Genomics assisted plant breeding

Christian Jung, Plant Breeding Institute, University of Kiel

The impact of genome research on plant breeding

Cultivar genotyping

Exploiting biodiversity

Selecting crossing paretns

Predicting hybrid performance

Molecular marker
Marker-assisted major gene
selection
Selecting homozygous
plants
Identifying elite alleles
Pyramiding elite alleles

Marker-assisted QTL selection

Functional markers

Genomic selection

ESTs

Genomic
sequences

Major genes, QTL

Genetic modification

Christian Jung, Plant Breeding Institute, University of Kiel

Population structure analysis

Mutant detection by genotype

Elite genome selection

Marker-assisted backcrossing
Broadening genetic variation
Resistances
Quality traits
Growth traits
Flowering time

Traditional and new breeding aims

Christian Jung, Plant Breeding Institute, University of Kiel

Major Aims in Plant Breeding

Increasing the yield potential
Yield stability
Biotic stress resistance (pests and diseases)
Abiotic stress tolerance (drought, heat, cold, water)
Herbicide tolerance

Advanced quality

Starch
Oil
Protein
Others

Nitrogen efficiency in corn (Z. mays)

Altered growth characters

Harvest organs
Shoot length
Flowering time
Vernalization requirement

Altered reproductive system

Parthenocarpy
Apomixis
Male sterility

Advanced nutrient uptake

Christian Jung, Plant Breeding Institute, University of Kiel

www.ipe.uni-hannover.de/.../brasil2_klein.jpg

high N efficiency

low N efficiency

Genetically modified plants: past, presence and future

Herbicide resistance/tolerance
Insect resistance
Virus resistance
Altered starch composition (amylopectin potato)
Nutritional quality

Altered storage oil composition

Omega-3 fatty acids from plants

Vitamin content

Male sterility (hybrids)

Altered growth characters and phenological development
New approaches for resistance to biotic stress (fungi,
Phytophthora, insects)

Tolerance to abiotic stress (drought, salt, cold stress)

New storage compounds (bioplastic, technical enzymes,
proteins, edible vaccines)
Phytase maize
Technical breakthroughs:
RNAi
Site directed gene targeting/mutagenesis (designer
nuclease technology)
Christian Jung, Plant Breeding Institute, University of Kiel

Increasing the yield potential: the yield

components in wheat
Yield (g/m): TGW x GPS x SPM

TGW: Thousand
grain weight (g)

Christian Jung, Plant Breeding Institute, University of Kiel

GPS: grains/
spike

SPM: spikes/m

How wheat has been affected by breeding in the last 70

years
Total dry mass:

+ 4%

Grain to straw ratio:

+ 69%

Spike/m:

+ 20%

Grain/spike:

+ 43%

Grain weight:

- 14%

Grain yield:

+ 47%

achieved by the introduction of semi-dwarf genes

(Rht, reduced height)
Christian Jung, Plant Breeding Institute, University of Kiel

Norman E. Borlaug 1943 and 1970

Quelle: Koebnik, R. (2003)

The importance of dwarfing genes in plant

breeding
dwarfing genes are of upmost importance in plant breeding
drastic yield increases during the 1960s
Only GA3-insensitive genes have been used in wheat breeding
dwarfing genes
Rht genes from wheat (reduced height)
Gai genes from A. thaliana (giberellic acid insensitive)
encode gibberellic acid (GA) metabolism/pathway genes

Rht genes
Wheat, rice, barley
Wheat: ca. 20 genes
Reduced height: mutation in GA-20-oxidase gene (17aa deletion) GA-signal
transduction

Christian Jung, Plant Breeding Institute, University of Kiel

Dwarfing genes and the green revolution

Christian Jung, Plant Breeding Institute, University of Kiel

3 OCTOBER 2003 VOL 302 SCIENCE ( 2003), pp72

The importance of dwarfing genes in green

revolution

Short straw rice and wheat

Sg1 (semi-dwarf1) rice
IR8= super rice
Rht-semi dwarf wheat
Yield increase by GA-20Oxidase gene knockdown
mutation
Conventional
rice

High-yield
rice

Super rice

Quelle: Khush, Geo (1995)

Christian Jung, Plant Breeding Institute, University of Kiel

Semidwarf lines are being used in oilseed rape

breeding
regular straw hybrid
Semi-dwarf hybrid

Christian Jung, Plant Breeding Institute, University of Kiel

http://www.feiffer-consult.de/images/Raps%20Halbzwerge.jpg

Yield stability: pest and pathogen resistance in sugar beet

(Beta vulgaris L.)
beet cyst nematode (Heterodera
schachtii)

Beet necrotic yellow vein virus

(BNYVV), Rizomania disease

susceptible
genotype
resistant
genotype

Christian Jung, Plant Breeding Institute, University of Kiel

photos: Strube Saatzucht

Fatty acid composition and glucosinolate content in old

vs. new (00) oilseed rape varieties
Fatty acids (% of total)
70
old variety
60

00-rapeseed

Glucosinolates (mol/g)

50

120
100
80
60
40
20
0

40

30

20

10

0
C12

C14

C16 C18:0 C18:1 C18:2 C18:3 C22:1 Rest

Lauricacid rapeseed with C:12-thioesterase from California bay

(Umbellularia californica)

Christian Jung, Plant Breeding Institute, University of Kiel

Rbbelen, G. (1999). "Die historische Entwicklung der

Pflanzenzchtung in Deutschland: Das Beispiel der lpflanze
Raps." Biologie in unserer Zeit 3/99: 132-141.

Leaf angle in corn

Christian Jung, Plant Breeding Institute, University of Kiel

erectophile leaf type

more planophile leaf

type

Glyphosate-tolerant corn (RR-corn)

Bacterium with glyphosate-tolerance
gene CP4 EPSPS

Transgenic corn cell

Transformation vector

Petunia transit
peptide for EPSPS

particle gun
transformation

Genetically engineered
glyphosate-tolerant corn

mRNA

CP4 EPSPS with

transit peptide
nucleus
matures Protein,
insensitive
sensitive EPSPS

Glyphosate (Round up)

Photos: Louisiana State University
Christian Jung, Plant Breeding Institute, University of Kiel

EPSPS: 5-Enolpyruvat-Shikimat-3-Phosphatsynthase

Genetic mapping of major genes

Christian Jung, Plant Breeding Institute, University of Kiel

Linkage

Not linked

linked

closely linked

The degree of linkage is indicated by the recombination frequency (R).

Christian Jung, Plant Breeding Institute, University of Kiel

Genes and chromosomes in a diploid organism

unlinked genes

A1

A2

Gene

Linked genes
B1

B1

heterozygous
Alleles
Homologous
chromosomes
Christian Jung, Plant Breeding Institute, University of Kiel

homozygous

Parallel observation of phenotypes of interest

and marker locus
phenotype
Target locus

Z2

Z1

Linkage group
M1

M2

DNA-Polymorphism on the marker locus

Flowering plants

Non-flowering plants
phenotype
Marker locus

M2
M1

Z1: target locus, allele 1

M1: Marker locus, allele 1

Z2: target locus, allele 2

M2: Marker locus, allele 2

Christian Jung, Plant Breeding Institute, University of Kiel

recombinant

Single crossover between 2 loci

during the meiosis prophase

A1

A1 A2

A2

A1

A1 A2

A2

Result:
recombinant
gametes

B1

B1 B2

B2

Christian Jung, Plant Breeding Institute, University of Kiel

B1

B2 B1

B2

Chiasm is the microscopic visible expression of

crossovers
1 Chiasma

1 crossover

frequency of recombination: R
0 R 0.5
Christian Jung, Plant Breeding Institute, University of Kiel

50% recombinant
chromatids

In a population many crossovers may occur

a

Genotype of a F1 plant

Recombination in pachytene meiosisI

Pollen mother cells, embryo sac mother cells

Resulted gametes from F1 plants at the end of meiosis II

a a

A A

b b

B B

c C c C
d D d D
Christian Jung, Plant Breeding Institute, University of Kiel

segregation patterns of a marker linked to a recessive

disease resistance gene in a DH population
M1
b

M1
b

M2
B

M2
B

M1M2Bb

DH-production

M1M1bb

M1M1BB

Christian Jung, Plant Breeding Institute, University of Kiel

M2M2BB

M2M2bb

X: complete linkage

QTL mapping

Christian Jung, Plant Breeding Institute, University of Kiel

QTL Analysis
QTL
quantitative trait locus (gene or genome segment with measurable effect
on quantitative trait)
single copy locus which contributes to a trait under quantitative genetic
control (Geldermann 1975)
QTL mapping
search for associations between marker genotypes and phenotypic traits
if mean of markers differs significantly this can be taken as evidence
for the presence of one or more QTL in the vicinity of the
corresponding marker

Christian Jung, Plant Breeding Institute, University of Kiel

QTL Mapping Strategies

Mapping with non-related accessions (multiallelic, measuring effects of all alleles)
high genetic diversity (measuring effects of all alleles in the evaluated accessions)
no need for development of mapping population
Population C

Population B

Population A

Population D

Mapping with structured populations (biallelic segregation, measuring effects of two alleles)
limited genetic diversity (only two segregating alleles per locus generated from two
parents)
large segregating populations required (to achieve high resolution desired for MAS or
cloning candidate genes)
Parent A

X
F1

F2
Christian Jung, Plant Breeding Institute, University of Kiel

Parent B

QTL mapping with structured populations

A

Parents

Office

Genotyping each plant

with molecular markers

QTL analysis
biallelic segregation, measuring effects of two alleles)
single point analysis: comparison of marker means (t-test),
ANOVA for each marker
single marker regression approach: divide population into
different genotypic classes (A1A1, A1A2, A2A2)
interval mapping
Composite interval mapping

Laboratory

phenotyping under different

environments with >1 repeat

LOD score

Field

Create biparental populations (DH, F3, RILs,

MAGIC): >>100 lines

Map position (cM)

Cross validation and bootstrapping

Christian Jung, Plant Breeding Institute, University of Kiel

N. Emrani

Single marker QTL analysis

calculates whether phenotype values differ among genotypes for a given
molecular marker (t-test, ANOVA, single marker regression approach)
QTL declared when t- or F-test is significant

Advantages:

Disadvantages:

simple
analysis can be performed using
basic statistical software
analysis does not require a
complete linkage map

difficult to detect the QTL far from

the marker
not very accurate at assigning QTL
position because of recombination
between marker and QTL
performing a t-test for every
marker results in many false
positives

Christian Jung, Plant Breeding Institute, University of Kiel

Single Marker QTL Analysis by

Mean Comparison at Different Marker Loci
whole population, marker co-dominant

P1 P2

all

QTL T
A

B
AA-group

aa-group

123456789

aa

AA

123456789

BB

Christian Jung, Plant Breeding Institute, University of Kiel

bb

123456789

Simple Interval Mapping (SIM)

Developed by Lander & Botstein (1989) to increase the power of QTL detection

Intervals formed by pairs of adjacent (flanking) markers are taken as the unit of
analysis, presence of QTL is tested along the chromosome by the LOD score
(>2.5)
L(QTL _ present )
LOD log10
L(QTL _ absent )

Effects of recombination between marker and QTL is compensated

Complete genetic maps are needed

Two approaches

likelihood approach: interval mapping based on maximum likelihood methods

linear regression approach

Christian Jung, Plant Breeding Institute, University of Kiel

Composite Interval Mapping (CIM)

Combines simple interval mapping for a single QTL in a given interval with
multiple linear regression analysis on markers associated with other QTLs
(cofactors)

Uses subset of markers and stepwise regression, considers all markers on a

chromosome

Determines both the location and effects size of QTL more accurately than
SIM

Estimates additive and dominance effects

Can give higher resolution of QTL location as SIM

Requires special QTL software (PLABQTL, R, MapQTL, Map Manager,)

Christian Jung, Plant Breeding Institute, University of Kiel

Practical example: QTLs for clubroot resistance in rapeseed

Chromosome
N03

DH population 263/11
Express
Mapping with 338 AFLP
and 156 SSR markers

P35M36_144
GMR012

P32M32_107
P35M36_241
P44M48_106

Na14G02_220
P35M61_158
HMR0085b
HMR0327b
P33M40_211
P32M36_440
P39M37_342
HMR1335
ReAmpT12O21ST
P33M40_90
P33M39_138

QTL

Chromosome
N19

Christian Jung, Plant Breeding Institute, University of Kiel

allele from parent Express

mediates susceptibility

P33M58_182

P33M61_330

Nineteen QTL for clubroot

resistance were identified

allele from parent 263/11

mediates resistance

P35M59_245
P35M59_282
P32M45_297
P39M44_137
P35M53_55
P32M45_118
P32M51_218
P35M36_100
P35M42_255
Na14E02_160
P39M45_100
P45M51_203
P39M52_110

P46M59_189
P32M45_67
Ra2E03a_285
P39M44_136
P34M53_265
P32M32_152
Ra2G09_318
Ol10A05_210
P33M35_359
P39M44_419
P39M45_340
P45M50_174
P45M50_173
P32M50_103
P46M48_190
P44M48_370
P39M52_55
P33M35_215
P35M59_448
P34M53_296
HMR0449c
P35M42_160
P45M50_183
P46M38_117
HMR0322a
P32M33_273
P35M59_184
P46M58_420
P39M45_192
P35M59_165
HMR0284
P32M50_163
HMR0933
P32M45_144

allele from parent 263/11

mediates susceptibility

allele from parent Express

mediates resistance

QTL

Werner, S., Diederichsen, E., Frauen, M., Schondelmaier, J. & Jung, C.

Genetic mapping of clubroot resistance genes in oilseed rape.
Theoretical and Applied Genetics 116, 363-372 (2008).

Application of molecular markers in plant breeding

Major genes
Genetic mapping of single genes
Monitoring homozygosity in progeny
Marker-assisted gene pyramiding
Back-crossing transgenes
Marker assisted selection
Polygenes
Genetic mapping of quantitative trait loci
Marker assisted selection
Whole genome genotyping
Genetic relationship analysis and phylogenetic studies
Fingerprinting progeny in recurrent selection programs
Genomic selection
Hybrid prediction
Christian Jung, Plant Breeding Institute, University of Kiel