Marcatori molecolari nelle piante

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1 Marcatori molecolari nelle piante Mappa genetica: rappresentazione grafica dei singoli gruppi di associazione con l indicazione dell ordine e della posizione relativa dei geni lungo il cromosoma determinata in base alle frequenze di ricombinazione. Marcatori genetici: qualsiasi elemento genetico che può essere individuato mediante analisi di tipo diverso che consentono di studiarne l eredità, quindi sono posizioni specifiche sul cromosoma che servono come punti di riferimento per l analisi genetica. Gruppi di associazione (linkage): tendenza di due o più geni ad essere trasmessi insieme in quanto ubicati sullo stesso cromosoma e vicini tra loro, non segregano in modo indipendente.

2 A linkage map is a representation, in the form of a table or a graphic, of the position of genes (or markers) within a linkage group. The map positions are inferred from estimates of recombination frequencies between genes. The earliest linkage maps contained, in most cases, only a few morphological markers, mostly representing genes for which mutant phenotypes were available.

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6 Suppose the recombination between loci 1 and 2 = 6%, that between loci 2 and 3 = 20%, and that between loci 1 and 3 = 24%, then we can order the loci along the chromosome: Recombination frequencies are not additive, as in the example given here: = 26 is the true distance between markers 1 and 3 (and not 24). The underestimated recombination frequency between loci 1 and 3 is due to double or multiple crossovers, which go undetected as recombinants

7 Ordering of markers within a linkage group Finding the correct order of markers within a linkage group and calculating the map distances between them is the major job for the construction of linkage maps. The most simple ordering problem arises when we have recombination estimates for each pair of a set of three markers. The unit of map distance is one centimorgan (1 cm). A distance of 100 cm means that over that distance an average of 1 recombination event (per chromatid) occurs.

8 At the DNA level the amount of variation is so vast that in a particular cross between genotypes over thousand markers may be segregating at the same time. Although the degree of DNA polymorphism between genotypes of a crop species varies from species to species, the number of DNA markers that can be obtained to discriminate between genotypes is in principle unlimited. An example of a marker linkage map. Chromosome 1 of tomato. The map contains 139 markers. Left of chromosome bar: map distances in centimorgans. Right of chromosome bar: marker names.

9 1. A genetic linkage map shows the relative locations of specific DNA markers along the chromosome. Any inherited physical or molecular characteristic that differs among individuals and is easily detectable in the laboratory is a potential genetic marker. 2. Markers can be expressed DNA regions (genes) or DNA segments that have no known coding function but whose inheritance pattern can be followed. DNA sequence differences are especially useful markers because they are plentiful and easy to characterize precisely. 3. Markers must be polymorphic to be useful in mapping; that is, alternative forms must exist among individuals so that they are detectable among different members in family studies. 4. Polymorphisms are variations in DNA sequence that occur on average once every 300 to 500 bp. Variations within exon sequences can lead to observable changes, such as differences in color, shape and disease susceptibility.

10 Morfologici e molecolari Morfologici: fenotipici, si analizzano in termini visivi. Limitati in numero. Molecolari: rivelano polimorfismo a livello di 1- proteine, 2- DNA. Proteine: marcatore = proteina separabile con elettroforesi, soprattutto isozimi. Rivelano differenze e sono codominanti. Limitati in numero e problemi di modificazioni post-traduzionali. DNA: due categorie, ibridazione-dipendenti e PCR-dipendenti.

11 Isoelectric focusing of rmco1. Larrondo L F et al. Appl. Environ. Microbiol. 2003,

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13 Ibridazione: RFLP, restriction fragment lenght polymorphism. Si utilizzano sonde anche ignote per ibridare DNA genomico digerito con vari enzimi. Differenze nelle bande sono dovute alla presenza o assenza di siti di taglio.

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16 Marker basati su PCR in base al tipo di primer usato si può avere:

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18 RAPD Random amplified polymorphic DNA: primer corti 9-10 nt.

19 Gel electrophoresis (1.5%) of amplification products obtained with RAPD primer PL33 in 20 accessions of Withania somnifera

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22 AFLP- Amplified fragment length polymorphism A DNA fingerprinting technique that combines features of RFLP and RAPD techniques for amplification of a subset of genomic restriction fragments using selective primers.

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24 Physical mapping Construction of a physical map which consists of continuous overlapping fragments of cloned DNA that has the same linear order as found on the chromosomes from which they were derived.

25 Create a BAC Library Bacterial Artificial Chromosome Key reagent for any major sequencing project A typical BAC contains 150,000 bp of DNA BAC library replicated and spotted onto filters

26 Physical Mapping a BAC clone genome map Get a set of large clones (BAC, ~150 Kbp) Map them on the genome

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