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Postharvest Technology andPostharvest Technology and Tropical Fruit Genomes

Robert E. Paull, Nancy J. Chen, Helen Turano, Beth Irikura and Pingfang Wug g

Department of Tropical Plant & Soil SciencesUniversity of Hawaii at Manoa

Postharvest Deals withFresh Fruit, Vegetables and OrnamentalsSteps from harvest to consumerReduction in losses – quantity and qualityEnsuring consistency of supply

Volume & quality

QualityAppearance – suitability for the marketNutritional ValueSafety - Microbial

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General Characteristics of Fresh Fruit, Vegetables & Flowers

Living TissueHigh Water ContentSubject to PathogensDiverse - Morphological Structurep g

- Composition- Physiology

Potential for contamination

Postharvest Supply Chain- Components

Grower

Packer

Transport

Distributor

Money &

Information

Marketer

Consumer

Value

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Postharvest Research and Biological Scales

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Genome Sequencing Goals Reconcile molecular biology and genes with function and phenotypeArabidopsis is the reference genome

BenefitsComparative sequencing – plant genome p q g p gstructure, dynamics & plant evolutionIdentification of genes and other functional elements (regulatory genes, small RNAs)Tool for gene isolation from new target species

Genome Sequencing CostsYear Human Genome2003 $ 300 M

2007 $ 1 M

2008 $ 60,000

2010 $ 5,000

454 Sequencing– One Run – 500 Mb up to 600 b long - cost $12,000

Data Generated- Raw Sequences - Tetra/Giga bytes- Text Sequences - Giga/Mega bytes- Quality File - Mega/Kilo bytes

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Plant GenomesSpecies Genome Size Genome Completed

Arabidopsis thaliana 125 Mb 2000

Poplar 430 Mb 2006

Grape 505 Mb 2007 December

Papaya 373 Mb 2008 April

Peach 280 Mb Started 2008

Apple 753 Mb 2009

St a be (F esca) 206 Mb In p og essStrawberry (F. vesca) 206 Mb In progress

Tomato 950 Mb 2010

Citrus – Sweet Orange 382 Mb 2010 (1.2x)

Banana (M. acuminata) 610 Mb 2011

Maize ~ 2,300 Mb

Tropical fruit

Papaya as a Model Systemp

A small genome of 372 MbDiploid inheritance (2n=18)Fully sequenced genome (6X)ESTs 77,246 in GenbankESTs 77,246 in GenbankTransformation system availableSets fruit all year around

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Phylogeneticrelationships between

species with completedspecies with completed genome sequencing

projects (bold) or those ongoing.

Barbazuk W. B. et.al. Genome Res.;2008;18:1381-1392

Genome Duplication and Evolution

Papaya lineage diverged from Arabidopsis about 72MYAp y g g p

Lyons et al. 2008 Tropical Plant Biol. 1:181-190. Tang et al. 2008 Genome Res. 18:1944-1954.Ming et al. 2008 Nature 452:991-996.

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Genomes – Steps & Approaches 1. Sequencing1. Sequencing

BAC by BAC – Hierarchical - ~120 kb Whole Shotgun 1 to 3 kb

2. Gene Structure Prediction3. Gene Functional Prediction – Annotation4. Gene to Phenotype (phenome) – Function

Greatest complexityApproaches • Genetics• Physiological• Phenotype

Reverse geneticsQTL mappingCloning and taggingComparative mapping

Mapping & GenomesGenetic Classical linkage mapGenetic Classical linkage map

Recombination of genes relative to each other

Physical Location on DNARestriction site, nucleotide sequence, genes,

regardless of inheritance

Cytogenetic Location of markers on chromosome

Genes Assigning DNA seq. to chromosomes

Gene Structure Single gene – introns, exons, untranslated regions, termination and regulatory elements

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Approaches to Sequencing

enet

ics

2: 5

73G

reen

200

1 N

atur

e Rev

Ge

Whole Genome Sequencing

Green 2001 Nature Rev Genetics 2: 573

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Papaya initially had 9 major & 3 minor

Genetic mapping

Papaya initially had 9 major & 3 minor linkage groups (LGs)

Based upon 706 WGS (Whole Genome Seq.) and BES (BAC end seq.) microsatellite markers

Linkage GroupsLGs 8 and 10 are on the same chromosome

Based on assembled supercontigs.LGs 9 and 11 same chromosome

Based on florescent in situ hybridization (FISH)LGs 7 and 12 same chromosome based on FISH

Cytogenetic Mapping LG 7 + LG 12 LG 9 + LG 10

Physical assignment of papaya linkage groups to chromosomes by fluorescence in situ

hybridization (FISH)DNA markers from three minor LGs and hybridized withchromosome-specific markers to observe the co-localization ofsignals on metaphase chromosomes. Ching Man Wai

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Assembled papaya genome

Linkage Group

Assembled

SSR Markers

Assembled genome (purple bars) anchored to the 12 papaya linkage groups (red bars) through SSR markers (blue lines).

Assembled Genome

Gene PredictionCpETR1 - Ethylene Receptor - EVM 151.32

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Genome Statistics Sequenced Fruit Genomes

Arabidopsis Papaya Grape Tomato PeachSize (Mbp) 140 372 487 950 280

Chromosomes 5 9 19 12 8G+C content total(%) 35 35.3 36.2 36.1

Gene number 31,114 24,746 30,434 ~40,000 ~24,129

AAverage gene length (bp/gene) 2,232 2,373 3,399Average intronlength (bp) 165 501 1502

Transposons (%) 10 52 41.4

Papaya Gene Statistics

Genome Papaya Relative ToPapaya Gene Content 24,746 genes

Arabidopsis < 10 to 20%

Rice < 34%

Poplar < 46%

Grape < 19%

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0 5 10 15 20 25 30 3505

10152025303540

evm no.

Average number of predicted genes per40

4550556065707580859095

100105110115120125130135140145

posi

tion

p g p100kb on papaya chromosome 1

145150155160165170175180185190195200205210215220225230

Functional Categories of Genes on Linkage Group 1 = Chromosome 1

Cellular communication2.8%

Protein fate

Cell type differentiation0.7%

Protein fate9.8%

Cellular transport5.6%

Development2.8%

Energy1.4%

Organ differentiation0.7%

Metabolism18.2%

Unclassified 7 7%

Cell fate1.4%

Cell cycle and DNA processing0.7%

Systemic interaction with the environment

1.4%

Tissue differentiation0.7%

Transcription1.4%

Transport facilities and transport routes

5.6%

Subcellular localization22.4%

Regulation of metabolism and protein function

0.7%

Protein with binding function16.1%

7.7%

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Papaya Ethylene Receptors

Paull et al., Tropical Plant Biology 1, 246-277

Papaya MicroarrayCustom papaya microarray (Version 1)Th (4 72k) h 24 421 di dThe arrays (4x72k) have 24,421 predicted genesEach gene represented by two to three probes

Probed Microarray with RNAExtracted RNA from mature green and ~30% ripeg p16,375 genes were detected above background976 genes were up-regulated by at least 3-fold857 genes were down-regulated by at least 3-fold

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Mature Green and 30% Ripe Microarrays

Gene Expression During RipeningCellulose synthase was highly expressed in green fruit and decreased 8-fold at 30% ripe stageIn the 30% ripe fruit cell-wall genes that increased

4x polygalacturonases, 3x pectinesterases, pectate lyase, 2x glucanases, endoxylanase, xyloglucan-endotransglycosylase, β-1,4-xylosidase, glucosidase, β-glucuronidase, and chitinase. A β- and α-galactosidase both g β gdeclined.

Four expansins (CpEXPA10, CpEXPA13, CpEXPA14, CpEXPA17) increased and two declined (CpEXPA6, CpEXPA9).

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Phytohormone ChangesCpETR2 & CpETR3 up-regulated CpETR1 not expressedCpETR2 & CpETR3 up regulated, CpETR1 not expressed in either stagesCpCTR1 and CpACS1 (ACC synthase) expression declined and CpEIN2 increased three-foldAuxin-related genes mostly declinedGA-related genes were mostly down-regulated g y gCytokinin gene in degradation up-regulated.ABA genes showed little change between the two stagesBrassinosteroid-related genes mostly down-regulated

Papaya Slow RipeningLoss of Firmness during Ripening

100

150

200

250

Line 4-16

SunsetFirmnessN

0

50

100

0 3 6 9 12 15 18 21Days from Harvest

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Slow-Ripening QTL

Crossed the inbreed lines (Line #8 x Psls), the F1 was selfed and F2 progeny evaluated.

One potential QTL was isolated – LG #1

Predicted to contain ~ 50 genes on supercontig2121.

Eleven show genes little or no expression at the

Slow-Ripening QTLEleven show genes little or no expression at the mature green or the 35% yellow stage of ripening

Seven genes were up-regulated more than two-fold

Twenty-five genes down-regulated

Remainder showed less than two-fold change between stages.

Next step - which gene is it??

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Papaya has fewer genes than Arabidopsis with reductions in most gene families and biosynthetic

Summary of Major Findings

reductions in most gene families and biosynthetic pathways.The lower gene number is largely due to lack of recent genome-wide duplication.Papaya has fewer members of gene families involved in fruit ripening, with the exceptions of starch synthase.Thousands of genes change during ripeningPhytohormone changes can be tracked during ripening

Postharvest disease resistance need to look at gene

Future ApplicationsPostharvest disease resistance need to look at gene

expression and modify.Can consider looking at chilling resistance in tropical

fruit.Look at fruit development relating to size, shape and

sugar and acid accumulation.Modify genes associated with fruit ripening; initiation and rate of ripening.Fruit quality as it relates to nutrient content, storage, color, texture and softening.

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Primary Funding Sources:University of Hawaii at Manoa & US DoD (81XWH0520013) -MA

Acknowledgements

Maui High Performance Computing Centre -MAHawaii Agriculture Research Centre -RM and QYNankai University, China -LW

Other support the USDA T-STAR program, a USDA-ARS cooperative agreement (CA 58-3020-8-134) with HARC, the University of Illinois and the NSF Plant Genome Research ProgramUniversity of Illinois, and the NSF Plant Genome Research Program.