RESUMO
This article presents methodology for the construction of a linkage map in an autotetraploid species, using either codominant or dominant molecular markers scored on two parents and their full-sib progeny. The steps of the analysis are as follows: identification of parental genotypes from the parental and offspring phenotypes; testing for independent segregation of markers; partition of markers into linkage groups using cluster analysis; maximum-likelihood estimation of the phase, recombination frequency, and LOD score for all pairs of markers in the same linkage group using the EM algorithm; ordering the markers and estimating distances between them; and reconstructing their linkage phases. The information from different marker configurations about the recombination frequency is examined and found to vary considerably, depending on the number of different alleles, the number of alleles shared by the parents, and the phase of the markers. The methods are applied to a simulated data set and to a small set of SSR and AFLP markers scored in a full-sib population of tetraploid potato.
Assuntos
Mapeamento Cromossômico/métodos , Ligação Genética , Marcadores Genéticos , Ploidias , Algoritmos , Alelos , Análise por Conglomerados , Cruzamentos Genéticos , Genes Dominantes , Genótipo , Funções Verossimilhança , Escore Lod , Modelos Genéticos , Modelos Estatísticos , Fenótipo , Recombinação Genética , Solanum tuberosum/genéticaRESUMO
The type and frequency of simple sequence repeats (SSRs) in plant genomes was investigated using the expanding quantity of DNA sequence data deposited in public databases. In Arabidopsis, 306 genomic DNA sequences longer than 10 kb and 36,199 EST sequences were searched for all possible mono- to pentanucleotide repeats. The average frequency of SSRs was one every 6.04 kb in genomic DNA, decreasing to one every 14 kb in ESTs. SSR frequency and type differed between coding, intronic, and intergenic DNA. Similar frequencies were found in other plant species. On the basis of these findings, an approach is proposed and demonstrated for the targeted isolation of single or multiple, physically clustered SSRs linked to any gene that has been mapped using low-copy DNA-based markers. The approach involves sample sequencing a small number of subclones of selected randomly sheared large insert DNA clones (e.g., BACs). It is shown to be both feasible and practicable, given the probability of fortuitously sequencing through an SSR. The approach is demonstrated in barley where sample sequencing 34 subclones of a single BAC selected by hybridization to the Big1 gene revealed three SSRs. These allowed Big1 to be located at the top of barley linkage group 6HS.
Assuntos
Arabidopsis/genética , DNA de Plantas/química , DNA de Plantas/genética , Genoma de Planta , Plantas/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Bases , Cromossomos Artificiais Bacterianos , Clonagem Molecular , Primers do DNA , Bases de Dados como Assunto , Etiquetas de Sequências Expressas , Hordeum/genética , ProbabilidadeRESUMO
Solanum tuberosum L. DNA sequences containing simple sequence repeat (SSR) motifs were extracted from the EMBL database, cDNA and selectively enriched small-insert DNA libraries. Enrichment was achieved using either triplex affinity capture or single-strand hybridisation selection. One hundred and twelve primer pairs which successfully amplified products of the correct size from potato DNA were ultimately designed and synthesised. Ninety-eight of these revealed length polymorphisms in a panel of four diploid and two tetraploid clones, in agreement with the high information content of this class of markers which has been found in other species. All of the markers were assigned a quality score of 1-5 based on their potential usefulness. Eighty-nine loci from 65 of the primer pairs were located on two genetic linkage maps of potato by segregation analysis of the amplified alleles. Fifty-two of the SSRs were clearly single locus. The maps were aligned using 23 SSR primer pairs and 13 RFLP loci mapped in both populations. The markers described constitute a class which should replace Restriction Fragment Length Polymorphisms (RFLP) as the markers of choice for future genetic studies in potato. The sequences of the primers, together with other information on these markers are provided.
Assuntos
Sequências Repetitivas de Ácido Nucleico , Solanum tuberosum/genética , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA/genética , DNA Complementar/genética , DNA de Plantas/genética , Bases de Dados Factuais , Biblioteca Gênica , Ligação Genética , Marcadores Genéticos , Hibridização de Ácido Nucleico , Polimorfismo de Fragmento de RestriçãoRESUMO
We have constructed a partial linkage map in tetraploid potato which integrates simplex, duplex and double-simplex AFLP markers. The map consists of 231 maternal and 106 paternal markers with total map lengths of 990.9 cM and 484.6 cM. The longer of the two cumulative map lengths represents approximately 25% coverage of the genome. In tetraploids, much of the polymorphism between parental clones is masked by 'dosage' which significantly reduces the number of individual markers that can be scored in a population. Consequently, the major advantage of using AFLPs--their high multiplex ratio--is reduced to the point where the use of alternative multi-allelic marker types would be significantly more efficient. The segregation data and map information have been used in a QTL analysis of late blight resistance, and a multi-allelic locus at the proximal end of chromosome VIII has been identified which contributes significantly to the expression of resistance. No late blight resistance genes or QTLs have previously been mapped to this location.
