Allele mining in orphan underutilized crops

1. PRESENTED
BY:
OM PRAKASH YADAV
Regd. No.: J-17-M-507Regd. No.: J-17-M-507
Division of Plant Breeding and Genetics
Sher-e-Kashmir University of Agricultural
Sciences & Technology, Jammu

2. The word ‘allele’ is derived from the
Greek word ‘allelos’ meaning each other.
This word is a short form of allelomorphThis word is a short form of allelomorph
(other form) which is used to describe
variant form of a gene detected as
different phenotypes.

3. Alleles are alternative DNA sequences at
the same physical locus, which may or
may not result in different phenotypic
traits.
For example, at the gene locus for ABOFor example, at the gene locus for ABO
blood type proteins in human; eye color
in fruit fly.

4. Allele mining is a research field
aimed at identifying allelic variation of
relevant traits within genetic resources
collections.
Allele mining is searching of new
alleles in wild germplasm i.e. finding of
superior allele from the natural population.

6. Orphan crops are diverse set of minor crops
like tef, finger millets, yam or tubers that tend to
be regionally important but not traded around
then world and receive no attention by researchthen world and receive no attention by research
networks but can play an important role in
regional food security.

7. Orphan, abandoned, new,
underutilized, neglected, lost, underused,
local, minor, traditional, forgotten,
alternative, promising, underdeveloped:
these and other terms are often used as
synonyms for Orphan crops.synonyms for Orphan crops.

8. Crop Scientific Name
A. PSEUDO-CEREALS
Grain amaranth Amaranthus spp.
Buckwheat Fagopyrum spp.
Chenopodium Chenopodium spp.
VARIOUS ORPHAN/UNDERUTILIZED CROPS
IN INDIA
Job’s tear Coix lacryma-job
B. FOOD LEGUMES/ PULSES
Rice bean Vigna umbellata
Winged bean Psophocarpus tetragonolobus
C. OILSEEDS
Perilla Perilla frutescens
Paradise tree Simarouba glauca

9. D. VEGETABLES
Kankoda Momordica dioica
Winged bean Psophocarpus tetragonolobus
E. FODDER CROPS
Amaranth Amaranthus spp.
Salt bush Atriplex spp.
F. ENERGY, HYDROCARBON ANDF. ENERGY, HYDROCARBON AND
INDUSTRIAL PLANTS
Jojoba Simmondsia chinensis
Guayule Parthenium argentatum
Jatropha Jatropha curcas
Tumba Citrullus colocynthis
Paradise Tree Siimarouba glauca
JoshiJoshi et al.et al. 20022002

10. Amaranth :-
The seed protein content has been reported to
vary from 8.86% to 19.6%.
Its grains have high protein with high lysineIts grains have high protein with high lysine
and a good balance of other essential amino
acids.
Its excellent source of iron (10.66 ppm) and ß-
carotene.
Misra et al. 1985

11. The presence of a higher amount of folic acid
can help in increasing the blood haemoglobin.
Chenopod
Grain protein quality equals that of milk and
contains high lysine (6 g 100 g1 protein),contains high lysine (6 g 100 g protein),
methionine (2.3 g 100 g1 protein) and cysteine
(1.2 g 100 g1 protein).

12. Rice bean
The seeds contain a high amount of protein
(20.9%), tryptophan (0.79–1.10%) and
methionine (0.45–1.18%).
Faba bean
faba bean is becoming important as a source offaba bean is becoming important as a source of
protein (26.2%).
Cold hardy cultivars tolerate temperatures of -
10°C without serious injury.

13. Winged bean
Winged bean is rich in protein (29.8–37.4%
in seeds and 10.9% in tubers) and oil.
Seeds are rich in the antioxidant, tocopherol,
which improves human utilization of vitamin
A.
Bambara groundnut
The average composition of the seed is 63
percent carbohydrate, 19 percent protein, and
6.5 percent oil (NRC, 2006).

14. Tumba
The seeds contain 30–34% pale yellow oil,
which contains an alkaloid, a glucoside and
a saponin.
The roots have purgative properties and are
used in jaundice, rheumatism and urinary
diseases.

15. Misra et al. 1985

16. It helps in tracing the evolution of alleles.
Identification of new haplotypes and
development of allele-specific markers for use
in marker-assisted selection.
This capability will be important for giving
plant breeders direct access to key allelesplant breeders direct access to key alleles
conferring
(1) Resistance to biotic stresses
(2) Resistance of abiotic stresses

17. (3) Greater nutrient use efficiency
(4) Enhanced yield
(5) Improved quality, including human nutrition
It can also provide insight into molecular
basis of novel trait variations and identify the
nucleotide sequence changes associated with
superior alleles. In addition, the rate of
evolution of alleles.
JadhaoJadhao,, 20152015

18. There are two major approaches for allele
mining:
TILLING or Eco-TILLINGTILLING or Eco-TILLING
PCR-based or Sequencing based allele mining

19. TILLING is a general reverse genetic technique that
combines chemical mutagenesis with PCR based
screening to identify point mutations in regions of
interest. (McCallum et al., 2000)
TILLING is a powerful technology that employedTILLING is a powerful technology that employed
heteroduplex analysis to detect which organism in a
population carry single nucleotide mutation in specific
genes.
TILLING can also be used to detect naturally occurring
SNP in genes among the accession, variety or cultivar.
To study the gene function, or to detect genetic marker
in population.

22. EcoTILLING is similar to TILLING, except
that its objective is to identify natural genetic
variation as opposed to induced mutations.
This approach allows one to rapidly screen
through many samples with a gene of interest
to identify naturally occurring SNPs and / or
small INs/DELS.

23. SNPs are found in coding (mostly) and
noncoding regions.
Occur with a very high frequency about 1 in
1000 bases.
SNPs close to particular gene acts as a marker
for that gene.
SNPs in coding regions may alter the protein
structure made by that coding region.

25. STEPS INVOLVED IN ALLELE MINING
Selection of target trait (Trait priority)
Identification of accessions associated with desired phenotypic trait
Selection of target trait (Trait priority)
Selection of genes underlying the chosen target trait (Gene targets)
Primer designing for whole length of gene
Cont…Cont…

26. PCR amplification from the identified accessions
Sequencing-based allele mining
Nuclease cleavageNuclease cleavageNuclease cleavageNuclease cleavage
HeteroduplexingHeteroduplexingHeteroduplexingHeteroduplexing
Eco-TILLING based allele mining
PCRPCR
LiLiLiLi----CorCorCorCor gels and SNPgels and SNPgels and SNPgels and SNP
identificationidentificationidentificationidentification
Comparison of sequence date with phenotypic data andComparison of sequence date with phenotypic data andComparison of sequence date with phenotypic data andComparison of sequence date with phenotypic data and
identification of superior allelesidentification of superior allelesidentification of superior allelesidentification of superior alleles
Nuclease cleavageNuclease cleavageNuclease cleavageNuclease cleavage
Confirmatory sequencingConfirmatory sequencingConfirmatory sequencingConfirmatory sequencing

28. CASE STUDIES

29. Allele Mining in Cassava (Manihot
esclunta C.) through TILLING and
Eco-TILLING Approaches
Till, B.J., Nakitandwe, J., Bado, S., Afza, R., Matijevic, M.,
Hunh, O.A., Fregene, M., Dixon, A., Hurtado, P.X., Ceballos,Hunh, O.A., Fregene, M., Dixon, A., Hurtado, P.X., Ceballos,
H. and Mba, C.*

30. ABSTRACT
Targeting Induced Local Lesions IN Genomes (TILLING)
uses mutagenesis and nucleotide polymorphism discovery methods
for a low cost, high throughput reverse genetic strategy that is
applicable to most organisms and can increase the efficiency of
using induced mutations to generate novel traits. In parallel, we are
developed the Ecotilling technique for the identification ofdeveloped the Ecotilling technique for the identification of
spontaneous nucleotide polymorphisms that are linked to or causing
phenotypic variations. We posit the utility of using SNPs obtained
from Ecotilling assays for genotyping, and using induced mutation
and TILLING to enhance the efficiency of isolating desired cassava
mutants prior to field trialling.

31. Result and Conclusion
The Joint FAO/IAEA Programme for Nuclear Techniques
in Food and Agriculture focuses on using induced mutations for
crop improvement in underutilized crops. Approaches such as
TILLING and Ecotilling are used in understudied crops but needs
more appropriate techniques and training methods for allelemore appropriate techniques and training methods for allele
mining in cassava. Further, developed optimize protocols for
cassava, and preliminary work is promising for crop
improvement in cassava.

34. Applying Allele Mining Approach - Genotyping
(Tilling) and Phenotyping Analyses to Elucidate
Gene Function in a Chemically Induced Sorghum
Mutant Population
Xin*, Z., Wang*, M.L., Barkley, N.A., Burow, G., Franks, C.,
Pederson,G. and Burke, J.Pederson,G. and Burke, J.
Published: 14 October 2008
BMC Plant Biology 2008, 8:103 doi:10.1186/1471-2229-8-103

35. Abstract
Sorghum [Sorghum bicolor (L.) Moench] is ranked as the
fifth most important grain crop and serves as a major food staple
and fodder resource for much of the world, especially in arid and
semi-arid regions. The recent surge in sorghum research is driven
by its tolerance to drought/heat stresses and its strong potential as
a bioenergy feedstock. Completion of the sorghum genome
sequence has opened new avenues for sorghum functional
genomics. However, the availability of genetic resources,genomics. However, the availability of genetic resources,
specifically mutant lines, is limited. Chemical mutagenesis of
sorghum germplasm, followed by screening for mutants altered in
important agronomic traits, represents a rapid and effective means
of addressing this limitation. Induced mutations in novel genes of
interest can be efficiently assessed using the technique known as
Targeting Induced Local Lesion IN Genomes (TILLING).

36. Results
A sorghum mutant population consisting of 1,600 lines
was generated from the inbred line BT×623 by treatment with
the chemical agent ethyl methanesulfonate (EMS). Numerous
phenotypes with altered morphological and agronomic traits
were observed from M2 and M3 lines in the field. A subset of
768 mutant lines was analyzed by TILLING using four target
genes. A total of five mutations were identified resulting in a
calculated mutation density of 1/526 kb. Two of the mutationscalculated mutation density of 1/526 kb. Two of the mutations
identified by TILLING and verified by sequencing were
detected in the gene encoding caffeic acid O-methyltransferase
(COMT) in two independent mutant lines. The two mutant
lines segregated for the expected brown midrib (bmr)
phenotype, a trait associated with altered lignin content and
increased digestibility.cal Lesion IN Genomes (TILLING).

37. Methodology
Mutant population generation

40. CONCLUSION
Allele mining is a promising approach to discover naturally
occurring allelic variation at candidate genes controlling
various biotic and abiotic traits which have potential
applications in crop improvement programs.
It can also provide insight into molecular basis of novel trait
variations and identify the nucleotide sequence changes
associated with superior alleles.

41. It can be visualized as a vital link between effective
utilization of genetic and genomic resources in
genomics.
It is certainly expected that sequencing based allele
mining would emerge as a method of choice in
revealing natural allelic variations in loci of interestrevealing natural allelic variations in loci of interest
and to discovery the novel and effective alleles.

42. Prospects and Future Consideration for Allele
Mining
ALLELE MINING POSSESSES GOOD POTENTIAL TO BE APPLIED IN
MOLECULAR PLANT BREEDING
Development of novel and efficient mining strategies to
screen GenBank collections more efficiently for DNAscreen GenBank collections more efficiently for DNA
sequence variation and the management of genome
resources.
Both approaches can provide higher efficiencies in
breeding program and will therefore be useful in meeting
expected demands.

43. Some standardize bioinformatics tools, advanced
allele mining strategies and additional efforts are
needed to develop as computational tools for efficient
allele identification.
Overall, to improve food security, to promote
fundamental research and practical breeding new
technologies must be developed and advanced methodstechnologies must be developed and advanced methods
for identification of novel alleles for functional
genomics and crop improvement may become an
effective tool for helping in feeding of the twenty-first
century world.

44. THANK YOUTHANK YOU