Molecular Basis of Inbreeding Depression and Heterosis in Crop Plants Surendra Singh

1. Molecular Basis of Inbreeding
Depression and Heterosis in
Crop Plants
Speaker : Surendra Singh
ID. NO.- 40925

2. Development of Heterosis Concept
• 1766 Kolreuter – Hybrid vigour in Nicotiana
• 1799 A. Knight – Principle of anti-inbreeding
• 1828 Wiegmann – Described heterosis in Crucifers
• 1876 Darwin- First reported inbreeding depression and heterosis
in maize
• 1880 Beal – First published report of 51% increase in yield over
parents between open pollinated varieties
• 1891 Johnson – Crossing gave better off-springs
• 1892 McCleur – Inbreeding imparted sterility crossing
imparted vigour
• 1908 Davenport - Dominance theory
• 1908 East and Shull - Overdominance theory
• 1914 Shull – Coined the term heterosis
• 1918 Jones – Practical utilization of heterosis
• 1922 Burr Leaming Dent first hybrid in maize released
in USA

3. INBREEDING DEPRESSION

4. Estimation of Inbreeding Depression
 Inbreeding depression defined as the reduction or loss in vigour and fertility by
the consequence of inbreeding.
 Estimation of inbreeding depression
 Inbreeding depression= (F1 –F2/F1)x 100
 Inbreeding
Inbreeding is refers as mating between individuals related by descent or ancestry.
a. Selfing
b. Sib mating

5. Consequences of Inbreeding
 Appearance of lethal and sub lethal genes
 Reduction in vigour
 Fixation of genotypes
 Increase homozygosity
 Reduction in reproductive ability
 Reduction in yield

6. Degree of Inbreeding Depression
• High inbreeding depression: e.g., Alfalfa, Carrot etc.
• Moderate inbreeding depression: e.g., Maize, Sorghum etc.
• Low inbreeding depression: e.g., Onion, Cucurbits, Sunflower
etc.
• No inbreeding depression; in self pollinated plants

7. Homozygous and Heterozygous Balance
 Cross fertilized species are highly heterozygous.
 These species carry a large number of unfavorable recessive alleles.
 Sum total of these unfavorable alleles constitutes genetic load of the species.
 Harmful effect of such recessive alleles masked by their dominant alleles.
 Genetic organization favours heterozygosity
Heterozygous balance.
 Self fertilized species are naturally homozygous.
 No genetic load because unfavorable genes become homozygous and
eliminated from the population.
 Genetic organisation is adapted to homozygosity –
Homozygous balance

8. HETEROSIS
Heterosis describes that the
superiority of heterozygous
hybrid over its both homozygous
parents in terms of yield and
some other characters such as
fertility, vigour and growth etc.

9. Terminology
 Hybrid Vigour (Jones, 1918) Synonyms to heterosis. But hybrid vigour
describes superiority of hybrids over their parents while heterosis describes
both negative and possitive situations.
 Heterobeltiosis (Fonesca and Patterson, 1968) heterobeltosis the F1 superior
to the better parent
 Economic heterosis or Standard heterosis; The heterosis in relation to the
best commercial variety of the crop.
 Euheterosis or true heterosis (Dobzhansky, 1950)
“True heterosis only when the hybrid possessed higher fitness than their
parents”.

10. Luxuriance (Dobzhansky, 1950). Luxuriance defined as increased vigour
and size of interspecific hybrids but not fitness. Principle difference between
heterosis and luxuriance lies in the reproductive ability of hybrids.
Positive and Negative Heterosis (Powers,1944). Superior expression of the
hybrid was termed as positive heterosis such as quality traits etc. Inferior
expression of the hybrid in relation to the parents is called negative heterosis
such as plant height, anti-nutritional factors etc.
Adaptive heterosis (Dobzhansky, 1950). Adaptive heterosis used in case of
adaptability of hybrids in different environmental conditions.
Conti……

11. QUANTITATIVE DEFINITION
MID-PARENT HETEROSIS: It
indicates that a trait displays hybrid
performance that is significantly better
than the average(mid-parent) valve.
BETTER-PARENT HETEROSIS:
Indicates that a hybrid trait performs
significantly better than the better of
two homozygous parents.
USEFUL HETEROSIS
Indicate superiority of hybrids over
the standard commercial check.
Meredith and Bridge, 1972
AA BB AB(case1) AB(case2)
Offspring
Parents
Mid –parent
heterosis
Better- parent
heterosis
Performance
Better-parent
Mid-parent
Mid parent Heterosis (MH) = [ (F1- MP)/ MP ] x 100
Better parent (BP) = [ (F1- BP)/ BP ] x 100

12. Effect of heterosis
 Increased yield
 Increased fertility
 Increase size and general vigour
 Earlier flowering and maturity
 Greater resistance to insect and pest
 Greater adaptability
 Faster growth rate

13. Theories of Heterosis
 Genetic basis of heterosis
Dominace hypothesis
Overdominance hyphothesis
Epistasis hyphothesis
 Biometrical genetic explanation of heterosis
 Physiological basis of heterosis
 Molecular basis of heterosis

14. Genetic Basis of Heterosis

15. Dominance Hypothesis
 Dominant allele have favorable effect while the recessive allele have
unfavourable effect
 In heterozygous state deleterious effects of alleles are masked by their
dominant allele
 Heterosis is due to masking of deleterious recessive alleles by dominant
alleles
 Heterosis not result the heterozygoity
Davenport, 1908

16. Inbred A Inbred B
AABBccdd x aabbCCDD
with 2 dominant gene with 2 dominant gene
F1 –AaBbCcDd
with 4 dominant gene
Heterosis: dominance hyphothesis
Conti….

17. Objections
• If the heterosis is true it should be possible to obtain pure heterotic
individuals in F2 which are homozygous for all the genes.
• In second objection is that heterosis due to dominance, F2 Curve should be
skewed towards dominant gene but the curve always found smooth and
symmetrical.
Explanations:
• Jones (1921) suggested that there may be linkage between some favourable
dominant and unfavourable recessive gene.
• Collins (1921) traits like yield is governed large number of genes or
polygenes which exhibit continuous variation.

18. 0
1
2
3
4
5
6
7
Progressive heterosis is a phenomenon in
polyploid plants that is critical to
developing a viable model of heterosis
In autotetraploids, crosses between
homozygous tetraploid lines will produce
single cross hybrids (AABB and CCDD)
that exhibit heterosis. However, if different
single cross hybrids are mated that have
originated from different parents (for a total
of four grandparental lines) to produce a
double cross hybrid (ABCD), the heterotic
response is almost always superior to the
single cross tetraploid hybrids.
PROGRESSIVE
HETEROSIS
Increased allelic diversity creates a more
robust heterotic response.
Birchler et al., 2010

19. OVER-DOMINANCE
Heterozygotes at atleast some of loci are superior to
both the relevant homozygotes.
Heterozygosity essential for heterosis and while
homozygosity occur due to inbreeding responsible
for inbreeding depression.
East (1936) postulated that divergence of alleles
brought together in heterozygotes, tend to increase
the vigour of heterozygote.
*
P1 P2
A a
B B
F1
A
A
a
a
b
b
b
B
A1A4>A1A3>A1A2------------- so on
OVER-DOMINANCE

20. Pseudo Over-dominance
 Jones (1917) first pointed out that linkage
could cause considerable problems when
attempting to identify overdominance,
which gives rise to pseudo-overdominance.
 In that case, the pair of linked loci would
mimic a single, over dominant locus, thus
skewing a measure of true overdominance
when combined in the hybrid.
 Pseudo-overdominance can dissipate in the
selfing progeny Semel et. al., 2006 .
A
A
B
A
B B
a a
b
b
a
b
PSEUDO-OVERDOMINANCE

21. EPISTASIS AS GENETIC MODEL FOR HETEROSIS

 The genetic background and allelic
interactions therein can have an
effect on the heterotic
contributions of individual loci.
 Nuisance for the plant breeder
regarding genetic base debate.
TYPES OF
EPISTASIS

22. Biometrical basis of heterosis
 Heterosis depends directly on the existence of dominance and intraction
involving dominance at different loci.
Additive- Dominace model = ∑h˃∑d
 Heterosis is maximum when
- all the genes show directional dominance to a trait.
- dominant allele dispersed in both the parents.
 Gene dispersion expressed in terms of rd value when rd=1 all the dominant genes
present in one parent or rd=0 both parents have equal no. of dominant genes.
 If epistasis is present then
 Heterosis = ∑h+∑l˃ ∑d+∑i
 for positive heterosis the estimate of ∑h+∑l must be positive and greater then
∑d+∑i heterosis is higher when complementary epistasis combine with
gene dispersion whereas duplicate epistasis with complete association lowers the
magnitude of heterosis
Mather and Jink ,1983

23. Physiological basis of heterosis
• In maize of early growth during the first 2 weeks of germination
demonstrated that the heterotic F1 seedling possessed higher growth rates
than the inbreds. The basic physiological activity during the early seedling
growth is the formation of enzyme patterns, translocation, transformation
and utilization of stored food material in the seed.
• Then in the building-up of the active protoplasmic base for further
physiological activity. (Srivastava, 1981)
• Studies in cotton and rice demonstrate that heterosis in leaf area index
during the early seedling stage manifested as sufficient advantage during
the later stage of crop growth. (Banga and Banga, 1998)

24. MOLECULAR BASIS OF HETEROSIS
AT MOLECULAR LEVEL TWO MODELS ARE USED TO EXPLAIN HETEROSIS
 In the hybrid, when the two different alleles of various genes are brought
together, there is a combined allelic expression.
In the second model, the combination of different alleles produces an
interaction that causes gene expression in the hybrid to deviate relative to the
midparent.
Birchler et al., 2003

25. 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 and would be consistent
with the dominance hypothesis (Fu and
Dooner 2002). The inbreds become fixed
for these alleles, resulting in inbreeding
depression.
There is evidence that a primary
source of the variation in genome
content, and potentially
transcriptome content
(Bennentenz,2005).
Complementation and heterosis
FU and DOONER(2002)

26. • Maize genome of different inbred lines display significant aberrations from
genetic colinearity
• Observed in maize 10 genes in the bz region of the McC inbred line and
only the proximal 6 gene have the counterparts in B73 inbred line.
Fu and Dooner, 2002
• 22 gene copies of α-zein storage protein subfamily z1C were detected in
the BSSS53, inbred lines and only the 15 z1C genes were present in the
B73 genomic region. Out of these only 7 of the BSSS53 and six of the B73
z1C had an intact coding region and only two intact genes were present in
both inbred line.
Hochholdinger and Hoecker, 2007
Conti…

27. Heterosis at Gene Expression Level
 Differences in gene expression thought be an importance source
of phenotypic diversity and complex traits. (knight, 2004)
 The pattern of gene expression changes in hybrids result from
unique regulatory interactions in hybrids which give rise in
quantitative variants that may be responsible for the heterosis.
(Hochholdinger and Hoecker, 2007)
 It is a complex action of many components including the timing
of the expression of various genes the magnitude and location of
their expression and interaction of their gene product. (Li et al.,
2007)

28. CAUSE OF ALTERD EXPRESSION LEVELS IN HYBRIDS
A locus may be cis acting on a second locus if it must
be on the same DNA molecule in order to have effect.
that they regulate, whereas a locus is ‘trans’ acting if
it can affect a second locus even when on different
molecule that is affect allelic expression on both
homologous chromosomes, examples of trans-acting
regulators include genes that encode transcription
factors, which may be located anywhere in the
genome. (Brem et al. 2002).
The expression levels of individual genes are
themselves controlled by other genes, acting in cis or
trans .These quantitative changes in gene expression
may be the result of cis- or trans variations in gene
regulation (Wittkopp et al. 2004).
Allelic imbalance in expression levels at heterozygous
loci directly demonstrates cis regulatory
polymorphism. In hybrids, both parental alleles have
the same cellular context and are equally exposed to
trans-acting factors. Thus, difference in expression
level between two parental alleles is directly due to
cis-acting variation (Wittkopp et al. 2004).
When trans-acting repressors or activators are
brought together in new combinations, gene
expression cascades might be altered, resulting in
dominance inheritance in the hybrids.
Cis- Regulation Trans-Regulation
Inbred A
Inbred B
Hybrid A*B
Inbred A
Inbred B
Hybrid A*B

29. TYPES OF HYBRID EXPRESSION PATTERNS.
Below low parent
Like low
Parent
Mid parent
High parent
like
Above high
parent like
Potential hybrid expression levels
Non additive
Non additive
Additive
EXPRESSION
LEVELS
INBREDS

30. CASE STUDIES

31. Relationship between gene differential expression of
leaves in full opening flower stages of hybrids & their
parents and heterosis in pest-resistant cotton
Zhu et al., 2006
Ratio of five gene expression patterns in pest-resistant
transgenic cotton crosses

32. CROSSES M1 % M2 % M3 % M4 % M5 % SUM %
P1 X P7 6.4 4.5 9.5 4.9 74.7 25.3
P1 X P8 3.3 6.O 4.9 7.8 88.0 22.0
P1 X P9 2.1 3.2 4.9 9.4 80.4 19.6
P1 X P10 1.5 4.1 5.5 9.7 79.2 20.8
P2 X P7 4.8 8.8 6.7 8.4 71.3 28.7
P2 X P8 1.0 8.4 2.4 14.4 73.8 26.2
P2 X P9 2.4 4.8 6.0 9.3 77.5 22.5
P2 X P10 2.2 6.7 5.0 9.3 72.2 23.8
P3 X P7 3.8 4.5 4.5 9.6 77.6 22.4
P3 X P8 4.2 4.1 7.1 9.6 75.0 25.0
P3 X P9 1.9 6.6 5.8 10.4 75.3 24.7
P3 X P10 2.3 7.6 5.3 9.6 75.2 24.8
P4 X P7 9.8 4.0 10.3 6.6 69.3 30.7
P4 X P 8 1.6 3.6 11.2 10.7 72.9 27.1
P4 X P 9 1.6 5.6 6.8 8.1 77.9 22.1
P4 X P 10 1.4 5.4 8.0 9.1 76.1 23.9
P5 X P7 1.9 8.3 3.9 11.1 74.4 25.6
P5 X P8 1.4 13.3 2.5 10.1 72.1 27.9
P5 X P9 1.5 7.8 5.0 8.7 77.0 23.0
P5 X P10 2.6 7.5 3.2 9.5 72.2 22.8
P5 X P7 9.7 4.2 9.2 7.7 79.2 30.8
P6 X P8 2.3 7.2 5.3 9.6 75.2 24.8
P6 X P9 1.4 7.O 6.9 10.2 74.5 25.5
P6 X P10 1.6 10.0 4.1 12.5 71.8 28.2
Average 3.0 (12.2%) 6.4(25.7%) 60.0(23.7%) 9.5(38.4%) 75.1 24.9

33. Correlations of five patterns of gene expression with
performance to five yield traits in cotton hybrids
Patterns Seed
cotton
yield
Lint yield Boll
number
Boll size Lint
percentage
M1 -0.0673 0.0202 -0.0660 -0.1372 0.3189
M2 0.0377 0.0902 -0.0741 0.1108 0.2266
M3 0.1016 0.0746 0.3040 0.0677 -0.0391
M4 0.0654 0.0727 0.4062* 0.0272 0.0691
M5 -0.2224 -0.3109 -0.3256 -0.2168 -0.5289**

34. Correlations of Five Patterns of Gene Expression with
Heterosis of Five Yield Traits in Cotton Hybrids
Patterns Seed
cotton
yield
Lint yield Boll
number
Boll size Lint
percentage
M1 -0.0660 0.0030 -0.0482 -0.1920 0.2524
M2 -0.2958 -0.3308 -0.4644* 0.0405 -0.2777
M3 0.1940 0.4361* 0.1158 0.1222 0.1834
M4 -0.2789 -0.2730 0.0133 -0.1429 -0.2308
M5 0.2353 O.1088 0.0224 0.1439 -0.2180

35. Relationship Between Differential Gene Expression
and Heterosis During Ear Development in Maize
(Zea mays L.)
Wang et al., 2007

36. • Relationship between differential gene expression during ear
development in maize studied with 12 inbred and 33 hybrid at 4
developmental stage
• With the help of 13 (three 3 prime and 5 prime anchored ) primer
for differential gene expression five pattern of gene expression
UNF1: genes expressed only in hybrid; DMP: genes expressed only in one parental
line and their hybrid but silenced in another line; ABF1; genes expressed in both
parental lines but silenced in hybrid; UNP: genes expressed in one parental line but
silenced in another line and their hybrid; MONO: genes expressed in both parental
lines and hybrid.

37. Ⅰ: spikelet branch primordial stage; Ⅱ: spikelet differentiation stage; Ⅲ: early flower differentiation
stage; Ⅳ: sexual organ maturation stage
Developme
ntal stage
UNF1(%) DMP(%) ABF1(%) UNP(%) Mono(%)
I 14.0 18.4 8.7 22.7 36.3
II 9.9 13.7 5.7 19.2 51.5
III 12.7 11.2 4.6 18.7 52.9
IV 4.5 13.7 6.2 19.1 56.6
Percentages of Different Expression Patterns at Each Developmental Stage
Ratios of differential gene expression patterns during four periods of ear development

38. Regulatory gene and dosage effect
• Heterosis was that the combination of different alleles produces an
interaction that causes gene expression In hybrid to deviate relative to the
mid parent predictions(by an up-regulation of many house keeping genes ).
• .Regulatory genes in multicellular organism often function as part of
complexes and exhibit some measure of dosage dependence whereas
housekeeping genes show less dosage effect.
• Quantitative traits are expected to controlled by dosage dependent
regulatory loci.
• Heterosis is the result of different alleles being present at loci they
contribute to the regulatory hierarchy that control quantitative traits
Birchler, 2001 and 2003

39. • Indicate that the expression of many genes does not exhibit the expected
midparent value.
• Zein gene expression was studied in the endosperm of two inbred lines and
their reciprocal hybrids. Many zein genes that contribute to the total storage
protein pool.
• The relative expression of the various zein genes were determined of the
10 genes studied, only in one case did the hybrid expression follow the
predictions of allelic dosage contributing to the genotype.
Song and Messing, 2003
Conti…..

40. A CAUSE OF HETEROSIS
EPIGENETICS AS
“Epigenetics” refers to heritable
(through mitosis or meiosis) alterations in
gene expression that are independent of
DNA sequence: different epigenetically
regulated forms of a gene are known as
epialleles.
Epigenetic information systems, could
generate epigenetic variation/epiallels that
had never been considered as the cause of
phenotypic variation (Tsaftaris and
Polidoros 2000).
TYPES
DNA
methylation
RNA
INTERFERANCE
siRNAs, miRNAs etc

41. DNA Methylation and Heterosis
• 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 its function, but
does change its expression level, referred as an epigenetic change.
• Associated with gene silencing, and genes with abundant 5-methylcytosine
in their promoter region are usually transcriptionally silent.
• Altered regulatory interactions could play a role in the increased vigor.
Zhang et al., 2008

42. Cont…..
DNA methylation was a possible molecular regulator of
heterosis and inbreeding depression.
Methylation gradually accumulated during inbreeding or
selfing , and it increasing inbreeding depression. which is then
released when these lines are crossed to generate hybrids.
 Therefore, it can be suggested that inbreeding depression
partly or primarily results from lower levels or fewer genes
expressed simply due to homozygosity of methylated DNA in
regulating factors, while heterosis is from higher levels or larger
number of genes expressed simply due to heterozygous
conditions between methylated and non-methylated DNA in the
F1 hybrid.

43.  DNA methylation plays a role in heterosis, compared at single-base-pair
resolution the DNA methylomes of Arabidopsis thaliana Landsberg erecta and
C24 parental lines and their reciprocal F1 hybrids which exhibited heterosis.
 In these studies both hybrids displayed increased DNA methylation across
their entire genomes, especially in transposable elements and small RNAs.
Increased methylation of the hybrid genomes predominantly occurred in
regions that were differentially methylated in the two parents hybrids.
 Find that 77 genes sensitive to methylome remodelling were transcriptionally
repressed in both reciprocal hybrids, including genes involved in flavonoid
biosynthesis and two circadian oscillator genes .
 Suggest that genome-wide remodelling of DNA methylation may play a role
in heterosis.
Shen et al., 2012
Cont……

44. RNA interference
 Small RNAs of 20–30 nt acting in gene-silencing systems falls into two
major categories, miRNAs and siRNAs (small interfering RNAs).
 They are essential regulatory molecules playing important roles in
developmental regulation, responses to biotic and abiotic stresses, and
epigenetic control of transposable elements in most eukaryotes ( Vaucheret,
2006).
 miRNAs are derived from long, single-stranded RNAs (ssRNAs), which
can fold and form a stem-loop structure, and then are processed by
Drosha/Dicer family proteins. However, siRNAs are produce from long
double-stranded RNAs (dsRNAs) that originate from products of
bidirectional transcription or RNA-dependent RNA polymerases (RDRs)
(kim, 2005).

45. Expression Analysis of miRNAs and Highly-expressed Small
RNAs in Two Rice Subspecies and their Reciprocal
Hybrids
Chen et al., 2010

46. • In these study investigate with 1141 miRNA microarray
techology in two rice subspecies (O. sativa ssp. japonica cv.
Nipponbare and O. sativa ssp indica cv. 93-11) and their
reciprocal hybrids.
• In these comparisons, approximately 11%–14% of small
RNAs showed a differential expression.
• Small RNAs usually work as negative regulators to repress
translation or cleave transcript of mRNA.

48. The true mechanism of heterosis has remained elusive to scientists
over the last century.
 large contingent of scientists, particularly those working in
maize, have utilized this phenomenon extensively in genetic
improvement, but have failed to solve the puzzle that is heterosis.
Heterosis is very likely organism dependent, population and trait
dependent.
Future experiments must be carefully designed to provide greater
evidence for a mechanism of heterosis.
CONCLUSION