MARKER-ASSISTED BACKCROSSING

WELCOME

Anilkumar, CPALB5062

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FLOW OF PRESENTATION

• Introduction • Utilization of markers in backcrossing

I. Foreground selectionII. Background selectionIII. Recombinant selection

•Case study•conclusion

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Introduction

• Plant breeding is an art of managing the variability.

• Existence of variability is the pre-requisite for any kind of breeding programme.

• Creation of variability can be done through mutation, hybridization, polyploidy and genetic engineering.

Backcrossing

Backcross breeding is a well-known procedure for the introgression of a target gene from a donor line into the genomic background of a recipient line.

The objective is to reduce the donor genome content (DGC) of the progenies by repeated back-crosses.

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Backcrossing is used

• To transfer a major gene

• In disease/pest resistance breeding

• To transfer alien cytoplasm or to transfer

cytoplasmic male sterility

• To transfer a transgene from already

developed transgenic line.

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Types of backcrossing when more than one gene is to be transferred from different

sources

• Stepwise transfer

• Simultaneous transfer

• Stepwise but parallel transfer

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Need to grow large number of plants for selection in each generation of backcrossing.

Introgression of quantitative traits is nearly not possible.

Recovery of recipient genome is less efficient. Poses difficulties in negative selection of undesirable

genes or avoiding the linkage drag problems.

Problems of conventional backcrossing

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• Selfing after alternative backcross generation requires more number of generations to select genotype with target gene having maximum recurrent parent background.

Landmarks in genome Not affected by

environment Not stage specific Not tissue specific More precise Acts as proxies and

helps in indirect selection

Molecular Markers

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MARKER ASSISTED SELECTION Marker assisted selection refers to the use of DNA

markers that are tightly-linked to target loci as a surrogate to phenotypes.

Assumption: DNA markers can reliably predict phenotype.

Marker-assisted backcross is of great practical interest in applied breeding schemes either to manipulate ‘classical’ genes between elite lines or from genetic resources, or to manipulate transgenic constructions.

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It is an approach that has been developed to avoid problems connected with conventional plant breeding by changing the selection criteria from selection of phenotypes towards selection of genes that control traits of interest, either directly or indirectly.

MAB is the process of using the results of DNA tests to assist in the selection of individuals to become the parents in the next generation of a genetic improvement programme.

The success of MAB depends upon:

Principle of MAB:

•The distance between the closest markers and the target gene,

• Number of target genes to be transferred,

• Genetic base of the trait,

• Number of individuals that can be analyzed and the genetic background in which the target gene has to be transferred,

•The type of molecular marker(s) used, and available technical facilities (Weeden et al., 1992; Francia et al., 2005). 11

In Backcross Breeding, Markers Can Be Used To:

I. Control the target gene (foreground selection)

II. Control the genetic background (background

selection).

III. Control the linkage drag (recombinant selection)

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Foreground selection: selection using single marker

Select for marker allele of donor genotype/target gene

Close linkage between marker locus and target locus is essential

The probability that the Q/Q genotype can be obtained through

selection of marker genotype M/M, that is, the probability for

selecting the correct individuals, is

P=(1-r)2

Where, r- recombination frequency of marker and gene

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This may be particularly useful for traits that have laborious or time-consuming phenotypic screening

procedures .

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15 Foreground selection: selection using multiple marker for

multiple targets This is helpful in resistance breeding for

disease and pest resistance. Marker-trait association can be used to

simultaneously select multiple resistances from different disease races and/or insect biotypes and pyramid them into a single line through MAS.

Background selection o The second level of MAB involves selecting BC progeny with

the greatest proportion of recurrent parent (RP) genome, using markers that are unlinked to the target locus refer to this as ‘background selection’.

o Background markers are markers that are unlinked to the target gene/QTL on all other chromosomes,

o In other words, markers that can be used to select against the donor genome.

o The use of background selection during MAB to accelerate the recovery of recurrent parent genome with additional (or a few) genes has been referred to as ‘complete line conversion’ (Ribaut et al. 2002). 05/01/20

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Donor/F1 BC1

c

BC3 BC10

TARGET LOCUS

RECURRENT PARENT CHROMOSOME

DONOR CHROMOSOME

TARGET LOCUS

LIN

KED

DON

OR

GEN

ES

Concept of ‘linkage drag’ • Large amounts of donor chromosome remain even after many backcrosses• Undesirable due to other donor genes that negatively affect agronomic performance

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Conventional backcrossing

Marker-assisted backcrossing

F1 BC1

c

BC2

c

BC3 BC10 BC20

F1

c

BC1 BC2

Markers can be used to greatly minimize the amount of donor chromosome….but how?

TARGET GENE

TARGET GENE

Ribaut, J.-M. & Hoisington, D. 1998 Marker-assisted selection: new tools and strategies. Trends Plant Sci. 3, 236-239.05/01/2023 19

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RECOMBINANT SELECTION:The third level involves selecting BC progeny with the target gene and recombination events between the target locus and linked flanking markers—refer to this as ‘recombinant selection’.

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21 Advantages of MAB:•When phenotypic screening is expensive, difficult or impossible.

• When the trait is of low heritability (incorporating genes that are highly affected by environment).

• When the selected trait is expressed late in plant development, like fruit and flower features or adult characters in species with a juvenile period.

• For incorporating genes for resistance to diseases or pests that cannot be easily screened for due to special requirement for the gene to be expressed.

• When the expression of the target gene is recessive.

• To accumulate multiple genes for one or more traits within the same cultivar, a process called gene pyramiding. 05/01/20

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Reasons for unexpected results in MAB:

•The putative QTL may be a false positive.

•QTL and environmental interactions (Ribaut et al.,)

•Epistasis between QTLs and QTL and genetic background.

•QTL contain several genes and recombination between those genes would modify the effect of the introgressed segment (Eshed and zamir, 1995;Monna et al.,2002)

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23Some considerations that encourages MAB

Main considerations: Cost Labour Resources Efficiency Timeframe

Strategies for optimization of MAB process important Number of BC generations Reducing marker data points (MDP) Strategies for 2 or more genes/QTLs

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Case study

Rice cultivation in eastern part of India is purely depends on rain. This necessitates the cultivars to be drought resistant.

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Selection of parents and target QTLs

• Recipient parent: Kalinga III (Indica rice)

• Donor parent: Azucena (Japonica rice)

• Total QTLs: 5

• Root QTLs: QTL2, QTL7, QTL9, QTL11

• Aroma QTLs: QTL8

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BACKCROSSING AND SELECTION :DEVELOPMENT OF NILS

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Pyramid crossing and selection

Foreground selection for target QTLs

RFLPs were used for first 3 backcrosses.

Done using RFLPs that had mapped in Bala × Azucena population and flanked, or were within, the regions containing the target QTLs.

In addition, the RFLP marker C570 was used. C570 was polymorphic between Azucena and Kalinga III but not between Bala and Azucena and mapped near to QTL9. 05/01/2023 29

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• Selection after 3rd backcross was done using flexible and cheaper PCR-based SSRs.

QTLs Trait of QTL RFLPs SSRs

QTL 2 Root thickness, root penetration G39, G57, C601 RM6, RM221, RM318

QTL 7 Deep root per shoot ratio, root length, deep root weight per tiller

RG351, RG650, C507 RM234, RM351

QTL 8 Aroma G1073 RM223

QTL 9 Deep root thickness G385, C570 RM 242

QTL 11 Root length, root penetration C189, G1465 RM229, RM206

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Recombinant selection: selection for recurrent parent allele near QTLs

QTLs Trait of QTL RFLPs SSRs

QTL 2 Root thickness RG171, G45 -

QTL 5 Root length, root thickness and penetration RG13, C43 -

QTL 7 Flowering time - RM248

QTL 8 Osmotic adjustment G56 -

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Negative selection was performed at these QTLs for Azucena alleles because these alleles had negative effect which are linked to targeted QTLs.

Background selection: selection for recurrent parent allele at other regions

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A- BC3F121-01-03B- BC3F142-01-05C-PY2F33-26-05-18

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Greenhouse and field experiments

Extensive greenhouse and field experiments were carried out at UAS(B)

I. To identify chromosomal regions from Azucena that delayed anthesis so that they could be selected against.

II. To test the presence of pleiotropic or linkage drag effects in developed NILs.

A segment on chromosome 1 that was introgressed unintentionally that had a significant effect on grain width.

Conclusion Five target regions is probably the limit of

efficient MABC breeding (Hospital and Chacosset 1997; Servin et al. 2004), but they successfully selected an ideotype with all five regions from Azucena introgressed into the predominantly Kalinga III genetic background, with almost complete line conversion.

The work would have less tedious if they i. Started with SSRs as number of assays would be

lessii. Started with pre-existing RILsiii. Tested more lines at each backcross generation

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