1. Gene Pyramiding and Its Role In Crop Improvement
DOCTORAL SEMINAR – I
(AGP-788)
Anil Kumar Bairwa id no

2. Contents Introduction Objectives of Gene Pyramiding
Types of Gene Pyramiding
Role in crop improvement
Conclusion

3. Introduction
Gene pyramiding is defined as a method aimed at assembling multiple desirable genes from multiple parents into a single genotype.
Watson & Singh (1953) first introduced the concept of gene pyramiding.
The end product of a gene pyramiding program is a genotype with all of the target genes.
Traditionally, gene pyramiding is mainly used to improve qualitative traits such as disease and insect resistance.

4. Objectives of Gene Pyramiding
1) Enhancing trait performance by combining two or more complementary genes 2) Remedying deficits by introgression of genes from other sources 3) Increasing the durability of disease resistance 4) Broadening the genetic base of released cultivars

5. Types of Gene Pyramiding
1. Conventional techniques
Serial gene pyramiding: Genes are deployed in same plant one after other
Backcross breeding
Pedigree breeding
Recurrent selection

6. Backcross breeding

7. Pedigree breeding

8. Recurrent selection

9. Disadvantages of Conventional Methods
1. Phenotypic evaluation
2. Linkage drag
3. Problems associated with recessive gene
Therefore, any improvement in the knowledge of the trait genetics (inheritance, genetic relationship, etc.) and techniques for inferring genotype-phenotype relationship will be useful.

10. 2. Molecular Techniques
Simultaneous gene pyramiding: Genes are deployed at a time in a single plant.
Marker assisted selection
Transgenic methods

11. 1. Marker Assisted Selection
1) Use of DNA markers that are tightly-linked to target loci as a substitute for phenotypic screening
2) A marker is a “genetic tag”
3) Use of molecular markers for indirect selection of different traits
4) Speeding up the process of conventional breeding
5) Facilitating the improvement of traits

12. Marker Assisted Selection
Marker-assisted selection(MAS) is a method of rapidly incorporating valuable traits into new cultivars.
Molecular markers, that have been shown to be linked to traits of interest are particularly useful for incorporating of genes.
One of the first wheat cultivars to be developed using MAS was the soft winter wheat cultivar “Madsen”, released in 1986 by the USDA-Agricultural Research Service (ARS) and Washington State University.
“Madsen” was developed using the isozyme marker from the endopeptidase protein, to incorporate a gene for resistance to eyespot (Allan et al. 1989).

13. MABC strategy for Gene Pyramiding
The concept of gene pyramiding by MABC was proposed by Nelson (1978) to develop crop varieties with durable resistance to diseases by bringing together few to several different oligogenes for disease resistance.
Introgression of two or more genes from a single DP is relatively simple, the DP is crossed with the RP and the F1 and the subsequent progeny are repeatedly backcrossed to the RP.
But when the genes to be pyramided are present in different DPs, they can be introgressed into an RP in one of the following two ways.
1) Separate Backcross Program
2) Single Backcross Program

14. 1) Separate Backcross Program

15. 2) Single Backcross Program

16. Effectiveness of marker-assisted backcrossing for gene pyramiding depends upon
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
Genetic background in which the target gene has to be transferred
Type of molecular marker used
(Weeden et al., 1992; Francia et al., 2005)

17. Minimum Population Size
This probability dictates the population size required to have a high probability of finding at least one plant with the desired combination of alleles.
Muller (1923) and Sedcole (1977) promote use of the following equation to determine the minimum population size required to recover an individual with the desired combination of traits:
N = loge(1-P)/loge(1-f)
where
N = minimum population size
P = desired probability of success (e.g. 99%, 95%, 90%)
f = frequency of the event (i.e., an individual plant having all desired alleles).
The frequency depends on the number of genes the breeder wants combined, whether the genes are genetically linked.

18. II. Transgenic methods
Iterative
Co-transformation
Linked transgenes

19. A. Iterative
A plant harboring one or more transgenes is cross with another plant containing other transgenes.
These techniques have been used, at the research level at least, to combine pre-existing transgenic traits.
There are two types of iterative process:
Crossing
Re -transformation

20. Crossing
Pyramiding two Bacillus thuringiensis (Bt) genes in the same plant on the production of Bt proteins and the control of diamondback moths (DBM) resistant to one or the other protein.
Broccoli lines carrying both cry1Ac and cry1C Bt genes were produced by crosses of cry1Ac and cry1C transgenic plants.

21. Production of cry1Ac + cry1C hybrid broccoli lines
We attempted to pyramid cry1Ac and cry1C genes in same plant by making crosses between cry1Ac and cry1C plants.
cry1C - female parent
cry1Ac - male parent
Stamens of cry1C flowers were removed before the buds opened and stigmas were immediately pollinated with cry1Ac pollen.

22. Selection cry1Ac + cry1C hybrids
To select cry1Ac + cry1C hybrids, seeds from the crosses were germinated on MS medium containing hygromycin (50 mg/l).
DNA was isolated from hygromycin- resistant seedlings, and PCR was carried out to identify those that contained the cry1Ac gene.
Alternatively, a leaf piece assay was performed to select cry1Ac +cry1C hybrid plants.
Half of a young leaf was then placed on MS medium containing hygromycin (20 mg/l) while the other half was placed on MS medium containing kanamycin (50 mg/l).
Plants, whose leaves formed callus in the presence of both antibiotics were subjected to PCR analyses to confirm that they carried both Bt genes.

23. Table. Production of Cry1A and Cry1C proteins in Bt-transgenic broccoli plants evaluated by ELISA
Fig. Detect the cry1Ac and cry1C mRNAs present in cry1Ac+cry1C hybrid plants.

24. Fig. a: Green Comet control; b: cry1Ac+cry1C hybrid (Q23 × H1);
c: cry1Ac plant (Q23); d: cry1C plant (H1)
Result :
Plants producing both cry1Ac and cry1C proteins caused rapid and complete mortality of DBM larvae resistant to cry1A or cry1C and suffered little or no leaf damage.

25. b) Re-transformation A plant harboring a transgene is
transformed with other transgenes.
Forsythia flower color by introducing genes for anthocyanin synthesis (Rosati et al 2003)
Dihydroflavonol 4-reductase from Antirrhinum majus (AmDFR)
Anthocyanidin synthase from Matthiola incana (MiANS)

26. Single transgenic Plant
Wild Plant
AmDFR
Single transgenic Plant
MiANS
Double transgenic plant
Wild type
Double transgenic

27. LIMITATIONS
Transgenes are not linked
Segregation in subsequent generation
Time consuming
Labor intensive
Accumulation of different marker gene

28. B) Co-transformation A plant is transformed with
two or more independent transgenes.
Co-transformation has been proven to be one of the most promising approaches taken to date for the introduction of multiple genes into plants.
This strategy is quick and can be used in a variety of species and with both direct and indirect transformation methods.
a) Direct
b) Indirect

29. Direct Simultaneous introduction of three insecticidal genes in rice
Maqbool et al (2001)
cry1AC
Leaf folder and stem borer
cry2A
Brown plant hopper
Lectin

30. b) Indirect Improving wood quality of Aspen (Li et al 2003)
FHL
CCL
FHL- ferulate 5-hydroxylase
CCL- 4-coumarate-CoA ligase

31. Advantages Limitations Quick both by direct and indirect method
Integrate at the same chromosomal location
Limitations
Silencing due to high copy number and tandem repeat
Variation in expression level of different transgene
The proportion of expressing all transgene decreases with increase in number of transgenes

32. C) Linked Transgenes
Two or more genes with their own promoter and terminator
4 transgenes (3 PHB synthesis+1 marker) linked within one T-DNA introduced into Arabidopsis LB P1 G1 T1 P2 G2 T2 P3 G3 T3 P4 G4 T4 RB

33. introduction
Transgenic Arabidopsis thaliana plants expressing the three enzymes encoding the biosynthetic route to polyhydroxybutyrate (PHB) are described.
Transgenic Arabidopsis plants generated using a triple construct, thus allowing the parallel transfer of all three genes necessary for PHB synthesis in a single transformation event.

34. Plant Material
The plasmid pBI ABC was electroporated into Agrobacterium tumefaciens C58C1 and used to transform Arabidopsis thaliana C24 plants via vacuum infiltration (Bent et al. 1994).
Transformed seeds were selected on MS medium containing1% sucrose and100 micro gram per ml kanamycin.
Confirmation of polymer accumulation:
Transmission electron microscopy of leaf samples of PHB-producing lines revealed that the polymer was accumulating as of electron-translucent granules with diameters of between 0.1 and 0.7 micro m.
These granules were located exclusively in the stroma of the plastids.

35. A) Chloroplast of a mesophyll cell of a mature wild-type leaf exhibiting a starch granule arrow but no PHB granule.
B ) Chloroplast of a mesophyll cell of a mature leaf of transgenic line 6 with agglomerates of electron-translucent granules
Fig. PHB content of transgenic Arabidopsis lines transformed with the construct pBI ABC in comparison to the wild type.

36. Result
Transgenic Arabidopsis thaliana plants accumulated more than 4% of their fresh weight (40% of their dry weight) in the form of PHB in leaf chloroplasts.

37. Limitations
Size of DNA to be introduced
Unique restriction sites to construct assembly
Silencing
Different level of linked gene expression

38. ROLE IN CROP IMPROVEMENT
1. Biotic and abiotic stress resistance
2. Biotic stress improvement
Bacterial
Viral
C. Insect
3. Inbred line development
4. Quality improvement

39. 1. Biotic and abiotic stress resistance
Introduction:
Severe yield loss due to various biotic stresses like bacterial blight, gallmidge and Blast and abiotic stresses like submergence and salinity area serious constraint to there productivity throughout the world.

40. Plant Material and breeding strategy
Recurrent parent
A well-adapted indica rice genotype Improved Lalat having four bacterial bligh tresistance genes ( Xa4, Xa21,xa13, and xa5) developed in CRRI.
Donor parent
Two cultivars, C1O1A51 and WHD-1S with blast resistance genes (Pi2 and Pi9)
Two cultivars, Kavya and Abhaya with Gall midge resistance genes (Gm1 and Gm4)
One cultivar, FR13A with submergence resistance QTL(Sub1)
One cultivar FL478 with salinity resistance QTL (Saltol) genes were chosen as the donor parents

41. Fig. The development of Improved Lalat pyramids through gene stacking

42. Result
Average lesion length ranging from 6.0 to 18 cm in susceptible but 0.1– 4.8 cm in the resistant plants ( BB)
Resistant verity so no disease symptoms in artificial diseased condition and high doses of nitrogen were applied (blast)
The appearance and percentage of galls (silver shoot) was recorded. In susceptible plants showed 90–100% plant damage with the appearance of gall but test seedlings with 0–20% of damage were considered as resistant (gall midge)
Average plant survival percent under 1.6m standing water over the top of the leaves. After 15 days of complete submergence (Submergence)
The spikelet fertility (%) was recorded during the reproductive stage at high salt concentration (Salinity)

43. 2. Biotic stress improvement
A. Bacterial resistance
Introduction :
Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice.
Three BB resistance genes, xa5, xa13 and Xa21, were pyramided into cv. PR106.

44. Planting material and methods
Three isogenic lines, IRBB5 (xa5),IRBB13 (xa13), IRBB21 (Xa21) and a line IRBB62 with all three genes in the background of IR24.
Crosses were made between PR106 and IRBB62, which carries 3 BB resistance genes.
F1 plants were backcrossed with PR106. Starting from the BC1F1 onward, polymerase chain reaction (PCR)-based molecular markers linked to xa5, xa13 and Xa21 were used to select plants with resistance alleles.
A similar strategy was used in the BC2F1 to obtain BC2F2 populations from which lines with pyramided genes were selected.
The BC2F2 plants were selfed and backcrossed again with PR106. The BC2F3 lines having 2 and 3 homozygous genes for resistance to BB were identified on the basis of molecular marker analysis and inoculated with BB races to determine disease reaction.

45. Fig. PCR analysis of the parental lines and BC2F2 plants.
PCR amplification
Three STS markers, RG556, RG136 and pTA248, tightly linked to resistance genes xa5, xa13 and Xa21, respectively.
Fig. PCR analysis of the parental lines and BC2F2 plants.

46. Result :
Large-scale and long-term cultivation of varieties with single genes may enable the pathogen to overcome BB resistance.
However, this can be delayed by pyramiding multiple resistance genes into rice cultivars.
The combination of genes provided a wider spectrum of resistance to the pathogen population prevalent in the region; Xa21 was the most effective, followed by xa5. Resistance gene xa13 was the least effective against Xoo.

47. B. Virus resistance Introduction:
Seven strains of Soybean mosaic virus (SMV) and three independent resistance loci (Rsv1, Rsv3, and Rsv4) have been identified in soybean.
The objective of this research was to pyramid Rsv1, Rsv3, and Rsv4 for SMV resistance by using molecular markers.
J05 carrying Rsv1 & Rsv3 and V carrying Rsv4 were used as the donor parents for gene pyramiding.

48. Plant materials and crossing procedure
Two soybean lines, ‘J05’ and V , were used as donor parents for pyramiding three SMV resistance genes, Rsv1, Rsv3, and Rsv4.
J05 is a Chinese cultivar containing two genes (Rsv1 and Rsv3) and resistant to seven SMV strains G1–G7 (Zheng et al. 2006).
V was derived from PI x Essex and carries Rsv4 for seedling resistance to SMV strains G1–G7.
J05 was crossed with V and the F1 plants were grown in the field and harvested individually.

49. Fig. Procedure of pyramiding Rsv1, Rsv3, and Rsv4 genes for SMV resistance

50. Marker selection and PCR-based assay
A total of 33 molecular markers surrounding three SMV resistance loci
12 for Rsv1,
7 for Rsv3, and
14 for Rsv4 were selected to screen J05 and V
The polymorphic markers between the two parents were further used to trace SMV resistance genes in each plant from the selected F2:3 lines and their advanced generations.
Linkage group for three SMV resistance loci
F for Rsv1,
B2 for Rsv3, and
D1b for Rsv4

51. Result
A series of F2:3, F3:4, and F4:5 lines derived from J05 9 V were developed for selecting individuals carrying all three genes.
Eight PCR-based markers linked to the three SMV resistance genes were used for marker-assisted selection.
Two SSR markers (Sat_154 and Satt510) and one gene-specific marker (Rsv1-f/r) were used for selecting plants containing Rsv1; Satt560 and Satt063 for Rsv3; and Satt266, AI856415, and AI g for Rsv4.
Five F4:5 lines were homozygous for all eight marker and all three SMV resistance genes that would potentially provide multiple and durable resistance to SMV.

52. C. Insect resistance
Introduction: Brown plant hopper (BPH) is the most destructive insect in rice production. Breeding of resistant cultivars is the most cost-effective and environment-friendly strategy for BPH management; however, resistant cultivars are currently hampered by the rapid breakdown of BPH resistance. Thus, there is an urgent need to use more effective BPH resistance genes or pyramiding different resistance genes to develop more durable resistant rice cultivars.

53. Introgression of Bph27(t) into japonica and indica cultivar rice
Ningjing3 (NJ3) is one of the most widely cultured elite japonica varieties in China, but it showed highly susceptible to BPH.
In order to develop BPH resistant Japonica cultivars,
NJ3 - recurrent parent
Ba - donor parent
Two markers (Q52 and Q31) reported by He et al. (2013) were used for tracking introgression of Bph27(t).
To introgress Bph27(t) into indica cultivar
recurrent parent
Two marker (RM471 and Q58) used for tracking introgression of Bph27(t).
Finally, a Bph27(t)- carrying japonica line R2256 and indica line R3-166 were selected from the BC6F2 populations.

54. A total of 321 and 218 evenly distributed and polymorphic markers were used to identify the background of the introgression linesR2256 and R3-166, respectively.
The results showed that NJ3 background of R2256 have arrived at
97.7 % and background of R3-166 also have arrived at 99.5 %. Therefore, these results demonstrated that Bph27(t) have been successfully introduced into NJ3 and through MAS.

56. Conclusion:
To pyramid the two BPH resistance genes and develop more durable BPH resistance rice, the plants with the two BPH resistance genes were screened from F2 population derived from R2381/R2256 F1, then Bph3 and Bph27(t) pyramided lines were selected.
Among them, eight lines with both homologous Bph3 and Bph27(t) were subsequently evaluated for the BPH resistance.
NJ3 were completely dead about 7 days after infestation with BPH, all three lines (R168-1, R230-2 and R339-4) with zero mortality rate displayed high BPH resistance.
When about 3-weeks after infestation, the survival rate of R2381 and R2256 arrived at about 5.1 % and 7.3 %, respectively, but the pyramided lines also showed invisible damaged symptom. These results indicate that Bph3 and Bph27(t) pyramided lines showed higher resistance than single gene introgression lines.

57. 3. Inbred line development
In a gene pyramiding scheme, strategy is to cumulate into a single genotype, genes that have been identified in multiple parents.
The use of DNA markers, which permits complete gene identification of the progeny at each generation, increases the speed of pyramiding process.
In general, the gene pyramiding aims at the derivation of an ideal genotype that is homozygous for the favorable alleles at all the loci.

58. Fig. gene pyramiding scheme cumulating six target genes
The gene pyramiding scheme can be divided into two parts
1. Crossing scheme
2. Fixing scheme
Fig. gene pyramiding scheme cumulating six target genes

59. Conclusion :
Marker-based breeding strategy reduces extensive phenotyping, provides more effective options to control linkage drag, makes the pyramiding of genes with very similar phenotypic effects possible, and reduces the breeding duration.
Marker- based gene pyramiding is now the method of choice for inbred line development targeted at improving traits controlled by major genes.

60. 4. Quality improvement
Introduction: An important goal of wheat breeding is to develop high-yielding varieties with better nutritional quality and resistance to all major diseases.
During the present study, in the background of a popular elite wheat cultivar PBW343, we pyramided eight quantitative trait loci (QTLs) for four grain quality traits (high grain weight, high grain protein content, pre-harvest sprouting tolerance, and desirable high molecular-weight glutenin subunits) and resistance against the three rusts.

61. Materials and methods
Four improved lines (in the background of wheat cv. PBW343) were used in the present study.
(1) PBW343 with resistance genes for LR, SR, YR and high GPC. (2) PBW343 with PHS tolerance QTL (3) PBW343 with genes for leaf and stem rust resistance and a QTL for high GW (4) PBW343 with two tightly linked genes for glutenin.

63. Result
Using marker-assisted selection in five consecutive generations (DCHF1–DCHF5), four pyramided lines (PYLs) were selected, each with all the eight desired QTLs in homozygous state.
The phenotypic characterization of the progenies of these PYLs suggested that the genetic background of PBW343 was retained in all these four PYLs.
Therefore, these PYLs should prove useful in future wheat breeding programs for improving not only the grain quality, but also the durability of resistance against all three rusts.
Multi-year/ multilocation trials are planned for these pyramided lines to evaluate their potential for release as a next-generation improved version of wheat cv. PBW343 for commercial cultivation.

64. Conclusion

65. Thank you