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Molecular markers: applications in forest breeding

M. Javed Iqbal, PhD

Slide 1 MJI2 I would like to tank------- for inviting me to give a presentation. The presentation will be general in nature with some emphasis on applications in tree improvement.

MJ Iqbal, 3/21/2006


Molecular markers:

· Generally refers to the assays that allow the detection of sequence differences between two or more individuals.



Slide 2 MJI3 Molecular markers is a group of assays that detects variations in sequences between two or more individuals. In case of isoenzymes, the variation is in the amino acid sequence that lead to different electrophoretic modility of the two alleles. In case of DNA based markers, the variation is in the nucleotide sequence of coding or non-coding regions of the genome.

MJ Iqbal, 3/21/2006


Steps involved in marker/trait association

Defining the target

· · · Is the trait important to breeding program or biological research? Do we need a molecular marker? Cost of bioassay vs marker, dominant vs recessive, quantitative trait, pyramiding resistance etc.

Identifying the germplasm for marker development

· Available germplasm with or without trait

Population structures

· · Knowledge of genetics (simply inherited or multigenic, heritability) Decide the best population type and size for the study

Phenotype evaluation Genotyping

· Identify polymorphic markers, screen population, develop maps or do BSA

Identify marker trait association

· Full maps/BSA/Association mapping

Developing markers for application-Marker validation

· · · Test marker/trait association in alternative populations Reliability of markers in predicting the phenotype Develop a palette of suitable markers and provide protocols and polymorphism data to breeding programs

Slide 3 MJI4 Here in this slide are the major steps involved in the developing an association of a marker with the trait of interest. The first step is to define the target, its importance and nature of the trait. Second step is to identify the germplasm for marker development Third step is to explore the population structure and the genetics of the trait of interest in that population After phenotype evaluation, the genotype is explored and markers are identified that are linked to the trait of interest. After establishing the marker trait association, suitable markers and protocols are provided to the breeders for incorporation in the breeding or marker assisted selection program

MJ Iqbal, 3/21/2006


Isoenzyme or protein-based

Isoenzymes are "electrophoretically separable variants of one enzyme system" (Bergmann et al. 1989)

· Bartels (1971) and Bergmann (1971) developed enzyme electrophoresis for Norway spruce

· They are products of structural genes whose roles in metabolism are known in most cases

Slide 4 MJI6 Isoenzymes or protein based markers are electrophoretically seperable variants of one or more enzyme systems. They were first developed for Norway spruce in 1971. They are the products of structural or metabolism related genes with known functions in most cases.

MJ Iqbal, 3/21/2006


Isozyme examples

(~60 references in PubMed search for tree isozymes)


Genetic variation study of Brazilian rosewood (Dalbergia nigra), an endangered tree species {Ribeiro et al 2005} Gene flow in European beech (Fagus sylvatica L.) {Wang 2004} Allozyme polymorphism and different seed productivity in pine trees (Pinus sylvestris L.) {Korshikov and Kalafat 2004} Allozyme analysis revealed strong relationships between height growth (a fitness trait) and the presence of different alleles in red spruce {Mosseler et al. 2003} Mating patterns in a Eucalyptus regnans seed orchard {Burczyk 2002}

· ·



Slide 5 MJI7 Isoenzyme are mostly used for genetic variation studies, gene flow analysis, seed productivity analysis, identify mating patterns and in some cases to determine if there is any correlation between the presence of different alleles and the height of the trees.

MJ Iqbal, 3/21/2006


DNA markers

SSR: Simple sequence repeat RAPD: Random amplified polymorphic DNA AFLP: Amplified fragment length Polymorphism RFLP: Restriction fragment length polymorphism SCAR: Sequence characterized amplified region STS: Sequence tagged site EST: Express sequence tags CAPS: Cleaved amplified polymorphic sequences SNP: Single nucleotide polymorphisms ·Purpose and objective are the same. ·Target application, throughput level and the availability of polymorphism determines the choice of marker for any specific R & D project.

Slide 6 MJI8 Compared to isozyme markers, there are a lrge variety of DNA markers available with a number of different applications. The choice of marker depends upon the objective of the study and the availability of resources, both financial and genetic resources such as DNA sequences information etc.

MJ Iqbal, 3/21/2006


A high throughput DNA isolation procedure used in our lab

DNA Isolation

Slide 7 MJI9 This slide shows some general steps involved in DNA isolation in highthrough put settings.

MJ Iqbal, 3/21/2006


Solutions are dispensed by this robot


P1 P2


P2 P1

Agarose gel electrophoresis or capillary electrophoresis with a DNA sequencer

Slide 8 MJI10 In automated highthroughput labs, robots used to decrease the labor costs fpr sample preparation and avoiding any technical variations introduced by manual handeling.

MJ Iqbal, 3/21/2006


RAPD Markers

A case study of DNA fingerprint of 6 F2 plants of Rhododendron "Adeles's Yellow" hybrid with two primer sets

Slide 9 MJI11 RAPD markers are the least expensive and can be used to start DNA fingerprinting of any genome or plant species. However, it produces dominant markers with some limitations.

MJ Iqbal, 3/21/2006


AFLP markers for genome analysis

Different varieties of the same species Different species

Slide 10 MJI12 For more highthroughput studies, AFLP markers can be used to scan the genome thoroughly to identify polymorphisms associated with the traits of interest. It generates large amount of data in short times, but hard to use in a marker assisted breeding program.

MJ Iqbal, 3/21/2006


Informed decisions based on genetic relatedness for breeding new varieties


MM has nice smell KJ has beautiful flower Cross these two instead to others that are at a greater distance from each other

Slide 11 MJI13 However, AFLP and RAPD markers can be used to identify genetic distances between different individuals, varieties and species. This information lead us to make informed decisions for the selection of suitable parents for developing germplasm with combination of traits.

MJ Iqbal, 3/21/2006



Application in fingerprinting

· · · DNA fingerprint is a "unique pattern of DNA fragments" DNA fingerprints must be reproducible High throughput, Can be automate

Applications: Protection of proprietary rights Genetic architecture of the species for making informed decisions in breeding Population studies of specific clonal/or natural stands

Slide 12 MJI14 RAPD markers are also very suitable for DNA fingerprinting studies that can be used for the protection of intelectual property rights.

MJ Iqbal, 3/21/2006


Germplasm purity

· · · Testing of nursery stock The fingerprint has to be consistent between clonally propagated plants of the nursery stock The method should detect any variability or contamination in the germplasm before it is too late

A DNA fingerprint of 9 randomly selected plants of Rhododendron arborescens

A DNA fingerprint of 10 randomly selected plants of Rhododendron yedoense var. poukhanense

Slide 13 MJI15 They are also very useful in determining the purity of the nursery stocks. In tree the generation time is very long, and if a not desirable stock is propagated, it can lead to serious financial losses. The genetic purity can be studied at early stages and, if there is contamination, the stocks can be eliminated.

MJ Iqbal, 3/21/2006


Develop linkage maps of molecular markers

Parents P1 P2 1 2 A B H A 3 4 B H 5 6 H A Mapping Population 7 8 A A 9 10 11 12 13 14 15 16 17 18 19 20 21 22 B A B A A A A A A B B A H B


Microsatellite markers or simple sequence repeat (SSR)

Slide 14 MJI16 Microsatellite markers, on the other hand are best for marker assisted breeding programs. They can be linked to the traits of interest and can distinguish between homozygous and heterozygous states of the alleles.

MJ Iqbal, 3/21/2006

SSR can be placed on a linkage map and be associated with the quantitatively inherited traits.


An overview of loblolly pine linkage map @

Slide 15 MJI17 The linkage maps shows genetic distance between two adjoining markers and when analyzed with the morphological or physiological traits, can identify marker-trait association. This information can be used in pyramiding loci for increased expression of the multi-loci effected traits.

MJ Iqbal, 3/21/2006


Markers can be linked to the traits of interest and used for MAS

C2 G

SATT214 Rfs3

11.8 cM


SATT309 Rfs2 & Rhg1



19.2 cM SDS SATT080 7.0 cM LOD=27


13.6 cM

SATT570 Rfs1

22.0 cM


17.3 cM SATT387 23 cM

OEO21000 Rfs

SATT371 Rfs4

SATT354 Rfs5


DNA markers linked to resistance to sudden death syndrome and cyst nematode diseases of soybean and are commonly used in MAS

Slide 16 MJI18 This slide shows an example of a microsatellite marker SATT309 associated with the resistaance to soybean cyst nematode and is routinely used for marker assisted selection of soybean varieties for resistance to SCN race 3.

MJ Iqbal, 3/21/2006



Populus is the model for tree genomics Whole genome has been sequenced A good genetic map is also available

Slide 17 MJI19 With our interest in trees, populus is the model tree that has been extensively studied. It's whole genome has been sequenced and a genetic map is alos available

MJ Iqbal, 3/21/2006


Among the other most studied tree are:

Loblolly Pines, Monterey Pine, Scots Pine, Slash Pine, Western White Pine, Douglas-Fir, Norway Spruce etc. A forest tree genome database

Slide 18 MJI20 After Populus, eucalyptus is being studied in Australia and several pine species are also being researched in different labs.

MJ Iqbal, 3/21/2006


What about hardwoods?

· Sweet gum (Liquidambar styraciflua)

· Only 38 sequences in the GenBank {CoreNucleotide (38), EST (0), GSS (0)}

· Yellow Poplar (Liriodendron tulipifera)

· Has ~9644 sequences in the GenBank {CoreNucleotide (111), EST (9533), GSS (0)}

· Red maple (Acer rubrum)

· Has only 120 sequences in the GenBank

· Compare to

· · · Populus trichocarpa: 163,977 sequences {CoreNucleotide (439), EST (163241), GSS (297)} Populus euphratica: 13,935 sequences {CoreNucleotide (29), EST (13906), GSS (0)} Arabidopsis thaliana: 1,382,091 sequences {CoreNucleotide (161570), EST (780804), GSS (439717)}

Slide 19 MJI21 If we compare the 3 hardwood species, the resources available are very limited compared to populus and Arabidopsis.

MJ Iqbal, 3/21/2006



· Study of forest population genetics · Use markers for trait based selection/MAS

· · · Enrichment of complex F1s Early generation selection Selection for qualitative and quantitative traits of economic importance

· Whole genome selection

· · · Choice of donor parent in backcrossing Recovery of recurrent parent genotype in backcrossing Linkage block analysis and selection

Slide 20 MJI22 So, in summary, molecular markers can be applied for population genetic studies, germplasm evaluation, selection of quqntitative and qualitative traits in breeding programs.

MJ Iqbal, 3/21/2006


· Molecular markers significantly reduces the time for breeding new and improved germplasm in plants · It guarantees the presence of incorporated traits confirmed by linked markers · Avoids surprises which can be very costly · TIME IS NOW TO START APPLYING THIS TECHNOLOGY IN TREE IMPROVEMENT


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