POLYPLOID CROP RESEARCH IN THE AGE OF GENOMICS

MAHESH R HAMPANNAVAR
PGS16AGR6978 119/07/2018 Mahesh R Hampannavar

POLYPLOIDCROP RESEARCH INTHE AGE
OF GENOMICS
219/07/2018 Mahesh R Hampannavar

Content
• Introduction
• Homeologous gene- in polyploidy
• Repeatable patterns - after allopolyploidization
• Common mechanisms in creation of variation
• Problems associated - polyploid crop improvement.
• Strategies for overcoming problems

Introduction
• Most of the species are polyploids
• Genome duplication
• Advantages of polyploidy over diploid crop
•Difficult and time-consuming to fully understand relationships
within a polyploid complex
•Complex genetic structure and gene regulation networks

The prevalence of Polyploid Complexes
• Polyploid complexes are common in certain families
• Polypodiaceae
• Trilliaceae
• Poaceae
• Brassicaceae
Grant, 1981

Classified based on the age of the polyploidy event(s).
Paleopolyploidy: Mesopolyploidy: Neopolyploidy:
• Ancient polyploidy
• Genetically diploid
• Result of WGD and Genome
re-organisation
• Diploidization
• 30 – 80 ………?
• Have more recent history
•Diploidized genomes
•Diploid like meiosis
•Disomic inheritance
•Quasidiploid complements
•But can be identified by
comparative genetic and
phylogenetic methods
• Young polyploids
• sometimes without
diploidized genomes
• Experimental and naturally
formed polyploids (nascent or
established)

Crop Species Ploidy type
Timing of
polyploidy
Rapeseed B napus Neo, allo (4x) 7500 years ago
Wheat T aestivum Neo, allo (6x) < 0.3 Mya
Strawberry Fragaria Neo, allo (8x) 0.37-2.05 Mya
Potato S tuberosum Neo, auto (4x) Recent
Banana Musa acuminata Neo, auto (3x) 7000 years ago
Cabbage B oleracea Meso (6x) 23 Mya
Soybean Glycine max Paleo (6x) 59 Mya
Sorghum Sorghum bicolor Paleo (4x) 70 Mya

Evolution importance of polyploidy
• Adaption to diverse environment
• Polyploidy have upper hand compare to diploids
• Less diversity in polyploidy crops

AH. Paterson et al. Nature 492, 423-427
(2012) doi:10.1038/nature11798
Evolution of spinnable cotton fibres.
AH. Paterson et al. Nature 492, 423-427 (2012) doi:10.1038/nature11798
Syntenic relationships among grape, cacao and cotton.

General effect of polyploidy
• Duplication leads to polyploidy
• Cell content and genetic material will vary.
• In paleopolyploid soybean,
nearly 75% of genes are present in multiple copies due to genome duplication
• Homoeologus sequences

Homeologous sequences on
polyploid
• Describe the relationship of similar chromosomes
or parts of chromosomes brought together
following inter-species hybridization and
allopolyploidization.
• whose relationship was completely homologous
in an ancestral species.
• the homoeologous chromosomes of the parental
genomes may be nearly as similar to one another
as the homologous chromosomes/gene.

• Origin of homeologus gene allopolyploidy in two path ways

whole genome duplication (WGD) in polyploidy
• Genome duplication underlies all polyploidization
• Genome duplication can also lead to birth of genes with new function and phenotypes
• WGD suffers from the meiotic challenges
• Duplication occurs only a time or several times.
• Cotton and brassica

Fractionation
• Refers to loss of duplicate genes in polyploids.
• genomic stresses, such as hybridization and polyploidization
• Genome shock
• Genome dominance- higher expression of one subgenome
• Less fractioned subgenome exhibits higher expression

Bias fractionation following WGD
Woodhouse et al., 2010 PloS Biology

• Homeologous genes - Additive or non additive
• Homeologous expression and silencing varied by gene and by plant
organ
• Differential expression homeologs may lead to functional plasticity
and phenotypic novelty,
• Allowing allopolyploids to adapt more readily to changing or variable
environments.
Why polyploidy are well
adopt to stress conditions

Difficulties in discriminating between homeologous sequences
• Discriminating between different homeologous genomic locations and alleles at a single
homologous locus is a major challenge.
• Developing homeolog-specific and allelic-specific markers is difficult
• Genome-wide quantification of homeolog expression can also be technically difficult due to high
sequence homology between homeologous gene pairs

Bread wheat (Triticum aestivum) has a large, complex and hexaploid genome consisting of A, B and D homoeologous
chromosome sets.
a novel approach combining wheat cytogenetic resources (chromosome substitution ‘nullisomic-tetrasomic’ lines) with
next generation deep sequencing of gene transcripts (RNA-Seq), to directly and accurately.
quantify the relative contribution of individual homoeoloci to gene expression

23
B > A = D
•Nullitertasomic – 1 , 5
•EST data base- Reference
•Chrom1 – 1123
Chrom 5 – 1247
•Chloroplast thylakoidal
peptidase like protien
(BE497595)- 24HSV
•Non random cDNA library
from root and shoots
•RNA-Seq read frequencies
from roots for the
nullitetrasomes
•Nulli B chrom
•Terta B chrom

Allopolyploidization-induced genomic variation leads to
phenotypic variation
• Allopolyploids may be more limited in genetic diversity than their diploid progenitors, but have the
complex genomes
• Increases in genome size and gene content- morphological and physiological differences between
allopolyploids and their diploid progenitors.

• Resynthesized Brassica napus allopolyploid lineage derived from a cross of diploid B. rapa and B.
oleracea
• Nine polyploid lines and their diploid parents were grown under four growth conditions and
measured for eight life-history traits.

30% were like one or the other
parent, 50% were intermediate, and
20% were transgressive 2719/07/2018 Mahesh R Hampannavar

Breeding scheme of N. obtusiata lines 1–5
and autotetraploids of N. attenuata and N.
obtusifolia. Allotetraploids: five emasculated
N. obtusifolia (No)
flowers were pollinated with excised N.
attenuata (Na) anthers

(A) leaves and (B) rosette-stage plants of N. attenuata (Na),
N. attenuata autotetraploid (NaT) (F5), N. obtusifolia (No), N.
obtusifolia autotetraploid (NoT) (F4), N. obtusiata (N o) (lines 1–5,
F5), N. clevelandii (Nc) and N. quadrivalvis (Nq). N o (lines 1–5)
leaves have long petioles and an intermediate parental shape. Nq and
Nc produce ovate-elliptical leaves with long and short petioles,
respectively. Synthetic polyploids rosettestage plants develop approx. 3
d before either parent (photographs were taken at the same stage).
Corolla limbs, flowers and seed morphologies of N. attenuata (Na), N. attenuata
autotetraploid (NaT) (F5), N. obtusifolia (No), N. obtusifolia autotetraploid
(NoT) (F4), N. obtusiata (N o) (lines 1–5, F5), N. clevelandii (Nc) and N. quadrivalvis (Nq). (A)

Changes in phenotype may be
• Subfunctionalization - evolution of partitioned ancestral functions among duplicate genes
• Neofunctionalization - evolution of novel functions among duplicate genes.

FUNCTIONALISATION
Neofunctionalization
• Neofunctionalization, one of the possible
outcomes of functional divergence, occurs when one
gene copy, or paralog, takes on a totally new
function after a gene duplication event.
• Neofunctionalization is an adaptive mutation
process; meaning one of the gene copies must
mutate to develop a function that was not present in
the ancestral gene.
Meso/sub functonalisation
one of the possible outcomes of functional
divergence that occurs after a gene
duplication event, in which pairs of genes that
originate from duplication, or paralogs, take on
separate functions. retaining different parts
(subfunctions) of their original ancestral function

AH. Paterson et al. Nature 492, 423-427 (2012) doi:10.1038/nature11798
Allelic changes between A- and D-genome diploid progenitors
and the At and Dt subgenomes of G. hirsutum cultivar Acala Maxxa.

Changes occurs - Soon after polyploidy
• newly synthesized allopolyploids usually exhibit greater genomic variation than natural allopolyploids.
• the frequency of lost parental sequences is generally much higher than the production of novel
sequences in newly synthesized allopolyploids.
• some sequences in neopolyploids change in a non-random fashion.

frequency of lost parental sequences is generally much higher than the production of novel sequences in newly
synthesized allopolyploids.
The overall genomic sequence changes of triticale were studied using AFLP and RFLP profiles of two octoploid and two
hexaploid triticales as well as their corresponding wheat and rye parents, F1 wheat–rye hybrids, and several early
generations of the re-synthesized triticale lines

• relationships between parental genomes are key factors in determining the direction, amount,
timing and rate of genomic sequence variation that occurred during inter-generic
allopolyploidization in this system.

Allopolyploidy-
induced
genomic
variation arises
from common
mechanisms
Homeologous
recombination
Homeologous
gene
conversion
small RNA
Transposon
activation

• It is a process that generates novel combinations of genetic material, eliminates deleterious
mutations and plays a role in DNA repair.
• During meiosis in plants, recombination predominantly occurs among allelic sequences on homologous
chromosomes.
• In allopolyploids which lack diploid pairing fidelity recombination may occur ectopically among
paralogous or homoeologous sequences.
• Homoeologous recombination may lead to reciprocal exchange and gene conversion, explaining many
of the small and large genetic changes detected in newly formed allopolyploids.
Homeologous gene Recombination

•Each of the four female parents, RV289, TO1147, MF216, and RV128,
were crossed with P1804. Two different F1 plants were used as the
sources for microspores for the (RV289 × P1804) population and a
single F1 plant was used for the other three populations.
•Haploid plants of each population were created from their respective
F1 through microspore culture
•The F1-derived DH populations are referred to as HUA (RV289 ×
P1804), MF (MF216 × P1804), and RV (RV128 × P1804), SYN (TO1147 ×
P1804).

• Gene conversion is the process by which one DNA sequence replaces a homoeologous sequence such that
the sequences become identical after the conversion event
• DNA strand transfer can occur followed by mismatch repair. This can alter the sequence of one of the
chromosomes, so that it is identical to the other.
• 1AA : 2Aa : 1aa
Homoeologous gene conversion
1AA : 2AA : 1aa
1AA : 2aa : 1aa

• Comparison of genomic sequences from several tetraploid
(AtDt) Gossypium species and genotypes with putative diploid A- and D-genome .
• Sequences of cotton D-genome v2 (G. raimondii), A-genome (G. herbaceum), and a tetraploid genome
(G. hirsutum, Acala ‘Maxxa’) are from Paterson et al. (2012).
• SNVs from the two progenitor genomes are identified by aligning reads from the A-genome to the
reference genome.
• For each of the SNV sites, the orthologous alleles in the tetraploid genomes are sorted into subsets
corresponding to the respective parental subgenomes

• Homeologous gene conversion events (HeGCEs)
• At-to-Dt conversion is far more abundant than the reciprocal, is enriched in heterochromatin.
• Dt-to-At conversion is abundant in euchromatin and genes, frequently reversing losses of gene
function.

• The long-standing observation that the
nonspinnable-fibered D-genome
contributes to the superior yield and quality
of tetraploid cotton fibers may be explained
by accelerated Dt to At conversion during
cotton domestication.

Role of repetitive sequence in polyploidy
● Red-centromeric tandem repeats;
● blue- telomeric repeats;
● yellow,-sub-telomeric tandem repeats;
● green,-intercalary tandem repeats;
● brown-ispersed repeats;
● white- genes and low-copy sequences.
Complexity in genomes : genome size, polyploidy and
repetitive DNA sequences, transposable elements
Repetitive DNA sequences comprise
- Large repeat sequences (>1Mbp)
- rDNA units (10 %),
- satellites (centromeres)
- Microsatellites/SSRs (1-10 bp)
-Telomeric sequences(short repeats

Estimated percentage of repetitive sequence in different crops
Crop Estimated repetitive
sequence
Rapeseed 35 %
Wheat 81 %
Strawberry 25-47 %
Potato 75 %
Banana 74 %
Cabbage 40 %
Soybean 59 %
• The key problem for current genome sequencing
is not the genome size per se, but the complexity of
the genomes, and in particular the number of short
repetitive sequences.
• difficult to uniquely place in a genome assembly.
• sequence collapse
• assembly of highly repetitive regions can results in
gaps and ambiguities that can lead to incorrect
interpretation of results

Role of small RNA in polyploidy
• Plant small non-coding RNAs include microRNAs (miRNAs),
small interfering RNAs (siRNAs) and trans-acting siRNAs.
• Small RNAs also function in RNA silencing and post-
transcriptional regulation of gene expression

• Among numerous rearrangements including inversions, duplications, deletions and insertions that
are stimulated in the genome as a result of polyploidization,
• inversions can easily produce the stem-loop structures that often form the basal structure of
miRNAs. As well, repetitive sequences are an important origin of small RNAs.
• small RNAs also can be produced by transposons.
• approximately 40% of miRNA families originated from tandem duplication events and/or
segmental duplication events.
Sun et al. (2012)

Role of Transposons in polyploidy
Transposons
Class I
(copy and paste)
LINE SINE
Class II
(cut and paste)
Subclass I
Tc1/mariner,
PIF/Harbinger, hAT,
Mutator, Merlin, Transib,
P, piggyBac and CACTA
Subclass II
Helitron and
Maverick
transposons

Sources of transposons in allopolyploidy
o From the diploid progenitors
o Normal evolutionary processes
o Transposable elements stimulated by allopolyploid formation

• Retrotransposons are a principal component of most
eukaryotic genomes, representing roughly 50–80% of
some grass genomes.
• RNA isolated from plants
• cDNA  PCR-AFLP  RNA bloting
Aegilopes
sharonensis
T.
monococcum
Synthetic
allotetraploidy

Analysis of the Wis 2-1A
retrotransposon activation Molecular characterization of chimeric transcripts

Current data on the reorganization of the transposable element (TE) genome fraction after allopolyploidy

Transposon activation may also lead to
• sequence losses in polyploid genomes, contributing to fractionation.
• High-frequency chromosomal recombination,
• Transposition
• repetitive-sequence-based changes.
• Formation of small RNA

Allopolyploid formation stimulates production of novel genomic
variation
Insertion or deletion of single nucleotides
chromosomal rearrangements
sequence loss and generation of novel sequences
Transposon activation
changes in cytosine methylation
 alteration of small RNAs,
 up- and down-regulation of gene expression
 gene silencing
 alternative splicing (AS)

Problems associated with genomic variation in
polyploidy crop improvement
• complicates measurements ….. phenotypic and genotypic variation.
• chromosome rearrangements severely interfere with the construction of
genetic maps, QTL mapping and marker assisted selection (MAS).
• hybridization and polyploidization disturb the positional relationships between
markers and hence affect the accuracy of genetic mapping.
• Formation of minor QTL

• Multiple mapping
• Use of cytogenetics
• Nanopore technology
• Multilocation testing
• Full reference sequences
Strategies for overcoming problems due to genomic
structural variation in polyploids

• Comparative genetic mapping approaches,
• where different genetic mapping populations are generated and compared using the same marker
set, can provide significant insight into genomic rearrangements based on shifts in marker location
between genotypes
1

•Each of the four female parents, RV289, TO1147, MF216, and RV128, were crossed with a male parent P1804.
Two different F1 plants were used as the sources for microspores for the (RV289 × P1804) population and a single
F1 plant was used for the other three populations.
•Haploid plants of each population were created from their respective F1 through microspore culture
•The F1-derived DH populations are referred to as HUA (RV289 × P1804), MF (MF216 × P1804), and RV (RV128 ×
P1804), SYN (TO1147 × P1804).
•RFLP and SSR markers

Characteristics of four genetic linkage maps and their
consensus map constructed using DH lines ofB. napus,
genotyped with RFLP and SSR markers

• Use of fluorescent in-situ hybridization to visualize the relative location of fluorescently-
labelled DNA probes can also help resolve chromosome rearrangements
2

• By using new cytogenetic tools to identify all
of the homoeologous chromosomes,
• conducted a cytological investigation of 50
resynthesized Brassica napus allopolyploids
across generations S0:1 to S5:6 and in the
S10:11 generation.

• Emerging technologies such as nanopore single molecule
• A nanopore-based device provides single-molecule detection and
analytical capabilities that are achieved by electrophoretically
driving molecules in solution through a nano-scale pore.
• The nanopore provides a highly confined space within which single
nucleic acid polymers can be analyzed at high throughput.
• Kilobase length polymers (single-stranded genomic DNA or RNA) or
small molecules (e.g., nucleosides) can be identified and
characterized without amplification or labeling
3
Nanopore single molecule sequence

• Principles for detection and base identification Nanopore sequencing uses electrophoresis to
transport an unknown sample through an orifice of diameter 10−9 meters, in magnitude.
• A nanopore system always contains an electrolytic solutions- when a constant electric field is applied,
an electric current can be observed in the system.
• The magnitude of the electric current density -
• nanopore's dimensions
• composition of DNA or RNA that is occupying the nanopore.
• samples cause characteristic changes in electric current density across
nanopore surfaces.

• Negative effect of repetitive sequences can be partially ameliorated by longer read
sequencing technology.
• When single sequence reads are longer, the chance that each read will contain a unique
sequence (“jumping” the repeat) will be greater.
• Nanopore sequencing technologies currently in development

• In order to detect and confirm minor QTL which result
from expression of homoelogs, repetitive testing and
large population sizes are required to enhance
phenotyping accuracy.
• As well as major QTL that can be more easily assessed,
minor QTL that are detected repeatedly in multiple
environments should be emphasized.
• Availability of fully completed reference genomes.
• Which helps to over come the currently problematic
with short-read sequencing technologies.
4

Conclusions
• Boon to agriculture
• Need of novel statistical and software tool
• Address the homeologous and repetitive sequences

Thank you

POLYPLOID CROP RESEARCH IN THE AGE OF GENOMICS

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