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Dec 16, 2012 (4 years and 8 months ago)

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Significance of Cytoplasmic
DNA in Plant breeding

Sudarshan S

MSc(Agri)

GPB

India

Outline

Introduction

Types of cytoplasmic DNA

Agronomic traits influenced

Breeding using cytoplasmic DNA

Case studies

conclusion

Introduction

DNA is the basic hereditary material

In most of the organisms DNA is the
genetic material.

In some viruses RNA is the genetic
material.

Eg . TMV


Classification

Based on Base per turn




1. B
-
DNA




2. A
-
DNA




3. C
-
DNA

Based on coiling Pattern




1. Right handed DNA




2. Left handed DNA

Based on location




1. Chromosomal DNA




2. Cytoplasmic DNA

A DNA, B DNA , C DNA

RIGHT HANDED

DNA

LEFT HANDED


DNA

C
-
DNA

A
-
DNA

B
-
DNA

Cytoplasmic DNA

DNA found in cytoplasm is called
cytoplasmic DNA.

Plasmone, Organellar DNA, Extra nuclear
DNA.

Size of these organellar DNA is small
compared to genomic DNA.

Traits inheritance
-

Cytoplasmic
inheritance.

Differences

Character

Nuclear DNA

Cytoplasmic DNA

Gene

Nuclear genes

Cytoplasmic

genes

Inheritance

Mendelian inheritance

Cytoplasmic inheritance

Mode of inheritance

Biparental


Uniparental

Size

More

Less

Mitochondrial DNA


Chloroplast DNA





Cytoplasmic DNA




Mitochondrial DNA (mtDNA)

Varies enormously in plants.

In plants it is as big as 2000kb.

Produces ATP by oxidative phosphorylation

mt DNA of higher plants has a large coding
capacity.



Site of Krebs cycle and oxidative
phosphorylation.


In mtDNA, mutation rate is very high 10 times
the nuclear rate



Arabidopsis
-

57 mt genes



The lower plant
liverwort

(
Marchantia
polymorpha)
is the first plant of which the
mitochondrial genome has been sequenced (Oda et
al. 1992)


Rice mitochondrial genome

Size: 490.520bp

Sizes of Plant mitochondrial genome

Plant

Genome size(kb)

Triticum

aestivum

440

Zea mays

570

Brassica campestris

218

Brassica

oleracea

219

Brassica napus

221

Raphanus sativa

242

Helianthus annus

300

Spinacea oleracea

327

Cucumis melo

2500

Nair, 1993

Chloroplast DNA


DNA found in the chloroplast is called
plastid DNA or cp DNA.

Chloroplast DNA is highly conserved.

Plastome encodes genes for photosynthesis.

Replicated by nuclear encoded proteins.

This may code for 70
-
90 proteins.



Contd…

Chloroplast genome contains inverted
repeats, short unique sequence and long
unique sequence.

Eg.

Crop

Inverted
sequence (bp)

SUS (bp)

LUS (bp)

Arabidopsis

26284

17780

84170

Spinach

25073

17860

82719

Maize

22748

12536

82355

Nair, 1993

Fig. 15.5


cpDNA of rice

Chloroplast DNA of Tobacco

Chloroplast DNA of Rice

Agronomic traits influenced

Cytoplasmic Male Sterility. Eg: Cotton, Maize etc.

Yield and quality parameters.

Disease resistance. Eg: Yellowberry in wheat

Combining ability. Eg: Pearl millet by Kumar
et
al.,

2010.

Breeding using cytoplasmic factors

Cytoplasmic factors are important in qualitative
traits such as Cytoplasmic Male Sterility and
disease resistance.

The contribution of cytoplasmic factors to
quantitative agronomic traits is probably of minor
significance.

In order to further enhance the performance of
cultivars breeders can exploit such minor effects.



Cytoplasmic factors should be
considered to


Characterize variability

Generate variability by recombination

Select favorable genotypes


Characterization of Plant
Material

The molecular basis of genetic diversity of
cytoplasm has revealed greater variability of
mitochondrial genome compared to
chloroplast genomes.

Characterization has been done in several
crops

In potato

Origin of cultivated tetraploid potato.

Loss
et al
. (1999) characterized different
mitochondrial types in 100 diploid and 144
tetraploid potato clones.

α,
β
,
ε

and
δ

Maize

Peiretti (2003) evaluated the plasmone of 30
dent and 32 flint lines using 23 mt
-
probes
and found clear polymorphisms.

A genetic distance analysis revealed three
clusters viz. dent, flint and both lines.

Since mt
-
probes result in simpler
restrictions offer an easy system for
characterization of inbred line diversity for
applied use.

Rice

Cytoplasmic variability of cultivated
species
Oryza sativa

and
Oryza glaberrima

indicates high conservation of cp and mt
genomes

Lin performed pedigree analysis to quantify
ancestral contributions to 27 rice cultivars
released at IARI

One ancestor was the ultimate cytoplasmic
parent for 22 of 27 cultivars

Creating new variability

It can be achieved by the following ways

Undirected processes

Directed alterations for plasmone
transformation

Undirected processes

Different kinds of variability need to be
considered

Generation of new nuclear cytoplasmic
combination

New combinations between unaltered
chloroplast and mitochondria

Creation of new mitochondrial or plastid
genotypes.

Significance of cytoplasmic
DNA

Cytoplasmic Male Sterility

They contribute in photosynthesis and respiration.

Evolution of plant species.

Thus mitochondria and chloroplast represent a
source of genetic diversity.

Phylogenetic studies.

Cytoplasmic DNA is used

Production and use of alloplasmic lines by
combination breeding.

Somatic cell fusion.

Direct transfer of cytoplasmic DNA genes
into organelles.


resulting in the case of plastids in
transplastomic plants.

Cytoplasmic Male Sterility

CMS is a maternally inherited form of anther dysfunction
or pollen sterility


It is determined by cytoplasm, result of mutation in
mitochondrial genome.


It is always associated with open reading frames


This method can not be used in crop plants where
economic part is seed.

S

F

S

rr

rr

rr

X

Sterile

Fertile

Sterile

CMS Utilization

It provides a possible mechanism of
pollination control in plants


Permit the easy production of commercial
quantities of hybrid seeds.

CMS is used in hybrid seed production viz.,
Miaze
, Redgram etc.


Significance

Hybrid seed production

Eg. Maize, Redgram

Pollen control

Reduces labor cost

Saves time.

Chloroplast transformation

Modification of chloroplast and/or
introduction into them of some selected
transgenes could improve photosynthesis
and even enhance crop yields.


Methods

Particle gun method

PEG treatment

Microinjection

Multigene

engineering

No gene


silencing

No

position


effect

No

Pleiotropic


effect

Gene

Containment

Advantages of Chloroplast


transformation


Herbicide

resistance

Gene containment by maternal
inheritance

Plastid genes in most of the angiospermic plant species
follows uniparental mode of inheritance in a strictly
maternal fashion.

Gene is not present in the pollen and minimizes the
possibility of


outcrossing transgenes to related weeds or
crops.


reduces the potential toxicity of transgenic
pollen to non target insects.





Multigene engineering in a single event

Phytoremediation

Introduced
mer
AB operon in a single event

Tolerance level to mercury increased
tremendously in transgenics.



Ruiz
et al.,
2003

Transplastomics


a convergence of
biotechnology and

evolution


This is the approach of placing transgenes in the chloroplast
genome of a recipient crop plant.



Consequently, pollen shed by a transplastomic plant should not
carry or transmit the transgene, since transmission is through the
female gamete.



Successful Chloroplast Transformation in

Carrot

Betaine aldehyde dehydrogenase (Badh) gene


High accumulation of glycine betaine [50x high] and
tolerate NaCl (400mM )

Soybean

First stable transformant by somatic embryogenesis was
developed expressing Bacillus thuringiensis Cry1Ab
protoxin



Transgenic

Chloroplast Genome

Nuclear

Genome

Transgene copy number

10
-
100 plastid per

cell

Two copies of each
chromosome per cell

Level of gene expression

High accumulation of
transgenes

Less

accumulation of
transgenes

Gene arrangement and
transcription

Genes are often arranged in
operons and transcribed into
polycistronic RNA

Independently inserted into
chromosome and transcribed

into monocistronic mRNA

Position effect

Absent

Present

Gene silencing

Not reported

Present

Gene containment

Maternal gene inheritance
results in natural gene
containment

Paternal inheritance results in
outcrossing

among crops and
weeds.

Comparison of chloroplast and nuclear genetic engineering

Daniell
et al.,
2002

Plant species

Methods

Selection

Expressed
genes

Literature cited

Nicotiana
tabacum

Bombardment

Spectinomycin

rrn16

Svab

et al., 1990

Tobacco

Bombardment

Kanamycin

ntpII

Carrer

et al., 1993

Tobacco


Bombardment


Spectinomycin

Bt

Coss et al., 2001

Arabidopsis
thaliana

Bombardment


Spectinomycin

aadA

Sikdar et al.,
1998

Rice

Bombardment

Spectinomycin

aadA

& gfp

Lee et al., 2006

Tomato

Bombardment


Spectinomycin

aadA

Rufet et al., 2001

Brassica napus

Bombardment


Spectinomycin

aadA

& cry1Aa10

Hou

et al., 2003

Gossypium
hirsutum

Bombardment


Kanamycin


aphA
-
6

Kumar et

al.,
2004

Chloroplast transformation methods and selection conditions for Plant Species

Wang
et al.,
2009

Case studies

Effect of cytoplasm on oil content

Crop : Sunflower

Objective : Influence of alloplasmic male
sterile lines on oil content

Materials used:


3 CMS sources viz. CMS 852A, FMS 852A,
IMS 852A



restorer lines viz. Acc. No. 1229, 232 and
TUB 365

Patil
et al.,

2003

Hybrid

Pl height
(cm)

Days to
flowering

Days to
maturity

Head
diameter (cm)

% Seed
set

100 seed
weight (g)

Oil

content
(%)

Seed
yield/
plt

(g)

CMS852A x 1229

162

61

85.7

11

77.4

2.7

31.6

11.8

FMS852A x 1229

154

62

89

11.1

74.8

2.8

34.8

13.5

IMS852A x 1229

153

62

87

10

66.7

2.5

33.9

11.4

CMS852A x Tub365

145

60

86

11.8

76.5

2.6

31.1

18.7

FMS852A x Tub365

147

60

86.7


10.6

80.2

2.7

31.4

13.8

IMS852A x Tub365

161

61

87.3

1.09

76.9

3.2

34.2

14.6

CMS852A x 232

140

60

85

10.2

84.5

3.1

34.2

13.8

FMS852A x 232

149

62

87.7

10.7

83.2

3.2

36.3

14.4

IMS852A x 232

157

60

87.7

10.7
81.8

3.1

30.2

15.2

IMS IB24Ax 1229

150

62

87.3

11

88.2

2.7

33.2

15.5

FMS IB24 x 1229

163

63


88.3

10.8

67.9

2.7

31.1

12.1

IMS IB24A x 232

164

63

87.3

10.3

82.5

2.8

34.4

14.9

FMS

IB24A x 232

168

62

87

11

85.2

3.1

32.3

16.2

IMS IB24A x Tub365

181

63

89.3

10.9

83.6

3.4

35.4

16.8

FMS IB24A x Tub365

194

63

89

11

79.1

3.1

35.9

15.7

IMS

IB24A x Tub346

168

68

88.3

11

82.2

2.4

34.1

16.2

FMS IB24A x Tub346

159

62

86.7

8.8

76.4

2.5

28.6

8.5

LSD at P= 0.05

29

3

3.4

2.2

13.5

0.7

1.7

7.5

Patil
et al.,

2003

Mean performance of hybrids and parents in respect of 8 quantitative characters

Effect of cytoplasm on oil content

Crop:
Brassica napus

Materials: Youcai 601, Gaoyou 605,
Huashang 3, Yunyou 8, Zhongyou 821,
Eyouchangjia, Zhong R
-
888 and Tower.

Experiment conducted in two environments.



Wu
et al.,

2006

Parent

Embryo additive
effect

Cytoplasmic effect

Maternal

additive
effect

G
t
I

G
t

II


A

AE I

AE II

C

CE I

CE II

Am

AmE I

AmE II

1999

2000

Youcai 601

0.16

3.29

-
2.60

1.55

1.73

-
0.43

-
0.78

-
4.77

3.66

1.19

1.56

Gaoyou 605

-
0.11

-
2.68

2.64

3.26

3.12

0.04

-
0.41

1.19

0.04

4.37

5.47

Huashuang 3

--
0.04

-
2.22

0.40

-
0.28

-
0.65

0.41

0.12

1.38

-
0.30

-
1.68

0.31

Yunyou 8

-
0.02

-
2.67

1.71

-
1.91

-
0.38

-
1.13

0.31

1.47

-
0.92

-
3.20

-
1.96

Zhongyou
821

-
0.04

1.90

-
2.05

-
0.33

-
2.09

1.93

0.14

-
0.58

0.30

-
0.99

-
0.05

Eyouchangjia

0.28

6.57

-
2.53

-
1.72

-
1.86

0.05

0.21

-
2.80

1.18

0.67

-
2.53

Zhong R
-
888

-
0.18

-
2.62

0.85

-
0.74

-
1.32

0.92

0.25

1.68

-
2.48

-
2.93

-
1.38

Tower

-
0.05

-
1.57

1.57

0.17

1.44

-
1.80

0.15

2.42

-
1.47

2.56

-
1.44

Wu
et al.,

2006

Genetic main effects and GE interaction effects for oil content (%) of rape seeds


Resistance to yellow berry disease in
wheat

Objective: to determine the effect of cytoplasm on
resistance to yellowberry in durum wheat.


Materials used: 28 populations including parental
lines, F1, RF1, F2, RF2, BC1P1 and BC1P2.


Design: RCBD

Bnejdi
et al.,
2010

Model

Om Rabi

x Cocorit 71

Ben Bachir x Karim

Ben Bachir

x Cocorit 71

Om Rabi x Karim

Three parameter

model

Mean

2.53

4.93

3.19

3.05

Additive

-
1.96

-
2.20

-
1.60

-
2.24

Dominance

-
1.35

-
3.88

-
1.82

-
1.77

A
P

P < 0.01

P < 0.01

P < 0.01

P < 0.01

Three parameter

model with maternal effect

Mean

1.77

2.28

2.30

2.11

Additive

-
2.6

-
1.85

-
1.17

-
2.50

Dominance

0.39

-
0.47

-
0.27

-
0.42

Maternal additive

1.31

0.72

0.44

0.77

Maternal

Dominance

-
0.03

1.64

0.24

0.86

Cytoplasmic genetic effects

-
0.18

-
0.29

-
0.57

-
0.09

A
P

P < 0.01

P < 0.01

P < 0.01

P < 0.01

Best Fit Model

Mean

3.54

8.35

3.73

6.14

Additive

-
0.86

-
1.28

-
0.78

-
1.51

Dominance

-
4.14

-
12.08

-
3.18

-
8.54

Additive x additive

-
1.46

-
5.79

-
1.40

-
3.84

Dominance x dominance

2.67

5.21

1.29

3.91

Additive x dominance

6.50

3.78

2.46

3.59

Cytoplasmic genetic effects

-
0.94

-
0.50

-
0.78

-
0.53

A
P

0.12

0.20

0.69

0.02

Estimates of gene effects for grain resistance to yellow berry in durum wheat

Bnejdi
et al.,
2010

Influence of cytoplasm on agronomic
performance

Crop: Sorghum

Cytoplasms used: A1(Milo) and A2 (non milo)

Materials used: alloplasmic male sterile lines viz. ICSA
11,
-
17,
-
26,
-
37, 38,
-
42,
-
88001, 88005,
-
18757,
PM17467A and PM 7061A and


R lines used: ICSR 93001, 92003 and 93031

Ramesh
et al.,
2006

Mean performance of isonuclear, alloplasmic(A
₁, A₂ a nd B
) sorghum hybrids for agronomic traits

Ramesh
et al.,
2006

Hybrids

Days to 50% flowering

Plant height (m)

Grain yield

(t/ha)

A
1

x R

A
2
x R

B x R

A
1
x R

A
2
x R

B x R

A
1
x R

A
2
x R

B x R

ICSA/B 17

x ICSR 92003

67

68

70

3.2

3.2

2.3

2.2

2.9

2.3

ICSA/B

88001 x ICSR 92003

66

66

67

3.3

3.2

2.4

3.0

2.4

1.9

PM 17467 x ICSR 92003

65

67

67

2.3

2.5

2.4

2.8

1.7

2.3

ICSA/B 11 x ICSR 93001

69

70

64

2.3

2.2

2.3

3.5

3.5

1.1

ICSA/B 17 x ICSR 93001

70

69

67

1.9

2.2

2.3

2.9

3.6

2.8

ICSA/B 26 x ICSR 93001

72

73

66

2.3

2.2

2.1

4.6

4.8

1.0

ICSA/B 37 x ICSA 93001

70

70

73

2.3

2.1

2.2

3.3

4.5

2.3

ICSA/B 38 x ICSA 93001

68

69

69

2.3

2.3

2.1

4.4

4.5

3.7

ICSA/B 42 x ICSA 93001

68

67

68

2.2

2.2

2.1

3.5

3.1

2.8

ICSA/B 88001 x ICSR

93031

68

68

67

2.4

2.2

3.2

4.0

3.8

1.4

ICSA/B 88005 x ICSR 93031

69

68

67

2.4

2.2

2.9

3.7

2.0

1.3

LSD (between

overall mean of
A
1
/
A
2
&

B

hybrids
) (P= 0.05)

0.30

0.05

0.15


CV(%)

1.60

7.10

20.70

Phylogenetic studies

Because of the low frequency of structural changes in
the chloroplast DNA molecule (cpDNA) a useful tool
for plant phylogenetic studies.


The sequences of the chloroplast
rbcL

gene (coding
the large subunit of Rubisco)


Other genes
-

matK, ndhF, psaB and trnL
-
trnF

can be
used

Phylogenetic studies done in

Fern

Oryza

Solanaceae

Sesamum

Phylogenetic tree of Solanaceae

Passarin
et al.,
2008

Transplastomics in Tobacco

Efficient plastid transformation in
Nicotiana tabacum

using plastid cloned DNA of
Solanum nigrum

carrying
mutations conferring spectinomycin and streptomycin
resistance.


Result: observed recombination frequency > expected

Conclusion: transformation is efficient & agriculturally
important traits can be transferred

Kavanagh
et al.,
1999


(A) The gel electrophoretic separation of
BamHI
-
digested plastid DNA of wild
-
type N.
tabacum (lane 1) and several plastid
transformants (lanes 2

4) reveals different
combinations of the Solanum specific cut sites.
Only fragments.2.6 kb are shown. The
transformant specific fragments are marked by
arrowheads. On the left a HindIII digest of
lambda DNA is also shown. (B) The fragment
patterns obtained with PstI
-
AatII double
-
digested
plastid DNA in wild
-
type N. tabacum (lane 1) and
several plastid transformants (lanes 2

4)
demonstrate the identity of the copies of the
inverted repeated region in the plastid
transformants for the spectinomycin resistance

Kavanagh
et al.,
1999

Genetic map of the pSSH1 plastid transformation
plasmid. The 7.8
-
kb S. nigrum plastid DNA insert
was cloned into the HindIII site of the pUC19
vector (thick line). The plastid genes (solid bands)
shown inside and outside the circle are
transcribed counterclockwise and clockwise,
respectively. The relative position and the
direction of transcription of the ampicillin
resistance gene in pUC19 are shown by a long
arrow. Asterisks mark intron containing genes.
BamHI restriction sites of the insert are shown.
Sites of the streptomycin (str
r
) and the
spectinomycin (spe
r
) resistance mutations are
shown by short arrows.

Kavanagh
et al.,
1999

Herbicide resistance

EPSPS

(
5

enol

pyruvyl

shikimate

3
-
phosphate

synthase)

is

nuclear

encoded

chloroplast

targeted

enzyme

whose

over

expression

provides

glyphosate

resistance

Glyphosate

insensitive

EPSPS

enzyme

from

bacteria

or

its

mutated

from

which

have

decrease

affinity

for

the

herbicide

can

be

used

for

chloroplast

genetic

engineering

High

accumulation

of

EPSPS

in

tobacco

chloroplast

conferred

glyphosate

resistance

Ye
et al.,

2001

Drought tolerance

Conferred by trehalose compound

Trehalose synthesis is mediated by trehalose 6 phosphate
phosphatase (T6P)

Yeast gene trehalose phosphate synthase gene(TPS 1) was
introduced into the tobacco chloroplast and nuclear
genomes

Result: chloroplast transgenic plants showed normal
growth where as nuclear transgenic plants showed stunted
growth



Daniell
et al.,
2002

Conclusion