markers that correlated in frequency with low mean January temperature of the geographical origin of the population, and another to identify AFLP markers correlating in frequency with the cold tolerance phenotype of the populations, as determined by LT50 values in freezing tests. In the first analysis six AFLP markers showed significant type II trends with mean January temperature, and in the second analysis 28 bands had a significant univariate relationship with the LT50 value of the accessions. In steps 2 and 3 of the stepwise analysis a further 4 and 5 bands, respectively, improved the fit significantly. The results of the two types of regression analysis are discussed in relation to ecogeography and cold tolerance phenotype of the populations.     

A high-density intervarietal map of the wheat genome enriched with markers derived from expressed sequence tags

Bread wheat (Triticum aestivum L.) is a hexaploid species with a large and complex genome. A reference genetic marker map, namely the International Triticeae Mapping Initiative (ITMI) map, has been constructed with the recombinant inbred line population derived from a cross involving a synthetic line. But it is not sufficient for a full understanding of the wheat genome under artificial selection without comparing it with intervarietal maps. Using an intervarietal mapping population derived by crossing Nanda2419 and Wangshuibai, we constructed a high-density genetic map of wheat. The total map length was 4,223.1 cM, comprising 887 loci, 345 of which were detected by markers derived from expressed sequence tags (ESTs). Two-thirds of the high marker density blocks were present in interstitial and telomeric regions. The map covered, mostly with the EST-derived markers, approximately 158 cM of telomeric regions absent in the ITMI map. The regions of low marker density were largely conserved among cultivars and between homoeologous subgenomes. The loci showing skewed segregation displayed a clustered distribution along chromosomes and some of the segregation distortion regions (SDR) are conserved in different mapping populations. This map enriched with EST-derived markers is important for structure and function analysis of wheat genome as well as in wheat gene mapping, cloning, and breeding programs.     

Four QTLs determine crown rust (Puccinia coronata f. sp. lolii) resistance in a perennial ryegrass (Lolium perenne) population

Crown rust resistance is an important selection criterion in ryegrass breeding. The disease, caused by the biotrophic fungus Puccinia coronata, causes yield losses and reduced quality. In this study, we used linkage mapping and QTL analysis to unravel the genomic organization of crown rust resistance in a Lolium perenne population. The progeny of a pair cross between a susceptible and a resistant plant were analysed for crown rust resistance. A linkage map, consisting of 227 loci (AFLP, SSR, RFLP and STS) and spanning 744 cM, was generated using the two-way pseudo-testcross approach from 252 individuals. QTL analysis revealed four genomic regions involved in crown rust resistance. Two QTLs were located on LG1 (LpPc4 and LpPc2) and two on LG2 (LpPc3 and LpPc1). They explain 12.5, 24.9, 5.5 and 2.6% of phenotypic variance, respectively. An STS marker, showing homology to R genes, maps in the proximity of LpPc2. Further research is, however, necessary to check the presence of functional R genes in this region. Synteny at the QTL level between homologous groups of chromosomes within the Gramineae was observed. LG1 and LG2 show homology with group A and B chromosomes of oat on which crown rust-resistance genes have been identified, and with the group 1 chromosomes of the Triticeae, on which leaf rust-resistance genes have been mapped. These results are of major importance for understanding the molecular background of crown rust resistance in ryegrasses. The identified markers linked to crown rust resistance have the potential for use in marker-assisted breeding.