We already have seed produced by the F1 plants created by the following cross:

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1 Mapping the position of traits on a chromosome: Determining the genetic distance between a morphological trait and DNA Markers (Restriction Fragment Length Polymorphisms/RLFPs) using the model plant, Arabidopsis thaliana. You will use the genetic model plant, Arabidopsis thaliana, to conduct a linkage analysis of known Restriction Fragment Length Polymorphisms (RFLPs) and a mutation that causes a morphological defect called virescent. Arabidopsis is a small flowering plant with a short life cycle that is used as a model experimental system for studying plant biology. The A. thaliana genome, which is about 125 Mb, has been sequenced and annotated. There are many DNA markers scattered throughout A. thaliana s five chromosomes, including the three RFLPs on chromosome I that we will use in our analysis. Information on A. thaliana can be obtained at the web site, The vir2 mutation: The recessive mutation, vir2, causes leaves to be a lighter green than wild type and also has effects on growth (vir2/vir2 homozygous mutant plants are slower to germinate, grow more slowly, and are smaller in stature as compared to wild type). VIR2 is dominant and results in more rapid germination, dark leaf coloration, and larger plant stature. You will identify plants that are homozygous for vir2 by inspection, using VIR2/VIR2 wild type and vir2/vir2 mutant plants as controls for comparison. vir2 is known to be on chromosome I but its exact location is unknown. Your data will contribute to the BIO 304 database for mapping vir2. The RFLP Loci: You will use two RFLP loci in your attempt to map the location of vir2. RFLP #1 (R1) is located near the end of the left arm of chromosome 1, RFLP #2 (R2) is located near the end of the right arm of chromosome I. There are two alleles of each RFLP locus in the F2 population of plants you will study. The alleles of a given RFLP differ by the presence/absence of a single restriction enzyme site. To determine which RFLP alleles are present in a plant, you will amplify the locus by PCR and then digest the PCR product with the appropriate restriction enzyme and analyze the results by agarose gel electrophoresis. Restriction maps and the corresponding DNA sequences of both alleles of each locus are attached to this document. You should use this information to interpret the results of your analysis. Experimental Overview: Each student will select one vir2/vir2 F2 progeny plant from the cross series described below and analyze the plants DNA by PCR. You ll collect tissue samples from leaves using a Harris UniCore tissue sampler. You ll place a tissue sample from each plant in two tubes containing PCR reaction mixes. Each tube contains a reaction mix with primer pairs for amplifying one RFLP locus. You ll verify that the PCR reaction worked by gel electrophoresis and then digest a sample of the PCR product with the appropriate restriction enzyme and analyze the results by gel electrophoresis. You will use the information provided on the RFLP alleles to interpret your results and deduce the genotype of each plant. We already have seed produced by the F1 plants created by the following cross: P vir2 R1C R2C / vir2 R1C R2C VIR2 R1L R2L /VIR2 R1L R2L NOTE: The original parents include a vir2/vir2 mutant, which is in a genetic background known as Columbia. Columbia plants have the RFLP alleles R1C and R2C, where C stands for the Columbia allele. The other parent is a VIR2/VIR2 wild type in the Landsberg genetic background, which has the RFLP alleles R1L and R2L, where L stands for Landsberg. F1 F2 vir2 R1C R2C / VIR2 R1L R2L (these plants were allowed to self-pollinate to produce F2 seed) You will plant them on Day 1 of this lab.

2 Mapping a Morphological Trait Using DNA Markers (RFLPs): Lab 1 1. Sowing seeds from F1 plants (seeds are the F2 population) Today you will sow Arabidopsis seeds collected from F1 plants created by Biology personnel. The seeds are mixed with sand in a 1.5 ml microfuge tube. You to sprinkle the sand/seed mixture over the surface of soil in order to get an even distribution of F2 plants growing in your tray. Think of lightly salting food as you sprinkle the seed/sand mixture over the soil and avoid dropping a big clump of seed/sand in one spot. 2. Sowing seeds for controls The TAs have sown seed from the original parents (the Columbia and Landsberg ecotypes) and they will serve as controls for you to use as you try to identify VIR2+/ and vir2/vir2 among the F2. 3. Cold stratification to break seed dormancy The trays will be placed in a refrigerator for 1 week to simulate cold stratification, which occurs during the cold, moist conditions of winter in nature. Cold stratification helps break seed dormancy and, under laboratory conditions, promotes synchronous germination. 4. Coming up Five weeks from today you will observe the plants, score the vir2/vir2 morphological phenotype and take tissue samples for DNA analysis.

3 Your name and group #: Mapping a Morphological Trait Using DNA Markers (RFLPs): Report after Lab 1 1. Write the genotypes of P1, P2, and the F1. 2. Predict VIR2 and R1 genotypes of the gametes that were produced by the F1 plants. Indicate the proportion of each gamete genotype under each of the following assumptions: a) VIR2 and R1 assort independently b) VIF2 and R1 are 40 cm apart. c) VIR2 and R1 are 30 cm apart d) VIR2 and R1 are 20 cm apart e) VIR2 and R1 are 10 cm apart. Show your work below and record the genotype frequencies in Table 1. TABLE 1 UNLINKED 40cM apart 30cM apart 20cM apart 10cM apart vir2 - R1C vir2 - R1L VIR2 + R1C VIR2 + R1L

4 3. Predict VIR2 and R2 genotypes of the gametes that were produced by the F1 plants. Indicate the proportion of each gamete genotype under each of the following assumptions: a) VIR2 and R2 assort independently b) VIF2 and R2 are 40 cm apart. c) VIR2 and R2 are 30 cm apart d) VIR2 and R2 are 20 cm apart e) VIR2 and R2 are 10 cm apart. Show your work below and record your results in Table 2. TABLE 2 UNLINKED 40cM apart 30cM apart 20cM apart 10cM apart vir2 - R2C vir2 - R2L VIR2 + R2C VIR2 + R2L

5 RsaI (128) RsaI (90) RsaI (226) R1C PCR Product 941 bp 1 GTCCTATCTC TACGATGTGG ATGATATTAT TGCGGCGGAA GGTTCGATGA CAGGATAGAG ATGCTACACC TACTATAATA ACGCCGCCTT CCAAGCTACT 51 AGGGTAAACA AAAGCTGCCA CACACTGAAC AATTCCGGTA CGATTCTTTT TCCCATTTGT TTTCGACGGT GTGTGACTTG TTAAGGCCAT GCTAAGAAAA 101 AATTAACTCA TCATTTGCTT TCCACGTACG ATCCTCAAAA CTTACAATGA TTAATTGAGT AGTAAACGAA AGGTGCATGC TAGGAGTTTT GAATGTTACT 151 TTTAAATTTT AAGATAAAAG TTTTAAAATT TTTCTTTTTC TTTTCCATTT AAATTTAAAA TTCTATTTTC AAAATTTTAA AAAGAAAAAG AAAAGGTAAA 201 AAGGGTTAAT TGAAATAATT GAGGTACTGG TTCCCTTAAT TAAATGTGAT TTCCCAATTA ACTTTATTAA CTCCATGACC AAGGGAATTA ATTTACACTA 251 AGTATCAGTA GACTTTAAAA ATGTGAAATT AAAAGTGGGG TTGTGTGGGT TCATAGTCAT CTGAAATTTT TACACTTTAA TTTTCACCCC AACACACCCA 301 GAGAAATAAC TCGAAATGCT CGTGTCCTTT AATTATAGTG GGACCGTAAA CTCTTTATTG AGCTTTACGA GCACAGGAAA TTAATATCAC CCTGGCATTT 351 TATTTCTCAT TATATGCCTA CTTGGCATTA GAAATCATTT TTTTTTGACA ATAAAGAGTA ATATACGGAT GAACCGTAAT CTTTAGTAAA AAAAAACTGT 401 GCCATTATAA ATTAATTAAA AGGTGTTGAA TATCTTTCTT TGTCTTTTGT CGGTAATATT TAATTAATTT TCCACAACTT ATAGAAAGAA ACAGAAAACA 451 TACGGGGAAA ATAAATTATT TTCATAATAT TATTACTCGA CCATACAGCG ATGCCCCTTT TATTTAATAA AAGTATTATA ATAATGAGCT GGTATGTCGC 501 AAACTGGTCC AGGATACGAA TTATACAGCT AATTACAACA TTTTTGGATT TTTGACCAGG TCCTATGCTT AATATGTCGA TTAATGTTGT AAAAACCTAA 551 TTTGGGTTCA AAGTCCCTAC TTTGGCATTT TATATTGTAT ATTTTTTCCT AAACCCAAGT TTCAGGGATG AAACCGTAAA ATATAACATA TAAAAAAGGA 601 TAAAATCCTA TGTGTGTGTG CATGTGTATA TCTTTGTTAG ATAACACGTT ATTTTAGGAT ACACACACAC GTACACATAT AGAAACAATC TATTGTGCAA 651 AAGTTCGGTT CATAGAATAG GTAGGAAAAT AAAAGTAGTC TCTCGGAATT TTCAAGCCAA GTATCTTATC CATCCTTTTA TTTTCATCAG AGAGCCTTAA 701 AGTCGGAAAG ATAAAGTAAT TCAGATAAAC AAATGAATAA GTATATGCAA TCAGCCTTTC TATTTCATTA AGTCTATTTG TTTACTTATT CATATACGTT 751 GTGATATCAA GTGGGGTAAA ATTCTCCATC TTTACTTTTT GCCTATAAAA CACTATAGTT CACCCCATTT TAAGAGGTAG AAATGAAAAA CGGATATTTT 801 AATTTCACAC CCACTCGTAG AATATTTGGT TTTTACAGAA AGAGTATCTT TTAAAGTGTG GGTGAGCATC TTATAAACCA AAAATGTCTT TCTCATAGAA 851 CATCATTGCG GAAAATTAAA TACACCAATT TGCTATTTTA TCTTTATTAT GTAGTAACGC CTTTTAATTT ATGTGGTTAA ACGATAAAAT AGAAATAATA RsaI RsaI RsaI 901 AGTATTATAA TTATGATTAT GATTCTAATG CCTTATAACT T TCATAATATT AATACTAATA CTAAGATTAC GGAATATTGA A

6 RsaI (128) RsaI (90) R1L PCR Product 941 bp 1 GTCCTATCTC TACGATGTGG ATGATATTAT TGCGGCGGAA GGTTCGATGA CAGGATAGAG ATGCTACACC TACTATAATA ACGCCGCCTT CCAAGCTACT 51 AGGGTAAACA AAAGCTGCCA CACACTGAAC AATTCCGGTA CGATTCTTTT TCCCATTTGT TTTCGACGGT GTGTGACTTG TTAAGGCCAT GCTAAGAAAA 101 AATTAACTCA TCATTTGCTT TCCACGTACG ATCCTCAAAA CTTACAATGA TTAATTGAGT AGTAAACGAA AGGTGCATGC TAGGAGTTTT GAATGTTACT 151 TTTAAATTTT AAGATAAAAG TTTTAAAATT TTTCTTTTTC TTTTCCATTT AAATTTAAAA TTCTATTTTC AAAATTTTAA AAAGAAAAAG AAAAGGTAAA 201 AAGGGTTAAT TGAAATAATT GAGGTGCTGG TTCCCTTAAT TAAATGTGAT TTCCCAATTA ACTTTATTAA CTCCACGACC AAGGGAATTA ATTTACACTA 251 AGTATCAGTA GACTTTAAAA ATGTGAAATT AAAAGTGGGG TTGTGTGGGT TCATAGTCAT CTGAAATTTT TACACTTTAA TTTTCACCCC AACACACCCA 301 GAGAAATAAC TCGAAATGCT CGTGTCCTTT AATTATAGTG GGACCGTAAA CTCTTTATTG AGCTTTACGA GCACAGGAAA TTAATATCAC CCTGGCATTT 351 TATTTCTCAT TATATGCCTA CTTGGCATTA GAAATCATTT TTTTTTGACA ATAAAGAGTA ATATACGGAT GAACCGTAAT CTTTAGTAAA AAAAAACTGT 401 GCCATTATAA ATTAATTAAA AGGTGTTGAA TATCTTTCTT TGTCTTTTGT CGGTAATATT TAATTAATTT TCCACAACTT ATAGAAAGAA ACAGAAAACA 451 TACGGGGAAA ATAAATTATT TTCATAATAT TATTACTCGA CCATACAGCG ATGCCCCTTT TATTTAATAA AAGTATTATA ATAATGAGCT GGTATGTCGC 501 AAACTGGTCC AGGATACGAA TTATACAGCT AATTACAACA TTTTTGGATT TTTGACCAGG TCCTATGCTT AATATGTCGA TTAATGTTGT AAAAACCTAA 551 TTTGGGTTCA AAGTCCCTAC TTTGGCATTT TATATTGTAT ATTTTTTCCT AAACCCAAGT TTCAGGGATG AAACCGTAAA ATATAACATA TAAAAAAGGA 601 TAAAATCCTA TGTGTGTGTG CATGTGTATA TCTTTGTTAG ATAACACGTT ATTTTAGGAT ACACACACAC GTACACATAT AGAAACAATC TATTGTGCAA 651 AAGTTCGGTT CATAGAATAG GTAGGAAAAT AAAAGTAGTC TCTCGGAATT TTCAAGCCAA GTATCTTATC CATCCTTTTA TTTTCATCAG AGAGCCTTAA 701 AGTCGGAAAG ATAAAGTAAT TCAGATAAAC AAATGAATAA GTATATGCAA TCAGCCTTTC TATTTCATTA AGTCTATTTG TTTACTTATT CATATACGTT 751 GTGATATCAA GTGGGGTAAA ATTCTCCATC TTTACTTTTT GCCTATAAAA CACTATAGTT CACCCCATTT TAAGAGGTAG AAATGAAAAA CGGATATTTT 801 AATTTCACAC CCACTCGTAG AATATTTGGT TTTTACAGAA AGAGTATCTT TTAAAGTGTG GGTGAGCATC TTATAAACCA AAAATGTCTT TCTCATAGAA 851 CATCATTGCG GAAAATTAAA TACACCAATT TGCTATTTTA TCTTTATTAT GTAGTAACGC CTTTTAATTT ATGTGGTTAA ACGATAAAAT AGAAATAATA RsaI RsaI 901 AGTATTATAA TTATGATTAT GATTCTAATG CCTTATAACT T TCATAATATT AATACTAATA CTAAGATTAC GGAATATTGA A

7 1 GCGTGACCAT CAAGACTAAT TAACTAAGAC CACATTTTAA AAAAACTATT CGCACTGGTA GTTCTGATTA ATTGATTCTG GTGTAAAATT TTTTTGATAA 51 AATAATTACT ACAATTTGTA ATTAAAAAGA TCAACGAGAA ATGCCACGTG TTATTAATGA TGTTAAACAT TAATTTTTCT AGTTGCTCTT TACGGTGCAC 101 GACGAATACT AGCAACGCCA AGTGGAAAGA GCGTTCGAGA GAACAAGGCA CTGCTTATGA TCGTTGCGGT TCACCTTTCT CGCAAGCTCT CTTGTTCCGT 151 AAACCAAATA CGCCCCTAGT ATTCTACAGA TGTCGACTGG ATAATTACAA TTTGGTTTAT GCGGGGATCA TAAGATGTCT ACAGCTGACC TATTAATGTT 201 AAGATTTCAA TAAACAGTAC TAATTAATTT CTAGTGGTGA GTTTTTGTAA TTCTAAAGTT ATTTGTCATG ATTAATTAAA GATCACCACT CAAAAACATT 251 ATATCTACTT CTTCCAATTA CCAGCTGCTA TATAAATCCC CTTCTCTGTT TATAGATGAA GAAGGTTAAT GGTCGACGAT ATATTTAGGG GAAGAGACAA 301 TCTCTTTTCT TACATCACAA TCACACAAAA CTAACAAAAG ATCAAAAGCA AGAGAAAAGA ATGTAGTGTT AGTGTGTTTT GATTGTTTTC TAGTTTTCGT 351 AGTTCTTCAC TGTTGATAAT GTCTACCACC GGACAGATTA TTCGATGCAA TCAAGAAGTG ACAACTATTA CAGATGGTGG CCTGTCTAAT AAGCTACGTT 401 AGGTTTTCTT TTTATTCTGT CTTTTTCCAA ATATTTATTG ATCGGTTACA TCCAAAAGAA AAATAAGACA GAAAAAGGTT TATAAATAAC TAGCCAATGT 451 TTTCTGTTGA GGTTTTTGTT ATGAATCCAC AATTTCTATG TTGAATTAAC AAAGACAACT CCAAAAACAA TACTTAGGTG TTAAAGATAC AACTTAATTG 501 AAAACCTGTG TCGTTTTTTT GTGGTGGTTG CAGCTGCTGT GGCATGGGAA TTTTGGACAC AGCAAAAAAA CACCACCAAC GTCGACGACA CCGTACCCTT 551 GCCGGAAAGC CACTGGTGAT CGAGGAAGTG GAGGTTGCTC CACCGCAGAA CGGCCTTTCG GTGACCACTA GCTCCTTCAC CTCCAACGAG GTGGCGTCTT 601 ACACGAAGTT CGTATCAAGA TTCTCTTCAC TTCTCTCTGT CACACCGATG TGTGCTTCAA GCATAGTTCT AAGAGAAGTG AAGAGAGACA GTGTGGCTAC 651 TTTACTTCTG GGAAGCTAAG GTAGAGTAAT CAATTTATTA CACTCCAAAT AAATGAAGAC CCTTCGATTC CATCTCATTA GTTAAATAAT GTGAGGTTTA 701 TCATAATCAA GTTCTAATTT TTTTAGAATT CTAATTTTTT ATCTAAAAAA AGTATTAGTT CAAGATTAAA AAAATCTTAA GATTAAAAAA TAGATTTTTT 751 ATTCAACCTT TTTGATTCCA CAGGGACAAA CACCGTTGTT TCCACGTATC TAAGTTGGAA AAACTAAGGT GTCCCTGTTT GTGGCAACAA AGGTGCATAG 801 TTCGGCCATG AAGCTGGAGG GTAATAGAAA CACTAATCTT CTTTGCTTCG AAGCCGGTAC TTCGACCTCC CATTATCTTT GTGATTAGAA GAAACGAAGC 851 TTTTGGATAT TTTTAAGGTT TTAGAGATTC AAGGTCGTTT TTTTTGTTGT AAAACCTATA AAAATTCCAA AATCTCTAAG TTCCAGCAAA AAAAACAACA 901 TGTGTAGGAT TGTTGAGAGT GTTGGAGAAG GAGTGACTGA TCTTCAGCCA ACACATCCTA ACAACTCTCA CAACCTCTTC CTCACTGACT AGAAGTCGGT 951 GGAGATCATG TGTTGCCGAT CTTTACCGGA GAATGTGGGG AGTGTCGTCA CCTCTAGTAC ACAACGGCTA GAAATGGCCT CTTACACCCC TCACAGCAGT 1001 TTGCCACTCG GAGGAATCAA ACATGTGTGA TCTTCTCAGG ATCAACACCG AACGGTGAGC CTCCTTAGTT TGTACACACT AGAAGAGTCC TAGTTGTGGC 1051 AGCGAGGAGG GATGATTCAC GATGGTGAAT CAAGATTCTC CATTAATGGC TCGCTCCTCC CTACTAAGTG CTACCACTTA GTTCTAAGAG GTAATTACCG 1101 AAACCAATTT ACCATTTCCT TGGGACTTCC ACGTTCAGTG AGTACACAGT TTTGGTTAAA TGGTAAAGGA ACCCTGAAGG TGCAAGTCAC TCATGTGTCA 1151 GGTTCACTCT GGTCAGGTTG CTAAGATCAA TCCGGATGCT CCTCTTGACA CCAAGTGAGA CCAGTCCAAC GATTCTAGTT AGGCCTACGA GGAGAACTGT 1201 AGGTCTGTAT TGTCAGTTGT GGTTTGTCTA CTGGGTTAGG AGCAACTTTG TCCAGACATA ACAGTCAACA CCAAACAGAT GACCCAATCC TCGTTGAAAC 1251 AATGTGGCTA AACCCAAGAA AGGTCAAAGT GTTGCCATTT TT TTACACCGAT TTGGGTTCTT TCCAGTTTCA CAACGGTAAA AA 1292 bp R2C PCR Product

8 1 GCGTGACCAT CAAGACTAAT TAACTAAGAC CACATTTTAA AAAAACTATT CGCACTGGTA GTTCTGATTA ATTGATTCTG GTGTAAAATT TTTTTGATAA 51 AATAATTACT ACAATTTGTA ATTAAAAAGA TCAACGAGAA ATGCCACGTG TTATTAATGA TGTTAAACAT TAATTTTTCT AGTTGCTCTT TACGGTGCAC 101 GACGAATACT AGCAACGCCA AGTGGAAAGA GCGTTCGAGA GAACAAGGCA CTGCTTATGA TCGTTGCGGT TCACCTTTCT CGCAAGCTCT CTTGTTCCGT 151 AAACCAAATA CGCCCCTAGT ATTCTACAGA TGTCGACTGG ATAATTACAA TTTGGTTTAT GCGGGGATCA TAAGATGTCT ACAGCTGACC TATTAATGTT 201 AAGATTTCAA TAAACAGTAC TAATTAATTT CTAGTGGTGA GTTTTTGTAA TTCTAAAGTT ATTTGTCATG ATTAATTAAA GATCACCACT CAAAAACATT 251 ATATCTACTT CTTCCAATTA CCAGCTGCTA TATAAATCCC CTTCTCTGTT TATAGATGAA GAAGGTTAAT GGTCGACGAT ATATTTAGGG GAAGAGACAA 301 TCTCTTTTCT TACATCACAA TCACACAAAA CTAACAAAAG ATCAAAAGCA AGAGAAAAGA ATGTAGTGTT AGTGTGTTTT GATTGTTTTC TAGTTTTCGT 351 AGTTCTTCAC TGTTGATAAT GTCTACCACC GGACAGATTA TTCGATGCAA TCAAGAAGTG ACAACTATTA CAGATGGTGG CCTGTCTAAT AAGCTACGTT 401 AGGTTTTCTT TTTATTCTGT CTTTTTCCAA ATATTTATTG ATCGGTTACA TCCAAAAGAA AAATAAGACA GAAAAAGGTT TATAAATAAC TAGCCAATGT 451 TTTCTGTTGA GGTTTTTGTT ATGAATCCAC AATTTCTATG TTGAATTAAC AAAGACAACT CCAAAAACAA TACTTAGGTG TTAAAGATAC AACTTAATTG 501 AAAACCTGTG TCGTTTTTTT GTGGTGGTTG CAGCTGCTGT GGCATGGGAA TTTTGGACAC AGCAAAAAAA CACCACCAAC GTCGACGACA CCGTACCCTT 551 GCCGGAAAGC CACTGGTGAT CGAGGAAGTG GAGGTTGCTC CACCGCAGAA CGGCCTTTCG GTGACCACTA GCTCCTTCAC CTCCAACGAG GTGGCGTCTT 601 ACACGAAGTT CGTATCAAGA TTCTCTTCAC TTCTCTCTGT CACACCGATG TGTGCTTCAA GCATAGTTCT AAGAGAAGTG AAGAGAGACA GTGTGGCTAC 651 TTTACTTCTG GGAAGCTAAG GTAGAGTAAT CAATTTATTA CACTCCAAAT AAATGAAGAC CCTTCGATTC CATCTCATTA GTTAAATAAT GTGAGGTTTA 701 TCATAATCAA GTTCTAATTT TTTTAGAATT CTAATTTTTT ATCTAAAAAA AGTATTAGTT CAAGATTAAA AAAATCTTAA GATTAAAAAA TAGATTTTTT 751 ATTCAACCTT TTTGATTCCA CAGGGACAAA CACCGTTGTT TCCACGTATC TAAGTTGGAA AAACTAAGGT GTCCCTGTTT GTGGCAACAA AGGTGCATAG 801 TTCGGCCATG AAGCTGGAGG GTAATAGAAA CACTAATCTT CTTTGCTTCG AAGCCGGTAC TTCGACCTCC CATTATCTTT GTGATTAGAA GAAACGAAGC 851 TTTTGGATAT TTTTAAGGTT TTAGAGATTC AAGGTCGTTT TTTTTGTTGT AAAACCTATA AAAATTCCAA AATCTCTAAG TTCCAGCAAA AAAAACAACA 901 TGTGTAGGAT TGTTGAGAGT GTTGGAGAAG GAGTGACTGA TCTTCAGCCA ACACATCCTA ACAACTCTCA CAACCTCTTC CTCACTGACT AGAAGTCGGT 951 GGAGATCATG TGTTGCCGAT CTTTACCGGA GAATGTGGGG AGTGTCGTCA CCTCTAGTAC ACAACGGCTA GAAATGGCCT CTTACACCCC TCACAGCAGT 1001 TTGCCACTCG GAGGAATCAA ACATGTGTGA TCTTCTCAGG ATCAACACCG AACGGTGAGC CTCCTTAGTT TGTACACACT AGAAGAGTCC TAGTTGTGGC 1051 AGCGAGGAGG GATGATTCAC GATGGTGAAT CTAGATTCTC CATTAATGGC TCGCTCCTCC CTACTAAGTG CTACCACTTA GATCTAAGAG GTAATTACCG 1101 AAACCAATTT ACCATTTCCT TGGGACTTCC ACGTTCAGTG AGTACACAGT TTTGGTTAAA TGGTAAAGGA ACCCTGAAGG TGCAAGTCAC TCATGTGTCA 1151 GGTTCACTCT GGTCAGGTTG CTAAGATCAA TCCGGATGCT CCTCTTGACA CCAAGTGAGA CCAGTCCAAC GATTCTAGTT AGGCCTACGA GGAGAACTGT 1201 AGGTCTGTAT TGTCAGTTGT GGTTTGTCTA CTGGGTTAGG AGCAACTTTG TCCAGACATA ACAGTCAACA CCAAACAGAT GACCCAATCC TCGTTGAAAC 1251 AATGTGGCTA AACCCAAGAA AGGTCAAAGT GTTGCCATTT TT TTACACCGAT TTGGGTTCTT TCCAGTTTCA CAACGGTAAA AA XbaI 1292 bp R2L PCR Product XbaI (1081)

9 Mapping a Morphological Trait Using DNA Markers (RFLPs): Lab 2 Today you will observe the phenotype of your F2 plants, count the number of wild type and vir2 mutant plants, and set up PCR reactions to amplify three RFLP loci from tissue sample of two vir2 mutant plants. A. On your table you will have: Two PCR reaction tubes R1 (blue) and R2 (orange/red) 1 ml of bleach 1ml of sterile distilled water A Harris 0.5 mm UniCore hole punch (don t touch it yet!) A cutting pad Plants PCR contents: 20 ul reaction total volume 10 ul 2X buffer 1 ul forward primer 1 ul reverse primer 7.6 ul dh 2 O 0.4 ul enzyme B. Count the number of wild type and mutant F2 plants in the tray. We ll take the average of the class data. Wild type: Mutant: Record your results in the table on the Smart Board and record class data. C. Obtain leaf tissue samples for PCR: EACH STUDENT ANALZES ONE PLANT You will take tissue plugs from a leaf on each plant you analyze and add it to a PCR reaction mixture provided to you by the TAs. You will do this by using a 0.5 mm Harris hole punch to obtain leaf tissue cores, using the technique illustrated by the instructor. PLEASE NOTE: 1. Do not stab yourself with the needle of the hole punch. You should analyze plant DNA, not your own. 2. Do not break the needle of the hole punch. Very little force/pressure is needed to push the needle through the leaf, so don t over exert yourself and break the hole punch. 3. Rinse the hole punch needle in water between punches of the same leaf a. Rinse the hole punch by submerging the needle in water and pressing the button ten times. 4. Wash and rinse the hole punch when you are done with one leaf and before you work on another leaf. a. Wash the hole punch needle in bleach by submerging the needle in bleach and pressing the button three times. b. After the bleach wash, rinse the hole punch by submerging the needle in water and pressing the button ten times.

10 Detailed Protocol for Setting up PCR Reactions: 1. Each student should select one (supposed) vir2/vir2 plant for analysis. 2. Take one leaf from one plant; place it on the cutting pad. Extract a tissue plug from the leaf using the Harris hole punch as demonstrated by the instructor and eject the plug into the blue R1 tube. Make sure the leaf plug is visible in the tube using the dissecting scope and then close the tube tightly. Label it R1 and your assigned student number (1 through 30). 3. Rinse the hole punch in water. Extract a second plug from the same leaf and eject it into the orange/red R2 tube. Make sure the leaf plug is visible in the tube using the dissecting scope and then close the tube tightly. Label it R2 and your assigned student number. 4. Wash the hole punch in bleach and then rinse in water. ALL BLEACH MUST BE RINSED OFF! 5. Let another student at your table use the punch and pad for their plant leaf. 6. When all students are done, wash the hole punch in bleach and rinse it in water and leave it on your bench for a TA to collect. 7. Inform the TAs that you are done and they will collect the PCR reactions, place them in a PCR machine and run the following PCR protocol: 1 Cycle of 98 o C for 5 minutes 40 Cycles of 98 o C for 5 seconds 58 o C for 5 seconds 72 o C for 30 seconds 1 Cycle of 72 o C for 1 minute You will analyze the PCR reactions in the next lab period.

11 Your name and group #: Mapping a Morphological Trait Using DNA Markers (RFLPs): Report after Lab 2 1. The seeds you plated a few weeks back were supposed to be F2 from the series of crosses presented at the beginning of this lab and which you wrote up for the lab 1. There is some doubt among the BIO 304 instructors and TA whether this is true because the mutant phenotype has not been obvious among potential F2 populations in semesters past. State the class observations and perform a chi-square analysis to determine whether the observed proportion of vir2/vir2 plants is consistent with the plants being F2. 2. The attached sequence pages show the R1 and R2 genetic loci that will be amplified by the PCR reactions you set up today. The PCR reaction includes a step to allow primers to anneal to the templates ( annealing step ), which is conducted at a temperature that is determined by the melting temperature of the primers in each reaction. The primers for the R1 locus correspond to the first 23 bp of R1 sequence and the last 27 bp of the sequence. The primers for the R2 locus correspond to the first 20 bp and last 20 bp of R2. Write the primer sequences and calculate the T m of these primers using the following equation: #G+#C-16.4 N o T m( C)= ( )

12 3. Use the sequence information for R1 and R2 to determine the expected size of each PCR product. Also indicate the size of restriction fragments that will be produced for the C and L alleles of each locus. Enter your information in the table below. Locus R1C R1L R2C R2L Size of Undigested PCR Product Size of Fragments After Restriction Digests 4. Since it was difficult to identify vir2/ vir2 mutants, it is possible that someone messed up and the seeds planted at the beginning of this lab were not F2. There are no other A. thaliana seed sources in the department, therefore the possible alternatives are P1 (Columbia ecotype), P2 (Landsberg ecotype), or the F1 hybrid. The class has performed PCR analysis of two RFLP loci of many plants. Predict the results of the RFLP analysis if the plants are P1, P2 or F1 using the information from step 3. Predicted RLFP data if the plants are P1: R1 locus: R2 locus: Predicted RFLP data if the plants are P2: R1 locus R2 locus Predicted RFLP data if the plants are F1: R1 locus R2 locus

13 Mapping a Morphological Trait Using DNA Markers (RFLPs): Lab 3 Today you will run samples of your PCR reactions from the previous lab on a gel and set up restriction digests of each PCR reactions. You will analyze the restriction digests during the next lab period. A. On your bench you will have: PCR reactions you set up in the previous lab period a tube with DNA molecular weight markers a tube with 6X gel loading buffer a 1.6% agarose gel in the gel tank Two tubes containing 15 ul of RsaI Reaction Mix (BLUE TUBES) Two tubes containing 15 ul of XbaI Reaction Mix (ORANGE TUBES) B. Set up restriction digests of a 5 ul sample of each PCR reaction by pipetting 5 ul of the PCR reaction into the appropriate Reaction Mix tube: 5 ul of the one student s R1 PCR reaction into one Blue Tube. Label it with the student # 5 ul of that same student s R2 PCR reaction into one Orange Tube. Label it with the student # 5 ul of the another student s R1 PCR reaction into one Blue Tube. Label it with the student # 5 ul of that same student s R2 PCR reaction into one Orange Tube. Label it with the student # NOTE: continue until each student has set up one digest for each of their PCR reaction. Incubate the reactions at 37 degrees for 30 minutes, and then prepare the reaction for gel electrophoresis by adding 5ul of 6X gel loading buffer. C. Load sample of your restriction digests on your gel as follows: 10 ul molecular weight marker in lane 1 20 ul of one student s Blue Tube in lane 2 20 ul of a second student s Blue Tube in lane 3 20 ul of a third student s Blue Tube in lane 4 20 ul of the first student s Orange Tube in lane 5 20 ul of the second student s Orange Tube in lane 6 20 ul of the third student s Orange Tube in lane 7 NOTE: The other two students on the bench should use the second gel to run markers and their digests using the same pattern as above. Run the gel at 90 volts for about 30 minutes. The TAs will photograph the gel and give you a copy of the photo. D. Analyze your results: Analyze your results using the information from your write up from plant lab 2. Plant 1: vir2 vir2, R1 R1, R2 R2

14 Record your results on the white board and record your data and the class data in Table 4 on the next page. You will use this information to determine whether there is evidence for linkage between the vir2 locus and each RFLP locus. Class results: Student # vir2 vir2 R1 R1 R2 R2 1 vir2 vir2 2 vir2 vir2 3 vir2 vir2 4 vir2 vir2 5 vir2 vir2 6 vir2 vir2 7 vir2 vir2 8 vir2 vir2 9 vir2 vir2 10 vir2 vir2 11 vir2 vir2 12 vir2 vir2 13 vir2 vir2 14 vir2 vir2 15 vir2 vir2 16 vir2 vir2 17 vir2 vir2 18 vir2 vir2 19 vir2 vir2 20 vir2 vir2 21 vir2 vir2 22 vir2 vir2 23 vir2 vir2 24 vir2 vir2 25 vir2 vir2 26 vir2 vir2 27 vir2 vir2 28 vir2 vir2 29 vir2 vir2 30 vir2 vir2 31 vir2 vir2 32 vir2 vir2 33 vir2 vir2 34 vir2 vir2 35 vir2 vir2

15 Your name and group #: Mapping a Morphological Trait Using DNA Markers (RFLPs): Report after Lab 4 1. Based on the class data, what is the percent recombination for vir2 and the R1 locus? 2. Perform a chi-square test to determine whether the evidence for linkage of vir2 and R1 is statistically significant. Show your work below. 3. Based on the class data, what is the percent recombination for vir2 and the R1 locus? 4. Perform a chi-square test to determine whether the evidence for linkage of vir2 and R1 is statistically significant. Show your work below. 5. Draw a map of chromosome I showing the locations of the R1, R2, and VIR2 loci.