Allele mining-Utilization of PGR in plant breeding.ppt

Allele mining novel approach to isolate gene based on sequence informations

1. Allele mining

2. Utilization of PGR in plant breeding
•Gene banks collectively hold huge number of
germplasm accessions
•Global germplasm collections are underutilized
for many crops
•Genetic resources are deemed to harbour a
wealth of undisclosed allelic variants
•Unlocking this variation is a challenge
- enormous size of collections
- resource limitations

3. Allele mining
….novel approach to isolate gene based on sequence
informations. It aims at identifying allelic
variation of relevant traits within genetic resource
collection. It involves….
•PCR based isolation of different versions of the gene
found in germplasm
•Deciphering the genetic variation in relation to trait or
performance of the accession
•Identification of favorable alleles for future experiments

4. Basic steps used in mining alleles
1.Identification of the crop spp. and target trait
2.Information of the gene controlling that trait (physical and functional
characterization)
3.Defining the germplasm set to be mined (trait-specific)
4.Screening of the germplasm set for the trait (Field/lab screening) and
detecting presence of the target gene in the germplasm using gene-
specific marker(s)
5.Cloning, sequence analysis and identification of alleles
6.Functional validation of alleles
Utilization of new functional alleles in crop improvement

5. Wheat – Powdery mildew
Pm3
•exists in 7 functionally distinct alleles (Pm3a….Pm3g)
•is the only powdery mildew resistance gene cloned to date
•high sequence conservation of Pm3 alleles suggest their
recent evolution from the ancestral sequence Pm3CS that
is a susceptible Pm3 allele

6. Defining germplasm to be mined
FIGS (Focused Identification of germplasm strategy) – an
approach that uses information about the environment
from which the accessions with specific traits have been
collected to predict where selection pressure for adaptive
traits may occur
On basis of this information, trait-specific sets then may be
assembled from large collections

7. Allele mining - Steps
•Total collections – 16089 accessions
•Identified 1320 accessions drawn from 323 geographic
sites with potentially high selection pressure for powdery
mildew resistance (FIGS powdery mildew set)
•Complete set of 1320 accessions screened with various
powdery mildew isolates
•211 accessions exhibiting complete or intermediate
resistance to at least one isolate identified,
• and analysed at molecular level

8. Allele mining – Steps ….
•first screened for the presence of a Pm3-like gene with a
diagnostic sequence-tagged site marker
•second for the presence of the already-known Pm3 alleles
•identification of 111 landraces as candidates for the isolation of
Pm3 alleles that were positive for the Pm3 diagnostic fragment
but lacked any of the known Pm3 alleles
•isolation of Pm3 alleles was performed on 56 landraces
completely resistant to at least one powdery mildew isolate,
whereas lines with intermediate resistance were not
considered further.
•Pm3 coding sequences were successfully amplified from 45
landraces, cloned, and sequenced, others …

9. Schematic representation of sequence alignment
of Pm3 alleles

10. Allele mining – Steps ….
•analysis of sequence diversity led to the identification of
16 previously unknown Pm3 allelic sequences, because
several landraces possessed identical alleles
•Among the 45 sequences, 9 were identical to the
susceptible Pm3CS, suggesting that ….
•Nine of the previously undescribed alleles were 4442 bp
long, corresponding to the size of the ancestral reference
sequence Pm3CS, whereas 7 alleles bear insertions and
deletions (InDels)

11. VIGS
Virus-Induced Gene Silencing
Virus-induced gene silencing (VIGS) is a recently developed
gene transcript suppression technique for characterizing
the function of plant genes. The approach involves
cloning a short sequence of a targeted plant gene into a
viral delivery vector. The vector is used to infect a young
plant, and in a few weeks natural defense mechanisms of
the plant directed at suppressing virus replication also
result in specific degradation of mRNAs from the
endogenous plant gene that is targeted for silencing

12. Functional analysis ….
….of Pm3 Candidate Resistance Genes using VIGS
•to determine whether the resistance observed in landraces
with previously undescribed alleles is based specifically
and solely on the Pm3 alleles
•VIGS was used to suppress Pm3 gene expression
•leaves of landraces carrying the alleles Pm342416 and
Pm342255 (IG42416 and IG42255) lost resistance to Bgt
isolate
•resistance was not altered in the landraces with the alleles
Pm342469, Pm342525, and Pm310963, indicating that ….

13. BSMV-mediated virus induced silencing of
Pm342416 and Pm342255

14. Identification of functional Pm3 alleles by
transient transformation
•Susceptible variety ‘Chancellor’ chosen for transformation
•Non-functional Pm3CS allele used a control
•Seven alleles (Pm342416, Pm342525, Pm323728,
Pm342255, Pm310963, Pm342277, and Pm342868) showed
a significant reduction in the haustorium index in
comparison with Pm3CS
•Transformation with the remaining 6 alleles (Pm342920,
Pm39939, Pm342469, Pm331594, Pm314442, and
Pm341606) did not result in a reduction of the haustorium
index…….. race specificity

15. (C) Microscopic view of cells from the landrace with Pm342416 at 2 dpi.
(D) The cells with haustoria are accompanied by HR at 5 dpi.

16. Macroscopic view of the infected leaf segments from landraces with
Pm342416 and Pm342255 at 8 dpi, respectively.

17. Conclusion ….
… the alleles Pm342416, Pm342255, Pm323728, Pm310963,
Pm342525, Pm342277, and Pm342868 are previously
uncharacterized, functionally active forms of Pm3
that are called Pm3l, Pm3m, Pm3n, Pm3o, Pm3p, Pm3q, and
Pm3r, respectively