WELCOMED

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Rahul Kumar

Roll no.-10477

Division of Vegetable Science

Indian Agricultural Research Institute

New Delhi

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Heterosis breeding-Classical and Molecular concepts

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Phenotypic manifestation of heterosis. In

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

HistoryIn

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Heterosis was first described by Charles Darwin (Darwin1876) and independently rediscovered by Shull (1908) andEast(1908).

Term coined by “SHULL” in (1952) as “ stimulation ofheterozygosity”.

After maize hybrid was first utilized in field on a largescale in USA in 1930s.

1st Hayes and Jones (1916) reported hybrid vigor forcucumber mainly contributed to notable increasing of fruitsize and number.

F1 hybrid of brinjal was utilized before 1925 in Japan(Kakizaki , 1931)

QUANTITATIVE DEFINITIOND

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a

1/2

P1 F1 P2

b

P1 Additive Partially dominant* Dominant* P2 Overdominant*

www.annualreviews.org • Heterosis in Crop Plants 75

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Midparent heterosisBetter-parent (or high-parent) heterosis

1/2

Heterosis and additive and non-additive gene expression

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Genomic and epigenetic insights into the molecular bases of heterosis•Z. Jeffrey Chen

Nature Reviews Genetics- 14, 471–482 (2013)

This explains high-parent or low-parent heterosis

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GENETIC MODELS FOR HETEROSISIn

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Complementing action ofsuperior dominant alleles fromboth parental inbred lines atmultiple loci over thecorresponding unfavorablealleles, leading to improvedvigor of hybrid plants

Allelic interactionsat one or multipleloci in hybridsthat result insuperior traitsTomato rin mutant

A simple case of dominance complementation, in which the two recessive mutations (‘a’ from P1 and ‘b’ from P2) are linked in trans, or ‘in repulsion’.

Epistatic model for heterosisIn

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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

(Powers 1945).

Discussion on dominance modelIn

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AA CC EEbb aadd cc ddee BB*

Aa Cc EeBb dd

P1 P2

F1

Cancelling of deleterious

or inferior alleles

Heterosis depend on

number of dominant

genes.

Both parents should

differ in dominant genes.

Complementation across

loci must be cumulative.

(Coors and Panday,1999), to

produce a superior

phenotype.

– Dominance is considered

more popular one (Charles

worth and Willis, 2009).

Dominance may be insufficientIn

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Rapid rate of inbreed—ingdepression in tetraploids(Dudley, 1974)

The progressive heterosis in tetraploids

(Groose, et al.1989)

Is the simple complementationresponsible for heterosis

Several evidences suggestthat mechanisms beyondsimple complementation maybe important in heterosis.

The absence of a decline in the magnitude of

heterosis from improved inbred parents

(Duvick 2001)

Is over-Dominance sufficient to explain heterosis

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EVIDENCES LIMITATIONS

Heterozygous individualmay have an advantage dueto the combination of bothallozymes (Falconer and Mackay,1996)

Role of single genes in themanifestation of heterosisfor various traits inArabidopsis and Tomato(Redei, 1962; Semel et. al., 2006;Krieger, 2010)

EXAMPLES OF ODO GENESSFT Gene in TomatoErecta mutant inArabidopsis

For ODO to produce superiorphenotypes, single gene orsmall genomic regions areneeded which seem contradictto the hybrid performance ofmany agronomic importanttraits controlled by multiplegenes (Lippman and Zamir, 2007)

Though evident as examples ofoverdominance, it is possiblethat they involve dosageeffects on regulatory networks(Birchler , 2010)

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teHemizygous

complementation

Complementation of present–

absent genes

Hemizygous complementation of

many such genes with minor

quantitative effects in hybrids

might thus lead to a significantly

increased performance of hybrid

plants.

Progressive heterosisIn

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Progressive heterosisrefers to the fact thatdouble cross hybridautotetraploids (ABCD)typically show greatervigor than single crosshybrids (AABB; CCDD andso on).

Increased allelic diversity creates a more robust heterotic response.

Quadruplex hybrid, whichcontains potentially fourdifferent alleles per locus,exceeds even that of thehybrids A–B and C–D.

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There is a genomic dosage effectoperating on heterosis

Relative gene expression levels in hybrids and regulation of allele-specific gene expression in hybrids

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te .cis-regulation reflect the

relative expression levels of

the parental inbred lines in

the allelic ratio of gene

expression in the hybrid.

• trans-acting factors show

equal expression of the two

alleles in the hybrid.

EPISTASIS AS GENETIC MODEL FOR HETEROSIS

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

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

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

Central role of the circadian clock in plant growth and development

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Internal time keepers or circadian clock Regulators CCA1 - CIRCADIAN CLOCK ASSOCIATED 1LHY- LATE ELONGATED HYPOCOTYL TOC1 TIMING OF CAB EXPRESSION 1

in a major negative feedback loopGI – Gigantia CK2- protein kinase

NADPH oxidases (NOX proteins) PSEUDORESPONSE REGULATOR (PRR) 3 5 7 and 9

ZEITLUPE (ZTL),Phytochromes (PHYs) and cryptochromes (CRYs)

JMJD5 encodes a histone demethylase and activates the morning-phased clock genes CCA1 and LHY

NADPH oxidases (NOX proteins) activate CCA1, LHY and GI, and PCL1 represses PRR9

Protein kinases affecting CCA1 binding affinity and function and leading to temperature compensation for the clock

The central clock regulators CCA1 and LHY mediate output pathways regulate genes in various biological pathways, such as flowering

The circadian clock also regulates hypocotylgrowth, through repression mediated by an evening protein complex

Growing around the clock: a molecular mechanism for hybrid vigor

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te Diagram of CCA1 and LHY (red line) and TOC1 (green line) expression rhythms in a 24-h clock with 16 h of light (open bar) and 8 h of darkness (filled bar).

Period is the time forcompleting one cycle of rhythms and is shown from one peak to another (or form one trough to another).

The expression amplitude of rhythm is defined as one-half the distance between the peak and trough.

Epigenetics asA cause of heterosis TYPES

DNA METHYLATION

HISTONEMODIFICATION

RNAINTERFERANCE

siRNAs, miRNAs etcCHROMATIN REMODLING

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19

“Epigenetics” refers toheritable (through mitosis ormeiosis) alterations in geneexpression that areindependent of DNA sequence:different epigeneticallyregulated forms of a gene areknown as epialleles.

Chromatin status, mediatedthrough epigeneticmodification, can potentiallyaffect gene expression in cis (atthe gene itself) or in trans (byregulating loci indirectly).

DNA methylation and heterosisIn

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Conversion of cytosine to 5 methyl cytosine.Could generate epigenetic variation/ Epialleles and creation

of hybrid vigour.

DNA methylation does not change the DNA sequence and itsfunction, but does change its expression level, referred asan epigenetic change.

Associated with gene silencing, and genes with abundant 5-methylcytosine in their promoter region are usuallytranscriptionally silent. (Jones and Takai, 2001; Dong etal,2006)

It can be suggested that inbreeding depression partly or primarily resultsfrom lower levels or fewer genes expressed simply due to homozygosityof methylated DNA in regulating factors.

Heterosis is from higher levels or larger number of genes expressedsimply due to heterozygous conditions between methylated and non-methylated DNA in the F1 hybrid.

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teMolecular changes at epigenetic, genomic, proteomic and

metabolic levels lead to heterosis traits

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Small RNAssmall interfering RNAs (siRNAs)

Mediate post-transcriptional gene silencing,RNA-directed DNA methylation, and chromatinremodeling.

These RNAS are negative regulators of targettranscript accumulation (Allen et al.,2005).

miRNAs and siRNAs are differentiallyexpressed between hybrid and its parentalinbred lines (Mica et al.,2006).

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teA model for small RNAs in the allelic expression of genes and

transposable elements in hybrids and allopolyploids.

Silenced

Expressed

RNA Directed DNA methylatin

Reduced vigour Increased amount of si RNA

cis and trans acting effect

TE –Transposable element, siRNA-Small interfering RNA ,

Gene activation of parent 1

Gene silencing of parent 2

QTL AND HETEROSISIn

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Molecular breeding may act one of the promising approach tounreveal genetic basis of heterosis.

Mainly used to identify genes or genomic regions that contributeheterosis for trait of interest, that may be used in MAS toincrease performance of hybrids.

Provide answer to certain questions.

Which genes are involved and their nature?

Epistatic properties of these genes?

Their interaction with environment?

How best to exploit heterosis fully?

Number of genes or genomic regions involved and their distribution?

(Coors and Panday,1999)

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teNumerous QTLs with different levels ofdominant, over dominant, and epistatic effects

have been mapped for heterosis in

Tomato (Semel et al., 2006),

A. thaliana (Hua et al. 2003;Kusterer et al., 2007;Melchinger et al., 2007;Meyer., et al 2010).

Besides the involvement of various gene actions found inthese studies, all the three gene actions may conditionheterosis in crops (Li et al, 2008; Swanson-Wagner, et al2006).

EMERGING MODEL BASED ON ENERGY USE EFFICIENCY

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EnergyBiomass = Energyinput -Energyconsumed

Mixing of two distant genomesbrings about cis, trans, andchromatin level changes in thehybrid.

Differential expression of genes.

Additive or non additive modes ofgene action

May affect major regulatorypathways

Regulate downstream metabolicpathways in either a positive or anegative manner.

Case study 1 In

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Precocious shoot termination in determinate tomatoes is partially suppressed by sft/+ mutant

heterozygosity.

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Indeterminate

leaf production to decrease

(Red: fully ripe fruit; orange: ripening fruit; green: unripe fruit; yellow: flowers) Arrows represent canonical axillary shoots.

Asterisks

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Delay in precocious termination.

Colored bars indicate average leaf numbers within sympodial units with standard deviations. Statisticalsignificance in B and C was tested by Wilcoxon rank sum test, and significance levels are indicated byasterisks (*P,0.05, **P,0.01, ***P,0.001). doi:10.1371/journal.pgen.1004043.g001

Continued….

sft/+ heterozygosity induces weak semi-dominant delays in both primary and sympodial

flowering transitions.

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Note the extremely delayed flowering of sft sp double mutants, indicating a weak semi-dominant effect for sft/+ heterozygosity.

sft/+ sp plants show slightly delayed primary shoot flowering time compared to sp as measured by leaf production before formation of the first inflorescence.

Statistical differences were tested by Wilcoxon rank sum tests and significance levels are marked by asterisks (***P,0.001). {sympodial inflorescence meristems (SIM)}

the SYM of transitioning or initiating the first SIM, indicating a developmental delay parallel to the PSM of

(B–G) Representative images and quantification of developmental progression (ontogeny) of meristems in the first inflorescence and sympodial shoot meristems (SYM) of sp (left images) and sft/+ sp plants (right images) at 20th DAG.

SYM of sp mutants completed the flowering transition and differentiated into the first or second FM and initiated the next SIM,

Delayed sim

Transcriptome profiling reveals a semi-dominant delay in meristem maturation from sft/+ heterozygosity.

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EVM- Early Vegetative MeristemsMVM -Middle Vegetative MeristemsLVM -Late Vegetative MeristemsTM - Transition MeristemFM-Flower Meristem

TM - Transition Meristem First sympodial shoot meristem (SYM)

DDI quantification of SYM maturation scores indicate an intermediate maturation

TM maturation state indicating sft/+ heterozygosity causes a semi-dominant delay in the primary flowering transition.

Semi dominant delay

Intermediate maturation

Case study 2In

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Distribution of QTL mode of inheritance for tomato traits.

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QTL341

QTL382

QTL118

QTL classified as dominant means that both the IL (homozygous for the S. pennellii

allele) and the ILH (heterozygous) were very similar to each otherA recessive QTL means that only the IL is significantly different from M82, whereas the ILH is similar to M82.Additivity reflects a situation in which the ILH is in between its parents

ODO is inferred where the ILH is significantly higher or lower than both its parents.

The frequency distribution of the mode-ofinheritanceindex for QTL in the reproductive and nonreproductive groups.

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The ‘‘reproductive’’ curve (QTL for increasing reproductive traits) has a peak in the ODO domain,indicatingthat many of the QTL fall within this mode of inheritance

In contrast, most of the QTL for the nonreproductive group and for the decreasing reproductive phenotypes resided in the recessive–additive domain.

Heterosis is partitioned, in part, into small genomic regions that convey advantage in the heterozygous state (ODO QTL), and, together, they contribute to the genome-wide effect

Seed no and fruit per plant =Reproductive fitness

CONCLUSIONIn

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te 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.