Genomics and Heterozygosity in Banana

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Genomics and Heterozygosity in Banana

• Pat Heslop-Harrison phh4_at_le.ac.ukwww.cytogenomics
  .org

Discussion meeting at 18 January 2008
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The Banana
0.2 Introduction Musa Diversity

• Pat Heslop-Harrison
• www.biobanana.com

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Genomics in Musa
0.3 Introduction - Outline

• Musa the genus
• Banana The chromosomes
• Heterozygosity and DNA variation
• The future of banana genomics

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The banana genome DNA and x11 Chromosomes
1.1 Chromosomes DAPI

• Haploid genome size
• 500 to 600 Mbp DNA(Rice 440 Mbp Arabidopsis
  150 Mbp Barley 5500 Mbp)

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1.2 Chromosomes Diploid
Musa acuminata Calcutta 4 AA genomes,
2n2x22
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1.3 Chromosomes Triploid

• Cavendish the most common dessert banana
  cultivar
• 2n3x33 AAA genomes

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1.4 Chromosomes Hybrid Musa

• Cultivars are parthenocarpic, sterile triploids,
  2n3x33
• AAB and ABB plantains, cooking

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BSV Expression in Banana
1.5 Chromosomes BSV ParaRetroVirus
Unexpected epidemiology Appears after tissue
culture or Low night temperatures
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Nuclear Copies of BSV in Banana
1.6 Chromosomes BSV In situ hybridization
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What is a banana?
2.1 Musa genus Taxonomic position

• Monocotyledon giant herb not a tree!

? ?
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What is a banana?
2.2 Musa genus APG

• Monocotyledon giant herb not a tree!

APG II 2003
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BAC sequencesSequence identification basedon
reference sequences
2.3 Musa genus Sequences
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2.4 Musa genus Reference genes

• Glycerol-3-phosphate dehydrogenase

Rice Rice Calcutta 4 Calcutta 4 Arabidopsis
Arabidopsis
Rice Rice Calcutta 4 Calcutta 4 Arabidopsis
Arabidopsis
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Diversity in Musa DNA
3.1 Diversity Questions

• How heterozygous is Musa at the nucleotide level?
• How much allelic variation is there?
• How much structural variation is there?
• How much variation is there between diploids,
  triploids and different species
• What markers are appropriate
• Could sequences be contigd?

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Teo, Schwarzacher
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IRAP diversity in Musa
3.3 Diversity IRAP gels
Teo, Tan, Ho, Faridah, Othman, HH, Kalendar,
Schulman 2005 J Plant Biol Nair, Teo,
Schwarzacher, HH 2005 Euphytica Desai, Maha,
HH et al. in prep.
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3.4 Diversity IRAP Genomes
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3.5 Diversity IRAP/SSR analysis by many genomes
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Heterozygosity and AlleleDiversity Analysis
Strategies
3.6 Diversity Genomics strategy

• Homologous BACs identified for sequencing using
  3-D pools and PCR primers, sequenced to identify
  heterozygotes
• Homoeologous BACs from different species
  identified and sequences
• Primers developed from completed BAC sequences,
  ESTs or reference species genomic/EST genic,
  SSRs and un-annotated regions were targeted
• DNA amplified by PCR from single plants of known
  genotype (vegetatively propagated)

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Heterozygosity and AlleleDiversity Analysis
Strategies
3.7 Diversity Data sources

• Six BAC pairs for analysis
• From 8 PCR products, a total of 5946 bases of
  Musa genomic sequence were analysed, with an
  average depth of sequencing of about 40-fold from
  4 lines, giving a total of gt500kb of sequence
  data

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3.8 Diversitiy Heterozygosity in A genome

• Homologous BAC sequences from Calcutta 4
  Homologous over the full length

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3.8 Diversity Heterozygosity in A genome

• Homologous BAC sequences from Calcutta 4
  Homologous over the full length
• except for a 5kb insert
• a Ty1-copia retroelement

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3.9 Diversity Single accession heterozygosity
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Comparison of homologous BACs excluding
microsatellite and transposon regions
3.10 Diversity Heterozygosity in C4

• 24650 bp
• 1.91 insert
• 1.11 deletion
• 1.87 transition
• 1.19 transversion
• Total 93.9 similar

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3.10 Diversity Heterozygosity
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Comparison of homoeologous BACs Musa balbisiana
to Musa acuminata
3.11 Diversity Heterozygosity in C4/PKW

• 46234 bp
• Total gaps 9.19 differences
• Outside gaps 3.24
• (Overall BLAST 93.27)
• MBP_91N22 vs MA4_54N07

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Polymorphisms
3.12 Diversity Polymorphisms
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500 to 800 bp regions
3.13 Diversity Single accession heterozygosity

• Calcutta 4 four of 7 heterozygous
• Pahang five of 7 heterozygous
• M. malaccensis two of 7 heterozygous
• One Calcutta 4 (both alleles different) to
  reference sequence polymorphism
• One polymorphism within Pahang Double Haploid
  (not found in parent)

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(No Transcript)
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3.15 Diversity Heterozygosity

• 3ACE Polymorphism in microsatellite-related
  regions

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Nature of Polymorphisms
3.16 Diversity Heterozygosity

• Same sites involved in variation
• Probably not identical-by-descent
• Relevance to comparative genomics including
  monocots to eudicots

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Sources ofGenomic Diversity
4.1 Summary Nature

• Transposon diversity active now
• SSR diversity
• In-del diversity frequent
• SNP diversity
• Duplications
• Translocations

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4.2 Summary SNPs and heterozygosity

• Identical polymorphism-pairs found in different
  species
• In Musa, same bases involved in polymorphisms in
  different lines (coding and non-coding)
• Within-line variation as high as at
  species/subspecies level here
• All genomic regions involved in polymorphisms

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4.3 Summary Sequencing Musa

• Musa highly heterozygous, consistent with
  outbreeding, hybridity and vegetative propagation
• Assembly of shot-gun reads from cultivars would
  be problematic
• In the Pahang Double Haploid, seven of the
  eight loci identical to a parental allele
  assembly of WGS straightforward
• Alleles of Pahang Double Haploid entirely
  representative of A and B genome bananas

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Musa Genomic Sequence
4.4 Summary Need for sequence

• A reference genome near the grasses
• Getting to a master reference sequence
• Inferring an ancestral monocot and then plant
  genome
• New diversity in alleles
• Non-host resistance
• New products in fibre
• Nature of variation over wider range
• New stepping stones between genomes/species
• Crops for domestication and new products
• No clear rules
• Exploitation of the genepool
• Use of the genepool
• Providing methods and diversity (variation) for
  breeders and geneticists to use

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Musa Genomic Sequence
4.4 Summary Need for sequence

• A reference genome near the grasses
• Getting to a master reference sequence
• Inferring an ancestral monocot and then plant
  genome
• New diversity in alleles
• Non-host resistance
• New products in fibre
• Nature of variation over wider range
• New stepping stones between genomes/species
• Crops for domestication and new products
• No clear rules
• Exploitation of the genepool
• Use of the genepool
• Providing methods and diversity (variation) for
  breeders and geneticists to use

StrategyDe-RiskingCollaborationsComplementary
expertiseStep-by-step strategyQuick routes to
exploitation (genomics and breeding)
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Acknowledgements
Isabelle Hippolyte, Franc Christophe Baurens, and
Frederic Bakry, CIRAD, Montpellier, France for
DNA and libraries
Foo Cheung, William A. Moskal, Jr, and
Christopher D. Town, JCVI, Rockville, USA
Mathieu Rouard and Nicolas Roux, Bioversity,
Montpellier, France
Jaroslav Dolezel, Musa Genomics Resource Centre,
Oloumec
Takuji Sasaki and Takashi Matsumoto, NIAS,
Tsukuba, Japan, for BAC sequencing
Luis Rodríguez Zapata and Andrew James, CICY,
Mexico
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Acknowledgements
Trude Schwarzacher
Keerthy Santhosh and Arathi Vijayan, University
of Kerala, India
Rebekah Burrows
Azhar Mohamad, Nuclear Malaysia
Julian Osuji, Nigeria Emmanuel Otwe, Ghana OJ
Benedict, Nigeria
Dhairyasheel Desai, India
Suneetha Eluru, Andhra Pradesh, India
Chee How Teo, Malaysia
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Genomics, Biodiversity and Heterozygosity in
Banana

• Pat Heslop-Harrison www.cytogenomics.orgUserid/P
  W visitor
• Heslop-Harrison JS, Schwarzacher T. 2007.
  Domestication, genomics and the future for
  banana. Annals of Botany 100(5)1073-1084.
  doi10.1093/aob/mcm191
• Vaughan DA, Balázs E, Heslop-Harrison JS. 2007.
  From crop domestication to super-domestication.
  Annals of Botany 100(5)893-901.
  doi10.1093/aob/mcm224

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• NBS-LRR disease resistance homologues
• Highly diverse gene family
• Azhar HH
• Cytogenet Genome Res
• In press 2008