This document discusses various methods for transferring genes from crop wild relatives into cultivated crops. It begins by defining alien introgression as the transfer of genes from unadapted species into breeding programs. It then discusses why introgression is useful given the loss of genetic diversity during domestication. Historical examples of introgression improving disease resistance in wheat and other crops are provided. Modern techniques discussed include marker-assisted backcrossing to introgress traits while retaining the cultivated background genome. The document also explores using next-generation sequencing to identify genes and develop markers for introgression programs.

1. Alien Introgression in Crop Improvement –
New Insights
Credit Seminar
Presented by :
Asmat Ara
Ph. D GPB
03/AG(GPB)/2016-D

2. Alien introgression
• Defined as transfer of one or more genes from exotic/un-
adapted/ crop wild relatives (CWR) to adapted breeding
populations.
(Edgar Anderson)
• CWR contain a wealth of genetically important traits due to
their adaptation to a diverse range of habitats and the fact that
they have not passed through the genetic bottlenecks of
domestication.
(Vollbrecht and Sigmon, 2005; FAO, 2008).

3. Why alien introgression ?
Loss in genetic diversity, due to :
 Selection for domestication related traits.
 Genetic drifts (domestication bottlenecks)
 Modern breeding practices.

4. Diagrammatic representation of loss of genetic diversity in crop species
The Symbol ( asterisk )
indicates the novel genetic variation (Mir et al,2014)

5. Looking for new sources of
variation ??

7. Historical background
• CWR were used in crop improvement in sugarcane in the
first half of the 20th century.
• Their utility was recognized in breeding programmes of
major crops in the 1940s and 1950s (Plucknett et al. 1987).
• Gene for resistance to leaf rust translocated onto Triticum
aestivum from Aegilopes umbellulata.( Sears, 1950)
• wild gene use in crop improvement gained in prominence by
the 1970s and 1980s with their use being investigated in an
increasing wide range of crops (Hoyt 1988).

8. Use of crop wild relatives in the past 20 years in released cultivars
of 13 crops of international importance
+ indicate number of wild relatives that have contributed beneficial traits to crop varieties
- indicates wild relatives have not contributed beneficial traits in that category

9. Important genes in wheat found in related species
System-wide Genetic Resources Program
(1996)

10. • Stem or black rust (Puccinia graminis f. sp. tritici) and brown or leaf rust
(caused by Puccinia triticina) continue to be a serious threat in many wheat
growing regions of the world.
• Leaf rust res. genes in D genome: Lr21, Lr22a, Lr32, Lr37, Lr39, Lr41, Lr42 &
Lr43
• Lr34 (Ae. tauschii) is a minor/slow rusting gene which provides durable
resistance & presently used extensively in wheat improvement programme in
India and worldwide
• Objective: Evaluation of genetic diversity for stem and leaf rust resistance in D
genome species of wild relatives of wheat

11. Response of accessions of D genome species of Aegilops to stem and leaf rust pathotypes

12. Alien gene transfer Non-sexual methods/
Horizontal transfer
Sexual
hybridisation/
Vertical transfer
 Interspecific
 Intergeneric
Alien gene
detection
Somatic hybridizationGenetic transformation
Genetic
transformation
Direct gene transfer
TransgenesisIntragenesis
& cisgenesis
Interspecific &
intergeneric SH

13. Limitations
 Vertical Gene Transfer
(VGT)
 Crossability barriers
 Chromosome Pairing
 Linkage drag
 Background effects
 Pleiotropic effects
 Horizontal Gene
Transfer (HGT)
 Regeneration protocol
 Isolation of genes from
wild species
 Expression of alien genes
 Gene flow
Moreover, they provide limited information on genetic basis of complex
traits chromosome locations of QTL.

14. Understanding genome
variation in crops through
use of new genomic
techniques.

15. Solution
• Evolution of Molecular markers
• High throughput marker genotyping
technologies.
Illumina’s GoldenGate assay
Whole genome genotyping infinium assay

16. Next Generation Sequencing
 Next Generation Sequencing (NGS), also known
as high- throughput sequencing ,allows mass
sequencing of genomes and transcriptomes much
more quickly and cheaply than the previously
used Sanger sequencing.
NGS technologies include :-
• Solexa / Illumina sequencing
• Roche / 454 sequencing
• AB SOLiD sequencing

17. Applications of Gene Sequencing in the context of
gene introgression
• Identification of genes and also in
development of novel molecular markers in
crop species. The markers derived from
sequence information may be used for:
• Characterization of genetic resources and association with
agronomic traits.
• Identification of new sources of biotic and abiotic stress
resistance genes

18. Genetic diversity analysis.

19. Allele Mining
• Promising approach
to dissect naturally
occuring allelic
variation at
candidate genes
controlling key
agronomic traits.
• Helps in tracing
evolution of alleles,
identification of
new haplotypes and
development of
allele specific
markers.

20. 1. TILLING :
(Targeting Induced Local Lesions IN Genomes)
 Reverse genetic strategy that works with a
mismatch- specific endonuclease to detect
induced DNA polymorphisms in genes of
interest.
 Developed by Colbert et al. 2001.
(Fred Hutchinson Cancer Research
Center, Seattle , Washington)
 American scientists ‘Comai’ &
‘Henikoff’ (2003) have initiated first
TILLING project in Arabidopsis.

21. MERITS of TILLING
 It is independent of genome size, reproductive system or generation
time.
 High throughput & data analysis can also be automated.
 Overcomes disadvantage of knockout gene therapy for a specific gene,
as eliminates the need for removal of gene from gene pool to detect its
function.
 TILLING can introduce genetic variation in an elite germplasm without need
to acquire variation & thus avoiding introduction of agriculturally
undesirable traits.
 Also overcomes problems of transgenic approach as it is independent of
transgene efficiency and regeneration of plant.

22. EcoTILLING
• It is a method that uses TILLING techniques to look for
natural variations in individuals, usually for population
genetics analysis.
• EcoTILLING can be performed more inexpensively
than full sequencing, the method currently used for most
single nucleotide polymorphism (SNP) discovery.
• But differs from TILLING in that natural polymorphisms
are detected rather than polymorphisms induced through
chemical mutagenesis.

23. TILLING and EcoTILLING

25. New genetic approaches for
harnessing the natural
variation

26. Introgression lines (ILs)
• Lines generated by introgression of favourable
gene/QTL/chromosomal segment by developing
isogenic lines using wild species and the
variety/genotype of interest.
Introgression/exotic libraries are constructed using
introgression lines each of which carries a fragment of
defined homozygous chromosomal segment from donor
exotic parent with a homozygous genetic background of
elite parent.

27. Parental survey to detect introgressed segments
from O. australiensis into BPH resistant
introgression line

28. RFLP pattern in F2 population of a cross of BPH resistant
introgression line (IR65482-4-136-2-2) with the recurrent parent
(IR31917-45-3-2).

29. AB-QTL analysis
Approach for simultaneous discovery and transfer of
QTL’s from wild species to crop variety.
Proposed by Tanksley and Nelson

30. Association mapping
 Also called as linkage disequilibrium mapping.
 A natural population survey to determine marker
trait associations using LINKAGE DISEQUILIBRIUM
 The power depends on the strength of this
correlation (i.e., on the degree of LD between the
genotyped marker and the functional variant.
Could be an answer and alternative to family based mapping
To dissect complex traits

31. What is linkage disequilibrium and how ?
 Non-random association of alleles at
adjacent loci..
Linkage disequilibrium
around an ancestral
mutation
Ancestral
chromosome
The closer two markers are, the stronger the LD
The resolution with which a QTL can be
mapped is a function of how quickly LD decays
over distance.

32. Linkage vs Association mapping: How it leads to
high resolution..
LINKAGE MAPPING
ASSOCIATION MAPPING
20 generations
6-7 generations
Cardon and John, 2001

33. Approaches for Association mapping
Candidate gene association
mapping
Genome wide association mapping
Relates polymorphism in selected candidate
genes
Genetic variation in whole genome
to find signals
Germplasm
Genotyping
Phenotyping
Population
Structure
LD
Association
mapping

34. Introgression or pyramiding of
gene/genomic regions responsible
for the trait of interest

35. 3
3
3
3
3
3
1
2
2
2
2
2
2
= Recurrent
parent allele
= Donor parent
allele
Target
gene
1. Select donor allele at markers linked to target gene.
2. Select recurrent parent allele at other linked markers.
3. Select recurrent parent allele at unlinked markers
throughout genome.
Marker assisted back-crossing

36. A case study:
Development of the
submergence tolerant
Swarna-Sub1 with
details of markers used
for foreground,
recombinant, and
background selection.
The numbers of plants
selected in each
generation are indicated
in parentheses.
Neeraja et al. (2007)

37. Genotype of the Swarna-Sub1 (BC2F2) plant that
is homozygous for the recipient genome except
for the Sub1 region on chromosome 9.
Neeraja et al. (2007

38. Introgressed
fragment
containing Sub1
in:
a) selected
BC3F2
plant and
b) b) selected
BC2F2
plant
Neeraja et al. (2007)

39. Marker- assisted recurrent selection
 Used for pyramiding of several genes /QTL’s (of minor effect) in a
single genotype.
 Utilised for selection of traits associated with multiple QTL’s
by increasing the frequency of favourable QTL’s or marker alleles.
This approach involves :
1. Identification of F2 progeny which contains favourable alleles for
most, if not all QTL’s.
2. Recombination of the selected progenies to the selfed ones
3. Repetition of these cycles.

40. Genome wide selection
• Approach used to pyramid favourable alleles for minor
effect QTLs at whole genome level.
• Genome selection (GS) considers marker effects across the
whole genome.
• The use of high-density markers is one of the features.
• Based on two distinct and related groups: training and
breeding populations.
• Phenotyping is a key informant in GS to build up accuracy
of statistical models.
• GS may revolutionize plant and tree breeding practices.
Highlights

41. Conclusion
• TILLING and ecoTILLING being swift polymorphism
detection and genotyping methods can be used for
determining the range of variation for genetic mapping
based on linkage analysis.
• Due to reduced costs on sequencing and genotyping
technologies combined with advances in biometrics and
bioinformatics, bright future on application of these novel
approaches is envisaged.

42. Contd…
• AB-QTL approach has to play an increased role in
time to breeding cultivars with wide genetic
background.
• Genomic selection aided by genotyping will help in
identification of more recombination events.
• Utilization of specific populations will help further
mapping of gene/QTL to make selection of desirable
recombinants towards development of superior
genotypes.

43. ….Thank You

44. Queries?
QUERIES….