Marcadores Moleculares

1. Marcadores Moleculares Introdu��o e Hist�rico Descri��o de Marcadores Compara��o entre Marcadores Moleculares Classifica��o de Marcadores Moleculares Caracter�sticas do Genoma de Planta Aplica��es diversidade gen�tica mapeamento e sele��o assistida

2. INTRODU��O E HIST�RICOMarcadores Moleculares

3. Gen�tica �arte� e sele��o inconsciente da inven��o da agricultura at� s�c. XIX 1900s - Descoberta dos princ�pios gen�ticos 1920-50 - Melhoramento gen�tico cient�fico gen�tica quantitativa e biometria (fen�tipo � previsor ruim do valor gen�tico!) 1970-80 - Utiliza��o de marcadores gen�ticos moleculares

4. Sucesso no melhoramento depende da capacidade de distinguir fatores gen�ticos herd�veis dos ambientais Marcadores gen�ticos s�o unidades herd�veis simples

5. Polimorfismo de DNA resulta ac�mulo de muta��es pontual ou inser��o/dele��o macro-rearranjos: transloca��es, invers�es, dele��es

7. Hist�rico de Marcadores 1. Karl Sax (1923): prop�s m�todo para localiza��o de QTLs liga��o entre genes de caracter�stica qualitativa (cor de semente) e quantitativa (peso de semente); Problema: aus�ncia de muta��es m�ltiplas em estoque de elite, baixa viabilidade 2. Hunter & Markert (1957) - marcas bioqu�micas desenvolveram isoenzimas em gel de amido

8. Hist�rico de Marcadores 3. Hubby & Lewotin (1966) demonstraram que 30% de loci de isoenzimas exibiam polimorfismo em popula��es selvagens de Drosophila; 4. 1970�s - ferramentas moleculares desenvolvimento de vetores de clonagem; enzimas de restri��o; polimerases; ligases; Southern (1977);

9. Hist�rico de Marcadores 5. RFLP proposto por Botstein et al. (1980) descrito para humanos 6. PCR proposto por Mullis & Faloona (1987) 7. VNTR por Jeffrey (1987) 8. RAPD por Rafalski et al. (1990)

10. Hist�rico de Marcadores 9. SSR em plantas por Akkaya et al. (1992) 10. AFLP por Zabeau & Vos (1993) 11. CAPS por Konieczny & Ausubel (1993) 12. SCAR por Paran & Michelmore (1993) 13. Cho et al. (1999) - SNPs em Arabidopsis

11. DESCRI��O DOS MARCADORES MOLECULARES

12. Marcadores Moleculares RFLP VNTR (minissat�lite) RAPD, AP-PCR, DAF PCR-espec�fico - SSR, ISSR, CAPS, SCARs AFLP SNPs

13. Restriction Fragment Length Polymorphism - RFLP RFLP examina diferen�a em tamanho de fragmentos de restri��o de DNA espec�ficos Polimorfismo deriva de muta��o pontual, inser��o, dele��o Utiliza-se DNA celular total Requer DNA puro de alto peso molecular

14. Metodologia de RFLP 1 . Digerir DNA em fragmentos pequenos 2. Separa��o dos fragmentos por gel eletroforese 3. Transfer�ncia de fragmentos de DNA para filtro

15. Metodologia de RFLP 4. Visualiza��o dos fragmentos de DNA sondas marcadas (32P) ou a frio 5. An�lise dos resultados bandas analisadas para alelos e/ou presen�a/aus�ncia diferen�as em padr�o de bandas reflete diferen�as gen�ticas A escolha de sonda/enzima de restri��o � crucial

16. Digest�o de DNA Gen�mico e Separa��o em Gel

17. Transfer�ncia para Membrana de Nylon ou Nitrocelulose

18. Hibridiza��o em Nylon ou Nitrocelulose

19. Constru��o de biblioteca gen�mica ou de cDNA

22. Interpreta��o de resultados

26. Heran�a de RFLPs

27. An�lise de Diversidade e Filogenia por RFLP

28. RFLP: sondas de locos �nico DNA Nuclear biblioteca gen�mica biblioteca de cDNA DNA Citoplasm�tico biblioteca de DNA cloropl�stico e mitocondrial Sondas de RFLP s�o: locos-espec�fica, co-dominante esp�cie-espec�fica

29. RFLP: sondas multi-locus Repeti��es em linha (tandem) - �til encontrada em v�rios loci altamente polim�rficas Sequ�ncia de Minissat�lite VNTR: variable number of tandem repeats uso em �DNA fingerprinting� uso de seq��ncias repetidas de fago M13

30. Interpreta��o dos resultados

31. Vantagens e Desvantagens de RFLP Reprodut�vel Marcadores co-dominantes Simples Trabalhoso Caro Uso de sondas radioativas*

32. Random Amplification of Polymorphic DNA - RAPD Amplifica seq��ncias an�nimas de DNA usando primers arbitr�rios 10 bases com >50% G+C PCR com um �nico primer M�todo r�pido para detec��o de polimorfismos Marcador dominante Problemas de reproducibilidade The random amplified polymorphic DNA (RAPD) technique is a PCR based method which uses one or sometimes two short arbitrary primers (usually 8-10 bases) to amplify anonymous stretches of DNA which are then separated and visualised by gel electrophoresis. The key point about this technique is that nothing is known about the identity of the amplification products. The amplification products are however extremely useful as markers in genetic diversity studies. Other important features of the technique are: The number of fragments. Many different fragments are normally amplified using each single primer, and the technique has therefore proved a fast method for detecting polymorphisms. The majority of commercially produced primers result in 6 to 12 fragments; some primers may fail to give any amplification fragments from some material. Simplicity of the technique. RAPD analysis does not involve hybridisation/autoradiography or high technical expertise. Only tiny quantities of target DNA are required. Arbitrary primers can be purchased. Unit costs per assay are low. This has made RAPD analysis very popular. RAPD markers are dominant. Amplification either occurs at a locus or it does not, leading to scores of band presence/absence; this means that homozygotes and heterozygotes cannot be distinguished. Problems of reproducibility - RAPD does suffer from a sensitivity to changes in PCR conditions resulting in changes to some of the amplified fragments. Reproducible results can be obtained if care is taken to standardise the conditions used (Munthali et al., 1992; Lowe et al., 1996). The random amplified polymorphic DNA (RAPD) technique is a PCR based method which uses one or sometimes two short arbitrary primers (usually 8-10 bases) to amplify anonymous stretches of DNA which are then separated and visualised by gel electrophoresis. The key point about this technique is that nothing is known about the identity of the amplification products. The amplification products are however extremely useful as markers in genetic diversity studies. Other important features of the technique are: The number of fragments. Many different fragments are normally amplified using each single primer, and the technique has therefore proved a fast method for detecting polymorphisms. The majority of commercially produced primers result in 6 to 12 fragments; some primers may fail to give any amplification fragments from some material. Simplicity of the technique. RAPD analysis does not involve hybridisation/autoradiography or high technical expertise. Only tiny quantities of target DNA are required. Arbitrary primers can be purchased. Unit costs per assay are low. This has made RAPD analysis very popular. RAPD markers are dominant. Amplification either occurs at a locus or it does not, leading to scores of band presence/absence; this means that homozygotes and heterozygotes cannot be distinguished. Problems of reproducibility - RAPD does suffer from a sensitivity to changes in PCR conditions resulting in changes to some of the amplified fragments. Reproducible results can be obtained if care is taken to standardise the conditions used (Munthali et al., 1992; Lowe et al., 1996).

33. RAPD The random amplified polymorphic DNA (RAPD) technique is a PCR based method which uses one or sometimes two short arbitrary primers (usually 8-10 bases) to amplify anonymous stretches of DNA which are then separated and visualised by gel electrophoresis. The key point about this technique is that nothing is known about the identity of the amplification products. The amplification products are however extremely useful as markers in genetic diversity studies. Other important features of the technique are: The number of fragments. Many different fragments are normally amplified using each single primer, and the technique has therefore proved a fast method for detecting polymorphisms. The majority of commercially produced primers result in 6 to 12 fragments; some primers may fail to give any amplification fragments from some material. Simplicity of the technique. RAPD analysis does not involve hybridisation/autoradiography or high technical expertise. Only tiny quantities of target DNA are required. Arbitrary primers can be purchased. Unit costs per assay are low. This has made RAPD analysis very popular. RAPD markers are dominant. Amplification either occurs at a locus or it does not, leading to scores of band presence/absence; this means that homozygotes and heterozygotes cannot be distinguished. Problems of reproducibility - RAPD does suffer from a sensitivity to changes in PCR conditions resulting in changes to some of the amplified fragments. Reproducible results can be obtained if care is taken to standardise the conditions used (Munthali et al., 1992; Lowe et al., 1996). The random amplified polymorphic DNA (RAPD) technique is a PCR based method which uses one or sometimes two short arbitrary primers (usually 8-10 bases) to amplify anonymous stretches of DNA which are then separated and visualised by gel electrophoresis. The key point about this technique is that nothing is known about the identity of the amplification products. The amplification products are however extremely useful as markers in genetic diversity studies. Other important features of the technique are: The number of fragments. Many different fragments are normally amplified using each single primer, and the technique has therefore proved a fast method for detecting polymorphisms. The majority of commercially produced primers result in 6 to 12 fragments; some primers may fail to give any amplification fragments from some material. Simplicity of the technique. RAPD analysis does not involve hybridisation/autoradiography or high technical expertise. Only tiny quantities of target DNA are required. Arbitrary primers can be purchased. Unit costs per assay are low. This has made RAPD analysis very popular. RAPD markers are dominant. Amplification either occurs at a locus or it does not, leading to scores of band presence/absence; this means that homozygotes and heterozygotes cannot be distinguished. Problems of reproducibility - RAPD does suffer from a sensitivity to changes in PCR conditions resulting in changes to some of the amplified fragments. Reproducible results can be obtained if care is taken to standardise the conditions used (Munthali et al., 1992; Lowe et al., 1996).

34. Interpreta��o de RAPDs Marcadores RAPD s�o an�nimos Dados bin�rios (presen�a x aus�ncia) RAPD s�o dominantes (AA = Aa) Problemas de co-migra��o mesma banda, mesmo fragmento? uma banda, um fragmento? Questionamento para filogenia banda hom�logas?

35. PCR com primers arbitr�rios: ac�mulo de siglas! RAPD Random Amplified Polymorphic DNA DAF DNA Amplification Fingerprinting AP-PCR Arbitrarily Primed Polymerase Chain Reaction MAAP Multiple Arbitrary Amplicon Profiling (sugerido por incluir todas as pequenas varia��es na t�cnica) All of the following techniques use one or two, short, GC-rich primers of arbitrary sequence. RAPD was the first to become available (Williams et al., 1990) and is by far the most commonly used of these techniques. DAF - DNA amplication fingerprinting Differences between DAF (Caetano-Anolles, et al., 1991a,b) and RAPD: higher primer concentrations in DAF shorter primers used in DAF (5-8 nucleotides) two-temperature cycle in DAF compared to 3-temperature cycle in RAPD DAF usually produces very complex banding patterns AP-PCR - arbitrarily primed polymerase chain reaction Differences between AP-PCR (Welsh and McClelland, 1990) and RAPD: in AP-PCR the amplification is in three parts each with its own stringency and concentrations of constituents high primer concentrations are used in the first PCR cycles primers of variable length, and often designed for other purposes are arbitrarily chosen for use (e.g. M13 universal sequencing primer) MAAP is only an acronym proposed by Caetano-Anolles et al. (1992) to encompass all of these closely related techniques, but which is not commonly used.All of the following techniques use one or two, short, GC-rich primers of arbitrary sequence. RAPD was the first to become available (Williams et al., 1990) and is by far the most commonly used of these techniques. DAF - DNA amplication fingerprinting Differences between DAF (Caetano-Anolles, et al., 1991a,b) and RAPD: higher primer concentrations in DAF shorter primers used in DAF (5-8 nucleotides) two-temperature cycle in DAF compared to 3-temperature cycle in RAPD DAF usually produces very complex banding patterns AP-PCR - arbitrarily primed polymerase chain reaction Differences between AP-PCR (Welsh and McClelland, 1990) and RAPD: in AP-PCR the amplification is in three parts each with its own stringency and concentrations of constituents high primer concentrations are used in the first PCR cycles primers of variable length, and often designed for other purposes are arbitrarily chosen for use (e.g. M13 universal sequencing primer) MAAP is only an acronym proposed by Caetano-Anolles et al. (1992) to encompass all of these closely related techniques, but which is not commonly used.

36. Diferen�as entre ensaios com primers arbitr�rios RAPD 10mers, gel de agarose corado com brometo DAF 5mers, gel de acrilamida e rea��o marcada 32P AP-PCR 10mers, gel de acrilamida e rea��o marcada 32P All of the following techniques use one or two, short, GC-rich primers of arbitrary sequence. RAPD was the first to become available (Williams et al., 1990) and is by far the most commonly used of these techniques. DAF - DNA amplication fingerprinting Differences between DAF (Caetano-Anolles, et al., 1991a,b) and RAPD: higher primer concentrations in DAF shorter primers used in DAF (5-8 nucleotides) two-temperature cycle in DAF compared to 3-temperature cycle in RAPD DAF usually produces very complex banding patterns AP-PCR - arbitrarily primed polymerase chain reaction Differences between AP-PCR (Welsh and McClelland, 1990) and RAPD: in AP-PCR the amplification is in three parts each with its own stringency and concentrations of constituents high primer concentrations are used in the first PCR cycles primers of variable length, and often designed for other purposes are arbitrarily chosen for use (e.g. M13 universal sequencing primer) MAAP is only an acronym proposed by Caetano-Anolles et al. (1992) to encompass all of these closely related techniques, but which is not commonly used.All of the following techniques use one or two, short, GC-rich primers of arbitrary sequence. RAPD was the first to become available (Williams et al., 1990) and is by far the most commonly used of these techniques. DAF - DNA amplication fingerprinting Differences between DAF (Caetano-Anolles, et al., 1991a,b) and RAPD: higher primer concentrations in DAF shorter primers used in DAF (5-8 nucleotides) two-temperature cycle in DAF compared to 3-temperature cycle in RAPD DAF usually produces very complex banding patterns AP-PCR - arbitrarily primed polymerase chain reaction Differences between AP-PCR (Welsh and McClelland, 1990) and RAPD: in AP-PCR the amplification is in three parts each with its own stringency and concentrations of constituents high primer concentrations are used in the first PCR cycles primers of variable length, and often designed for other purposes are arbitrarily chosen for use (e.g. M13 universal sequencing primer) MAAP is only an acronym proposed by Caetano-Anolles et al. (1992) to encompass all of these closely related techniques, but which is not commonly used.

37. RAPD - resumo R�pido Simples Baixo custo Sem uso de radio-is�topos Marcador dominante Problemas de reproducibilidade Problemas de interpreta��o

40. S�tio de Seq��ncia Dirigida(Sequence-tagged sites) Sequence-Tagged Microssat�lites (STMS) ou SSR ou Microssat�lites Microssat�lites ancorados Inter-Simple Sequence Repeat (ISSR) Sequence-characterized amplified regions (SCARs) Cleaved amplified polymorphic sequence (CAPS) � PCR-RFLP More and more sequence information is becoming available from different sources and can be located in widely available databases. This information is extremely useful for developing new strategies for the analysis of genetic variation. A sequence-tagged site (STS) is the general term given to a marker which is defined by its primer sequences (Olsen et al., 1989). STSs have been used extensively for mapping of the human genome. Examples of STSs are given in the following slides, namely: Sequence-tagged microsatellites (STMS) also known as Simple Sequence Repeat Polymorphisms (SSRP) Anchored microsatellite oligonucleotides including inter-simple sequence repeat (ISSR) primers Sequence-characterised amplified regions (SCARs) Cleaved amplified polymorphic sequence (CAPS)More and more sequence information is becoming available from different sources and can be located in widely available databases. This information is extremely useful for developing new strategies for the analysis of genetic variation. A sequence-tagged site (STS) is the general term given to a marker which is defined by its primer sequences (Olsen et al., 1989). STSs have been used extensively for mapping of the human genome. Examples of STSs are given in the following slides, namely: Sequence-tagged microsatellites (STMS) also known as Simple Sequence Repeat Polymorphisms (SSRP) Anchored microsatellite oligonucleotides including inter-simple sequence repeat (ISSR) primers Sequence-characterised amplified regions (SCARs) Cleaved amplified polymorphic sequence (CAPS)

41. Microssat�lites (SSR) Sequence-Tagged Microsat�lites (STMS) tamb�m conhecido como microssat�lite ou Simple Sequence Repeat (SSR) Normalmente locus simples e multi-al�lico Co-dominante Altamente reprodut�vel

42. Microssat�lites STMS ou SSRs Seq��ncias curtas (1 a 6 bases) repetidas em tandem Presentes em procariotos e eucariotos Presentes em regi�es codificantes e n�o codificantes Maioria das repeti��es s�o dinucleot�deos (AC) n (AG) n (AT)n

43. Polimorfismo devido a diferen�as no n�mero de repeti��es Escorregamento da DNA polimerase durante a replica��o Crossing-over desigual entre crom�tides irm�s Codominantes Normalmente locos simples e multi-al�lico Microssat�lites

44. Microssat�lites (SSR) altamente informativo - v�rios alelos por locos detec��o por PCR facilmente transfer�vel entre labs distribui��o homog�nea no genoma

45. Microssat�lites (SSR)

46. Microssat�lites (SSR) Obten��o de seq��ncias: a partir de banco de dados de genoma ou cDNA hibrida��o com biblioteca gen�mica, identifica��o de clones e seq�enciamento constru��o de biblioteca enriquecida por afinidade com seq��ncia da matriz

47. Detec��o do polimorfismo G�is de agarose G�is de acrilamida (detecta diferen�as de at� 2pb) colora��o direta: nitrato de prata (barato) Colora��o indireta: marca��o radioativa ou fluorescente Microssat�lites

48. Problemas Custo e trabalho envolvidos no desenvolvimento dos primers Constru��o de bibliotecas gen�mica sequenciamento Triagem dos melhores primers ?Possibilidade de se usar seq��ncias depositadas em banco de dados EST � SSR funcional x SSR gen�mico Microssat�lites

49. Microssat�lites (SSR)

55. Microssat�lites (SSR)

56. Microssat�lites (SSR)

58. Microssat�litesBananeira 3x e 4xCir 24.25

59. Microssat�lites

60. Microssat�lites Ancorados ISSR Amplifica��o de segmentos gen�micos flanqueados por repeti��es Anelamento locos-espec�fico Inter-simple sequence repeats (ISSR) ancorados na extremidade 3� ou 5� Marcadores dominantes Microssat�lites mais �teis que minissat�lites

61. Microssat�lites Ancorados ISSR

62. ISSRUBC 811 UBC 816

63. SCARs SCARs - sequence-characterised amplified regions proposto por Paran & Michelmore (1993) marcador locus-�nico derivado de fragmentos sequenciados de RAPD, ISSR, AFLP maior estabilidade - primers espec�ficos analisado para presen�a/aus�ncia possibilidade de simplifica��o de an�lise e automa��o

64. SCARs

66. CAPS ou PCR-RFLP CAPS - cleaved amplified polymorphic sequence marcador locus-espec�fico produto amplificado por PCR e analisado por RFLP seq��ncia de banco de dados, clones de cDNA ou gen�mico codominante

67. CAPS

70. Amplified Fragment Length Polymorphism - AFLP Combina��o de RFLP e PCR Resulta em padr�es muito informativos Marcador dominante M�todo cada vez mais usado

74. AFLP de cana com 33P

75. AFLP de feij�ogel desnaturante corado com prata

77. COMPARA��O ENTRE MARCADORES MOLECULARES

78. Escolha de Marcadores Caracter�stica RFLP RAPD SSR AFLP ISSR CAPS Polimorfismo Pontual Pontual # Pontual Pontual Pontual InDel InDel Rep. InDel InDel InDel N�vel de Polimorfismo m�dio m�dio alto m�dio m�dio baixo Abund�ncia alta m.alta m�dia m.alta m�dia alta Domin�ncia CoDom Dom CoDom Dom Dom CoDom [DNA] 10 mg 25 ng 50 ng 500 ng 25 ng 25 ng Seq��ncia n�o n�o sim n�o n�o sim Marca��o sim/n�o n�o n�o sim/n�o n�o n�o Repetibilidade alta baixa alta m�dia baixa alta

79. CLASSIFICA��O DE MARCADORES MOLECULARES

80. Classifica��o por Tipo de T�cnica M�todos sem uso de PCR RFLP VNTR M�todos com uso de PCR PCR com primers arbitr�rios RAPD, AP-PCR, DAF, MAAP; Polimorfismo de Tamanho de Fragmento Amplificado AFLP; ISSR PCR s�tio-espec�fico CAPS, SCAR SSRs (microssat�lites) TGGE, SSCP, DGGE

81. Classifica��o por N�mero de C�pias da Seq��ncia Alvo Seq��ncia de poucas c�pias - codificante RFLP Seq��ncia com c�pias repetidas VNTR SSRs (microssat�lites) ISSR Seq��ncia com n�mero de c�pias indefinido RAPD, AP-PCR, DAF, MAAP; AFLP; CAPS, SCAR