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Norwich Interveiw (Potentail Questions (Career (Research - acedemia or…

- - - - Floresece tagged PLVR genome - good for exploring the inital stages
      - SCAR narker liked to RIadg on solanum - helps to predict resistance - would help show its presence
      - solarum adgena has lots of PLRV resistnace - resnatce controlle dby multiple complents and multilple genes
        
        LOP-868 varity
      - Ri adg found using AFLP map- all major compents were at a single linakge group
      - Tranfered the Riadg gene with SCAR marker form LOP-868 to solarum tumberiom with sucess on infection
  - - - Rejoin va HR homolous remcombination
      - Rejoin via NHEJ non homologous end joining
        
        Involves Ku heterdoiner protien
        
        Ku70 mut
        
        MRX complex
        
        hterodrimeric DNA ligase 4 complex
        
        lig4 mut
        
        Ku heterdimer binds to DNA ends then endbriding by MRX then Lig4 complex seals the gap
        
        delta ku70 and delta lig4 gives enchanced GT efficenies
  - - - Easier for genetic maniptulation
      - Have mutated Sli gene to overcome self-incompatibitly
      - Yeild = tetraploids
    - - Long 22hour days
      - Reached flowering and harvets much faster
      - Sucessfully on ceral crops
      - Rapid gernation cycling through single seed decent
    - - can use mutations in 3 arabidopsis pathwasy that cna increase crossover rates
      - important as increase crossovers = increaed gentic diverty and increased recombination of alleles/different allelic combinatiosn which is v impaornt in plant breeding/ quicker to get to elite lines
  - - - knock out S-RNase
        
        are cytotoxins that stop pollen with S-haplotype matchaing with pistil
      - selfing and selcetion leads to high homozygoity
    - - dwarf DELLA allele
      - CRISPR used with gemniviral replicaiton to replace genes
        
        marker gene was not needed
        
        CRISPR for DSB, added donar template adn amplified via geminviral repiclaion
    - - clustered regularly spaced short palindromic repeats
      - Cas9/12 nucasles cause DSB
      - Design gudie RNAs
        
        can traget to very specific places in the genome
        
        can target muliple places at once
        
        spacer: complimentry DNA giving target speicficty in cRNA
        
        cRNA - sall trans-encoded RNA
        
        encode gRNA
        
        PAM
        
        protospacer adjecnt motif
        
        type of gRNA
        
        PAM in DNA matchest PAM in cas9 complex. check spacer on cRNA is comp to sequ next to PAM
      - DSB repaired by non-honlogous end joinging (NHEJ) - ligation of free ends
      - came from prokarytic defense mechaniusm agaisnt bacterophages
- - - - Vital for human surval
      - Vital for planet care
    - - pushing forwards iwht leading research
      - oppertuniy to spread awareness and importace
- - - - Test and amplify prmers
      - Gel extractfragements and quantiy
      - Gibson to join 2 fragmeent
      - Transfrom into NEB 5-alpha cells
      - 5.Grow up and select sucessful colonies on LB miller broth adn Amp resistaance
      - GenElute Plasmid miniprep
      - RedTaq PCR to check gibson sucessful
      - :8. Bluk up sucessful PCr and DNA persciptate
      - Transfrom DNA into fusarium cells
      - grow up spores of PH-1 fg then perpare them
      - DIgest spores into protoplast and check health
      - Transform constructs into fg, grow overnigth and then plate on regeneration agar
      - Not done yet
        
        Check homologous recombination
        
        Extracting DNA from fungal tissue
        
        RedTaq PCR to check for fragements
        
        inncolutlate wheat and disease phenotype
        
        assay of diff mutants grown under zinc limiting conditions
- - - - PPD – professional development – so much more, communication, networking, giving talks, convey research and stress its importance
    - - PIPs – 3 months, maybe teaching, maybe management
- - - - R genes encode NLR immune rescptors
        
        wild relatives have R genes for late blight, PLRV and nematodes
      - Use crispr/cas9 and guide RNA to cuase BSD which allow knock in breeeding by HR.
    - - tet X dip = dihaploid (2n from tet)
      - CIP found r genes for PLRV and JHI put in andigena lines
        
        Only have rough map form CIP
        
        WIll make more dihapliods, screeen for res, backcross into hoska and them capture sequences
        
        FInd NLR genes
        
        score using the GFP floescnce
    - - Colab with JHI
        
        We make dihaploids with res genes
        
        JHI restance screens them
        
        Susceptable lines renseq and NLR alleles identifed
  - - - match crispr with recq4
        
        lines made homozygous and crispr t-dna seg away
      - rgene backcrosses into recq4 then into diploid/hosaka
      - use ku70 mutant to reduce NHEJ increaing HR
- - - - looks for NGG on genome
    - - from complex and seach for PAM
        
        gRNA homolgous to genome
        
        cas9 cuts
    - - different endonuclase
      - Has a different PAM downstream of gRNA
      - gives staggered cuts
- - - - Recognise Avrps4 and popp2
    - - highly replicated, duplicated repeats
      - ~3400 NLR genes in potatoes which is good as pahtogens are always eveloving
      - NLR genes make u very small amount of the pot genome
- - - - no crossing or linkage drag with KIB
      - KIB is VERY VERY specifc
    - - Solarerum americam WT pot realtive, cant cross but has lots of useful geens