1. Using mutants to clone genes Objectives 1. What is positional cloning? 2.What is insertional tagging? 3.How can one confirm that the gene cloned is the same one that is mutated to give the phenotype of interest?

2. Reading References: Westhoff et. al. Molecular Plant Development: from gene to plant. Chapter 3: 52-65.

3. Positional (map-based) Cloning Map based cloning is a dependable method of cloning a gene using a mutant phenotype, molecular genetic markers and genetic recombination. This method is most easily done in organisms where the necessary tools (genetic map, physical map and or sequence of the genome) are available.

4. Positional (map-based) Cloning 1. Use the mutant phenotype and DNA-based genetic markers of known position to map, using recombination, the gene of interest to a site on a specific chromosome.

5. DNA-Based Genetic Markers The genomes of two individuals of the same species are rarely identical and can have many nucleotide differences between them. These variations in DNA sequences often do not alter the function of a gene but can be used as phenotypes in genetic mapping by detecting the differences using: 1. PCR amplification (simple sequence-length polymorphism = SSLP) 2. a combination of both PCR amplification followed by restriction endonuclease digestion (cleaved amplified polymorphic sequences = CAPS).

6. Chromosome of Individual #1 PCR primers amplify this region Homozygote #1 Small Insertions and deletions (SSLP) in DNA sequence can be identified using PCR and gel electrophoresis Chromosome of Individual #2 These simple sequence length polymorphisms SSLP can be used as co-dominant markers for specific positions on a chromosome. Homozygote #2 Heterzygote CTGGACTACTACGAGTTACC GACCTGATGATGCTCAATGG CTGTTACC GACAATGG

7. Chromosome of Individual #2 CTGG GAATTC TTACC Chromosome of Individual #1 CTGG GAAGTC TTACC Amplify by PCR Restrict amplified fragments with EcoR1 and separate on an electrophoretic gel. EcoR1 site Ind #1 Ind #2 #1 x #2 F1 DNA single nucleotide polymorphism may be identified using CAPS

8. Mapping to DNA-Based Genetic Markers The genomes of individuals form different populations of the same species differ in a large number of SSLPs. This variation can be detected and used as genetic markers for specific positions on chromosomes. When two such individuals are crossed all the differences will segregate in the F2 progeny and can be mapped relative to one another or any novel phenotype in one of the parents. Eg. Arabidopsis populations from different parts of the world are called ecotypes: Columbia (ecotype from southern US)(Col) Landsberg erecta (ecotype from Germany)(Ler)

9. PCR primers amplify this region Col SSLP 8 Small Insertions and deletions in DNA sequence can be identified using PCR and gel electrophoresis These microsatellites (simple sequence length polymorphisms SSLP) can be used as co-dominant markers for specific positions on a chromosome. Ler SSLP 8 Heterzygote CTGTTACC GACAATGG Chromosome of Columbia (Col) ecotype homozygous for SSLP allele at locus 8, chromoso me 1 Chromosome of Landsberg erecta (Ler) ecotype homozygous for SSLP allele at locus 8, chromosome 1 CTGGACTACTACGAGTTACC GACCTGATGATGCTCAATGG

10. SSLP markers can be mapped using recombination just like genes In a cross Columbia and Landsberg erecta, the resulting F1 progeny will be heterozygous at all SSLP loci that were identified between the two: SSLP 16 C /SSLP 16 L ; SSLP 72 C /SSLP 72 L ; SSLP 8 C /SSLP 8 L Therefore in the F2 generation they can be mapped relative to one another

11. Chromosome 1 of Landsberg erecta ecotype showing SSLP markers Chromosome 1 of Columbia ecotype showing SSLP markers SSLP14 L SSLP16 L SSLP41 L SSLP83 L SSLP8 L SSLP8 C SSLP83 C SSLP41 C SSLP16 C SSLP14 C SSLP markers can be mapped using recombination just like genes

12. Arabidopsis genetic map showing the position of SSLP markers. 12345 SSLP 8 SSLP 83 SSLP 14 SSLP 25 SSLP 68 SSLP 102 SSLP 43 SSLP 95 SSLP 24 SSLP 71 SSLP 4 SSLP 39

13. Apetala2 mutant has flowers where the sepals and petals are replaced by reproductive organs

14. AP2 normal flowers > ap2 flowers AP2 protein is required to make sure that the proper organs are made in the outer part of the flower. We are studying how floral morphogenesis is controlled during development and would like to determine what kind of protein is encoded by AP2. ie Which of the 30,000 Arabidopsis genes known by DNA sequence (entire genome has been sequenced) is AP2. Apetala2 mutant has flowers where the sepals and petals are replaced by reproductive organs

15. Procedure For Mapping a Mutant Phenotype Relative to Defined DNA Markers Chromosome ?ap2/ap2 (Col) AP2/AP2 (Ler) chromosome 1 SSLP 16 C /SSLP 16 C SSLP 16 L /SSLP 16 L chromosome 4 SSLP 72 C /SSLP 72 C SSLP 72 L /SSLP 72 L F 1 AP2/ap2 SSLP 16 C /SSLP 16 L SSLP 72 C /SSLP 72 L F2F2 See how often the Columbia allele of the AP2 gene (ap2) segregates with the Columbia alleles of SSLP 16; SSLP 72 and all other mapped SSLP loci. X

16. Procedure For Mapping a Mutant Phenotype Relative to Defined DNA Markers ap2/ap2 (Col) x AP2/AP2 (Ler) F 1 AP2/ap2 Ap2 Mutants isolated from the F2, DNA extracted from each and tested for different molecular markers. Plant 1 Plant 2 Plant 3 Plant 4 Plant5 Plant 6 Plant 7 Plant 8 F2 ap2/ap2 ap2/ap2 ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2 What is the expected frequencies of the alleles for one molecular marker in these F2 progeny assuming no linkage to AP2? Ler Col SSLP8 C SSLP83 C SSLP41 C SSLP14 L SSLP16 L SSLP1 L SSLP83 L SSLP8 L SSLP16 C SSLP14 C

17. PCR primers amplify this region SSLP 8 C SSLP genotypes in DNA sequence of the ap2 mutants can be identified using PCR and gel electrophoresis These microsatellites (simple sequence length polymorphisms SSLP) can be used as co-dominant markers for specific positions on a chromosome. rSSLP 8 L Heterzygote CTGTTACC GACAATGG Chromosome 1 of Columbia (Col) ecotype homo-zygous for SSLP allele at locus 8, Chromosome1 of Landsberg erecta (Ler) ecotype homozygous for SSLP allele at locus 8, CTGGACTACTACGAGTTACC GACCTGATGATGCTCAATGG

18. Procedure For Mapping a Mutant Phenotype Relative to Defined DNA Markers ap2/ap2 (Col) x AP2/AP2 (Ler) F 1 AP2/ap2 Ap2 Mutants isolated from the F2 and DNA extracted F2 ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2 SSLP 71 #4 C/L C/C L/L C/L C/L L/L C/L C/C SSLP 83 #1 C/C C/C L/C C/C C/C C/C C/C C/C SSLP 8 #1 C/C C/C C/C C/C C/L C/C C/C C/C SSLP 16 #1 C/C C/L L/C L/L C/L L/L C/C L/C Chromosome # Ler Col SSLP8 C SSLP83 C SSLP41 C SSLP14 L SSLP16 L SSLP41 L SSLP83 L SSLP8 L SSLP16 C SSLP14 C

19. Chromosome of Landsberg erecta Chromosome of Columbia ecotype with ap2-1 mutation ap2-1 AP2 SSLP8 C SSLP83 C SSLP41 C SSLP14 L SSLP16 L The ap2-1 mutant phenotype is found to segregate with SSLP83 C and SSLP8 C but no others. The map distance from each of these two to AP2 is 1/16 = 6.25 map units. If there is 12.5 map units between SSLP8 SSLP83 the AP2 gene must lie between these two SSLP sites. SSLP41 L SSLP83 L SSLP8 L SSLP16 C SSLP14 C [ ]

20. Positional Cloning AP2 [ ] AP2 DNA from the AP2 locus With 10 genes = Genetic map recombination SSLP8 C Genes in the AP2 locus SSLP83 C

21. The Sequences For All Annotated Genes Are Available Sequence: AT1G30960.1 Date last modified 2003-05-27 Name AT1G30960.1 Tair Accession Sequence:2015773 GenBank Accession NM_102835 Sequence Length (bp) 1314 Sequence NM_102835 1 ATGAAAGCTT TTAGATCTCT ACGTATACTA ATTTCCATCT CACGAACGAC 51 GACGAAGACA ACACCTCGTA ATCCCCATCA AGCACAAAAC TTTCTCCGCC 101 GATTTTACTC AGCGCAGCCG AATCTAGACG AACCCACTTC CATCAATGAA 151 GACGGATCAA GCAGCGACTC TGTTTTCGAT AGTAGTCAAT ACCCAATCGA 201 CGATTCCAAT GTAGATTCCG TGAAGAAGCC CAAGGAAGCA ACTTGGGATA 251 AAGGGTACAG AGAAAGAGTA AACAAAGCCT TCTTTGGAAA CTTGACAGAG 301 AAAGGTAAAG TGAAAGTTGC AGAAGAAGAG AGTTCTGAAG ATGATGAGGA 351 TAGTGTTGAT AGGTCAAGGA TTCTCGCTAA GGCTCTCTTA GAGGCTGCGT 401 TAGAGTCACC AGATGAAGAA CTTGGTGAAG GTGAAGTTAG AGAAGAAGAT 451 CAGAAGTCGC TTAATGTCGG CATCATCGGT CCACCTAATG CAGGAAAATC 501 TTCGCTGACT AATTTCATGG TTGGAACAAA GGTTGCTGCT GCTTCACGGA 551 AGACTAACAC GACGACACAT GAAGTGTTAG GAGTATTGAC AAAAGGAGAT 601 ACACAAGTCT GTTTCTTCGA TACTCCGGGT CTGATGCTGA AGAAAAGCGG 651 ATATGGTTAC AAAGACATCA AGGCTCGTGT GCAAAATGCT TGGACTTCTG 701 TTGACCTGTT TGATGTCCTC ATTGTTATGT TTGATGTCCA TAGGCATCTC 751 ATGAGTCCCG ATTCAAGAGT GGTACGCTTG ATCAAATACA TGGGAGAAGA 801 AGAAAATCCG AAACAAAAGC GCGTTTTATG TATGAACAAA GTTGATCTGG 851 TTGAGAAGAA AAAGGATCTA TTAAAGGTTG CTGAGGAGTT CCAAGATCTT 901 CCGGCATATG AAAGATACTT CATGATATCG GGACTTAAGG GATCAGGAGT 951 GAAAGATCTT TCCCAATACT TAATGGATCA GGCTGTTAAA AAACCATGGG 1001 AAGAAGATGC ATTCACGATG AGTGAAGAAG TCTTGAAGAA CATTTCTCTT 1051 GAAGTTGTTA GGGAGAGATT ACTAGACCAT GTCCATCAGG AAATACCATA 1101 TGGTCTGGAG CACCGTCTAG TGGACTGGAA AGAGCTGCGT GACGGGTCTC 1151 TTAGAATTGA ACAGCATCTC ATCACTCCTA AACTTAGCCA ACGCAAGATT 1201 CTTGTAGGCA AGGGCGGTTG CAAGATCGGG AGGATAGGAA TTGAGGCCAA 1251 TGAAGAACTC AGGAGAATAA TGAACCGCAA AGTTCATCTC ATTCTCCAGG 1301 TTAAGCTCAA GTGA Comments (shows only the most recent comments by default) Attribution type name date submitted_by AGI-TIGR 2001-03-06submitted_by GenBank 2002-08-20 General comments or questions: curator@arabidopsis.org Seed or DNA stock questions (donations, availability, orders, etc): abrc@arabidopsis.orgAGI-TIGRGenBank curator@arabidopsis.org abrc@arabidopsis.org

22. Positional (map-based) Cloning 1. Use the mutant phenotype and DNA-based genetic markers to map, using recombination, the gene of interest to a region on a specific chromosome. 2. Examine the sequence of chromosomal DNA from that region to determine the number of annotated genes. 3. Narrow down to correct gene using predicted function, mutant allele sequence, complementation, expression analysis etc.

23. Insertional Tagging 1. Isolate mutant phenotype of interest from an insertional mutagenized population of plants. (Insertion DNA must be cloned: eg TDNA or Transposon). 2. Check that the transposon or TDNA in the mutant segregates with the mutant phenotype. ---The segregation of an insert can often be followed using the phenotype of a gene encoded in the insert (eg Kanamycin resistance), a probe for the insert or PCR primers that can amplify part of the insert. ---repetitive elements (eg. transposons) may complicate such an analysis.

24. Insertional Tagging 1. Isolate mutant phenotype of interest from an insertional mutagenized population of plants. (Insertion DNA must be cloned: eg TDNA or Transposon). 2. Check that the transposon or TDNA in the mutant segregates with the mutant phenotype. 3.Clone or amplify the chromosomal DNA at the site of insertion using the known sequence of the TDNA or transposon.

25. Insertional Tagging P  coding region P  coding region Gene X Gene X with insert Portion of a chromosome with genes including the one with insert

26. Digest genomic DNA with restriction endonuclease Identify the fragment carrying the insert: Eg. 1. Make a library and probe with the insertion sequences or 2. Ligate the DNA into circles and amplify using divergent insert primers (inverse PCR) Insertional Tagging

27. Inverse PCR ligate Clone into vector Amplify by PCR

28. Insertional Tagging 1. Isolate mutant phenotype of interest from an insertional mutagenized population of plants. (Insertion DNA must be cloned: eg TDNA or Transposon). 2. Check that the transposon or TDNA in the mutant segregates with the mutant phenotype. 3.Clone or amplify the chromosomal DNA at the site of insertion using the known sequence of the TDNA or transposon. 4. Sequence the DNA flanking the TDNA or transposon from the mutant and use the sequence to identify the wild type gene.

29. Insertional Tagging Clone into vector Use sequences from gene X to identify the wild type allele. P  coding region Gene X

30. Connecting a cloned gene with a mutant phenotype Despite the method of cloning, one must confirm that the gene cloned (X) is the same gene that is mutated in mutant M (gene M). 1. Transgene complementation. The wild type fragment carrying gene X should be able to complement the recessive mutant M phenotype. This hypothesis can be tested by transforming the homozygous mutant with the wild type gene to check if it will restore the wild type phenotype. Eg. Transform pea rr plants with the SBEI gene to see if the gene will complement the mutant phenotype.

31. Connecting a cloned gene with a mutant phenotype 1. Transgene complementation. 2. Sequence gene X from several mutants homozygous for different alleles of gene M. If the ‘ M ’ gene and X gene are the same then one should find a gene X mutation in every M mutant. This hypothesis can be tested by sequencing gene X from several M mutants each carrying a different allele of the gene of interest. Eg. Sequence the SBEI gene in several different ‘r’ pea mutants each homozygous for a different r mutant allele. One should find a different mutation in the SBEI gene in every such ‘r’ mutant.

32. Connecting a cloned gene with a mutant phenotype 1. Transgene complementation. 2. Sequence gene X from several mutants homozygous for different alleles of gene M. 3. Cosegregation analysis. DNA-based markers (RFLP) identifying gene X should cosegregate with the mutant phenotype M in genetic crosses. This hypothesis can be tested by crossing mutant M to a wild type plant, self-fertilizing the F1 progeny to produce F2 progeny and scoring F2 plants for the mutant phenotype and the gene X molecular marker. Eg. Follow the segregation of an RFLP for the SBEI gene with the wrinkled seed phenotype of rr.

33. Connecting a cloned gene with a mutant phenotype Genotype of plants homozygous for different alleles of the AP2 gene: AP2/AP2 ap2-1/ap2-1 ap2-2/ap2-2 -cloned a wild type gene, MYB83, encoding a transcription factor. Is MYB83 gene AP2? Clone MYB83 from each of the three plants above by PCR amplification. If MYB83 is AP2 then MYB83 from AP2/AP2 will have a wild type sequence. MYB83 from ap2-1/ap2-1 will have a mutation. MYB83 from ap2-2/ap2-2 will also have a mutation but different from that of ap2-1.

34. Connecting a cloned gene with a mutant phenotype 1. Transgene complementation. 2. Sequence gene X from several mutants homozygous for different alleles of gene M. 3. Cosegregation analysis. 4. Reverse genetics. Identify mutant alleles of gene X using reverse genetics. Mutations in gene X should have the same phenotype as mutant M and fail to complement the M mutant phenotype. Eg. A loss of function mutation in the SBEI gene should have a wrinkled seed phenotype.

35. A population of plants has been transformed with a fragment of DNA carrying a gene that confers antibiotic resistance (resistance is a dominant phenotype. One plant from the population has an ap2 mutant phenotype and fails to complement a known ap2 mutant. You hypothesize that the transformed DNA has inserted into the AP2 gene resulting in a loss of function mutation. If so you can use the line to clone the AP2 gene. To check whether the transformed DNA fragment is actually in the AP2 gene you cross the new ap2 mutant from the transformed population to wild type and select 30 ap2 mutants from the F 2 population. Seed from each of the ap2 mutants is tested for resistance to the antibiotic. One hundred percent of the seed from 27 plants was resistant to the antibiotic. Seed from the other 3 plants was 75% resistant and 25% sensitive to the antibiotic. Is the new ap2 mutant caused by an insertion of the transformed DNA into the AP2 gene?