Synthetic hexploid wheat

IndianAgriculturalResearchInstitute
Vijay Kamal Meena
Division of Genetics
Indian Agricultural Research Institute
New Delhi

Synthetic Hexaploid Wheat

 Synthetic hexaploid wheat is an artificial hybridization between tetraploid
wheat and an accession of Aegilops tauschii.
Introduction
 Numerous SHWs have been produced globally by various institutions
including CIMMYT-Mexico, ICARDA-Syria, Department of Primary
Industries (DPI), Victoria-Australia, IPK-Germany, Kyoto University-Japan,
and USDA-ARS.
 First primary synthetic between a tetraploid wheat and Aegilops tauschii
occurred in 1946 (Mc Fadden and Sears)
Source: Mujeeb-Kazi et al-Plant Breeding Reviews, 35-122, 2013

Evolution of Wheat

Cont..
 Natural hexaploid wheat is estimated to have lost 10,000 to 16,000
genes following polyploidization, compared with the three diploid
progenitors. (Brenchley et al., 2012)
 The second neohexaploidization event led to a supradominance, with
the D subgenome dominant over the tetraploid (subgenomes A and B).
(Pont et al. 2013)
 The first neotetraploidization event resulted in subgenome dominance
where in the A subgenome was dominant over the B subgenome.

Cont..
 Kamut (durum wheat) eg- Triticum turgidum; ssp. polonicum or
Triticum turgidum; ssp. Turanicum.
 Emmer are tetraploids (2n = 4x = 28) eg- Triticum dicoccoides, Triticum
dicoccum.
 Enkorns are diploid (2n = 2x = 14) `eg- Triticum boeticum, which
includes T. aegilopoides, T. thaoudar, and T. urartu, Triticum
monococcum
 Dinkels are hexaploid (2n = 6x = 42) eg- Triticum aestivum (AABBDD)
Source: Parvaiz Ahmad et al

A puzzle showing puzzle piecing together the wild relatives of wheat
Source: Parvaiz Ahmad et al

F1 hybrid
(2n=3x=21, ABD)
Colchicine treatment or
meiotic restitution
Synthetic hexaploid wheat
(SHW) (2n=6x=42, AABBDD)
1. Interspecific cross between Ae. tauschii And T. turgidium
Production and Utilization of
Source: Mujeeb-Kazi et al-Plant Breeding Reviews, 35-122-2013

wheat
(AABBDD)
Aegilops tauschii
(2n=2x=14, DD)
F1 hybrid* (ABDD)
* Embryo rescue
Hexaploid wheat
Hexaploid wheat
(2n=6x=42, AABBDD)
Hexaploid wheat
Hexaploid wheat
(2n=6x=42, AABBDD)
2. Direct cross for genetic transfer from diploid Ae. tauschii into hexaploid wheat

 Advantage- no colchicine treatment is necessary in the cross between
wheat and the Ae. tauschii hybrid.
 Disadvantage- segregating for the D genome, and exhibit instability because of
aneuploidy, potentially making genetic analysis more difficult.
Cont..

3. Direct Cross of Tetraploid Wheat to Common Wheat
Hexaploid wheat
(2n=6x=42, AABBDD)
Triticum turgidum
(2n=4x=28, AABB)
F1 hybrid
(AABBD)
Hexaploid
wheat
(AABBDD)
F2 --- Fn
Hexaploid
wheat
(AABBDD)
BC1F1--- BC1Fn
Hexaploid wheat
(AABBDD)

1. Genetic Analysis via Crosses and Backcrossing.
Triticum turgidum
(2n=4x=28, AABB)
Aegilops tauschii
(2n=2x=14, DD)
F1 hybrid
(2n=3x=21, ABD)
Bread wheat
(2n=6x=42, AABBDD)
F1 hybrid Bread wheat
BC1F1 --- BC1Fn
Synthetic backcross derived lines
(SBL)
Colchicine treatment or meiotic restitution
Current Strategies for Using Synthetic
Hexaploid Wheat in Breeding

Bread wheat
(2n=6x=42, AABBDD)
F1 hybrid Bread wheat
BC1 Bread wheat
BC2 QTL MAPPING
2. Advanced Backcross-Quantitative Trait Loci (AB-QTL) Analysis.

 Advanced backcross QTL analysis’ by Tanksley & Nelson (1996).
 QTL analysis in BC2 or BC3 stage
 Combine QTL mapping and development of improved breeding lines
 Use backcross mapping populations
Cont..

 DNA elimination, gene silencing and duplication are associated with
allopolyploidization in wheat
 The D genome of SHW suffered more disruption during the
allopolyploidization process than the A and B genomes.
 In plants, allopolyploidization is an important process arising from
interspecific or inter-generic hybridization followed by chromosome
doubling.
Allopolylpoidy - Induced
Genomic Changes In SHW
Source: Ma Yu et al- Biotechnology & Biotechnological Equipment-2017

 Four types of processes during hybridization may be responsible for
elimination of the genotyped loci and genomic restructuring.
 Either loss of a sequence from one or two parents
 Gain of a novel sequence that is different from those of the parents
 Pericentric rearrangements
 Transposable elements activity

Linkage map of SHW-L1 and chromosomal regions from AS60 or AS2255 eliminated in SHW-L1
 Most of the eliminated loci tightly linked with expressed
QTLs are very important to alterations of adaption and
yield during wheat evolution.

 Allopolyploids shows important changes at the genetic, gene
expression, and epigenetic levels.
 Allelic heterozygosity, increased genome dosage and intergenomic
interactions leads to heterosis. (Chen, 2007; Ni et al., 2009).
 Genes located on dominant genome shows higher expression
levels. (Schnable et al., 2011; Cheng et al., 2012; Garsmeur et al., 2014).
Molecular Characterization of
Allohexaploid SHW as a Model for Heterosis Studies
Source: Aili Li et al. Plant Cell 2014

A. Chromosomes in root tip cells. Green arrows indicate the 4A/7B chromosome
translocations in synthetic hexaploid and its T. turgidum parent PI 94655 (4x).
B. Seven-day-old seedlings of T. turgidum (4x), synthetic wheat (6x), and Ae. tauschii
(2x).
C. Heading stage spikes of the third generation of self-pollinated allohexaploid
wheat (S3, 6x) and its parents.
D. Developing seeds of S3 allohexaploid plants and their progenitors. 4x, T.
turgidum; 2x, Ae. tauschii; 6x, newly synthesized allohexaploid wheat.
E. Sampling schema. Samples in oval circles have biological replicates- T. turgidum,
Ae. Tauschii, S1 to S4- generation of selfed allohexaploid wheat.

Parental Expression Level Dominance (ELD) and Nonadditively
activated expression in SHW
 Common Wheat differs from SHW -
 newly synthesized SHW represent an early genetic stage of common
wheat that may potentially confer hybrid vigor.
 Homoeologs in nascent SHW should represent their initial expression
state, possibly maintained by epigenetic mechanisms.
 ELD - Genes are differentially expressed between the parental lines and
exhibit expression level in the progeny that is statistically similar to that
of one parent.
 Elongation of Spike Cells in SHW shows presence of nonadditively
activated expression of auxin pathway related genes in young spikes.

SHW retained more ELD-ab genes than ELD-d genes-
 ELD-ab Floral development genes PI, AP3, and AG homologs, which
may contribute to spike and flower organ renovation, plant height,
spike shape, and grain length
 ELD-d - Stress responses and photoperiod adaptability, genes
encoding chitinase, and HSP90 that may be involved in biotic stress
responses, AKT1 homolog (salt tolerance), LHY and CO homologs
(flexibility in flowering condition)
Pistillata (PI), Apetala3 (AP3), Agamous (AG), Late Elongated Hypocotyl (LHY),
Constans(CO), Arabidopsis potassium channel(AKT1)

Gene Expression Patterns among Three Tissues in SHW and Its Progenitors
C- T. turgidum
D- A. tauschii
E- SHW

 Seedling-specific genes are significantly enriched for
secondary metabolic process and pathways for stress
responses.
 Spike-specific genes are significantly enriched for transport
and cell growth pathways.
 Seed-specific genes are functionally enriched in the
embryonic development pathway.
 GO is a major bioinformatics initiative to unify the representation
of gene and gene product attributes across all species.
S3 plants and its two progenitors GO -
Gene ontology(GO) Analysis

miRNAs are Important for Growth Vigor and Adaptation in SHW
 miRNAs may play important roles in heterosis for nascent SHW
 miR169 and miR319 - involved in drought, salt, and cold responses
 miR5200, miR9006, and miR9009- biotic resistance and flowering
control via regulating their targets, such like RGAs(R gene analogs)
and FT (Flowering locus T)
 miR167 and its putative targets (including several auxin response
factors) like miR167-ARF8-GH3.2 auxin signaling pathway is
relevant to spike growth vigor.

Effect of Allohexaploidization on Expression of miRNAs and Their Targets.

Genetic Buffering of siRNAs and Homoeolog Expression Regulation in SHW
 siRNA-mediated epigenetic mechanisms are involved in the
maintenance of genome stability of interspecific hybrids and
allopolyploids (Ha et al., 2009b; Kenan-Eichler et al., 2011; Greaves et al., 2012).
 > 70% of wheat genes have TEs (Transposable elements) in their
neighboring regions, siRNA-mediated DNA methylation may also
contribute to interactive homoeolog expression through associated TE
sequences.
 SHW contains more higher density of siRNA at upstream and
downstream TE associated D homelogos, leads to biased repression
of D homeologos.

A Small RNA–Dominated Model for Homoeolog Expression Regulation during Wheat
Allohexaploidization.

Biotic Stress Disease or Pest Source of Resistance Reference
Rust Leaf Rust
Stem Rust
Stripe Rust
Ae. tauschii
Synthetic Hexaploid
Ae. tauschii (Lr21,Lr39)
Synthetic Hexaploid
Ae. tauschii
Synthetic Hexaploid
Ae. tauschii
Synthetic Hexaploid
Both parents of SH
Synthetic Hexaploid
Innes & Kerber 1994
Kerber & Dyck 1969
Cox et al. 1992
Ogbonnaya et al. 2008
Innes & Kerber 1994
Assefa & Fehrmann 2004
Cox et al. 1992
Friesen et al. 2008
Ma et al. 1995
Nematodes Cereal Cyst Nematode Synthetic Hexaploid (Cre3) Ogbonnaya et al. 2008
Virus Barley Yellow Dwarf Synthetic Hexaploid Saffdar et. al 2009
Leaf Spot Diseases Spot Blotch
Septoria Tritici blotch
Ae. Tauschii
Synthetic Hexaploid (stb5)
Mujeeb-Kazi et al. 2007
Other Fungal Diseases Powdery Mildew
Karnal Bunt
Ae. Tauschii
Synthetic Hexaploid
Cox et al. 1992
Mujeeb-Kazi et al. 2006
Insects Hessian Fly Synthetic Hexaploid
( H22, H23, H26, H32)
Friesen et al. 2008
IMPACT OF SYNTHETIC HEXAPLOID IN WHEAT IMPROVEMENT
A. Disease and Pests Resistance

B. New genetic variability for tolerance to abiotic stress
 Ae. tauschii and the wild and cultivated emmers have evolved over thousands
of years in some of the harshest environments on earth across North Africa
and western Asia.
 Utilization of D genome for improving Salinity Tolerance-
 A major locus, Kna1, (controlling K+/Na+ uptake) present on Chr. 4DL
SBLs showing significantly
enhanced Na+ exclusion
Source: Mujeeb-Kazi et al-Plant Breeding Reviews, 35-122, 2013

Case Study 1

 Boron tolerance in Wheat- controlled by two root specific boron
transporter genes
 Bo4 on Chr. 4AL
 Bo1 on Chr. 7BL
 Aim of the Study -
 To identify novel genomic regions in primary synthetic wheat
for boron toxicity tolerance.

 Method
Material
1. Tolerant checks- Halberd and Frame
2. Sensitive checks- Meering
3. 333 SHW accessions
 Phenotyping of Root growth done by filter paper method in
boron toxicity and control conditions.
 Genotyping done by using DArT markers for identification of
novel genomic regions for tolerance to boron toxicity in SHW.

 Result

 In Checks level of tolerance to boron toxicity- based on relative root
length
Halberd > Frame > Meering
 Some of SHW accessions show higher boron toxicity tolerance than
the tolerant checks
 In SHW DArT analysis identified –
 wPt-8886 on 4A near to Bo4 locus
 wPt-4886,wPt-2847 on 1AL – novel regions for boron
toxicity tolerance in SHW.

Conclusion
 Hexaploid wheat can be reconstituted by natural intercrossing, induced
chromosome doubling, and embryo rescue to produce primary “synthetic”
wheat.
 Combining variability from both modern durum wheat and ancestral
tetraploids with Aegilops tauschii has produced new genetic variation for a
range of biotic, abiotic, and quality-related traits.
 Direct and indirect evidence indicates that much of the newly observed
genetic diversity in synthetic wheat is novel. Synthetic derivatives, developed
by crossing primary synthetics with adapted cultivars, have been developed
with enhanced resistance to biotic and abiotic stresses.
 The exploitation of synthetic wheat is still in its infancy. In the future,
combining novel genetic diversity in synthetic wheat with that existing in the
wheat gene pool can be expected to significantly enhance the adaptation and
marketability of wheat.

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Synthetic hexploid wheat

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