Chapter 19

Genomics and Agriculture

Applications of genomics approaches to agriculture

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Contents
Background Agriculturally related sequencing projects
Crop plant Farm animal Pathogens

Genomics applied to trait improvement

Breeding Transgenics, biotechnology and clones
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Background
Agriculture looks to genomics for the next green revolution (Gene revolution?) Reasons: POPULATION EXPLOSION (7 billion and 800 million malnourished)
Pace of traditional breeding is slow Identify genes for useful traits Relate a trait to genetic and physical maps and whole genome sequencing Protect food chain and security (pathogens)
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Genomics applied to agriculture

Approaches
Similar for crop plants and farm animals Relating traits to genes
Relating genetic maps to physical maps Rarely monogenic QTL analysis (Quantitative Trait Loci) Problem of large genome size So use syntenic relationships ESTs

DNA sequence

Genes can be manipulated, either through breeding or through genetic engineering, to remove deleterious traits and enhance desirable traits
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Sequencing of plant genomes

Reference plant: Arabidopsis thaliana
No direct agricultural value
Related to cabbage and mustard

Reference for all plants First plant genome sequenced (Dec. 2000)
Size: 130 Mbp Number of genes: 28,000 Segmental duplications segmental duplications larger than 100 kb make up nearly 60% of the Arabidopsis genome
Evidence for past increase in ploidy Only 35% of Arabidopsis genes are unique, while 38% belong to families of more than five members
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Sequencing of crop-plant genomes

Reasons for sequencing rice first
Importance as crop Largest food source for poor Feeds half of worlds population (3 billion) Demand likely to increase dramatically 80% of daily calories in Asia come from rice In Asia alone, demand will increase by at least 35%.
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Rice genome
Smallest among grass genomes (Wheat, oat, rye, Barley, corn)
Few repetitive elements

Synteny with other grasses (recent evolution from a common ancestor approximately 5070 million years ago) Genetic and physical maps Genomic resources
Over million ESTs

Efficient transformation
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Efforts to sequence the rice genome

International Rice Genome Sequencing Project (IRGSP)

Different efforts
Public: IRGSP: clone by clone/ Beijing Genomics Institute: shotgun Private: Monsanto/Syngenta shot gun
sufficient coverage to be able to map certain phenotypic traits such as plant size and fertility

Public performed 10x coverage

Two strains: Indica and Japonica

Gold standard for other cereal genomes Microarray of rice can be used on maize RNA
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Facts about the rice genome

Size: 430 Mbp (3.3 X Arabidopsis) Number of genes: approximately 60,000 Repetitive elements: Most in intergenic regions versus in introns in humans Animals use alternative splicing and plants gene duplication?
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Rice and Arabidopsis genomes

No large areas of synteny 80% of Arabidopsis genes have homologs in rice Reverse not true Only 50% of rice genes have homologs in Arabidopsis 150200 million years of divergence (Quick change)
Rice

Arabidopsis

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Genomics of other cereals

Maize: 3,000 Mbp Wheat: 5,000 Mbp Barley: 16,000 Mbp Genome organization
Genic or gene-rich islands in the sea of retroposons

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Sequencing strategies for grass genomes

Alternative approaches to genome sequencing
Methylation based: use non-methylated DNA by digesting genomic DNA with bacterial enzyme that destroys methylated DNA Hybridization based: repetitive DNA fraction removed

EST collections based on synteny but private companies do not share the data

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Genomics of other crop plants

Tomato, potato, soybean
EST collections

Woody species
Poplar and pine Genome organization
No genic islands

Candidate-gene approach Because the genomes of most of the woody species are larger than even that of maize, genomic sequencing will probably have to wait until drastic reductions in sequencing 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 costs arrive (How wrong was that prediction?).

Genomics of farm animals

Livestock farming = 3040% of world agriculture farm animals provide much of the protein in the daily diet in developed world Poultry and livestock sales in United States > $70 billion Disadvantages of genomics
Large sizes of farm-animal genomes like humans Long gestation times Difficulty of doing genetics

Nevertheless, genomics programs have been initiated for most of the major farm-animal groups, including pigs, cows, sheep, and poultry.
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Genomics of farm mammals

Pig, cow, sheep Draft sequencing
Compare to mouse, rat, and human

BAC libraries Physical maps EST libraries

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Genomics of poultry
No reference genome Different than humans BAC libraries > 300,000 ESTs EST Microarrays 22 K genes

http://www.chick.umist.ac.uk

http://www.genome.org/cgi/content/full/15/12/1692
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Sequencing of agricultural pathogens

Animal pathogens
Brucella suis (bacterium) Infects animals, but can affect humans Genome revealed to be similar function to plant pathogen Agrobacterium

Plant pathogens
Problem: large size of genomes Agrobacterium, Phytophtora (potato blight), Fusarium

bacterial pathogen that has invaded a cows milk-secreting cells, 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 causing mastitis (inflammation of the mammal breast ).

Bioterrorism issues: Food security

Intensive agriculture raises risks of disease spread
Example: outbreak of footand-mouth disease Costed 48 billion to UK

Knowledge of pathogen genomes

Helps identify disease agent Could be used in rapiddetection technologies Could also be used a biological weapon by the US military who considered using Brucella in 1950s

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Breeding
Domestication of plants and animals selected for valuable traits
e.g., temperament allowing bovines to be kept in captivity

Later, other traits selected for

e.g., milk production

Traits controlled by several genes

For each gene, different alleles

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Quantitative traits
Major traits in continuous gradient (not yes and no like monogenic traits) Controlled by QTLs Infinitesimal model
Many genes, each with small effect

Major-gene model
A few genes, each with large effect

Genomic nature of QTLs: transcriptional control

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QTL analysis
QTL analysis requires genetic and physical maps Similar to association mapping in humans
Relate traits to markers

Or cross two subspecies with different traits

Both domesticated: cows Wild plus domesticated boars but takes time to introgress

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Genome scan for QTLs using crosses

Genome scan for QTLs in progeny: cosegregation Relate trait to markers Identifies interval on chromosome

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QTL for tomato fruit size

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Marker-assisted breeding
Once a QTL is found, it can be used to assist breeding
Even if the nature/function of gene is unknown

Markers on either side of the QTL can be followed during the breeding program Introgress the QTL from one subspecies into another Markers have to be very closely linked

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QTL to gene
If genome sequenced: Candidate genes in interval If genome not sequenced: Find syntenic region in sequenced genome In QTL analysis of fat and lean chickens by scientists at the Roslin Institute, a QTL was identified that accounts for over 40% of the genetic variability in this trait. When this portion of the chicken genome was compared with the human genome, five genes that play a role in lipid metabolism were identified in the QTL interval. To confirm identity: Look for mutations Microarrays and 2-D gels for expression analysis Transfer gene and determine consequences

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Genomics tools for breeding

Polymorphic markers
Microsatellites SNPs

Expression approaches
Microarrays 2-D gels

Bioinformatics
Databases
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Improvement of plant traits

Stress resistance
Abiotic
Salinity Drought

Biotic
Pathogens

Increased yield per acre Decreased fertilizer utilization: negative effects on downstream waterways Improved value-added traits: oil absorption by potato during frying or color of grains
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Examples of plant breeding

Hybrid vigor
Corn with improved yield Compare inbred parental lines with hybrids

Size of tomato fruit

Comparisons of wild relatives with crop plants and found a single gene for that
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Improvement of animal traits

Growth rate Meat quality Disease resistance Reproductive performance Behavior

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Example of animal breeding

Meat quality in pigs
Meat-to-fat ratio

Cross Chinese Meishan pigs with European Large White pigs Meishan much fatter than European variety Identified QTL for lean meat

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Transgenic technologies
Goal: rapid modification of genes responsible for traits in plants and animals Gain of function:
Overexpression Ectopic expression

Loss of function:
Homologous recombination Antisense or RNAi

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Reverse-genetics example
Gene that encodes muscle-specific transcription factor in mouse
Myogenin required
neo targeting vector Tk

genome locus

myogenin
selection

Homologous recombination used to delete gene Mice born, but cant make muscle

neo product of homologous recombination selectable marker disrupts myogenin gene

RNAi and antisense RNA

Double-stranded RNA able to disrupt gene expression
Cells have machinery that destroy doublestranded RNA: viruses/ cDNA

Appears to be basis for the following:

Interfering RNA (RNAi)
Double-stranded RNA introduced into cells

Antisense RNA
Introduce complementary RNA Forms double-stranded RNA in cells

Gene knockout techniques

Homologous recombination: low efficiency RNAi leaky

Deleted in KO
AGL5 wt genomic

AGL5 KO construct

KanR

agl5 KO genomic

KanR

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Transgenic plant technology

Tumor production Nopaline synthesis

Agrobacteriummediated transformation Gene inserted into Ti plasmid Agrobacterium cocultivated with plant Ti plasmid transferred into plant genome Selection with antibiotics

T-DNA

T-DNA Transfer functions

Ti plasmid

Nopaline utilization

Origin of replication

Agrobacterium gall on a cherry tree

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Transformation of rice

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Transgenic animal technologies

Introduction of genes directly into nucleus
Microinjection 1980s Used to produce transgenic pigs, cattle, and sheep Problems: inefficiency, random chromosomal insertion, tandem duplication

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Nuclear transfer
The term Nuclear transfer or cell nuclear replacement better than cloning Technique known since 1950s Transfer of nucleus from adult cell to unfertilized egg with nucleus removed Or fusion of adult cell with enucleated egg Problem: abnormal development due to imprinting where one of the parental gene is shut off in embryogenesis A cloned elite cow sold recently for over $40,000
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Animal cloning (1997)

Finn Dorset
Udder cell

Scottish Blackface
Egg cell

Fuse cells with electric shock Fused cell grows into an embryo

Remove nucleus from egg cell

Embryo is placed in foster mother

Cloned lamb is born

Dolly

Finn Dorset
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Xenotransplantation (the transplantation of animal organs into humans)

Pharmaceutical proteins

Alpha-1-antitrypsin Applications in emphysema and cystic fibrosis Problems in isolating from humans (contamination), yeast, or bacteria (processing) Target in cell and then perform nuclear transfer Produce as Milk protein and purify
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Pet cloning
A cat named Little Nicky, produced in 2004 by Genetic Savings & Clone for a north Texas woman for the fee of US$50,000 (CC or copy cat) A family dog Missy was cloned in 2008 (Bestfriends again) A dog named Booger for its Californian owner was cloned into 5 clones by SNL (Korea) 50K Goats, sheep, pigs, Cattle Horses? Cloned in 2006 for $150K Humans? Dinosaurs? Mammoths? Risks?

Summary I
Need for genomics approaches in agriculture Genomic sequencing
Crop plant
Rice

Farm animal
Livestock and poultry

Pathogens Bioterrorism

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Summary II
Genomics and breeding
QTLs Traits

Transgenic technologies
Plant Animal

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Questions
What is your opinion about genetic engineering of bacteria, plants, animals and human?

Write an essay and submit by November 18th 2011

2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458