Development and mapping of SSR markers for maize

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.     

Recombinant inbreds for molecular mapping in maize: theoretical and practical considerations

Molecular mapping is rapidly being extended to more species as one result of the current emphasis on genome initiatives. The choice of the population used for mapping can have important consequences on the efficiency and accessibility of the mapping information. Recombinant inbred lines offer certain advantages over other mapping populations for many species. These more or less permanent populations permit many geneticists to contribute to the mapping effort and to profit from each other's work. Recombinant inbred lines are used extensively in mouse genetics and have been used in maize to compile a detailed molecular map.     

Linkage group alignment of sorghum RFLP maps using a RIL mapping population.

Several molecular maps have been constructed in sorghum (Sorghum bicolor L. Moench) using a variety of probes from different grass species such as sorghum, maize, sugarcane, rice, oat, and barley. In order to enhance the utility of the existing mapping information by the sorghum research community, alignment and integration of all major molecular maps is necessary. To achieve this objective, a genetic map of 214 loci with a total map distance of 1200 cM was constructed using 98 F7 sorghum recombinant inbred lines (RILs) from a cross between two inbred lines, B35 and Tx7000. Few cDNA clones of sorghum and maize related to photosynthesis and drought stress were mapped on this map for the first time. Five major restriction fragment length polymorphism (RFLP) maps independently developed in this species were used for alignment purpose. The distributions of previously mapped markers were compared with their respective sorghum maps to align each of the linkage groups. In general, consistent linear order among markers was maintained in all the linkage maps. The successful alignment of these RFLP maps will now allow selection of a large number of markers for any region of the sorghum genome with many potential applications ranging from fine mapping and marker-assisted selection to map-based cloning for the improvement of sorghum and related species.     

Molecular mapping in tropical maize (Zea mays L.) using microsatellite markers. 1. Map construction and localization of loci showing distorted segregation

Microsatellites have become the most important class of markers for mapping procedures. Primarily based on restriction fragment length polymorphism (RFLP) markers, several molecular genetic maps of maize have been developed, mainly using temperate inbred maize lines. To characterize the level of polymorphism of microsatellite loci and construct a genetic map in tropical maize, two elite inbred lines, L-08-05F and L-14-4B, were crossed to produce 400 F(2) individuals that were used as a mapping population. A survey of 859 primer pair sequences of microsatellites was used. The polymorphism screens of each microsatellite and genotype assignment were performed using high-resolution agarose gels. About 54 % of the primer sets gave clearly scorable amplification products, 13 % did not amplify and 33 % could not be scored on agarose gels. A total of 213 polymorphic markers were identified and used to genotype the mapping population. Among the polymorphic markers, 40 showed loci deviating from expected Mendelian ratios and clusters of deviating markers were located in three chromosome regions. Non-Mendelian scoring was present in 19 markers. The final genetic map with 117 markers spanned 1634 cM in length with an average interval of 14 cM between adjacent markers.     

<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.     

Genetic dissection of intermated recombinant inbred lines using a new genetic map of maize

A new genetic map of maize, ISU-IBM Map4, that integrates 2029 existing markers with 1329 new indel polymorphism (IDP) markers has been developed using intermated recombinant inbred lines (IRILs) from the intermated B73xMo17 (IBM) population. The website http://magi.plantgenomics.iastate.edu provides access to IDP primer sequences, sequences from which IDP primers were designed, optimized marker-specific PCR conditions, and polymorphism data for all IDP markers. This new gene-based genetic map will facilitate a wide variety of genetic and genomic research projects, including map-based genome sequencing and gene cloning. The mosaic structures of the genomes of 91 IRILs, an important resource for identifying and mapping QTL and eQTL, were defined. Analyses of segregation data associated with markers genotyped in three B73/Mo17-derived mapping populations (F2, Syn5, and IBM) demonstrate that allele frequencies were significantly altered during the development of the IBM IRILs. The observations that two segregation distortion regions overlap with maize flowering-time QTL suggest that the altered allele frequencies were a consequence of inadvertent selection. Detection of two-locus gamete disequilibrium provides another means to extract functional genomic data from well-characterized plant RILs.     

Distribution of Activator (Ac) throughout the maize genome for use in regional mutagenesis

A collection of Activator (Ac)-containing, near-isogenic W22 inbred lines has been generated for use in regional mutagenesis experiments. Each line is homozygous for a single, precisely positioned Ac element and the Ds reporter, r1-sc:m3. Through classical and molecular genetic techniques, 158 transposed Ac elements (tr-Acs) were distributed throughout the maize genome and 41 were precisely placed on the linkage map utilizing multiple recombinant inbred populations. Several PCR techniques were utilized to amplify DNA fragments flanking tr-Ac insertions up to 8 kb in length. Sequencing and database searches of flanking DNA revealed that the majority of insertions are in hypomethylated, low- or single-copy sequences, indicating an insertion site preference for genic sequences in the genome. However, a number of Ac transposition events were to highly repetitive sequences in the genome. We present evidence that suggests Ac expression is regulated by genomic context resulting in subtle variations in Ac-mediated excision patterns. These tr-Ac lines can be utilized to isolate genes with unknown function, to conduct fine-scale genetic mapping experiments, and to generate novel allelic diversity in applied breeding programs.     

Breeding designs for recombinant inbred advanced intercross lines

Recombinant inbred lines derived from an advanced intercross, in which multiple generations of mating have increased the density of recombination breakpoints, are powerful tools for mapping the loci underlying complex traits. We investigated the effects of intercross breeding designs on the utility of such lines for mapping. The simplest design, random pair mating with each pair contributing exactly two offspring to the next generation, performed as well as the most extreme inbreeding avoidance scheme at expanding the genetic map, increasing fine-mapping resolution, and controlling genetic drift. Circular mating designs offer negligible advantages for controlling drift and exhibit greatly reduced map expansion. Random-mating designs with variance in offspring number are also poor at increasing mapping resolution. Given equal contributions of each parent to the next generation, the constraint of monogamy has no impact on the qualities of the final population of inbred lines. We find that the easiest crosses to perform are well suited to the task of generating populations of highly recombinant inbred lines.     

A large maize (Zea mays L.) SNP genotyping array: development and germplasm genotyping, and genetic mapping to compare with the B73 reference genome

SNP genotyping arrays have been useful for many applications that require a large number of molecular markers such as high-density genetic mapping, genome-wide association studies (GWAS), and genomic selection. We report the establishment of a large maize SNP array and its use for diversity analysis and high density linkage mapping. The markers, taken from more than 800,000 SNPs, were selected to be preferentially located in genes and evenly distributed across the genome. The array was tested with a set of maize germplasm including North American and European inbred lines, parent/F1 combinations, and distantly related teosinte material. A total of 49,585 markers, including 33,417 within 17,520 different genes and 16,168 outside genes, were of good quality for genotyping, with an average failure rate of 4% and rates up to 8% in specific germplasm. To demonstrate this array's use in genetic mapping and for the independent validation of the B73 sequence assembly, two intermated maize recombinant inbred line populations - IBM (B73×Mo17) and LHRF (F2×F252) - were genotyped to establish two high density linkage maps with 20,913 and 14,524 markers respectively. 172 mapped markers were absent in the current B73 assembly and their placement can be used for future improvements of the B73 reference sequence. Colinearity of the genetic and physical maps was mostly conserved with some exceptions that suggest errors in the B73 assembly. Five major regions containing non-colinearities were identified on chromosomes 2, 3, 6, 7 and 9, and are supported by both independent genetic maps. Four additional non-colinear regions were found on the LHRF map only; they may be due to a lower density of IBM markers in those regions or to true structural rearrangements between lines. Given the array's high quality, it will be a valuable resource for maize genetics and many aspects of maize breeding.     

Precise centromere mapping using a combination of repeat junction markers and chromatin immunoprecipitation-polymerase chain reaction

Centromeres are difficult to map even in species where genetic resolution is excellent. Here we show that junctions between repeats provide reliable single-copy markers for recombinant inbred mapping within centromeres and pericentromeric heterochromatin. Repeat junction mapping was combined with anti-CENH3-mediated ChIP to provide a definitive map position for maize centromere 8.     

A molecular marker map in 'Kanota' x 'Ogle' hexaploid oat (Avena spp.) enhanced by additional markers and a robust framework.

Molecular mapping of cultivated oats was conducted to update the previous reference map constructed using a recombinant inbred (RI) population derived from Avena byzantina C. Koch cv. Kanota x Avena sativa L. cv. Ogle. In the current work, 607 new markers were scored, many on a larger set of RI lines (133 vs. 71) than previously reported. A robust, updated framework map was developed to resolve linkage associations among 286 markers. The remaining 880 markers were placed individually within the most likely framework interval using chi2 tests. This molecular framework incorporates and builds on previous studies, including physical mapping and linkage mapping in additional oat populations. The resulting map provides a common tool for use by oat researchers concerned with structural genomics, functional genomics, and molecular breeding.     

Maize chromosomal knobs are located in gene-dense areas and suppress local recombination

Knobs are conspicuous heterochromatic regions found on the chromosomes of maize and its relatives. The number, locations, and sizes of knobs vary dramatically, with most lines containing between four and eight knobs in mid-arm positions. Prior data suggest that some knobs may reduce recombination. However, comprehensive tests have not been carried out, primarily because most knobs have not been placed on the genetic map. We used fluorescent in situ hybridization and two recombinant inbred populations to map seven knobs and to accurately place three knobs from the B73 inbred on the genomic sequence assembly. The data show that knobs lie in gene-dense regions of the maize genome. Comparisons to 23 other recombinant inbred populations segregating for knobs at the same sites confirm that large knobs can locally reduce crossing over by as much as twofold on a cM/Mb scale. These effects do not extend beyond regions ~10 cM to either side of knobs and do not appear to affect linkage disequilibrium among genes within and near knob repeat regions of the B73 RefGen_v2 assembly.     

Distributed simple sequence repeat markers for efficient mapping from maize public mutagenesis populations

The genome sequence of the B73 maize inbred enables map-based cloning of genetic variants underlying phenotypes. In parallel to sequencing efforts, multiple public mutagenesis resources are being developed predominantly in the W22 and B73 inbreds. Efficient platforms to map mutants in these genetic backgrounds would aid molecular genetic analysis of the public resources. We screened 505 simple sequence repeat markers for polymorphisms between the B73, Mo17, and W22 inbreds. Using common thermocycling conditions, 47.1% of the markers showed co-dominant polymorphisms in at least one pair of inbreds. Based on these results, we identified 85 distributed markers for mapping in all three inbred pairs. For each inbred pair, the distributed set has 64–71 polymorphic markers with a mean distance of 27–29 cM between markers. The distributed markers give nearly complete coverage of the genetic map for each inbred pair. We demonstrate the utility of the marker set for efficient placement of mutants on the maize genetic map with an example mapping experiment of a seed mutant from the UniformMu mutagenesis resource. We conclude that these distributed molecular markers enable rapid mapping of phenotypic variants from public mutagenesis populations.     

The suitability of restriction fragment length polymorphisms as genetic markers in maize

Summary Strain identification in Zea mays by restriction fragment length polymorphism should be feasible due to the high degree of polymorphism found at many loci. The polymorphism in maize is apparently higher than that currently known for any other organism. Five randomly selected maize inbred lines were examined by Southern filter hybridization with probes of cloned low copy sequences. Typically, several alleles could be distinguished among the inbred lines with any one probe and an appropriately selected restriction enzyme. Despite considerable polymorphism at the DNA level, 16 RFLP markers in three inbred lines of maize were examined for six to 11 generations and found be stable. Mapping of RFLP markers in maize can be accelerated by the use of B-A translocation stocks, which enable localization of a marker to chromosome arm in one generation. The use of recombinant inbred lines in further refinement of the map is discussed.     

Description of 31 YAC contigs spanning the majority of Arabidopsis thaliana chromosome 5

In order to generate a physical map of Arabidopsis thaliana chromosome 5, 142 molecular markers mapping to chromosome 5 have been used in colony hybridization experiments with four Arabidopsis, ecotype Columbia, yeast artificial chromosome (YAC) libraries. This resulted in 634 YAC clones being anchored on chromosome 5. Southern blot analysis confirmed their positioning and provided data, which along with knowledge of the sizes of all the YAC clones, enabled the clones to be arranged into 31 contigs. Genetic mapping of markers located within 29 of these contigs on the Landsberg erecta/Columbia recombinant inbred lines allowed positioning of the contigs along the chromosome. A high proportion of the YAC clones were found to contain chimaeric inserts. The availability of this YAC contig map will accelerate chromosome-walking experiments, provide substrates for large-scale genomic sequencing projects and facilitate the mapping of new probes to this chromosome.     

玉米相对饱和遗传连锁图谱构建与一种新的AFLPs共显性分析方法探讨

利用一个F2作图群体(X178×B73),首先构建了一个含有130个SSRs的玉米连锁框架图,然后用119个AFLPs位点增加图谱密度,得到一个全长1659·3cM,标记间平均间距6·66cM的玉米相对饱和连锁图。同时,对SSRs和AFLPs的一些遗传特性进行了分析,探讨了AFLP标记进行共显性分析的一种新方法。分析表明SSRs和AFLPs分子标记具有多态性和可靠性高等特点,是构建高密度分子标记遗传连锁图的有效技术。加密的玉米遗传连锁图谱为比较基因组研究、数量性状位点(quantitativetraitloci,QTLs)克隆、杂种优势机理研究以及标记辅助选择等提供了技术基础。     

Localization of zein genes in maize

The band patterns of zein polypeptides were determined for many commercial inbred corn lines and maize stocks using isofocusing in agarose gels and sodium dodecyl sulfate (SDS)-urea gels. Each inbred line or homozygous maize strain genotype has a distinct zein profile which has been catalogued according to the distance of charge migration and molecular weight (kilodaltons). Several zein polypeptides were mapped to chromosomes 4 and 10 with the use of reciprocal translocations. The mapping of at least two polypeptides on distal 4L and 10L had not been previously reported. The general methods used in the present research will permit the mapping of all the zein polypeptides to chromosomal sites.Pioneer Hi-Bred International provided financial support.     

Mapping of genome-wide resistance gene analogs (RGAs) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Isolation and mapping of genome-wide resistance (R) gene analogs (RGAs) is of importance in identifying candidate(s) for a particular resistance gene/QTL. Here we reported our result in mapping totally 228 genome-wide RGAs in maize. By developing RGA-tagged markers and subsequent genotyping a population consisting of 294 recombinant inbred lines (RILs), 67 RGAs were genetically mapped on maize genome. Meanwhile, in silico mapping was conducted to anchor 113 RGAs by comparing all 228 RGAs to those anchored EST and BAC/BAC-end sequences via tblastx search (E-value < 10⁻²⁰). All RGAs from different mapping efforts were integrated into the existing SSR linkage map. After accounting for redundancy, the resultant RGA linkage map was composed of 153 RGAs that were mapped onto 172 loci on maize genome, and the mapped RGAs accounted for approximate three quarters of the genome-wide RGAs in maize. The extensive co-localizations were observed between mapped RGAs and resistance gene/QTL loci, implying the usefulness of this RGA linkage map in R gene cloning via candidate gene approach. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Wenkai Xiao, Jing Zhao and Shengci Fan have contributed equally to this research.     

An improved genetic linkage map for cowpea (Vigna unguiculata L.) combining AFLP, RFLP, RAPD, biochemical markers, and biological resistance traits.

An improved genetic linkage map has been constructed for cowpea (Vigna unguiculata L. Walp.) based on the segregation of various molecular markers and biological resistance traits in a population of 94 recombinant inbred lines (RILs) derived from the cross between 'IT84S-2049' and '524B'. A set of 242 molecular markers, mostly amplified fragment length polymorphism (AFLP), linked to 17 biological resistance traits, resistance genes, and resistance gene analogs (RGAs) were scored for segregation within the parental and recombinant inbred lines. These data were used in conjunction with the 181 random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), AFLP, and biochemical markers previously mapped to construct an integrated linkage map for cowpea. The new genetic map of cowpea consists of 11 linkage groups (LGs) spanning a total of 2670 cM, with an average distance of 6.43 cM between markers. Astonishingly, a large, contiguous portion of LG1 that had been undetected in previous mapping work was discovered. This region, spanning about 580 cM, is composed entirely of AFLP markers (54 in total). In addition to the construction of a new map, molecular markers associated with various biological resistance and (or) tolerance traits, resistance genes, and RGAs were also placed on the map, including markers for resistance to Striga gesnerioides races 1 and 3, CPMV, CPSMV, B1CMV, SBMV, Fusarium wilt, and root-knot nematodes. These markers will be useful for the development of tools for marker-assisted selection in cowpea breeding, as well as for subsequent map-based cloning of the various resistance genes.     

Genetics of symbiosis in Lotus japonicus: recombinant inbred lines, comparative genetic maps, and map position of 35 symbiotic loci

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.