sch l of marine sciences june - 24 - 2014 mikhmoret marina conservation of green sea turtles through...
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Sch l of Marine Sch l of Marine SciencesSciences
June - 24 - 2014June - 24 - 2014Mikhmoret marinaMikhmoret marina
Conservation of Green Sea Turtles through Genetics and Genomics
Dr. Yaron Tikochinski
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Green Sea Turtles in Israel:On the verge of extinction
About 10 nesting females along the Israeli shore (about 200 Km)
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About Sea Turtles:
•Philopatric
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About Sea Turtles:
•polyandry
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The Sea Turtle Rescue Center
•Locate
•Heal
•Release
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Sea Turtles Rescue Center:Save and Heal
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Sea Turtles Rescue Center: Feed and Bread
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Let’s Increase the Numbers
“I can make my own people”
Jerry Seinfeld
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Breeding Stock
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The Sea Turtle Rescue Center
Chelonia mydas
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A Large Variable Population
“Population with no Variation will not survive Evolution”
C. T. Urtle
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A Stable, Strong Population
“My boys can swim!”George Costanza(marine biologist)
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Genetic Variability of Green Turtles
Chelonia mydas
•Mitochondrial DNA D-Loop
•600bp at the 5’
•70 haplotypes worldwide
•All Mediterranean (but 2)
CM-A13
•Genomic STR’s show
variability
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Genomic STR’s
Chelonia mydas
•Genomic STR’s show variability
•Difficult to analyze – can’t tell a
mother’s genotype by her
offspring
•Back to mtDNA?
•Longer Fragments?
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The Mitochondrial D-loop
Chelonia mydas
ACACAGGAATAAAAGTGTCCACACAAACTAACTACCTAAATTCTCTGCCGTGCCCAACAGAACAATACCC GCAATACCTATCTATGTATTATTGTACATCTACTTATTTACCAATAGCATATGACCAGTAATGTTAACAG TTGATTTGGCCCTAAACATAAAAAATCATTGAATTTACATAAATATTTTAACAACATGAATATTAAGCAG AGGATTAAAAGTGAAATGACATAGGACATAAAATTAAACTATTATACTCAACCATGAATATCGTCACAGT AATTGGTTATTTCCTAAATAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCA GTTTCAAGCCCATTCAGTCTGTGGCGTACATAATTTGATCTATTCTGGCCTCTGGTTAGTTTTTCAGGCA CATACAAGTAACGACGTTCATTCGTTCCCCTTTAAAAGGCCTTTGGTTGAATGAGTTCTATACATTAAAT TTATAACCTGGCATACGGTAGTTTTACTTGCATATAGTAGTTTTTTTTCTCTCTGTGTTCTCAGGCCCAC ATAACTGATACCTGCCGATTCAGTGAAACTGGACTTACGTTTAAATATGATTGGCCGTGCAAACTGATTA ATGGTATTATTAAGTTAATGCTTATAAGACATAGAATTTCACAATTAAACCTAAACAATGATCTACAACC TAACTCATTATTAACTGTACTTTTTAGCTAAACCCCCCTACCCCCGTTAAAGTCAACACCAGCCCGCTAT AGCCATTTACTTCTCGCCAAACCCCTAAATCCGAGACTGACCAAACTGACATAATATCAACTGCATAAGC ATCACACAAATCAATAGGATACTTACACTAATATTTAAAAAGTACTATACAATTCAAAACACCTCTACCA CACCTCAACCAATATATATATATATTATACATTATATATATATATATATTATATATATTATATATATAAT AT
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DNA Repeats – a Source for Polymorphism
Chelonia mydas
• Mutations’ hot spots
• Evolutionary shortcuts
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Polymorphism Emerging
The mitochondrial D-loop:PCR with fluorescent primers.
The 3’ end has length polymorphism:115, 117, 119, 121, 123, 125, 127 bp
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AT Repeats - Aligned
STR 1 STR 2 STR 3 STR 4
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New HaplotypingSTR1 STR 2 STR 3 STR 4 Stranded Local*
8 8 8 4 0 18 7 7 4 16 477 9 7 4 0 17 8 5 4 3 16 9 6 4 4 166 8 9 4 6 126 8 8 4 37 116 7 7 4 0 15 9 6 4 0 25 8 8 4 3 38 8 7 4 28 8 5 4 18 7 6 4 27 10 5 4 27 8 8 4 57 8 7 4 27 8 6 4 47 7 8 4 47 7 7 4 27 7 6 4 46 10 5 5 16 9 8 4 16 8 10 4 26 8 7 4 86 8 6 4 146 8 5 5 26 8 5 4 546 7 6 4 36 7 5 4 25 8 7 4 15 8 6 4 45 8 5 4 45 7 6 4 1
194 95
•34 Haplotypes (+1)(mostly non-Israeli)
•Can we use them?
• Polymorphic
• Reliable
• Reproducible
• Kinship
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Mediterranean/Israeli Green Turtles Tree
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Mediterranean/Israeli Green Turtles Tree
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Does it Tell a True Story?
•DNA never lies
•Scientists should always doubt
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Analysis
Close Linkage*
Stranded turtles
8 8 8 4 5 08 7 7 4 4 167 9 7 4 2 07 8 5 4 7 36 9 6 4 4 46 8 9 4 5 66 8 8 4 9 376 7 7 4 5 05 9 6 4 3 05 8 8 4 6 38 8 7 4 6 28 8 5 4 5 18 7 6 4 4 27 10 5 4 1 27 8 8 4 7 57 8 7 4 8 27 8 6 4 6 47 7 8 4 3 47 7 7 4 6 27 7 6 4 6 46 10 5 5 1 16 9 8 4 2 16 8 10 4 5 26 8 7 4 9 86 8 6 4 9 146 8 5 5 2 26 8 5 4 10 546 7 6 4 7 36 7 5 4 3 25 8 7 4 6 15 8 6 4 7 45 8 5 4 6 4
HaplotypeClose
Linkage*Stranded turtles
8 8 8 4 5 08 7 7 4 4 167 9 7 4 2 07 8 5 4 7 36 9 6 4 4 46 8 9 4 5 66 8 8 4 9 376 7 7 4 5 05 9 6 4 3 0
8 8 7 4 6 28 8 5 4 5 18 7 6 4 4 27 10 5 4 1 27 8 8 4 7 57 8 7 4 8 27 8 6 4 6 47 7 8 4 3 47 7 7 4 6 27 7 6 4 6 46 10 5 5 1 16 9 8 4 2 16 8 10 4 5 26 8 7 4 9 86 8 6 4 9 146 8 5 5 2 26 8 5 4 10 546 7 6 4 7 36 7 5 4 3 25 8 7 4 6 15 8 6 4 7 45 8 5 4 6 4
Haplotype
8 8 8 4 5 08 7 7 4 4 167 9 7 4 2 07 8 5 4 7 36 9 6 4 4 46 8 9 4 5 66 8 8 4 9 376 7 7 4 5 05 9 6 4 3 0
8 8 7 4 6 28 8 5 4 5 18 7 6 4 4 27 10 5 4 1 27 8 8 4 7 57 8 7 4 8 27 8 6 4 6 47 7 8 4 3 47 7 7 4 6 27 7 6 4 6 46 10 5 5 1 16 9 8 4 2 16 8 10 4 5 26 8 7 4 9 86 8 6 4 9 146 8 5 5 2 26 8 5 4 10 546 7 6 4 7 36 7 5 4 3 25 8 7 4 6 15 8 6 4 7 45 8 5 4 6 45 7 6 4 5 1
Israeli Other
125
69
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Mediterranean Green Turtles New Haplotyping
Haplotype Israel Stranded Turkey N. Cyprus S. Cyprus
6 8 8 4 1 37 17 12 20
8 7 7 4 1 16 6 0 0
6 8 7 4 1 8 8 4 0
6 8 9 4 1 6 0 0 0
6 9 6 4 1 4 1 5 1
7 8 5 4 1 3 3 0 0
5 8 8 4 1 3 0 0 0
8 8 8 4 1 0 0 0 0
7 9 7 4 1 0 0 0 0
6 7 7 4 1 0 0 0 0
5 9 6 4 1 0 0 0 0
6 8 5 4 0 54 27 0 0
6 8 6 4 0 14 11 16 0
7 8 8 4 0 5 1 1 1
7 8 6 4 0 4 1 1 0
7 7 8 4 0 4 0 0 0
7 7 6 4 0 4 2 0 0
5 8 6 4 0 4 0 0 0
5 8 5 4 0 4 0 0 0
6 7 6 4 0 3 1 1 0
8 8 7 4 0 2 0 0 0
8 7 6 4 0 2 2 0 0
7 10 5 4 0 2 0 0 0
7 8 7 4 0 2 0 0 0
7 7 7 4 0 2 3 0 0
6 8 10 4 0 2 0 0 0
6 8 5 5 0 2 0 0 0
6 7 5 4 0 2 2 0 0
8 8 5 4 0 1 0 0 0
6 10 5 5 0 1 0 0 0
6 9 8 4 0 1 2 0 1
5 8 7 4 0 1 0 0 0
5 7 6 4 0 1 0 0 0
7 10 6 4 0 0 0 1 0
11 194 87 41 23
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Mediterranean Green Turtles Satellite Tracking
Broderick et al., 2007
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Mediterranean Green Turtles Satellite Tracking
Rees et al., 2008
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Can We Use This Storyteller Outside the Mediterranean?
Atlantic – same pattern
Pacific - ?
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Genetic Variability of Indo-Pacific Green Turtles
STR 1 STR 2 STR 3 STR 4 STR 5 STR 6STR 1 STR 2 STR 3 STR 4 STR 5 STR 6
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Repeats in Other Sea Turtles
Loggerhead:ATATT
Conventional: 3 HaplotypesRepeat Haplotyping: 48-108 repeats 30 Haplotypes
(250 turtles)
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Repeats in Other Sea Turtles
Hawksbill:CATATATAT
Conventional: ? HaplotypesRepeat Haplotyping: 10,23,30 repeats 3 Haplotypes
(8 turtles)
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Repeats in Other Sea Turtles
Olive Ridley:ATATT and ATATTATT
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Defining Aims
Why do we look at the DNA?
Conservation of Biodiversity
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Defining Aims
Sea turtles need our help in order to
survive as species
A stable population needs genetic
variation
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Defining Aims
We look at DNA in order to evaluate
genetic polymorphism
This is just a glimpse!
500bp +300bp +STR’s (genomic and
mitochondrial)
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Sea Turtle Genome - Status
We have completed 2X coverage
BGI (China) Published the genome
We are starting a transcriptome
We look for collaborators
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What Can We Do With the Sea Turtle Genome
A lot:
Easily find polymorphic sites (STR’s)
Genes Traits
Genes Diseases
Gene expression
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Population Studies
Variability Variability Variability
Mitochondrial D-loop
Short Tandem Repeats
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Library construction for STRs
Can we do it better?
Extract
Digest
Clone
Find STRs
Screen Population
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The Alternative
Rational:
One individual will show population
polymorphism
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The Alternative
2x Genome of 1 specimen
Isolate all STR
Locate site specific pairs
Isolate heterozygote sites
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The Algorithm
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Primer design
60 8_>gnl|ti|1262525131_175488122 AT TCTTCAAGTAACTTACTGGTGCCTTTAAGTCCTGTTCAGGCCCAGTCTCatatatatGTATATAAAATTTCTATTTCATGAAAGAGTGCTGCTGTGCATGCCCAACT 68_>gnl|ti|1203695794_169077269 AT TCTTCAAGTAACTTACTGGTGCCTTTAAGTCCTGTTCAGGCCCAGTCTCatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatGTATATAAAATTTCTATTTCATGAAAGAGTGCTGCTGTGCATGCCCAACT Forward Primer TTAAGTCCTGTTCAGGCCCAGTCT Reverse Primer GCATGCACAGCAGCACTCTTTCA // 58 88_>gnl|ti|1249504736_174204011 AC AGCTGATTTTGTGTACTTTTGATAGTATCATCACATTCCTAACAAGTTTacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacacTTCCATCCTGCAAATTTGAAGTGAAAGCACCCAGACTTTCTTCTCTACCC 30_>gnl|ti|1203651562_168979272 AC AGCTGATTTTGTGTACTTTTGATAGTATCATCACATTCCTAACAAGTTTacacacacacacacacacacacacacacacTTCCATCCTGCAAATTTGAAGTGAAAGCACCCAGACTTTCTTCTCTACCC Forward Primer AGTATCATCACATTCCTAACAAGT Reverse Primer GAAAGTCTGGGTGCTTTCACTTC //
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Thank you for your attention
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Thank you
Looking beyond the horizonLooking beyond the horizon
Sch l of Marine Sch l of Marine SciencesSciences
Thanks:Yaniv Levy Yakup Kaska
Adi Barash Lucy Wright
Raphael Bendelac Prof. Brendan Godley
Alon Daya Annette Broderick
Adam Friedmann Andreas Demetropoulos
Uzi Motro
Marina Friling Genome Project:
Renanel Pickholtz Jeremy Edwards