Understanding Complex Traits in Maize through Structural and Functional Genomics Georgia Davis.

Understanding Complex Traits Understanding Complex Traits in Maize through Structural in Maize through Structural and Functional Genomicsand Functional Genomics

Georgia DavisGeorgia Davis

Structural genomicsStructural genomics

Science related to discovery of gene Science related to discovery of gene order and organization across the order and organization across the genome.genome.

MutationMutation

Changes in DNA sequence lead to…Changes in DNA sequence lead to… Changes in amino acid sequence lead Changes in amino acid sequence lead

to…to… Changes in protein or lack of protein.Changes in protein or lack of protein.

AlleleAllele

One of two to many alternative One of two to many alternative forms of the same gene (eg., round forms of the same gene (eg., round allele vs. wrinkled allele).allele vs. wrinkled allele).

Alleles have different DNA Alleles have different DNA sequences that cause the different sequences that cause the different appearances we see. appearances we see.

MendelMendel

Monk at the St. Thomas monastery in Monk at the St. Thomas monastery in the Czech Republic.the Czech Republic.

Performed several experiments between Performed several experiments between 1856 and 1863 that were the basis for 1856 and 1863 that were the basis for what we know about heredity today.what we know about heredity today.

Used garden peas for his research.Used garden peas for his research. Published his work in 1866.Published his work in 1866.

MendelMendel

Results are remarkably accurate and Results are remarkably accurate and some have said they were too good to some have said they were too good to be unbiased.be unbiased.

His papers were largely ignored for His papers were largely ignored for more than 30 years until other more than 30 years until other researchers appreciated its researchers appreciated its significance.significance.

Garden PeaGarden Pea

Pisum sativumPisum sativum DiploidDiploid Differed in seed shape, seed color, Differed in seed shape, seed color,

flower color, pod shape, plant height, flower color, pod shape, plant height, etc.etc.

Each phenotype Mendel studied was Each phenotype Mendel studied was controlled by a single gene.controlled by a single gene.

TermsTerms

Wild-type is the phenotype that would Wild-type is the phenotype that would normally be expected.normally be expected.

Mutant is the phenotype that deviates from Mutant is the phenotype that deviates from the norm, is unexpected but heritable.the norm, is unexpected but heritable.

Notice that this definition does not imply that Notice that this definition does not imply that all mutants are bad in fact many beneficial all mutants are bad in fact many beneficial mutations have been selected by plant mutations have been selected by plant breeders.breeders.

Advantages of plantsAdvantages of plants

Can make controlled hybrids.Can make controlled hybrids. Less costly and time consuming to Less costly and time consuming to

maintain than animals.maintain than animals. Can store their seed for long periods of Can store their seed for long periods of

time.time. One plant can produce tens to One plant can produce tens to

hundreds of progeny.hundreds of progeny.

Advantages of plantsAdvantages of plants

Can make inbreds in many plant Can make inbreds in many plant species without severe effects that are species without severe effects that are typically seen in animals.typically seen in animals.

Generation time is often much less than Generation time is often much less than for animals.for animals.– Fast plants (Brassica sp.)Fast plants (Brassica sp.)– ArabidopsisArabidopsis

Round vs. wrinkledRound vs. wrinkled

The SBEI causes the round vs. wrinkled The SBEI causes the round vs. wrinkled phenotype.phenotype.

SBEI = starch-branching enzymeSBEI = starch-branching enzyme Wrinkled peas result from absence of Wrinkled peas result from absence of

the branched form of starch called the branched form of starch called amylopectin.amylopectin.

When dried round peas shrink uniformly When dried round peas shrink uniformly and wrinkled do not.and wrinkled do not.

Round vs. wrinkledRound vs. wrinkled

The non-mutant or wild-type round allele is The non-mutant or wild-type round allele is designated W.designated W.

The mutant, wrinkled allele is designated w.The mutant, wrinkled allele is designated w. Seeds that are Ww have half the SBEI of wild-Seeds that are Ww have half the SBEI of wild-

type WW seeds but this is enough to make type WW seeds but this is enough to make the seeds shrink uniformly.the seeds shrink uniformly.

W is dominant over w.W is dominant over w.

Round vs. wrinkledRound vs. wrinkled

An extra DNA sequence is present in An extra DNA sequence is present in the wrinkled allele that produces a non-the wrinkled allele that produces a non-functional SBEI and blocks the starch functional SBEI and blocks the starch synthesis pathway at this step resulting synthesis pathway at this step resulting in a lack of amylopectin.in a lack of amylopectin.

A Molecular View A Molecular View

Parents F1 F2 Progeny

WW ww Ww ¼WW ¼Ww ¼wW ¼ww

1: 2 : 1 Genotype = 3: 1 Phenotype

Xeroderma pigmentosaXeroderma pigmentosa

Autosomal recessive.Autosomal recessive. UV exposure damages DNA.UV exposure damages DNA. Defect in DNA damage repair.Defect in DNA damage repair. Risks include cancer, telangiectasia, Risks include cancer, telangiectasia,

disfigurement.disfigurement. Can be diagnosed before birth.Can be diagnosed before birth. Take total protection measures from Take total protection measures from

sun/fluorescent light.sun/fluorescent light.

Xeroderma pigmentosaXeroderma pigmentosa

UV damages tissue that UV damages tissue that contains molecules that can contains molecules that can

absorb light.absorb light.

Mechanisms of UV damageMechanisms of UV damage

Low penetration into tissues.Low penetration into tissues. Molecular fragmentation—proteins, Molecular fragmentation—proteins,

enzymes, and nucleic acids contain enzymes, and nucleic acids contain double bonds that can be ruptured by UV.double bonds that can be ruptured by UV.

Free radical generation—molecules of Free radical generation—molecules of susceptible tissues absorb UV and eject susceptible tissues absorb UV and eject an electron, which is taken up by oxygen, an electron, which is taken up by oxygen, then termed superoxide, a free radical.then termed superoxide, a free radical.

Free radicalsFree radicals

Are scavenged by superoxide Are scavenged by superoxide dismutase, vitamin C, vitamin E, dismutase, vitamin C, vitamin E, glutathione peroxidase, carotene.glutathione peroxidase, carotene.

Lesion mutant in maizeLesion mutant in maize

Mutants across organismsMutants across organisms

Sometimes mutations in the same Sometimes mutations in the same gene in different organisms have gene in different organisms have similar phenotype.similar phenotype.

This allows researchers to choose the This allows researchers to choose the organism with the best genetic organism with the best genetic resources to study the normal function resources to study the normal function of that gene. of that gene.

This also allows researchers to identify This also allows researchers to identify prospective genes for a phenotype in prospective genes for a phenotype in one species, based on another.one species, based on another.

Structural GenomicsStructural Genomics

Genetic mappingGenetic mapping Physical mappingPhysical mapping In situIn situ hybridization hybridization

Functional GenomicsFunctional Genomics

Science related to the discovery and Science related to the discovery and definition of the function of genesdefinition of the function of genes

Whole plant Cellular Biochemical RNA

C16

C18

C20

C22

C24

C26

Aldehydes Alcohols

Wax Esters

Functional GenomicsFunctional Genomics

Trait analysisTrait analysis Gene disruption Gene disruption

strategiesstrategies– TargetedTargeted– RandomRandom

DNA microarraysDNA microarrays

RNA profilingRNA profiling Protein chips Protein chips Protein prediction Protein prediction

algorithmsalgorithms

Functional GenomicsFunctional Genomics

Insect ResistanceInsect Resistance– QTL AnalysisQTL Analysis– Biochemical Pathway Identified.Biochemical Pathway Identified.– Major QTL With Unknown Function.Major QTL With Unknown Function.

Aflatoxin ReductionAflatoxin Reduction– QTL AnalysisQTL Analysis– Major QTL With Unknown Function.Major QTL With Unknown Function.– One Biochemical Pathway Identified.One Biochemical Pathway Identified.

Fall armywormFall armyworm

Present east of Present east of Rockies and South of Rockies and South of Canada.Canada.

Egg-adult in 4 weeksEgg-adult in 4 weeks One female lays One female lays

several hundred several hundred eggs. eggs.

Broad host rangeBroad host range

Hosts of Fall ArmywormHosts of Fall Armyworm

Southwestern Corn BorerSouthwestern Corn Borer

Narrow host range.Narrow host range. Mainly in southeastern Mainly in southeastern

US.US.

Southwestern Corn Borer Southwestern Corn Borer HostsHosts

GermplasmGermplasm

Fall armyworm damage 10 days after infestationwith 30 larvae per plant

Lepidopteran ResistanceLepidopteran Resistance

Beet armyworm

European corn borer

Corn ear wormFall armyworm

Southwestern corn borer

Juvenile traitsJuvenile traits

Dull epicuticular wax layerDull epicuticular wax layer– Fatty alcoholsFatty alcohols

No leaf hairsNo leaf hairs Adventitious roots presentAdventitious roots present No buliforme cellsNo buliforme cells Cells are roundedCells are rounded

Juvenile vs. Adult LeavesJuvenile vs. Adult Leaves

Leaves maintain their phase status Leaves maintain their phase status throughout development.throughout development.

Number of juvenile leaves varies Number of juvenile leaves varies among inbreds.among inbreds.

A number of mutants in juvenile to A number of mutants in juvenile to adult phase change have been adult phase change have been identified.identified.

43%

Summary of QTL StudySummary of QTL Study

66%

SWCBFAWJuvenility

7%

51%

27%11% 59%

1 2 3 4 5 6 7 8 9 10gl15

Gl15Gl15

Apetala2Apetala2-type transcription -type transcription

factor.factor. Regulates long-chain wax Regulates long-chain wax

ester synthesis.ester synthesis. Controls epidermal juvenile Controls epidermal juvenile

to adult phase change.to adult phase change.

Functional GenomicsFunctional Genomics

Insect ResistanceInsect Resistance– QTL AnalysisQTL Analysis– Biochemical Pathway Identified.Biochemical Pathway Identified.– Major QTL With Unknown Function. Major QTL With Unknown Function.

Aflatoxin ReductionAflatoxin Reduction– QTL AnalysisQTL Analysis– Major QTL With Unknown Function.Major QTL With Unknown Function.– One Biochemical Pathway Identified.One Biochemical Pathway Identified.

Hosts of Hosts of Aspergillus flavusAspergillus flavus

Aspergillus flavusAspergillus flavus

Infection via silk channel wounds in kernels cracks in kernels

AflatoxinAflatoxin

Aflatoxin B1

Potent carcinogen.Associated with liver cancer

in humans.

Health concernsHealth concerns

Aflatoxin contaminationAflatoxin contamination

Large environmental influence.Large environmental influence.– Drought stress increases aflatoxin level.Drought stress increases aflatoxin level.

Correct genes in both fungus and host Correct genes in both fungus and host required for high levels of toxin required for high levels of toxin formation.formation.

Synthesis is biochemically similar to Synthesis is biochemically similar to anthocyanin biosyntheis in maize.anthocyanin biosyntheis in maize.

Aflatoxin QTL SummaryAflatoxin QTL Summary

1L7%

2L8% 3C

7%

4L24%

6S8%

unexplained25%

4L21%

Mp313E favorable allele

Va35 favorable allele

DAD A

Anthocyanin vs. aflatoxinAnthocyanin vs. aflatoxin

a2

bz1

c1-p

a1

a1 sh2

a2 bt pr

bz2 c1-n

c sh wx

in1

pr

r-g

sh bz wx

su c2

c2

C2I-df

23X toxin

9X toxin

7X toxin