Practical application of back cross bree

“Practical Application of Back Cross Breeding Programme” : 

“Practical Application of Back Cross Breeding Programme” 5/18/2010 1 Yanal Alkuddsi Ph.D (Agri) Dept. of Genetics and Plant Breeding University of Agricultural Sciences Dharwad, Karnataka, India, 580005

Introduction : 

Introduction A cross between a hybrid and one of its parents Suggested by HARLAN and POPE (1922) Hybrid and progenies in subsequent generations are repeatedly backcrosed to one of the parents of F1 Objective is to improve one or two specific defects of a HYV End result of backcross programme is a well adapted variety with one or two improved characters 5/18/2010 2

Terminology : 

Terminology Recurrent parent (RP) - parent you are transferring trait to Donor or nonrecurrent parent (DP) - source of desirable trait Progeny test - when trait is recessive 5/18/2010 3

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1. A suitable recurrent parent must be available, which lacks one or two characters. 2. A suitable donor parent must be available and should have character to be transferred in an intense form. 3. The character under transfer must have high heritability preferably one or few genes. 4. A sufficient number of backcrosses should be made so that genotype of recurrent parent is recovered in full usually 6-7backcross are sufficient. Requirments of A Backcross Programme 5/18/2010 4

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X Donor variety Resistance Gene Commercial Variety New Variety Backcross Breeding Scheme –Transgene 5/18/2010 5

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Donor species Resistance Gene Transformable Plant Transformed Plant gene insertion 5/18/2010 6

Transfer of A Dominant Gene : 

Transfer of A Dominant Gene First year Non-recurrent parent B (RR) * Recurrent parent A (rr) Second year Rr * rr Third year rr Rr * rr Fourth year rr Rr * rr Fifth year rr Rr * rr Six year rr Rr * rr F1 BC1 BC2 BC3 BC4 5/18/2010 7

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Seven year rr Rr * rr Eighth year rr Rr Ninth year Tenth year BC5 BC6 BC6F2 BC6F3 Eleventh year Twelfth year 5/18/2010 8

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1st year Non recurrent parent B ( rr) * Recurrent parent A(RR) 2nd Rr * RR (Recurrent parent A) 3rd BC1 RR,Rr Selfed 4th BC1F2 RR Rr rr * RR Test 5th BC2 Rr * RR 6th BC3 RR , Rr Transfer of A Recessive Gene F1 5/18/2010 9 Selfed

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7tH BC3F2 RR Rr rr * RR 8th BC4 Rr * RR 9th BC5 RR , Rr 10th BC5F2 RR Rr rr 11th BC5F3 12th Yield trial Yield trial 5/18/2010 10

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GENETIC CONSEQUENSES OF REPEATED BACK CROSSING Reduction in heterozygosity: Repeated back crossing leads to an increase in homozygosity at the same rate as does selfing. 5/18/2010 11 Increased similarity with the Recurrent Parent In the back cross progeny the frequency of homozygotes for the allele from the recurrent parent is ½, while in F2 it is only ¼. Selection for the Gene under Transfer

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The gene under transfer must be maintained by an efficient selection in the backcross generations. Otherwise the gene will also be replaced by its allele from the recurrent parent. 5/18/2010 12 Selection for the Recurrent Parent Type Often a selection for the recurrent parent type is done during the backcross generations. Extended opportunity for Breaking Undesirable Linkage The transferred gene and the gene tightly linked to it would thus remain in heterozygous state in the backcross generations. Therefore, there would be opportunity in each backcross generation for crossing over to occur between the gene being transferred and the genes tightly linked with it.

Effect of repeated backcrossing on frequency of homo and heterozygotes : 

Effect of repeated backcrossing on frequency of homo and heterozygotes 5/18/2010 13

Average proportion of genes from the recurrent parent in the different generations : 

Average proportion of genes from the recurrent parent in the different generations 5/18/2010 14

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Intervarietall Transfer of Simply Inherited Characters Intervarietal Transfer of Quantitative Characters Interspecific Transfer of Simply Inherited Characters Transfer of Cytoplasm Transgressive Segregation Production of Near- Isogenic Lines Germplasm Conversion Applications of Backcross Method 5/18/2010 15

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For certain traits (e.g. male sterility) it is important that a certain cytoplasm be retained In wheat, to convert a line to a male sterile version the first cross should be made as follows: Triticum timopheevi (male sterile) x Triticum aestivum (male fertile)wheat line. From that point on, the recurrent parent should always be used as the male. × 5/18/2010 16

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Parents: Tift 23A × African and Indian lines F1 × Tift 23 A (RP) BC1 50% × BC2 75% BC3 87.5% BC8 99.609% BC9 99.805% RP RP RP RP RP Tift 23A Resistant to DM 5/18/2010 17 × × × Ex 2. In Pear millet To transfer CMS and of restorer genes into new inbreds. Tift 23A from U. S.A.,highly susceptible to Downy Mildew. So DM resistant male sterile have been produced by BC Tift 23A to some African and Indian lines

Ex 2. In Sorghum development of Combine Khafir 60 by six successive backcrosses of male sterile plants in a Milo x combine Kafir lines. : 

Ex 2. In Sorghum development of Combine Khafir 60 by six successive backcrosses of male sterile plants in a Milo x combine Kafir lines. msc msc Msc Msc msc Msc CK 60 Fertile Milo Fertile F1 Hybrid Fertile msc Msc msc Msc msc msc Msc Msc Fertile Fertile Fertile Sterile msc msc msc msc msc msc msc msc msc msc msc msc F S S S S S F F F S S S 6th Backcross 2nd Backcross 1st Backcross CK 60 Fertile Sterile segregant × × × 3rd, 4th and 5th Backcross CK 60 Fertile Sterile CK60 A 5/18/2010 18

Example 3 Transfer of resistance traits through backcrossing in Sorghum : 

Example 3 Transfer of resistance traits through backcrossing in Sorghum Resistance source × high –yielding line ( R – or B line) F1 × HYL C1F1 BCF1F2, F3/F4 BC1F3 × HYL BC2F1 BC2F2, F3/F4 BC3F1 BC2F3 × HYL BC3F2, F3/F4 BC2F3 × HYL BC4F1 BC4F2, F3/F4 BC4F3 BC5F1 BC5F2 BC5F3 BC5F4 BC5F6 BC5F7 BC5F6 As in BC2F2 Selection under insect infestation for resistance Selection under insect infestation for resistance As in F2 HYL × As in F2 Select among and within families for resistance under infestation Select for resistance As in previous generation Select for resistance As in previous generation Replicated trials Replicated trials 5/18/2010 19

Example 4. Using Backcrossing for the production of monosomics and other aneuploids : 

Example 4. Using Backcrossing for the production of monosomics and other aneuploids 1. In bread Wheat Chinese Spring Wheat × (Triticum aestivum) n=21 Rye (Secale cereal) n=7 Haploid (2n=21) Restitution nucleus (with n=20) × Triticum aestivum n=21 2n=41 (20II +1I ) monosomics selfed 2n= 20II (nullisomics) + 2n= 20II +1I II (Trisomics) 2n= 20II +1I v (tetrasomics) 5/18/2010 20

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Example 5. Using Backcrossing for the development of isogenic lines . In Wheat Awned Wheat Parent (aa) Awnless Wheat Parent (AA) × Aa Chromosome reassortment F2 aa Aa AA F1 progeny are tip awned due to partial dominance F2 generation segregates in a 1:2:1 ratio ( awned, discard) Tip awned ( awned, discard) Backcross to both parents Aa × aa Aa × Aa A A A a A a a a Aa Aa aa aa Aa Aa AA AA 5/18/2010 21

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Probability of transferring genes To increase the probability to 99% and the number of Rr plants to 3, you must grow 14 progeny. If germination is only 80%, you must grow 14/0.8 = 18 progeny. Ave. recovery of RP = 1- (1/2)n+1, where n is the number of backcrosses to RP. The percentage recovery of RP varies among the backcross progeny For example, in the BC3, if the DP and RP differ by 10 loci, 26% of the plants will be homozygous for the 10 alleles of the RP; remainder will vary. 5/18/2010 22

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In Rice Practical Application in field crops Marker-assisted backcrossing for submergence tolerance IRRI MAS CASE STUDY 1. David Mackill et al., (2005) 5/18/2010 23

Abiotic stresses are major constraints to rice production in SE Asia : 

Abiotic stresses are major constraints to rice production in SE Asia Rice is often grown in unfavourable environments in Asia Major abiotic constraints include: Drought Submergence Salinity Phosphorus deficiency High priority at IRRI Sources of tolerance for all traits in germplasm and major QTLs and tightly-linked DNA markers have been identified for several traits Objectives: 5/18/2010 24

‘Mega varieties’ : 

‘Mega varieties’ Many popular and widely-grown rice varieties - “Mega varieties” Extremely popular with farmers Traditional varieties with levels of abiotic stress tolerance exist however, farmers are reluctant to use other varieties poor agronomic and quality characteristics 1-10 Million hectares Material and Methods: 5/18/2010 25

Backcrossing strategy : 

Backcrossing strategy Adopt backcrossing strategy for incorporating genes/QTLs into ‘mega varieties’ Utilize DNA markers for backcrossing for greater efficiency – marker assisted backcrossing (MAB) 5/18/2010 26

MAB: 1ST LEVEL OF SELECTION – FOREGROUND SELECTION : 

MAB: 1ST LEVEL OF SELECTION – FOREGROUND SELECTION Selection for target gene or QTL Useful for traits that are difficult to evaluate Also useful for recessive genes 5/18/2010 27

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Donor/F1 BC1 c BC3 BC10 TARGET LOCUS 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 5/18/2010 28

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Conventional backcrossing Marker-assisted backcrossing BC1 c BC2 c BC3 BC10 BC20 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. 5/18/2010 29

MAB: 2ND LEVEL OF SELECTION - RECOMBINANT SELECTION : 

MAB: 2ND LEVEL OF SELECTION - RECOMBINANT SELECTION Use flanking markers to select recombinants between the target locus and flanking marker Linkage drag is minimized Require large population sizes depends on distance of flanking markers from target locus) Important when donor is a traditional variety 5/18/2010 30

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OR Step 1 – select target locus Step 2 – select recombinant on either side of target locus BC1 * Marker locus is fixed for recurrent parent (i.e. homozygous) so does not need to be selected for in BC2 5/18/2010 31

MAB: 3RD LEVEL OF SELECTION - BACKGROUND SELECTION : 

MAB: 3RD LEVEL OF SELECTION - BACKGROUND SELECTION Use unlinked markers to select against donor Accelerates the recovery of the recurrent parent genome Savings of 2, 3 or even 4 backcross generations may be possible 5/18/2010 32

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P1 x F1 P1 x P2 CONVENTIONAL BACKCROSSING BC1 VISUAL SELECTION OF BC1 PLANTS THAT MOST CLOSELY RESEMBLE RECURRENT PARENT BC2 5/18/2010 33

Breeding for submergence tolerance : 

Breeding for submergence tolerance Large areas of rainfed lowland rice have short-term submergence (eastern India to SE Asia); > 10 m ha Even favorable areas have short-term flooding problems in some years Distinguished from other types of flooding tolerance elongation ability anaerobic germination tolerance 5/18/2010 34

Screening for submergence tolerance : 

Screening for submergence tolerance 5/18/2010 35

A major QTL on chrom. 9 for submergence tolerance – Sub1 QTL : 

A major QTL on chrom. 9 for submergence tolerance – Sub1 QTL Segregation in an F3 population Xu and Mackill (1996) Mol Breed 2: 219 5/18/2010 36

Make the backcrosses : 

Make the backcrosses Swarna Popular variety X IR49830 Sub1 donor F1 X Swarna BC1F1 5/18/2010 37

Seeding BC1F1s : 

Pre-germinate the F1 seeds and seed them in the seedboxes Seeding BC1F1s 5/18/2010 38

Collect the leaf samples - 10 days after transplanting for marker analysis : 

Collect the leaf samples - 10 days after transplanting for marker analysis 5/18/2010 39

Genotyping to select the BC1F1 plants with a desired character for crosses : 

Genotyping to select the BC1F1 plants with a desired character for crosses 5/18/2010 40

Selection for Swarna+Sub1 : 

Selection for Swarna+Sub1 Swarna/ IR49830 F1 Swarna BC1F1 697 plants Plant #242 Swarna X X 376 had Sub1 21 recombinant Select plant with fewest donor alleles 158 had Sub1 5 recombinant Swarna X Plant #227 BC3F1 18 plants 1 plant Sub1 with 2 donor segments BC2F1 320 plants Plants #246 and #81 Plant 237 BC2F2 BC2F2 937 plants 5/18/2010 41

Swarna with Sub1 : 

Swarna with Sub1 5/18/2010 42

Graphical genotype of Swarna-Sub1 : 

Graphical genotype of Swarna-Sub1 BC3F2 line Approximately 2.9 MB of donor DNA 5/18/2010 43

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Achievements of MAS 5/18/2010 45

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Some considerations for MAB : 

Some considerations for MAB IRRI’s goal: several “enhanced Mega varieties” 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 5/18/2010 47

Increasing kernel Density of two inbred lines of Maize. : 

Increasing kernel Density of two inbred lines of Maize. 5/18/2010 48 Thompson et. al., ( 2006) Objective: To evaluate sp. gravity and sinkers for their effectiveness in increasing the kernel density of IBL B 73G and A 632 by backcrossing.

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5/18/2010 49 Materials and Method. Inbreds - B73G and A632. Donor parents- TG , SYN and 9A High kernel density. a) B73G enhancement: B73G X TG B73G X SYN F1 X RP F1X RP up to BC4 BC3

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5/18/2010 50 b) A632. enhancement A632 X TG A632 X SYN A632 X 9A BC3 BC3 BC3 Backcross of B73G were grown in 2 states ie S. Dakota and N. Carolina.

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5/18/2010 51 Result: Table 1. Means for Sp.gravity and sinkers of backcross inbreds and inbred ** significant at 1% level

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5/18/2010 52 Table 2. Means for Sp.gravity and sinkers of backcross inbreds and A632 ** significant at 1% level

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5/18/2010 53 Conclusion: BC inbreds had greater kernel Density than RP B73G Sp. gravity is a measure of the collective density of all kernels in a sample. Sinkers technique identifies the kernels with highest density , which then can be saved for the next generation.

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Genetics of resistance to Fusarium udum in Pigeonpea [Cajanus cajan (L.) Millsp] Okiror M.A (2002) 5/18/2010 54

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Materials and methods Parents : NPP 725, NPP 726( resistant) : NPP 718 (Susceptible) Populations developed: F1, F2, F3 and Backcross Screening done in sick plots Number of plants wilted were recorded at pod maturity Data analyzed using chi square test 5/18/2010 55

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Results 5/18/2010 56

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When backcross done to NPP 718, all susceptible 5/18/2010 57

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Segregation ratio of F2 indicate trigenic segregation Backcross populations also segregated comparable to F2 5/18/2010 58

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Conclusion Segregation in backcross was attributed to effect of modifiers and or complementary genes Results demonstrated that several different genes are involved. Segregation in NPP 725 x NPp 726 was attributed to modifying genes and either of the complementary genes. Multigenic resistance : durable, beneficial and broad Points to remember Variability reported in Fusarium udum (chavan et al 1995) But performance of all genotypes resistant to wilt is similar to all pathogens. (Pandey et al 1996) 5/18/2010 59

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MERITS OF BACKCROSS METHODS1.The genotype of new variety is identical to the recurrent parent 2. Extensive testing for yield and adaptability is not needed. 3. Off season nurseries and green houses can be utilised. 4. Much smaller population is needed than pedigree. 5. Defects,can be solved without affecting the performance of recurrent variety. 6. only method for interspecific gene transfer and transfer of cytoplasm. 5/18/2010 60

DEMERITS OF BACKCROSS METHOD : 

DEMERITS OF BACKCROSS METHOD The new variety cannot be superior to recurrent parent. Undesirable linked genes also transferred to new variety. More number of backcrosses to be done time consuming process. By the time backcross programme improve it RP may have replaced by other superior variety. 5/18/2010 61

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ACHIEVEMENTS IN BACKCROSSING

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5/18/2010 63 CONCLUSION