Plant Molecular Cytogenetics - Postgenomics, Chromosomes and Domestication

Chromosomes, Crops and Superdomestication in Katowice

Pat Heslop-Harrisonphh4@le.ac.uk

www.molcyt.comUserID/PW ‘visitor’

Pathh1:

Twitter #PMC .

Slideshare pathh1

From Chromosome to Nucleus

Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com

Genome evolution• How do genomes evolve?

–Gene mutation very rarely (human: 10−8/site/generation)

–Chromosome evolution–Polyploidy and genome duplication (ancient &

modern)–Repetitive sequences: mobility & copy number

(10−4/generation in µsat)–Recombination–Epigenetic aspects: centromeres & expression

Genome evolution• How do genomes evolve?

– Gene mutation very rarely– Chromosome evolution– Polyploidy and genome duplication (ancient and modern)– Repetitive sequences: mobility & copy number– Recombination– Epigenetic aspects – centromeres & expression

• How can we exploit knowledge of genome evolution?– Biodiversity– Chromosome and genome engineering– Breeding– Markers

Musa biodiversity and genomes: x=11Red - AAA 2n=3x=33 – M. acuminata Palayam codan AAB (two bunch yellow, one green) Musa x Peyan ABB (green cooking banana) Njalipoovan AB (yellow) 2n=2x=22 M. acuminata x M. balbisiana Robusta AAA (green ripe) Nendran AAB Poovan AAB (one yellow bunch) Red AAA Peyan ABBVarkala, Kerala, India

RetrotransposonsClass I transposable elementsRNA intermediate

DNA transposonsClass II transposable elements

Cut-and-paste

RetroelementsSequences which amplify through an RNA intermediate

• 50% of all the DNA!

Retroelements

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

Alignment of two homologous Musa BACs shows gaps in both B genome M. balbisiana and A genome M. acuminata

MA4_82I11

MBP_81C12

MuhAT1

MuhAT2a

XX TE (SINGLE)XX TE MITE

XX TE (AGNABI)

MuhAT3 MuhAT4 MITE(MBIR)

XX TE XX TE (MBT)

272 bp 102,190 bp

26, 410 bp 128,068 bp

DNA transposons hAT are particularly frequent

8 bp TSD, and short TIRs of 5–27 bptransposase (sometimes degenerate) including a DDE site.Non-autonomous (MITE) derivatives of hAT with deletion coding sequence

Menzel, Schmidt, Nouroz, HH Chr Res subject minor revision 2015

13/04/2023 12

Sr. No. Primer Pairs Product Size (bp)

Sequence

1. hAT18486hAT19037

560 ACCCACCTGGCTCTTGTGTCAGCGAATGTGTTTTGACCAC

MBP 81C12 (M. balbisiana) x MA4 82I11 (M. acuminata) BACs.

Musa balbisiana (MBP 81C12)M

usa

acu

min

ata

(MA

4 82

I11)

Transposed Element

hAT 1

hAT 2

hAT 4

Microsatellite (AT)

hAT 3621 bp MBT

384 bp TE + 781 MITE

1676 TE

Microsatellite (AT)

4192 bp TE

13/04/2023 13

Sr. No. Primer Pairs Product Size (bp)

Sequence

1. hAT18486hAT19037

560 ACCCACCTGGCTCTTGTGTCAGCGAATGTGTTTTGACCAC

MBP 81C12 (M. balbisiana) x MA4 82I11 (M. acuminata) BACs.

Musa balbisiana (MBP 81C12)M

usa

acu

min

ata

(MA

4 82

I11)

Transposed Element

hAT 1

hAT 2

hAT 4

Microsatellite (AT)

hAT 3621 bp MBT

384 bp TE + 781 MITE

1676 TE

Microsatellite (AT)

4192 bp TE

A-genome specific hAT in three Musa accessions

(2n=3x=33)

Musa ‘WilliamsCavendish’ (AAA)

Musa (ABB)

Musa (ABB)

13/04/2023 15Dot plot showing the complete Inverted repeat.

13/04/2023 16

HP-1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

1KB800600400200

hAT1 insertion sites in Musa diversity collectionhAT486F and hAT037R

Top bands (560-bp) amplified hAT elementLower bands amplifying the flanking sequences only

Menzel, Nouroz, Heslop-Harrison, Schmidt 2014

Retroelement Markers

Retrotransposon LTRLTR

Retrotransposon LTRLTR

RetrotransposonLTR LTR

Retrotransposon LTRLTR

Insertion

IRAP – InterRetroelement PCR

Retrotransposon LTRLTR

RetrotransposonLTR LTR

IRAP diversity in Musa

Teo, Tan, Ho, Faridah, Othman, HH, Kalendar, Schulman 2005 J Plant BiolNair, Teo, Schwarzacher, HH 2006 Euphytica Teo, Schwarzacher et al. in prep.

13/04/2023 19

Phylogenetic analysis of Musa genomes – separating species. Teo, Schwarzacher et al.

BSV Expression in Banana

Double stranded DNA is infective: Insect vectorUnexpected epidemiology: Appearance after cold or tissue culture

Nuclear Copies of Banana Streak Virus in Banana

Nuclear Copiesof BSV in banana

DNA Fibre in situ hybridization

Harper, HH et al., Virology 1999 … cf D’Hont et al., Nature, 2012

Whole genome shotgun sequencing

• Changing all cytogenomics (.org) work

• Easily obtaining several-fold sequence coverage

D’Hont et al. Nature 2012 doi:10.1038/nature11241

Musa Bananan=11

Sequence:D’Hont, inc HH et al. Nature 2012

Haploid: Nair, HH 2013

Whole genome duplications

• The surprise to the sequencers: conserved synteny and relatively few breakpoints

• The surprise to the cytogeneticists: sequencing shows whole genome duplications (=polyploidy) deep in the phylogenetic tree

• The surprise to everyone: so few genes but multifunctional

A D’Hont et al. Nature 2012doi:10.1038/nature11241

BrachiariaLTR element families

Fabíola Carvalho SantosAndré Luiz Laforga VanzelaSee poster

Forage/pastureUrbanSavanna/cerrado

ForestSugar caneSoybean/corn

Brazil land use

Chromosomal evolution and the organization of repetitive DNA sequences in diploid and polyploid

Brachiaria forage grasses

Some probes show less hybridization to some chromosomes, perhaps indicating genome specificity.

Fabíola Carvalho SantosAndré Luiz Laforga VanzelaSee poster

From Chromosome to Nucleus

Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com

Wheat evolution and hybridsTriticum uratu

2n=2x=14AA

EinkornTriticum monococcum

2n=2x=14AA

Bread wheatTriticum aestivum

2n=6x=42AABBDD

Durum/SpaghettiTriticum turgidum ssp durum

2n=4x=28AABB

Triticum dicoccoides2n=4x=28

AABB

Aegilops speltoidesrelative

2n=2x=14BB Triticum tauschii

(Aegilops squarrosa)2n=2x=14

DD

TriticalexTriticosecale

2n=6x=42AABBRR

RyeSecale cereale

2n=2x=14RR

Copyright restrictions may apply.

Saeidi, H. et al. Ann Bot 2008 101:855-861; doi:10.1093/aob/mcn042

Inter-retroelement (IRAP) analysis of Triticum tauschii ssp tauschii from Iran

SSR/Microsats: all are different and no tree is supported

Different sequence classes evolve at different rates

Crop standing

Lodging in cereals

Crop fallen

Use of repetitive DNA sequences as chromosome markers

dpTa1pSc119.2Genomic Ae.ventricosa

Inheritance of Chromosome 5DAegilops ventricosaDDNN

ABDN

AABBDDNN MarneAABBDD

CWW1176-4

Rendezvous

Piko

VPM1 Dwarf A

96ST61

Virtue

×

×

×

×

Hobbit

× {Kraka × (Huntsman × Fruhgold)}

Triticum persicum Ac.1510AABB

Wheat Streak Mosaic Virus in North AmericaBob Graybosch, USDA

Wsm-1: only highly effective source of resistance to WSMV

Mace wheatGraybosch et al. 2009In situ: Niaz Ali & Schwarzacher

Chromosome evolution - Polyploidy

• Selected natural– Wheat– Banana – Brachiaria – Proso millet

• Synthetic– Triticale– Nicotiana

Proso millet (Panicum miliaceum): origins, genomic studies and prospects

Pat Heslop-Harrison, Farah Badakshi and Harriet Hunt

Panicum sensu stricto c. 100 species; x=9Evolution of Panicum miliaceum Proso millet

P. miliaceum 2n=4x=36

P. capillare2n=2x=18

P. repens2n=4x=36

also 2n=18 to 54

P. sumatrense2n=2x=18 or 4x=36

Global North-temperateLow genetic diverstiyWeedy forms

P. virgatum2n=4x=36 or 2x=18

? ? ? ? ??

• Hunt , HH et al. 2014. Reticulate evolution in Panicum (Poaceae): the origin of tetraploid broomcorn millet, P. miliaceum. J Exp Bot. 2014

• P. miliaceum: allotetraploid with maternal ancestor P. capillare and one genome shared with P. repens (also allotetraploid)

Hunt , HH et al. 2014. Reticulate evolution in Panicum (Poaceae): the origin of tetraploid broomcorn millet, P. miliaceum. J Exp Bot. March 2014

Chromosome and genome engineering

Cell fusionhybrid of two4x tetraploidtobaccospecies

Patel, Badakshi, HH, Davey et al 2011 Annals of Botany

Nicotiana hybrid4x + 4x

cell fusions

Each of 4chromosome

sets hasdistinctiverepetitiveDNA when

probed withgenomic DNA

Patel et alAnn Bot 2011

Cell fusionhybrid of two4x tetraploidtobaccospecies

Four genomesdifferentiallylabelled

Patel, Badakshi, HH, Davey et al 2011 Annals Botany

Arachis hypogaea - PeanutTetraploid of recent origin,

ancestors separated only 3 My ago

• Ana Claudia Araujo, David Bertioli, TS & PHH EMBRAPA, Brasília. Annals Botany 2013

•Arachis hypogea 2n=4x=40 probed with •(green) A. duranensis; (red) A. ipaënsis

Bertioli et al. Annals of Botany 2013

BAC in situ hybridization

Primula BAC mapping

Gilmartin, Lu, HH & Badakshi 2015?

Size and location of chromosome regions from radish (Raphanus sativus) carrying the fertility restorer Rfk1 gene and transfer to spring turnip rape (Brassica rapa)

DAPI metaphase blueRadish genomic red (2 radish chromosomes) far-red 45S rDNARfk1 carrying BAC green labels sites on radish and homoeologous pair in Brassica

Tarja Niemelä, Seppänen, Badakshi, Rokka HHChromosome Research 2012

BACs from different species have different repeat distributions – and hence different patterns of hybridization

Organelle sequencesfrom chloroplasts or

mitochondria

Sequences from viruses, Agrobacterium or other

vectors

Transgenes introduced with molecular biology

methods

Genes, regulatory and non-coding single copy sequences

Dispersed repeats:Transposable Elements

Repetitive DNA sequences

Nuclear Genome

Tandem repeats

DNA transposons copied and

moved via DNA

Retrotransposons amplifying via an RNA intermediate

Centromeric repeats

Structural components of chromosomes

Telomeric repeats

Simple sequence repeats or

microsatellites

Repeated genes

Subtelomeric repeats

45S and 5S rRNA genes

Blocks of tandem repeats at discrete chromosomal loci

DNA sequence components of the nuclear genomeHeslop-Harrison & Schmidt 2012. Encyclopedia of Life Sciences

Other genes

X MuTRR

MuTRF220 bp

• The original 177bp repeat fits nicely around the nucleosome allowing a tight coiling

• The repeat unit with the retroelement foot print, the 63bp box, has a much more open configuration

• It is maintained as it brings a CG and CNG site that allows control via methylation

MuTRR

MuTRF180 bp

Insertion and subsequent loss

Monkey retroelement

C.H Teo and Schwarzacher

A

B

C

Centromere

DNA sequenceTE

Tandem repeat monomerTE Transposable element

Single copy DNA

Spindle microtubules pulling apart chromatids

Metaphase chromosome

147bp plus 5-70bp linker = 150-220bp

100bp plus 55bp linker = 155bp

D

E

F

G

H

I

Kinetochore

Heslop-Harrison & Schwarzacher 2013. Nucleosomes and centromeric DNA packaging. Proc Nat Acad Sci USA. http://dx.doi.org/10.1073/pnas.1319945110. See also http://wp.me/p2Ewqp-7h

Henikoff et al 2013

C: antibody to CENH3 variant

Domestication

• Most species domesticated 10,000 years ago: cereals, legumes/pulses, brassicas, fruits, cows/sheep/pigs, silkworm/bees)

• Few species more recently (rabbits, fish; trees, biofuel crops)

• A few dropped out of production

• First steps: productive, reproduce easily, disease-free, edible/tasty, harvestable …

Heslop-Harrison & Schwarzacher Domestication genomics www.tinyurl.com/domest and review of rabbits www.tinyurl.com/rabdom

Domestication

• …• A few dropped out of production

• Second steps: more productive, harvestable

• Third step: fitting for sustainable intensification• Proso millet: the most water-efficient cereal• Superdomestication and design of crops

Heslop-Harrison & Schwarzacher Domestication genomics www.tinyurl.com/domest and review of rabbits www.tinyurl.com/rabdom www.tinyurl.com/superdom

Outputs–CROPS

– Fixed energy Inputs

–Light–Heat–Water–Gasses–Nutrients

–Light–Heat

–Water–Gasses

–Nutrients

(Ecosystem services)

Conventional Breeding

Superdomestication

• Cross the best with the best and hope for something better

• Decide what is wanted and then plan how to get it– Variety crosses– Mutations– Hybrids (sexual or cell-fusion)– Genepool– Transformation

Economic growth

• Separate into increases in inputs (resources, labour and capital) and technical progress

• 90% of the growth in US output per worker is attributable to technical progress

Robert Solow – Economist

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 530

200000000

400000000

600000000

800000000

1000000000

1200000000

Maize Rice, paddy

Wheat Population /10

1961 1970 1980 1990 2000 2010 2013

52 years of plant breeding progress

Agronomy

Genetics

GM maize

United Nations Millennium Development Goals-MDGs1990 to 2015

• Goal 1 – Eradicate extreme poverty and hunger

•Goal 2 – Achieve universal primary education

• Goal 3 – Promote gender equity and empower women

• Goal 4 – Reduce child mortality•

Goal 5 – Improve maternal health •

Goal 6- Combat HIV/AIDS, malaria and other diseases

• Goal 7 - Ensure environmental sustainability

• Goal 8 - Develop a global partnership for development

From Chromosome to Nucleus

Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com

Genome evolution• How do genomes evolve?

– Gene mutation very rarely – Chromosome evolution– Polyploidy and genome duplication (ancient and modern)– Repetitive sequences: mobility & copy number– Recombination– Epigenetic aspects – centromeres & expression

• How can we exploit knowledge of genome evolution?– Biodiversity– Chromosome and genome engineering– Breeding– Markers Pat Heslop-Harrison & Trude Schwarzacher

www.molcyt.comPathh1 on slideshare

Chromosomes, Crops and Superdomestication in Katowice

Pat Heslop-Harrisonphh4@le.ac.uk

www.molcyt.comUserID/PW ‘visitor’

Pathh1:

Twitter #PMC .

Slideshare pathh1

Major Genomic Components

• Tandem Repeats• Simple Sequence Repeats• Dispersed Repeats• Functional Repeats• Retroelements• Genes

Typical Fraction10%5%10%15%50%10%

A D’Hont et al. Nature 2012

doi:10.1038/nature11241

Whole-genome duplication events.

Satellite DNA probe green

• 45S rDNA

Differences between genomesMajor differences in the nature and amount of repetitive DNA

• dpTa1 tandem repeat

146 bp around histones

From Chromosome to Nucleus

Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com

• Three copies of the Arabidopsis 180 bp repeat showing (dark purple, stepped line) GC content of the sequence and (red, smooth line) sequence curvature. While GC and AT rich regions of a sequence generally correlate with curvature, the kinked region shows curvature with low GC content.

• How do genomes evolve?• How can we exploit knowledge of genome

evolution?– Biodiversity– Chromosome engineering– Markers

Genome engineering

• Introgression of chromosomes– Brassica – Raphanus– Wheat – Thinopyrum

Chromatin

• Packaging

UK Wheat 1948-200752,909 data points, 308 varieties

From Ian Mackay, NIAB, UK. 2009. Re-analyses of historical series of variety trials: lessons from the past and opportunities for the future. SCRI website.

Rules for successful domestication

• There aren’t any!

• Crops come from anywhere (new/old world; temperate/tropical; dry/humid)

• They might be grown worldwide• Polyploids and diploids (big genomes-small

genomes, many chromosomes-few chromosomes)

• Seeds, stems, tubers, fruits, leaves

10 m

DNA methylation is unevenly distributed on Musa chromosomes

copia elements

in methylated regions, but also in some low methylated regions (arrows)

5MeC

10 m

C.H Teo and Schwarzacher

5MeC

DNA methylation is unevenly distributed on Musa chromosomes

gypsy elements

in methylated regions, but also in some low methylated regions (arrows)

Teo & Schwarzacher in prep 2013

Genome evolution• How do genomes evolve?

– Mutation very rarely (human: 10−8/site/generation)

– Chromosome evolution– Polyploidy and genome duplication (ancient and modern)– Repetitive sequences – mobility & copy number (10−4 µsat)

– Recombination– Epigenetic aspects – centromeres & expression

• How can we exploit knowledge of genome evolution?– Biodiversity– Chromosome engineering– Breeding– Markers

Outputs

– Crops(Chemical energy)

– Food– Feed– Fuel

– Fibre– Flowers

– Pharmaceuticals– Fun 85

Molecular cytogenetics …

The genepool has the diversity to address these challenges …

New methods to exploit and characterize germplasm let use make better and sustainable use of the genepool

How to use diversity• Cross two varieties

• Genome manipulations• Cross two species and make a new one• Cell fusion hybrids• Chromosome manipulation• Backcross a new species

• Generate recombinants• Chromosome recombinations

• Transgenic approaches

• Use a new species

Nothing special about crop genomes?Crop Genome size 2n Ploidy Food

Rice 400 Mb 24 2 3x endosperm

Wheat 17,000 Mbp 42 6 3x endosperm

Maize 950 Mbp 10 4 (palaeo-tetraploid) 3x endosperm

Rapeseed B. napus

1125 Mbp 38 4 Cotyledon oil/protein

Sugar beet 758 Mbp 18 2 Modified root

Cassava 770 Mbp 36 2 Tuber

Soybean 1,100 Mbp 40 4 Seed cotyledon

Oil palm 3,400 Mbp 32 2 Fruit mesocarp

Banana 500 Mbp 33 3 Fruit mesocarp

Heslop-Harrison & Schwarzacher 2012. Genetics and genomics of crop domestication. In Altman & Hasegawa Plant Biotech & Agriculture. 10.1016/B978-0-12-381466-1.00001-8 Tinyurl.com/domest

Centromere

DNA sequenceTE

Tandem repeat monomerTE Transposable element

Single copy DNA

Spindle microtubules pulling apart chromatids

Metaphase chromosome

147bp plus 5-70bp linker = 150-220bp

Kinetochore

Heslop-Harrison JS, Schwarzacher T. 2013. Nucleosomes and centromeric DNA packaging. Proc Nat Acad Sci USA. http://dx.doi.org/10.1073/pnas.1319945110. See also http://molcyt.org (Dec 2013)

Genes!

EvolutionEpigeneticsDevelopment

PhenotypeMultiple abnormalities

Genetic changes  non-reverting

Changes seen, some reverting

(Male/Female)Normal Differentiation

CauseChromosomal loss, deletion or

translocationGene mutation / base pair

changesTelomere shortening

(Retro)transposon insertion Retrotransposon activation

SSR expansionMethylation

HeterochromatinizationChromatin remodelling

Histone modification

Outputs–CROPS

– Fixed energy Inputs

–Light–Heat–Water–Gasses–Nutrients

Outputs–CROPS

– Fixed energy

93

Inputs

–Light–Heat–Water–Gasses–Nutrients

– Light– Heat

– Water– Gasses

– Nutrients

Chromosomes, Crops and

Superdomestication What do we want?

What have we done?