Comparative Genomics of Fagaceae

Transcription

1 Fagaceae Images.google.com Linkage Map TM

2 Selection of mapping parents SM2 SM1 Predominant pollinator?

3 Progeny Exclusion for Full Sib Linkage Mapping Year Acorns genotyped SM2 pollinated All acorns genotyped across 6 microsatellite markers. Also all potential pollinators within 200 m of SM1 Exclude all acorns except those pollinated by SM2 Parentage analysis done with CERVUS.

4 Pollination rates in SM1 by SM2 across years SM2 paternity (%) across years Our current mapping population composed of 510 full sibs.

5 SSR markers developed and mapped Developing codominant SSRs Primary amplification test. Temperature gradient. Test of informativeness in parents. Informative markers configurations ab x cd, ef x eg, hk x hk, nn x np, lm x ll SSR Mapping 37 gssrs ( 26 with CA repeats, 11 with GA repeats) 39 Q. rubra EST-SSRs from FGP ( 181 screened & 148 amplified) 31 Q. robur EST-SSRs ( 113 screened & 99 amplified) 1 EST-SSR from Castanea mollissima.

6 SSR Framework Map of Q.rubra 1 (6) 2 (8) 3 (1) 4 (2)

7 5 (5) 6 (12) 7 (4) 8 (7)

8 9 (3) 10 (9) 11 (10) 12 (11)

9 Comparative Mapping Marker Name Linkage Gr. in Q. rubra Linkage Gr. in Q. robur FIR 021, FIR027, GOT040, GOT063, PIE111, VIT PIE099,PIE PIE FIR065, GOT066,POR FIR053, PIE FIR008,GOT009, PIE039, PIE126, PIE PIE002, PIE228, WAG FIR030, FIR095, GOT004, PIE PIE FIR026, PIE POR FIR

10 Fagaceae Mapping Parents Screening Panel Screening Panel included mapping parents from: American white oak (Quercus alba) American beech (Fagus grandifolia) Chinese chestnut (Castanea mollissima) American chestnut (Castanea dentata) English oak (Quercus robur) Sessile oak (Quercus petraea) Interspecific cross of Quercus robur x Quercus petraea

11 % markers amplified Amplification of Q.rubra markers across Fagaceae Mapping Parent Test Panel CA_gSSRs EST-SSRs Quercus alba Fagus grandifolia Castanea mollissima Castanea dentata Quercus robur Quercus petraea Q.robur x Q.patrea

12 High density RAD-tag Mapping in Quercus rubra Experimental approach: High quality DNA was extracted using QIAGEN for 250 full sibs. Genomic DNA was double digested with EcoRI-MseI for producing RAD-tags. Illumina HiSeq platform used for sequencing. High coverage parent lane (one lane/2 parents) 5 lanes of 50 progeny each. Bioinformatics: Reference loci identified from high coverage parent allele calls. Full sibs genotyped relative to parental allele calls. Data formatted and changed into a JoinMap readable format.

13 JoinMap 4.1 Pre-mapping quality control Missing data Extreme deviation from Mendelian expectation Similarity of loci. Grouping LOD value of for grouping test statistic. Grouping tree visually checked for selection of groups. Q. rubra and Q. robur gssr and EST-SSR markers used as anchor tags for identifying LGs. Mapping Maximum likelihood and Regression mapping algorithm with default criteria.

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15 Construction of RAD-tag pipeline SAM Tools GATK Oak2 Total # markers : 32,976 >30% missing data: >10 chi square value: 7994 Total # markers : 8,607 >30% missing data: 3529 > 10 chi square value : 1367 Oak3 Total # markers : 6977 >30% missing data: 1740 >10 chi square value: 6 Total # markers : 5,844 >30% missing data: 1432 > 10 chi square value : 100

16 Mapping RAD-tag markers 7994 RAD TAG SSRs 1 st : 108 SSRs +392 RAD 2 nd : 108 SSRs +200 old RAD +200 new RAD 3 rd :108 SSRs 4th:108 SSRs +200 RAD from +200 RAD from 2nd 3rd +200 new RAD +200 new RAD END

17 In silico Comparative Mapping Quercus rubra Quercus robur Mapped EST-SSRs Mapped SNP Sequence WGS Castanea mollissima Mapped EST-SSRs EST-SSRs X X X X RAD- tags X X X X X WGS

18 Phenotypes Bud burst Marcescence Leaf morphometrics Leaf nitrogen and carbon content QTL Mapping Phenotypic Data Collection Bud burst and marcescence 2012, 2013 and 2014 Leaf morphometrics 2013 and 2014 Clonally propagated Q.rubra mapping population SM1 SM2

19 Paper Submission Development and cross species transferability of genomic and EST microsatellite markers in Quercus rubra. L for comparative mapping in the Fagaceae ready for submission in Plant molecular biology reporter. First high density northern red oak mapping paper Fall 2014 Bioinformatics' RAD-tag paper Fall 2014 Comparative mapping papers Spring 2015 QTL paper 2015

20 JRS lab and ND Acknowledgements Professor. Jeanne Romero Severson Lab Technician: Lauren Fiedler Graduate Students: Daniel Borkowski, Andi Noakes Undergraduate: Rebecca Bullis Collaborators: Dr. Scott Emrich, Olivia Choudhury Notre Dame Genomics and Bioinformatics core facility: Brent Harker Melissa Pullins Grant # TRPGR IOS