Marker assisted backcross breeding
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This document discusses marker-assisted backcrossing (MAB) for introgressing traits from a donor parent into a recipient line. MAB uses DNA markers linked to target genes/QTLs to aid in selection. Markers can be used for foreground selection of target genes, background selection to recover the recipient genome, and recombinant selection to minimize linkage drag. A case study is described where MAB was used over multiple generations to introgress 5 drought resistance QTLs from a donor rice variety into a recipient variety. Through foreground, background, and recombinant selection using DNA markers, lines were developed with the target QTLs and most of the recipient genetic background.
Marker assisted backcross breeding
- 2. FLOW OF PRESENTATION • Introduction • Utilization of markers in backcrossing I. Foreground selection II. Background selection III. Recombinant selection •Case study •conclusion 9/14/2016 2
- 3. Introduction 9/14/2016 3 • 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.
- 4. 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. 9/14/2016 4
- 5. 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. 9/14/2016 5
- 6. Types of backcrossing when more than one gene is to be transferred from different sources • Stepwise transfer • Simultaneous transfer • Stepwise but parallel transfer 9/14/2016 6
- 7. 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 9/14/2016 7
- 8. • Selfing after alternative backcross generation requires more number of generations to select genotype with target gene having maximum recurrent parent background. 9/14/2016 8
- 9. 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 9/14/201 6 9
- 10. 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. 9/14/2016 10
- 11. 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). 119/14/2016
- 12. 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) 9/14/2016 12
- 13. 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 9/14/2016 13
- 14. This may be particularly useful for traits that have laborious or time-consuming phenotypic screening procedures . 9/14/2016 14
- 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. 9/14/2016 15
- 16. 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). 9/14/2016 16
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- 18. Donor/F1 BC1 c BC3 BC10 TARGET LOCUS RECURRENT PARENT CHROMOSOME DONOR CHROMOSOME TARGET LOCUS LINKEDDONOR GENES 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 9/14/2016 18
- 19. 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. 9/14/2016 19
- 20. 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’. 9/14/2016 20
- 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. 9/14/2016 21
- 22. 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) 9/14/2016 22
- 23. Some 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 9/14/2016 23
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- 25. Rice cultivation in eastern part of India is purely depends on rain. This necessitates the cultivars to be drought resistant.
- 26. 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 9/14/2016 26
- 27. BACKCROSSING AND SELECTION :DEVELOPMENT OF NILS 9/14/2016 27
- 28. Pyramid crossing and selection 9/14/2016 28
- 29. 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. 9/14/2016 29
- 30. • 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 9/14/2016 30
- 31. 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 - 9/14/2016 31 Negative selection was performed at these QTLs for Azucena alleles because these alleles had negative effect which are linked to targeted QTLs.
- 32. Background selection: selection for recurrent parent allele at other regions 9/14/2016 32 A- BC3F121-01-03 B- BC3F142-01-05 C-PY2F33-26-05-18
- 33. 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. 9/14/2016 33
- 34. 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 less ii. Started with pre-existing RILs iii. Tested more lines at each backcross generation 9/14/201 6 34
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