MOLECULAR BASIS OF HETEROSIS IN CROP PLANTS

ManjappaII Ph. D

Dept. of Genetics and Plant Breeding UAS GKVK Bangalore

II Seminar on

CONTENT

INTRODUCTION

HISTORY

MODERN VEIW OF HYBRID VIGOUR

GENETIC MODELS/THEORIES OF HETEROSIS

OMICS STUDIES OF HETEROSIS

MOLECULAR CLOCK MODEL FOR GROWTH VIGOUR

ROLE OF SMALL RNAs

EPIGENETIC REGULATION

PARENT OF ORIGIN EFFECT

EMERGING MODELS BASED ON ENERGY USE EFFICIENCY

CURRENT STATUS OF UNDERSTANDING OF MOLECULAR BASIS OF HETEROSIS

CONCLUSION

INTRODUCTION

Superior performance of heterozygous F1 hybrid plants in terms of increased biomass, size, yield, speed of development, fertility, resistance to disease and insect pest, or to climatic rigors of any kind compared to the average of their homozygous parental inbred lines (Shull, 1952 & Falconer, 1996)

Evolutionary definition: The heterozygotes have higher fitness in a population than the homozygotes

History

‘‘I raised close together two large beds of self-fertilised and crossed seedlings from the same plant of Linaria vulgaris. To my surprise, the crossed plants when fully grown were plainly taller and more vigorous than the self-fertilized ones.’’ - Charles Darwin

(The Effects of Cross and Self Fertilisation in the Vegetable Kingdom, 1876).

Documented growth, development, and seed fertility of cross-pollinated plants compared with the parents for more than 60 different species of plants.

Results: Inbreeding was generally deleterious & cross-fertilization was generally beneficial.

• George H. Shull: published ‘The composition of a field of maize (1908)’ which marked the rediscovery of hybrid vigor or heterois and the beginning of applying heterosis in plant breeding.

• Selfing maize plants led to reduction of overall growth vigor and yield.

• This was supported from maize inbreeding experiments by Edward M. East (1908)

• Term coined by “SHULL” in (1914) as “stimulation of heterozygosis ”• 1920s maize yield was increased by 6 fold.• 1964, Yuan, Long Ping, initiated research on hybrid rice and heterosis

in China• 1875: Wilson A S reported first hybrid between Wheat and Rhy in

Scotland, decade later W. Rhimpau produced first doubled fertile hybrid- Triticale.

• 1927: Karpchenko developed new species from hybrid between Raphanus and Brassica

In cotton AA progenitor species produce both lint fibers (long) and fuzz, where as DD progenitor species produce only few shorter lint fibers. The resulting allotetraploid G. hirsutum produce more abundant & higher quality fiber.

In Brassica napus has higher oil content and better oil composition than parents.

For viable hybrids, degree of heterosis is proportional to the parental divergence

Interspecific hybrids > Intraspecific hybrids Intersubspp. hybrid > intrasubssp. (rice) Not generalized to all hybrids (Maize) Genetic mechanism of heterosis different

between the species that are Self P or CP

Modern view of Hybrid vigor

The degree of heterosis may shift during different stages of growth and development

Eg: If growth vigor shown at seedling stage may show at reproductive stage also, but in some plants it is not.

Because they are controlled by different sets of genes and regulatory pathways.

Biomass heterosis is largely dependent on flowering time In B. napus late flowering is heterotic, whereas in maize

hybrids early flowering is heterotic, suggesting different effects of gene actions (repression or activation) on heterosis

single-locus heterosis in tomato could be controlled by a flowering locus T (FT)-like locus that regulates the transition from definite to indefinite inflorescence

Genetic, epigenetic & genomic changes

QUANTITATIVE DEFINITION

MID-PARENT HETEROSIS: It indicates that a trait displays hybrid performance that is significantly better than the average (mid-parent) value

Mid parent Heterosis (MH) = [ (F1- MP)/ MP ] x 100

BEST-PARENT HETEROSIS: Indicates that a hybrid trait performs significantly better than the better of two homozygous parents

Better parent Heterosis (BH) = [ (F1- BP)/ BP ] x 100

GENETIC MODELS FOR HETEROSIS

1. DOMINANCE MODEL: The dominance hypothesis explains heterosis by the complementing action of superior dominant alleles from both parental inbred lines at multiple loci over the corresponding unfavorable alleles, leading to improved vigor of hybrid plants (Davenport, 1908; Bruce, Keeble and Pellow 1910; Jones, 1917)

2. OVERDOMINANCE MODEL: Over-dominance hypothesis attributes heterosis to allelic interactions at one or multiple loci in hybrids that result in superior traits as compared to parents (Shull and East, 1908; Crown, 1948; Stuber, 1994)

East (1936): at a locus, A1, A2, A3, A4….. Each have different functions. A1A4 > A1A2 & it perform both allele function

3.PSEUDO-OVERDOMINANCE: The genetic intermediate of dominance and ODO is Pseudo-ODO, which is actually a case of simple dominance complementation, because of tight repulsion phase linkage and appears to be ODO (Stuber et. al., 1992; Graham et. al., 1997)Heterosis associated with this can dissipate in selfing progeny due to recombinationThis can arise from numerous alleles of recombination suppression regions

4. EPISTASIS MODEL : The epistasis hypothesis considers epistatic interactions between nonallelic genes at two or more loci as the main factor for the superior phenotypic expression of a trait in hybrids (Power, 1945)

Contd…

GENETIC MODELS FOR HETEROSIS

The absence of a decline in the magnitude of heterosis (Duvick, 2001) from improved inbred parents

In tetraploids inbreeding depression progresses much faster than homozygocity for recessive alleles (Dudley, 1974), allelic dosage have imp. Role.

The progressive heterosis in tetraploids: A1A1A2A2, A1A2A2A3 & A1A2A3A4 (Groose, 1989)

DOMINANCE MAY BE INSUFFICIENT

EVIDENCES LIMITATIONS

Heterozygous individual may have an advantage due to the combination of both allozymes (Falconer and Mackay, 1996)

Role of single genes in the manifestation of heterosis for various traits in Arabidopsis, Cereals and Tomato (Redei, 1962; Gustafson,1946; Dollinger, 1985; Semel et. al., 2006; Krieger, 2010)

EXAMPLES OF ODO GENESSFT Gene in TomatoErecta mutant in Arabidopsis

For ODO to produce superior phenotypes, single gene or small genomic regions are needed which seem contradict to the hybrid performance of many agronomic important traits controlled by multiple genes (Lippman and Zamir, 2007)

Though evident as examples of overdominance, it is possible that they involve dosage effects on regulatory networks that are not incompatible with the concept of multigenic control (Birchler , 2010)

IS OVER-DOMINANCE SUFFICIENT TO EXPLAIN HETEROSIS

The interaction of favorable alleles at different loci contributed by the two parents, which themselves may show additive, dominant and overdominant action (Powers, 1945, Yu et. al., 1997; Monforte and Tanksley, 2000; Li et. al., 2001; Luo et. al., 2001)

The genetic background and allelic interactions can have an effect on the heterotic contributions of individual loci

Recently demonstrated in tomato introgression lines that heterosis is manifested even in the absence of epistasis (Semel, et al. 2006)

EPISTASIS AS GENETIC MODEL FOR HETEROSIS

Except few examples of heterozygote advantage and single locus heterosis none of the genetic models on their own adequately explain the evidence regarding heterosis

sickle cell anaemia: homozygous recessive mutant- disease, heterozygous individuals: some susceptibility & more tolerant to malaria infection than normal individual allele

A. thaliana: erecta and augustifolia loci, encode regulatory proteins: erecta encodes a receptor-like kinase and augustifolia encodes a transcription factor. These have pleiotropic effects on phenotypes, including ‘erect stem’ and early flowering in the erecta mutant.

Single-locus heterosis

The flowering gene SINGLE FLOWER TRUSSdrives heterosis for yield in tomato

Kriwger et al, Nature Genetics,2010, 42, 459–463

First example of a single overdominant gene for yield Heterozygosity for tomato loss-of-function alleles

of SINGLE FLOWER TRUSS (SFT), which is the genetic originator of the flowering hormone florigen, increases yield by up to 60%

OD is robust, occurring in distinct genetic backgrounds and environments

• several traits integrate pleiotropically to drive heterosis in a multiplicative manner, and these effects derive from a suppression of growth termination mediated by SELF PRUNING (SP), an antagonist of SFT.

SFT-dependent heterosis arises from multiple phenotypic changes on component traits that integrate to improve yield.

• Improved heterosis as heterozygosity levels between the parents increase

• allopolyploids have higher heterosis than autopolyploids, and tetraploids have higher heterosis than diploids

• Effects in tetraploid hybrids could be due to an increased genome dosage, allelic heterozygosity and/or epigenetic changes

• Current genetic models cannot explain progressive heterosis

Polyploidy and progressive heterosis

Omics studies of heterosis

Transcriptomics Proteomics Metabolomics

Intraspecific hybridsA. thaliana, rice, maize wheatallopolyploid Arabidopsis cotton wheat, Senecio TragopoganResults:Some changes are additive and others are non-additive

TranscriptomicsGenome-wide changes in gene expression

• Although transcriptomic changes are complex, Some trends emerge,

1. Additive and non-additive gene expression changes are more correlated with genetic distance than genomic dosage:

• Non-additive gene expression is more common in interspecific hybrids than in intraspecific hybrids

• Nucleolar dominance, epigenetic silencing of the rRNA genes from one parent in interspecific hybrids of plants and animals

• Expression analysis was conducted by using AFLP cDNA in synthsised allopolyploid, natural Allopolyploid and their proginaters A. thaliana and A. arenosa

• rRNA genes from one parent are silenced in allopolyploid by mechanisms including DNA methylation, histone modifications and small RNAs

• Direction of transcriptomic dominance: A. arenosa genes are dominant over A. thaliana genes in Arabidopsis allotetraploids

Gene silence

Novel expression pattern

differential gene expression

Random expression change

2. Alteration in bilological networksIn Arabidopsis allotetraploids, non-additively expressed genes are enriched in fallowing pathways

• The majority of nonadditively expressed genes in the synthetic allotetraploids displayed expression divergence between the parents and were involved in various biological pathways

• Material: A. thaliana isogenic autotetraploids, diploids, A arenosa & synthetic allotetraploid lines

• used spotted 70-mer oligo-gene microarrays• Results:• 15% transcriptome divergence between the progenitors• 1362 (5.2%) and 1469 (5.6%) genes were expressed

nonadditively in Allo733 and Allo738, respectively

High level expression

Number of genes withexpression divergence

Chromosomal distribution of the 820 genes displaying nonadditive expression in both allotetraploids

Transcriptome divergence and nonadditive gene expression between allotetraploids and their progenitors

• Majority of nonadditively expressed genes in the allotetraploids display expression changes between the parents

• 65% of the nonadditively expressed genes in the allotetraploids are repressed

• 94% of the repressed genes in the allotetraploids match the genes that are expressed at higher levels in A. thaliana than in A. arenosa

• This is consistent with the silencing of A. thaliana rRNA genes subjected to nucleolar dominance and with overall suppression of the A. thaliana phenotype in the synthetic allotetraploids and natural A. suecica.

• This suggests transcriptome & phenotypic dominance of A. aerinosa over A thaliana

The 820 genes detected in both Allo733 and Allo738 lines (Allos) were classified into 15 functional categories

Nonadditive gene regulation occurs in various pathways

Progenitor-dependent repression of the genes involved in the ethylenebiosynthesis pathway

Fold change in expressionAllos

Allos-738Allos-733

Proginaters

3. Expression of genes involved in cell ageing, stress response and phytohormoe signalling pathways: These have changed in allopolyploids of

Arabidopsis, wheat and cotton, and in maize hybrids

A. thaliana: negative correlation between defence responses and growth and fitness.

overexpressing ACCELERATED CELL DEATH 6 (ACD6) reduces growth, whereas biomass is increased in the acd6 mutant

Arabidopsis allotetraploids: Repression of stress-responsive genes, (ACD6) and ethylene signalling and regulatory genes is observed

4. Genome-wide changes in gene expression in interspecific hybrids and allopolyploids can result from cis- and trans-regulatory divergence between hybridizing species Arabidopsis F1 allotetraploids and their progenitors: more genes

have cis-regulatory changes than trans-regulatory changes • lllly In interspecific hybrids of Drosophila & yeast & in

intraspecific hybrids of maize • A. arenosa trans-acting factors have greater effects than A.

thaliana factors on allelic expression in F1 allotetraploids • >90% of genes, regulated by both cis and trans effects, the

effects are ‘compensating’ (that is, reducing expression divergence) rather than ‘enhancing’ (that is, increasing expression divergence)

• Some genes with enhancing effects are associated with stress responses

• genes with compensating effect are related to biosynthetic and metabolic processes

PROTEOMICS• Additive and non-additive proteomic patterns have been

found in the embryos, roots and nuclei and mitochondria of ear shoots of maize hybrids, in mature embryos of rice hybrids and in the leaves of Arabidopsis autopolyploids and allopolyploids.

• Findings: Majority of these belongs to functional classes of stress response,

• Maize hybrid Zong3/87-1 exhibited an earlier onset or heterosis in radicle emergence

• The dry and 24 h imbibed embryos detached from seeds were used for protein extraction

• Differential proteomic analysis between hybrid and its parental lines was performed

additive expression

++: above high parent expression

+: high parent expression

–: below low parent expression

–: low parent expression

+/2: partial dominance expression

D: different from additivity

• differentially expressed protein spots in dry and 24 h imbibed seed embryos were 134 and 191.

• 47.01% (63/134) and 34.55% (66/191) protein spots displayed nonadditively expressed pattern.

• 54.55% of nonadditively accumulated proteins in 24 h imbibed seed embryos displayed above or equal to the level of the higher parent patterns

• 155 differentially expressed protein spots were grouped into eight functional classes, energy & metabolism, signal transduction, disease & defense, storage protein etc.

• Other Reports • More isoforms or allelic variants exist in maize hybrids with

high or low levels of heterosis than in their parents, suggesting transgressive effects. Some are respond to stresses (Dahal et al, 2012)

• Non-additively accumulated proteins or peptides do not necessarily match non-additively expressed genes. Suggests there are changes in post-transcriptional and translational regulation in hybrids and polyploids

• In A. thaliana intraspecific hybrids, biomass heterosis is correlated with increased levels of metabolic activity

METABOLOMICS

The maternal contribution of nutrients and metabolites to growth vigour is consistent with the parent-of origin effect on biomass vigour in ‑reciprocal hybrids

• 14–20 metabolites were sufficient to predict freezing tolerance among different F1 hybrids, and they explained 60% of the variance

• compatible solutes in the pathway leading to raffinose are crucial indicators of freezing tolerance heterosis

• Limited numbers of particular metabolites provide useful ‘biomarkers’ for the prediction of heterosis (Korn et al, 2010)

Central oscillators of circadian clock and their diurnal expression pattern

CCA1 BINDING SITE-AAAATATCT OR EVENING ELEMENT

POR a & POR b STARCH METEBOLISM

•Daytime Specific Repression-CCA1 & LHY & Upregulation- TOC1 & GI

•Histone Modification (H3K9 & H3K4)

•CCA1 like genes in Maize Hybrids

CCA1 Function under the control of TOC1

Flowering time, Metabolism & Plant growth

(Chen et . al., 2010)

MOLECULER CLOCK MODEL FOR GROWTH VIGOR

Circadian clock

Time keepers

Regulates - 15% genes 90% of transcriptome

Arabidopsis Allopolyploid: 130 genes

(A MOLECULAR MECHANISM FOR HYBRID VIGOR)

Input signals Allelic expression variation

Induced expression of CCGs

Fine Tuning of Expression Amplitude

GROWING AROUND THE CLOCK

Chl synthesis & starch metabolism

•Many life history traits map at CCA1 element. (Pl ht., leaf no., leaf length)•MAIZE HYBRIDS:CRY2 locus lies in the vicinity of CCA1-candidate of fruit length & ovule number•Indicates the role of epistatic interaction among CCA1, CHY and CRY2•Gene Regulation•Domestication of maize-evolution of Apical dominance from teosente

Locus teosente (tb1)

Represses axillary organ

Promotes female inflorescence

•Domestication of tomato--------- s locus mutant & an locus mutant

Compound inflorescence and highly branched

Wild Tomato: Few flowers

Apical dominance and branch formation are controlled by few regulatory genes, suggesting molecular basis of single locus heterosis

Do changes in circadian clock genes affect other traits in hybrids ?

Most siRNAs are derived from TEs & repeats, thus have diverged between sp.

Differences in siRNA levels between hybrids or allopolyploids and their

parents could alter allelic patterns of expression, RNA directed DNA

methylation (RdDM) and overall genomic stability

siRNAs generated by the RNA interference pathway can target homologous

genomic DNA sequences for cytosine methylation through a process called

RdDM (Henderson and Jacobsen, 2007; Law and Jacobsen, 2009, 2010)

siRNA show expressional variation in allotetraploids and hybrid as compared

to their parents.

24-nucleotide siRNAs guide the de novo methyltransferase DRM2 to

homologous loci to establish DNA methylation, which leads to transcriptional

silencing (Law and Jacobsen, 2009, 2010)

ROLE OF SMALL RNAS

H Addition of methyl group to cytosine residue (CG, CHG & CHH)

(H= A,C or G )

In plants, CG and CHG methylation is maintained by DNA

METHYLTRANSFERASE1 (MET1) and CHROMOMETHYLASE3 (CMT3)

CHH methylation by DOMAINS REARRANGED METHYLTRANSFERASE

(DRM2)

Greatest increase at CG site is 18-26% in parents to 36-37% in

hybrids

Degree of methylation changes in hybrids depends on parental

divergence (higher in diverse parents)

(Law and Jacobsen, 2010)

EPIGENETIC REGULATION

DNA Methylation

H Silencing mechanism predominant in transposons and repetitive

DNA elements

>96% of methylation increase in hybrids corresponds to siRNA

generating regions that are divergent between parents (RdDM)

In maize, pericentromeric regions with recombination

suppression are under strong selection in inbred lines, and the

genetic divergence between these regions is predicted to affect

heterosis (this region probably contain divergent TEs & genes

affected by siRNA & RdDM) ---(Gore et al, 2009)

Clock genes including CCA1 & LHY are unregulated in DNA

methylation mutants (CCA1 & LHY repressed normally)

• In Arabidopsis allotetraploids, expression peaks of CCA1 and LHY and of their regulators TOC1 and GI are altered relative to the progenitors and are positively associated with levels of histone H3 lysine 9 acetylation (H3K9ac) and H3K4 dimethylation (H3K4me2)

siRNA (RdDM)

HISTONE MODIFICATION

• These effects could be associated with a major group of Pol IV dependent siRNAs (p4 siRNAs), as these siRNAs are ‑ ‑maternally transmitted.

• The expression of maternal p4 siRNAs correlates negatively ‑with the expression of a group of AGAMOUS-LIKE (AGL) genes that encode type I MADS-box transcription factors which are expressed in endosperm and are involved in regulating seed size

x x

PARENT-OF‑ORIGIN EFFECT

Tetraploid Diploid Diploid Tetraploid

Small Seed Large Seed

High siRNA level Low siRNA levelAGL AGL

Molecular changes at epigenetic, genomic, proteomic and metabolic levels lead to heterosis traits.

Mixing of two distant genomes brings about cis, trans, and chromatin level changes in the hybrid, which results in differential expression of genes. These expression patterns, which might represent additive or non additive modes of gene action, may affect major regulatory pathways which, in turn, send out regulatory cues that cumulatively affect a number of downstream metabolic pathways in either a positive or a negative manner. These individual pathways, whether placed on the input side or the consumption side of the energy equation, affect various aspects of growth and development. The net positivity or negativity in the system, therefore, would define the state and extent of heterosis

EMERGING MODEL BASED ON ENERGY USE EFFICIENCY

(Goff et al., 2011)

EnergyBiomass = Energyinput - Energyconsumed

CURRENT STATUS OF UNDERSTANDING OF THE MOLECULER BASIS OF

HETROSIS

(Baranwal et al., 2012)

Heterosis is result of interacting genomes, resulting in complex changes

at the genetic, epigenetic, biochemical and regulatory network levels

Epigenetic regulation of circadian-mediated changes in chlorophyll

biosynthesis and starch metabolism offers one of the direct links to

growth vigor in plant hybrids

Availability of novel genetic and genomic tools, that allow for the

integrated study of the complex interactions between genome

organization and expression might contribute to a better

understanding of heterosis.

CONCLUSION

Thanks for patience listening