prethesis submission seminar on 9th may 2012

ADVISORY BOARD

• CHAIRMAN :- DR. A. K. ROY

• MEMBER:- DR. D. R. MALAVIYA

• MEMBER:- DR. P. KAUSHAL

• MEMBER:- DR. A. CHANDRA

• MEMBER:- DR. SURESH KUMAR

2

Assessment of molecular genetic diversity

among different species of Trifolium and

ecotypes of T. alexandrinum.

Study on interspecific/intervarietal

compatibility and inheritance of traits like

regeneration ability.

3

• Genus: Trifolium Tourn (L.).

• Family: Leguminosae.

• Sub-family: Palpilionaceae.

• It comprises of 290 annual and perennial herbs

commonly called clovers including several

cultivated and pasture species of high fodder value.

4

The important perennial pasture clover T.

repens, T. hybridum, T. pratense and T.

ambiguum are widely distributed in the

temperate and sub-temperate regions of the

world.

The annual types such as T. resupinatum, T.

subterraneum and T. alexandrinum are

commonly cultivated as winter annuals in

the tropical and subtropical regions. 5

The genus name Trifolium refers to the distinctive

leaves usually composed of three leaflets (trifoliolate).

The Mediterranean region is the centre of diversity

of the genus Trifolium (Zohary and Heller, 1984).

Berseem is widely adapted by the farmers of north,

northwest, eastern and central India.

Nearly 2 million hectare arable land is under

cultivation of Berseem with an average national

productivity of 85 t/ha.

6

The importance of crop is known for:-

Its high green fodder production (up to 100t/ha).

Its multicut nature (6-7 cuts).

Long duration of green fodder availability

(November to April).

High crude protein content (20-24%).

High digestability.

High palatability .

High nitrogen fixation ability.

7

1: Characterization of Trifolium species based on

morphology:-

8

To incorporate desirable traits like early flowering

from Fahli group and regeneration ability from

Mescavi group, inter-varietal crosses were

attempted among three ecotypes of T. alexandrinum

(Berseem) i.e., Mescavi (cv Wardan and BB-2),

Saidi (Ex-8) and Fahli (FAO-5).

F1 hybrids were crossed to male parent and female

parents to obtain BC1 seed.

9

All F1s and their parents, were

characterized for 12 qualitative and

quantitative traits.

F2 and BC1 populations were characterized

for 10 different morphological attributes

including metric and non-metric traits.

10

Hybrids and their parents were analyzed for

esterase and protein native banding pattern

along with their parents.

F2 progenies and their respective parents

were compared only for esterase isozyme

banding pattern.

11

BSA (Michelmore et al, 1991) was used to identify

RAPD markers linked with regeneration and non-

regeneration trait.

BC1 population from contrasting parents Wardan

(regenerating genotype) & FAO-5 (non regenerating

genotype) was developed by backcrossing F1 progenies

with Wardan.

4 bulks representing viz., P1 bulk (Wardan bulk), P1

type bulk (Wardan type plant progenies), P2 bulk (FAO-

5) & P2 type bulk (FAO-5 type plant progenies) were

prepared and screened with 110 RAPD primers for

identification of putative marker linked to regeneration. 12

DNA extracted from fresh leaves and PCR

reaction carried out as described by Williams

(1993).

18 decamer RAPD primers were used in analysis

of diversity within and between different ecotypes

of T. alexandrinum. 13

A total of 38 SSR primer pairs were used to estimate

polymorphism among 24 species of genus Trifolium.

DNA isolated and PCR reaction was performed in 10µl

volume containing template DNA, Taq polymerase enzyme,

Taq polymerase buffer, dNTPs, forward and reverse

primers.

The thermocycle parameters were used as described by

Barret (2004) with minor modifications.

The PCR products were electrophoresed on 3.3%

agarose gel.

14

To identify the stages of pre and post fertilization

barriers where 3 genotypes of T. alexandrinum

(EC511781, Wardan and BB-2) were used as

female parent and crosses were made with

different Trifolium species.

Pollen tube growth was studied at regular

interval of 1hr, 3hrs, 5hrs and 8hrs after

pollination.

The karyotype will be prepared using root –tip

mitotic chromosomes studies based on standard

root –tip squash method after pretreatment,

fixation, hydrolysis and squash method.

15

The metric data was analyzed statistically to group

the plants of similar types based on their similarity.

Euclidian cluster analysis for grouping of genotypes

and Mahalanobis Euclidean2 analysis was used.

16

Intra and Interspecific variation analyzed in 51

accessions representing 19 species of genus Trifolium.

Wide range of variation observed for most of the

traits like plant height (19-143.89 cm), number of

branches (5.67-55.11), number of node (5.0-16.33) etc.

18

S.N. Species Name T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

1 T. alexandrinum Mean 6.5 9.56 4.78 0.36 67.28 1.22 3.34 1.06 1.34 6.22

Range 4.0-8.67 8.33-13.0 3.77-5.7 0.3-0.4 59.57-

71.33 0.93-1.8

2.97-

4.13

0.83-

1.47

0.93-

2.23 6.0-6.67

SD 1.55 1.73 0.75 0.04 4.08 0.34 0.43 0.22 0.57 0.27

2 T. compestre Mean 55.11 16.11 1.3 0.25 63.22 0.79 1.44 0.8 0.62 8.33

Range 43.33-

62.67

14.67-

17.67

1.03-

1.47 0.2-0.27

59.67-

67.33

0.43-

1.33 1.3-1.53

0.73-

0.83 0.6-0.67 12-Jun

SD 10.34 1.5 0.24 0.04 3.86 0.48 0.13 0.06 0.04 3.21

3 T. apertum Mean 9.67 16.33 4.17 0.47 90.33 1.23 2.83 0.8 1.9 7.33

Range 8.0-11.00 19-Dec 4.0-4.5 0.4-0.5 87-93 1.2-1.3 2.5-3.1 0.8-0.8 1.7-2 8-Jun

SD 1.53 3.79 0.29 0.06 3.06 0.06 0.31 0 0.17 1.15

4 T.angustifolium Mean 17.5 9.5 2.35 0.315 57.415 1.62 3.83 0.7 2.7 8.5

Range 9.67-25.33 7.33-11.67 1.9-2.8 0.23-0.4 19.83-95 1.27-

1.97

3.63-

4.03

0.47-

0.93 2.6-2.8

7.67-

9.33

SD 11.07 3.07 0.64 0.12 53.15 0.49 0.28 0.33 0.14 1.17

5 T. spumosum Mean 5.67 5 1.37 0.25 19 2.79 1.12 0.99 1.04 6.17

Range 5.33-6.00 4.67-5.33 1.17-

1.57

0.23-

0.27

15.33-

22.67

2.67-

2.90

1.10-

1.13

0.97-

1.00 1.0-1.07 6.0-6.33

SD 0.47 0.47 0.28 0.03 5.19 0.16 0.02 0.02 0.05 0.23

6 T. diffusum Mean 10 11 2.27 0.27 49.33 1.33 2.1 0.77 1.6 4

Range 10-Oct 12-Oct 2.0-2.5 0.2-0.3 45-53 1.3-1.4 2.0-2.2 0.7-0.8 1.5-1.7 4-Apr

SD 0 1 0.25 0.06 4.04 0.06 0.1 0.06 0.1 0

7 T. grandiflorum Mean 5.67 7.67 2.2 0.2 57 0.37 1.77 0.83 0.8 6

Range 6-May 9-Jul 2.0-2.4 0.2-0.2 46-68 0.3-0.4 1.6-2 0.8-0.9 0.8-0.8 6-Jun

SD 0.58 1.15 0.2 0 11 0.06 0.21 0.06 0 0 19

8 T. hirtum Mean 11.33 6.5 1.1 0.25 32.33 1.97 1.55 1.115 1 6

Range 10.33-

12.33 6.33-6.67 1.03-1.17 0.23-0.27

31.33-

33.33 1.97-1.97 1.5-1.6 1.1-1.13 0.93-1.07 6-Jun

SD 1.41 0.24 0.1 0.03 1.41 0 0.07 0.02 0.1 0

9 T. echinatum Mean 16.93 10.93 2.87 0.31 91.67 0.45 1.85 1.01 1.07 6.07

Range 11.33-

25.67 7.33-12.33 1.77-5.07 0.3-0.33 81-105 0.3-0.77 1.73-1.97 0.93-1.23 1-1.2 5.33-7.33

SD 6.62 2.07 1.39 0.01 9 0.19 0.09 0.13 0.08 0.76

10 T. repens Mean 15.96 13.92 1.89 0.24 71.38 5.51 1.53 1.16 0.94 11.58

Range 13.67-

18.83

13.33-

15.17 1.67-2.37 0.2-0.27 62.33-76 4.1-6.98 1.33-1.72 1.0-1.33 0.87-1 9.33-15

SD 2.4 0.85 0.33 0.03 6.24 1.2 0.18 0.13 0.06 2.55

11 T .hybridum Mean 12.33 10.78 1.61 0.34 84.45 2.62 2.49 1.48 1.66 5.33

Max 15.33 12 1.7 0.37 91.67 2.83 2.5 1.57 1.77 6

Mini 10.67 9.33 1.43 0.33 76 2.47 2.47 1.4 1.57 4.67

SD 2.6 1.35 0.16 0.02 7.91 0.19 0.02 0.09 0.1 0.67

12 T. resupinatum Mean 11.39 13.89 2.28 0.47 143.89 1.93 1.98 1.1 0.96 4.56

Range 8.67-13.67 12.67-

15.33 2.13-2.7 0.37-0.57

89.0-

193.33 1.57-2.27 1.67-2.23 0.87-1.3 0.83-1.03 4.0-6.67

SD 1.81 1.15 0.21 0.09 41.32 0.29 0.2 0.19 0.08 1.07 20

13 T. subterraneum Mean 27.67 13.67 1.55 0.28 61.33 1.66 1.01 0.82 0.59 6.89

Range 25.67-

29.67

12.33-

16.00 0.50-2.17 0.23-0.30 24.0-81.00 0.83-2.07 0.87-1.13 0.77-0.90 0.37-0.70 2.0-10.00

SD 2 2.03 0.91 0.04 32.35 0.72 0.13 0.07 0.19 4.29

T .pilulare Mean 15.67 14 2.2 0.27 60.33 0.33 0.97 0.8 0.37 7

14 Range 10.0-24.0 13.0-15.0 1.90-2.50 0.20-0.30 54.0-65.0 0.30-0.40 0.90-1.0 0.70-0.90 0.30-0.50 6.0-8.0

SD 7.37 1 0.3 0.06 5.69 0.06 0.06 0.1 0.12 1

15 T. balansae Mean 8.33 10 2.67 0.2 61.33 0.37 1.7 0.67 0.97 6

Range 6.0-11.00 9.0-11.00 2.5-2.90 0.20-0.20 52.0-70.00 0.30-0.50 1.70-1.70 0.60-0.70 0.90-1.10 6.0-6.00

SD 2.52 1 0.21 0 9.02 0.12 0 0.06 0.12 0

16 T .ligusticum Mean 7 11.33 2.33 0.27 67.33 0.3 1.9 0.87 1.03 6.67

Range 7-Jul 12-Nov 2.0-2.7 0.2-0.3 60-77 0.2-0.4 1.7-2.1 0.7-1 0.9-1.1 6.0-8

SD 0 0.58 0.35 0.06 8.74 0.1 0.2 0.15 0.12 1.15

17 T. lappaceum Mean 18.34 12.17 2.18 0.3 84.5 0.37 1.72 0.85 0.9 7.34

Range 15.67-21 11.0-13.33 2.13-2.23 0.3-0.3 79.67-

89.33 0.37-0.37 1.67-1.77 0.83-0.87 0.87-0.93 6.67-8

SD 3.77 1.65 0.07 0 6.83 0 0.07 0.03 0.04 0.94

18 T. clypeatum Mean 10.5 7.335 3.75 0.47 83.335 5.5 2.635 2.03 1.935 8

Range 9.33-11.67 7.0-7.67 3.67-3.83 0.47-0.47 82.0-84.67 5.47-5.53 2.5-2.77 2.03-2.03 1.97-1.97 8-Aug

SD 1.65 0.47 0.11 0 1.89 0.04 0.19 0 0.05 0

19 T. dasyrum Mean 47.5 6.335 1.685 0.335 54.835 1.6 2.32 0.985 1.83 4.335

Range 35.33-

59.67 6.0-6.67 1.4-1.97 0.3-0.37 49.67-60 1.17-2.03 2.17-2.47 0.9-1.07 1.63-2.03 4.0-4.67

SD 17.21 0.47 0.4 0.05 7.3 0.61 0.21 0.12 0.28 0.47

Total Accession=48

Total Species=19

T1-No.of Branch T2-No of Node T3-Internodal Length

T4-Stem Diameter T5-Height./ Rossete Diameter T6-Petiole Length

T7-Leaflet Length T8-Leaflet Width T9-Stipule Length

T10-No. of Veins on Stipule 21

0

10

20

30

40

50

60

T.a

lexa

nd

rin

um

T.c

om

pes

tre

T.a

per

tum

T.a

ng

ust

i

T.s

pu

mo

sum

T.d

iffu

sum

T.g

ran

dif

loru

m

T.h

irtu

m

T.e

chin

atu

m

T.r

epen

s

T.h

ybri

du

m

T.r

esu

pin

atu

m

T.s

ub

terr

an

eum

T.p

ilu

lare

T.b

ala

nsa

e

T.li

gu

stic

um

T.la

pp

ace

um

T.c

lyp

eatu

m

T. d

asy

rum

No. of branches

0

2

4

6

8

10

12

14

16

18

T.a

lexa

nd

rin

um

T.c

om

pes

tre

T.a

per

tum

T.a

ng

ust

i

T.s

pu

mo

sum

T.d

iffu

sum

T.g

ran

dif

loru

m

T.h

irtu

m

T.e

chin

atu

m

T.r

epen

s

T.h

ybri

du

m

T.r

esu

pin

atu

m

T.s

ub

terr

an

eum

T.p

ilu

lare

T.b

ala

nsa

e

T.li

gu

stic

um

T.la

pp

ace

um

T.c

lyp

eatu

m

T. d

asy

rum

0

1

2

3

4

5

6

T.al

exa

nd

rin

um

T.co

mp

est

re

T.ap

ert

um

T.an

gust

i

T.sp

um

osu

m

T.d

iffu

sum

T.gr

and

iflo

rum

T.h

irtu

m

T.ec

hin

atu

m

T.re

pe

ns

T.h

ybri

du

m

T.re

sup

inat

um

T.su

bte

rran

eu

m

T.p

ilula

re

T.b

alan

sae

T.lig

ust

icu

m

T.la

pp

aceu

m

T.cl

ype

atu

m

T. d

asyr

um

0

0

0

0

0

0

0

0

0

0

1

T.a

lexa

nd

rin

um

T.c

om

pe

stre

T.a

pe

rtu

m

T.a

ng

ust

i

T.s

pu

mo

sum

T.d

iffu

sum

T.g

ran

dif

loru

m

T.h

irtu

m

T.e

chin

atu

m

T.r

ep

en

s

T.h

yb

rid

um

T.r

esu

pin

atu

m

T.s

ub

terr

an

eu

m

T.p

ilu

lare

T.b

ala

nsa

e

T.l

igu

stic

um

T.l

ap

pa

ceu

m

T.c

lyp

eatu

m

T. d

asy

rum

22

Stem diameter Internodal length

No. of nodes

0

20

40

60

80

100

120

140

160

T.ale

xand

rinu

m

T.co

mpest

re

T.apert

um

T.ang

ust

i

T.sp

um

osu

m

T.diffu

sum

T.gra

ndiflo

rum

T.hi

rtum

T.ech

inatu

m

T.re

pens

T.hy

bridum

T.re

supin

atu

m

T.su

bte

rrane

um

T.pilu

lare

T.bala

nsae

T.lig

ust

icum

T.la

ppace

um

T.cl

ypeatu

m

T. d

asy

rum 0

1

2

3

4

5

6

T.a

lexa

nd

rin

um

T.co

mp

estr

e

T.a

per

tum

T.a

ng

ust

i

T.sp

um

osu

m

T.d

iffu

sum

T.g

ran

dif

loru

m

T.h

irtu

m

T.ec

hin

atu

m

T.re

pen

s

T.h

ybri

du

m

T.re

sup

ina

tum

T.su

bte

rra

neu

m

T.p

ilula

re

T.b

ala

nsa

e

T.lig

ust

icu

m

T.la

pp

ace

um

T.cl

ypea

tum

T. d

asy

rum

0

1

1

2

2

3

3

4

4

5

T.a

lexa

nd

rin

um

T.co

mp

estr

e

T.a

per

tum

T.a

ng

ust

i

T.sp

um

osu

m

T.d

iffu

sum

T.g

ran

dif

loru

m

T.h

irtu

m

T.ec

hin

atu

m

T.re

pen

s

T.h

ybri

du

m

T.re

sup

ina

tum

T.su

bte

rra

neu

m

T.p

ilula

re

T.b

ala

nsa

e

T.lig

ust

icu

m

T.la

pp

ace

um

T.cl

ypea

tum

T. d

asy

rum 0

1

1

2

2

3

T.ale

xand

rinu

m

T.co

mpest

re

T.apert

um

T.ang

ust

i

T.sp

um

osu

m

T.diffu

sum

T.gra

ndiflo

rum

T.hi

rtum

T.ech

inatu

m

T.re

pens

T.hy

bridum

T.re

supin

atu

m

T.su

bte

rrane

um

T.pilu

lare

T.bala

nsae

T.lig

ust

icum

T.la

ppace

um

T.cl

ypeatu

m

T. d

asy

rum

23

Leaflet length

Plant height Petiole length

Leaflet width

0

1

1

2

2

3

3

T.a

lexa

nd

rin

um

T.co

mp

estr

e

T.a

per

tum

T.a

ng

ust

i

T.sp

um

osu

m

T.d

iffu

sum

T.g

ran

dif

loru

m

T.h

irtu

m

T.ec

hin

atu

m

T.re

pen

s

T.h

ybri

du

m

T.re

sup

ina

tum

T.su

bte

rra

neu

m

T.p

ilula

re

T.b

ala

nsa

e

T.lig

ust

icu

m

T.la

pp

ace

um

T.cl

ypea

tum

T. d

asy

rum

0

2

4

6

8

10

12

14

T.a

lexa

nd

rin

um

T.co

mp

estr

e

T.a

per

tum

T.a

ng

ust

i

T.sp

um

osu

m

T.d

iffu

sum

T.g

ran

dif

loru

m

T.h

irtu

m

T.ec

hin

atu

m

T.re

pen

s

T.h

ybri

du

m

T.re

sup

ina

tum

T.su

bte

rra

neu

m

T.p

ilula

re

T.b

ala

nsa

e

T.lig

ust

icu

m

T.la

pp

ace

um

T.cl

ypea

tum

T. d

asy

rum

24

No. of veins on stipule Stipule length

Wide variation was recorded for various

qualitative traits among the species studied.

Growth habit:- Most of the Trifolium species were

prostrate in nature while T. alexandrinum showed

erect or semi-erect growth habit.

Flowering period: T. clypeatum and T. dasyrum

flowered in 2nd week of February while T. hybridum

EC425029 followed by T. diffusum EC528538

flowered in 1st week of May. Most of the species

flowered in the month of March.

25

26

Fig: Variation for growth habit in Trifolium species.

Petiole hairiness: varied from hairy to non hairy.

T. spumosum and T. resupinatum had non hairy or glabrous

petiole while dense hairs in few species viz., T. diffusum, T.

hirtum, T. pilulare, T. balansae, T. clypeatum and T.

alexandrinum EC539057.

Leaflet hairiness:- Hairy or dense hairy leaflets were

found in most of the species viz., T. apertum, T.

angustifolium, T. diffusum, T. hirtum, T. balansae and T.

dasyrum but species like T. spumosum, T. repens, T. hybridum

and T. resupinatum had non hairy or glabrous leaflets.

27

Leaflet apex shape:- Round leaflet apex was found

to be common however the species such as T.

apertum, T. angustifolium, T. resupinatum, T. pilulare

and T. dasyrum had only tapering leaflet apex.

Leaf shape:-This trait varied from lanceolate,

oval, ovate, elongated, obovate, to rhomboid where

lanceolate leaf shape was seen predominantly.

28

29

Fig: Variation for leaf shape & leaf margin in different species of genus Trifolium.

Leaflet margin:-Leaflet had either serrate or entire

where entire margin was quite frequent.

Inflorescence apex shape:- Round was observed to

be predominant with some conical or tapering apex.

Flower colour:- A wide range of variation has been

observed for flower colour which varied from white to

dark purple colour, white flower (in 15 accessions) was

most common among the species followed by light

pink flower (in 14 accessions).

30

31

Fig: Variation for flower colour in different species of genus Trifolium.

T. compestre, had yellow flower and T.

clypeatum, T. hirtum, T. echinatum had

flowers with white + pink petals. Flowers

of T. clypeatum were largest in size as

compared to other species. 32

51 accessions of 19 Trifolium species were grouped

into 7 clusters based on similarities of Mahalanobis’s

D2 values.

Cluster-1 was the largest cluster with 30 accessions

followed by cluster-2 comprising of 8 accessions,

cluster-4 comprising of 6 accessions and cluster-3

comprising of 4 accessions.

Cluster-5 (T. alexandrinum EC418538), 6 (T.

angustifolium EC539064) & 7 (T. angustifolium

EC539058) were monotypic.

33

34

Cluster

No.

No. of accessions

per cluster

Species Name

1 30 T.echinatum EC425075, EC425076, T.lappaceum EC402165, T.echinatum EC425078, T.ligusticum

EC528544, T.lappaceum EC528542, T.resupinatum EC425071, T.echinatum EC425077, T.balansae

EC511775, T.grandiflorum EC528540, T.resupinatum EC425072, T.hybridum EC425029, EC401702,

T.hirtum EC425037, EC425039, T.hybridum EC401701, T.pilulare EC528544, T.diffusum EC528538,

T.subterraneum EC401717, T.spumosum EC528549, EC518781, T.repens EC400984, T. dasyrum

EC511808, T.subterraneum EC401718, T.echinatum EC401714, T.subterraneum EC539068,

T.alexandrinum EC528530, EC539057, EC511781 & EC400773

2 8 T.clypeatum EC511807, EC528536, T.repens EC402155 A, EC400385 B, EC400987 A, EC402155 B,

EC400987 B & EC400385 A

3 4 T.compestre EC425028, EC5425027, EC402155 & T. dasyrum EC528537

4 6 T.resupinatum EC425036, EC425074, EC425073, EC401715, T.apertum EC401712 & T.alexandrinum

EC400976

5 1 T.alexandrinum EC418538

6 1 T.angustifolium EC539064

7 1 T.angustifolium EC539058

Total clusters=7 Total Accession=51 Total species=19

35

Cluster-1 Cluster-2 Cluster-3 Cluster-4 Cluster-5 Cluster-6 Cluster-7

Cluster-1 90.49 218.94 226.45 199.94 202.83 265.53 432.91

Cluster-2 98.92 301.67 288.95 409.07 424.08 728.9

Cluster-3 107.35 402.47 440.78 341.85 545.03

Cluster-4 166.62 289.35 331.88 641.07

Cluster-5 0 104.94 187.68

Cluster-6 0 145.47

Cluster-7 0

Table:2 Mahalanobis Euclidean2 Cluster Distances (Genetic

distance) based on Tocher’s clustering method between nineteen

Trifolium species:-

36

No. of

Branch

No. of

Node

Internodal

length

Stem

Diameter

Ht./ Rossete

Diameter

Petiole

Length

Leaflet

Length

Leaflet

Width

Stipule

Length

No. of Veins

on Stipule

Cluster-1 14.17 10.52 2.37 0.3 67.87 1.38 1.88 0.98 1.07 6.10

Cluster-2 15.00 12.33 2.41 0.31 76.63 5.86 1.86 1.42 1.18 11.25

Cluster-3 56.25 13.58 1.47 0.28 62.42 1.10 1.70 0.87 0.98 7.25

Cluster-4 9.94 13.61 3.21 0.49 139.39 1.68 2.46 1.12 1.10 5.44

Cluster-5 4.00 9.33 4.00 0.30 59.67 1.80 4.13 1.47 2.23 6.00

Cluster-6 25.33 11.67 2.80 0.40 95.00 1.97 3.63 0.93 2.28 7.67

Cluster-7 9.67 7.33 1.90 0.23 19.83 1.27 4.03 0.47 2.60 9.33

Table:3 Clusters mean value for 10 quantitative characters in 7

clusters based on morphological variations in 19 Trifolium species:-

F1 hybrid: 5 hybrids (named as C-552-1, C-552-2, C-

552-3, 380 and 468) were produced.

Hybrid varied from parents for growth habit,

axillary bud, flowering initiation, leaf wrinkles and

most of the quantitative traits which confirmed the

hybridity.

37

38 0.70 0.77 0.85 0.93 1.00

Wardan

468

C-552-2

C-552-1

FAO5

C-552-3

380

Cluster-1

(79%)

Cluster-3 (85%)

Sub-cluster-2-2

Sub-cluster-2-1

(83%)

The similarity was estimated between 5 hybrids of Wardan

x FAO-5 combinations & their parents using the Jaccard

similarity matrix based on total 20 bands of esterase (10

bands) and native protein (10 bands).

78% 76%

71%

39

Fig:- Morphological variation in different intervarietal hybrids.

40

Fig:- Morphological variation in different intervarietal hybrids.

Only 3 F1 hybrid i.e., C-552-1, C-552-2 and 380

successfully survived and produced 23 F2 individuals

representing 9 F2 individuals of C-552-1, 12 of C-552-2

and 2 of 380 survived.

These 23 F2 individuals were evaluated for

morphological characterization.

All F2 individuals showed segregation of traits.

41

In general, these F2 individuals were less vigorous then

F1s.

All F2 individuals showed different vigour and

regeneration.

Out of 23 F2 individuals, 7 were intermediate between the

parents, 7 were P1 type (female type branching) however

remaining 9 individuals were significantly more close to

male parent (P2) regarding flowering and branching.

F2 individuals, showed significant variation for days to

flower initiation ranging from 62 to 100 days as compared to

early flowering (66 to 70 days) in F1 hybrids.

42

9 F2 progenies of this combination including their

respective parents were analyzed for esterase isozyme

system. Dendrogram generated, based on 11 polymorphic and

1 monomorphic band, divided the progenies in 3 clusters.

43 0.60 0.70 0.80 0.90 1.00

Wardan

1

2

5

FAO-5

9

7

3

6

8

4

Cluster-1

Cluster-2

Cluster-3

(72%)

(72%)

The genetic similarity was estimated between 11 F2

progenies of hybrid C-552-1 and their parents using the

Jaccard similarity matrix.

Dendrogram revealed presence of 3 main clusters.

44 0.60 0.70 0.80 0.90 1.00

Wardan

1

2

3

8

10

4

5

6

FAO-5

7

11

9

Cluster-2

Cluster-1

(62.1 %)

(66.4% to 100%)

Cluster-3

(61%)

33 BC1 plants were survived and evaluated for different

morphological attributes.

For general vigour, BC1 plants were significantly similar to F2

plants.

The days to flowering ranged from 66 to 70 days with an average

of 67.33 days in F1 hybrids while in F2 and BC1 plants, it was 62 to

100 (average 80.78) and 82 to 98 days (average 89.24) respectively.

The traits like early flowering, dense secondary branching and

leaf wrinkles were noticed in F1 hybrids as in Fahli group (male

parent). These traits were more prominent in BC1 plants as

compared to F2 plants.

45

Only 7 BC1 plants survived successfully and were evaluated

for various morphological traits.

Vigour of BC1 plants was quite similar to F2 plants.

The broad range for the character days to flowering

initiation was recorded in F2 plants ranging from 62 to 100

days. It was quite contrasting to BC1 plants, where days to

flowering ranged from 97 to 100 days and quite similar to P1

female parent (Wardan) showing a range of 90 to 94 days. In

P2 male parent (FAO-5) days to flowering initiation was

between 62 to 64 days whereas in F1 hybrids the range was 66

to 91 days.

46

The regeneration was the characteristic feature of

female parent P1 (Wardan) segregated in F2 plants

(three poor, thirteen medium good and seven good

regeneration) as well as in BC1 plants (two good, two

medium good and two very good regeneration).

F1 hybrids showed dense branching and early

flowering while BC1 plants did not show early

flowering and dense branching, showing their

closeness to P1 parent (Female: Wardan). 47

In this combination 4 hybrid plants numbered as C-

579-1, C-579-2, 482 and C-10 were produced.

These hybrids varied significantly for all qualitative

traits except leaflet margin which was entire in all the

hybrids.

However, semi-erect growth habit, hairy leaflet and

dense hairy stipule pubescence were similar in C-579-1,

C-579-2 and 482, while C-10 had erect growth, dense

hairy leaflet and hairy stipule.

Traits like petiole hairiness, leaflet apex shape and

leaflet colour were similar in all the hybrids except C-

579-1.

Variation was observed for quantitative traits among

the hybrids.

BB-2 x FAO-5 Combination:-

48

Biochemical characterization of BB-2 x FAO-5 hybrids

4 hybrids of BB-2 x FAO-5 were analyzed for native

protein banding pattern along with parents.

The dendrogram based on 11 bands of native protein

was divided in 3 main clusters.

49 0.70 0.77 0.85 0.93 1.00

BB-2

C-579-1

C-579-2

C-10

FAO-5

482

Sub-cluster 1-1

Sub-cluster 1-2

Cluster -2 (100%)

Cluster -3

(74%)

(88%)

7 F2 individuals representing 5 F2 individuals of

C-10 hybrid and 2 F2 individuals of hybrid 482

survived successfully.

F2 progenies also differed for regeneration (one

very good, four good and two medium).

Flowering initiation was similar in F1 hybrids

and F2 individuals.

50

3 resembled male parent, 1 individual had

male type of high secondary branching and 1

individual was maternal type having less

secondary branches whereas 1 individual was

intermediate among both the parents.

51

52

Similarity among F2 populations of BB-2 x FAO-5

UPGMA dendrogram was formed on the basis of 15 scorable

bands of esterase isozyme in 7 F2 progenies and their parents .

The population was divided in 2 main clusters; cluster-1 was

comparatively larger where intra-cluster similarity ranged from

66% to 100%.

0.50 0.63 0.75 0.88 1.00

BB-2

6

7

1

4

5

2

3

FAO-5

(66%-100%)

Cluster-2

Sub-Cluster-1-1

Sub-Cluster-1-2

Sub-Cluster-1-3

(55%)

3 hybrid were obtained and named as C-560, C-564

and 203 which were similar for qualitative traits like

leaflet margin, leaflet shape, leaflet apex shape, leaflet

colour, stipule pubescence and flower colour.

The hybrid C-560 was more similar to hybrid C-564

than hybrid 203 for qualitative traits.

The hybrid C-564 was more vigorous having higher

values than other hybrids and the parents for most of the

quantitative traits.

On comparing the hybrids with parents, it was

observed that leaflet margin, leaflet shape, axillary bud

and flower colour were similar among the parents and the

hybrids while rest of the qualitative traits varied.

Ex-8 x FAO-5 combination

53

The dendrogram was based on 24 bands representing

10 bands of esterase and 14 bands of protein native.

54 0.70 0.77 0.85 0.93 1.00

Ex-8

FAO-5

203

C-560

C-564

Cluster-1-1

Cluster-2

Cluster-1-2

88%

79%

74%

16 F2 individuals representing 4 F2 individuals from

C-560 and 12 from C-564 were compared with F1

hybrids for some morphological attributes.

Both F1 hybrids showed oval bud apex and dark

green leaf colour whereas in F2 individuals both the

traits varied.

Most of the F2 individuals flowered early like their

respective parents.

55

7 F2 progenies resembled P1 parent, 7

intermediate and 2 were P2 type with respect to

branching behavior.

The range of number of nodes on secondary

branch of 4th node and length of secondary branch

of 4th node was 0 to 8 and 0 to 55cm in F2

individuals respectively.

The average of number of primary branches per

plant was 2.5 in all F2 individuals. 56

Dendrogram was based on 5 monomorphic and 6

polymorphic bands and generated by using the

UPGMA algorithm.

57

0.75 0.81 0.88 0.94 1.00

Ex-8

2

1

3

4

FAO-5

Cluster-1-1 (94.1%)

Cluster-2

(77%)

Cluster-1-2

(85%)

In this combination 2 hybrid viz, 79-1 and 79-2

were produced which were similar for almost all

qualitative traits.

Growth habit, petiole hairiness and axillary bud

in the hybrids were more similar to the female

parent than the male parent.

Hybrids were early flowering, quite similar to

the female parent while male parent flowered late.

58

Both the hybrids had oval inflorescence apex and

pentafoliate leaves while in the parents it was

elongated trifoliate respectively.

No. of branches, no. of nodes, plant height, leaflet

length, leaflet width, leaflet no. and stipule length were

higher in the hybrids than the parents while rest of the

traits varied among the parents and the hybrids. 59

The genetic similarity was estimated between 4hybrids

(213, 378, 79-1 & 79-2) along with their parents.

Based on 14 bands of protein native, dendrogram was

divided in 2 clusters.

60 Coefficient 0.80 0.85 0.90 0.95 1.00

Ex8

213

79-1

79-2

Wardan

378

Cluster-1-1 92%

Cluster-1-2

Cluster-2

83%

78%

12 F2 individuals (11 from 79-1 and 1 from 79-2 hybrid)

successfully survived. The trait, vigour and regeneration

showed variation among the F2 individuals.

A range of 0 to 6 and 0 to 24cm has been found for the

trait, number of nodes on secondary branch of 4th node and

length of secondary branch of 4th node respectively.

7 were similar to male parent for branching behavior and

1 was more close to female parent while 4 were

intermediate among both the parents.

Flowering initiation was quite late in F2 individuals

(ranging from 51 to 119 days) as compared to F1 hybrids

which flowered after 67 days.

61

4 bulks representing viz., P1 bulk (Wardan

bulk), P1 type bulk (Wardan type plant

progenies), P2 bulk (FAO-5) & P2 type bulk

(FAO-5 type plant progenies) were screened

with 110 RAPD primers.

62

Total primers 110

Total amplified primers 92

Total yielded bands 623

Monomorhpic bands 403 (64.68%)

Polymorphic bands 220 (35.32%)

Range of yielded bands

1 band (OPH-1, OPH-8, OPH-15 & OPJ-16) - 14 bands (KITAB-18)

Average of bands/primer 6.77

Not Amplified 18

Monomorphic primers 17

Polymorphic primers 75

Primer generate nonspecific bands 46

Primer generate specific bands 29 63

64

Fig: Screening of four bulks with twenty-nine RAPD primer

generating specific bands:-

S.No. Primers Specific band no.

1 OPH-2 B5 for regeneration

2 OPH-3 B5 for regeneration and B3 for non-regeneration

3 OPH-6 Three bands, B3, B9 & B10 for non-regeneration

4 OPH-7 B1 for regeneration

5 OPH-19 B8 for non-regneration

6 OPD-2 B8 for regeneration


8 OPD-5 B5 for regeneration, B13 for non-regeneration

9 OPD-7 B6 for non-regeneration



12 OPD-19 B3 for non-regeneration

13 OPD-20 B3 &B6 for regeneration

14 KITAB-4 B2 & B7 for non-regeneration, B8 for regeneration

15 KITAB-5 B9 for regeneration


17 KITAB-9 B3 for regeneration);

18 KITAB-11 B10 for non-regeneration, B7 & B8 for regeneration




22 OPE-15 B1 for regeneration

23 OPJ-9 B3 for regeneration

24 OPJ-7 B2 for non-regeneration, B1 for regeneration

25 OPJ-13 B3 for non-regeneration, B5 for regeneration

26 OPJ-17 B5 for regeneration

27 OPC-4 B7 for regeneration

28 OPC-12 B4 for non-regeneratio

29 OPC-20 B4 for regeneration

65

Dendogram based on 623 bands generated by 92

primers was developed.

The dendrogram showed that the genetic similarity

among the bulks was quite high, ranging from 87% to

92%.

66 0.85 0.89 0.93 0.96 1.00

P1 bulk

P1 type bulk

P2 type bulk

P2 bulkbulk

Sub-cluster 1-1

(92%)

Sub-cluster 1-2

Cluster -2

88%

87%

67

S.No. Ecotype Primers Amplified bands

Monomorphic bands

Polymorphic bands

Range of amplified bands

1 Ex-8 12 76 21 55 2-11

27.63% 72.37% OPD-11 - OPD-2

2 Wardan 13 94 27 67 1-12

28.73% 71.27% OPD-10 - OPD-2

3 FAO-5 14 85 22 63 2-15

25.88% 74.12% OPD-12, 15 - OPD-11

4 BB-3 13 79 25 54 3-11

31.65% 68.35% OPD-13, 14, 16 - OPD-11

5 BB-2 14 115 24 91 3-14

20.87% 79.13% OPD-12 - OPD-11

Genetic variability was estimated among 24 plants each of Ex-8,

Wardan, FAO-5, BB-3 & BB-2 by using different oligonucleotide primers.

68

Coefficient

0.48 0.58 0.69 0.80 0.90

1 3 4 6 2 5 7 10 13 11 14 15 16 21 19 8 9 20 18 12 17 22 23 24

Coefficient 0.68 0.75 0.81 0.88 0.95

1 2 3 4 6 7 8 10 9 20 12 14 15 16 17 19 24 21 22 11 5 13 18 23

Cluster-1

Cluster-4

Cluster-3

Cluster-2

Cluster-3

Cluster-1

Cluster-2

69

Coefficient

0.64 0.71 0.77 0.83 0.90

1 2 5 6 7 8 15 16 23 14 17 18 19 20 21 22 24 9 11 13 10 12 3 4

Coefficient 0.70 0.76 0.81 0.86 0.92

1 2 3 4 5 11 10 6 7 8 15 16 17 13 9 14 12 19 20 21 22 23 24 18

Cluster-2

Cluster-1

Cluster-4

Cluster-3

Cluster-1

Cluster-2

Cluster-3

Cluster-4

Cluster-5

Cluster-6

Cluster-7

70

Coefficient

0.64 0.71 0.77 0.83 0.90

1

2

3

5

4

7

6

9

10

17

22

21

23

11

18

8

12

16

13

19

24

14

15

20

Cluster-3

Cluster-2

Cluster-5

Cluster-4

Cluster-6

Cluster-1

71

Fig:-RAPD profile of different ecotypes of Berseem.

BB-2 (OPD-11) FAO-5 (OPD-11)

Wardan (OPD-11)

FAO-5 (OPD-18) BB-2 (OPD-18)

BB-2 (OPD-5)

Wardan (OPD-18) BB-3 (OPD-18)

Wardan (OPD-8) BB-3 (OPD-8)

Wardan (OPD-13)

Genetic variability was calculated among 120 plants

comprising of 24 plants each of 5 genotypes viz., BB-2,

BB-3, Wardan, Ex-8 and FAO-5 by using 13

oligonucleotide primers.

The dendogram showed the presence of 11 main

clusters, which were again divided in total 17 sub-

clusters.

Inter-cluster similarity among 11 clusters was

ranging from 47.0% to 92.0%.

72

73 0.46 0.58 0.70 0.81 0.93

C1 C2 C3 C4 C5 C11 C10 C6 C7 C8 C15 C16 C17 C13 C9 C14 C19 C20 C108 C110 C111 C112 C113 C115 C120 C116 C117 C118 C12 C101 C21 C22 C23 C24 C100 C102 C103 C104 C106 C105 C107 C97 C98 C99 C38 C41 C42 C43 C39 C40 C47 C49 C50 C51 C53 C52 C55 C54 C57 C58 C65 C70 C69 C71 C59 C66 C56 C61 C67 C72 C109 C114 C119 C18 C68 C25 C26 C27 C28 C30 C33 C35 C37 C34 C36 C29 C60 C64 C44 C45 C46 C48 C31 C32 C62 C63 C73 C75 C77 C76 C78 C79 C74 C91 C80 C81 C92 C84 C82 C90 C85 C89 C94 C83 C86 C87 C88 C93 C95 C96

A total of 38 SSR primer pairs were used to screen for

amplification and polymorphism among 75 accessions

belonging to 24 species of genus Trifolium.

Out of 38 SSR primer pairs, 34 primer pairs amplified

generating 130 polymorphic bands while remaining 4

primer pairs were non-informative.

The number of fragments amplified by individual

primers ranged from 2 to 6. The amplified products size

ranged from 100 bp to 280 bp. 74

75

Fig: SSR profile generated by primer PR-27 (433) in 24 species of genus Trifolium:-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 M 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M 1 2 3 4 5 M

The genetic distance between 75 accessions belonging to

24 species was estimated using the dendogram formed by

SAHN agglomerative clustering.

The grouping based on 130 bands showed the presence of

11 main clusters.

Main clusters were further divided in 25 sub-clusters and

4 sub-sub-clusters.

Inter-cluster similarity ranged from 28% to 96% among

11 clusters. 76

77 Coefficient

0.08 0.31 0.54 0.77 1.00

P1 P5 P7 P4 P8 P3 P9 P10 P2 P6 P13 P14 P17 P18 P15 P16 P19 P20 P23 P21 P24 P11 P22 P56 P57 P33 P35 P32 P34 P39 P46 P48 P49 P55 P47 P27 P53 P54 P58 P59 P60 P29 P71 P72 P73 P76 P75 P74 P50 P51 P52 P63 P65 P66 P67 P68 P69 P70 P61 P62 P25 P26 P28 P30 P31 P40 P41 P42 P43 P44 P45 P36 P38 P37 P64 P12

Cluster-1

Cluster-2

Cluster-3

Cluster-5

Cluster-4

Cluster-6

Cluster-7

Cluster-8

Cluster-9 Cluster-10 Cluster-11

Cluster Sub-cluster Total accessions Species

1

1-1

1-1-1 14 T. alexandrinum (Wardan, BB-3, Giza-6, BB-2, Sakha-4, FAO-5, Serw-1, Helaly, Ex-8,

Fahli original, EC400976, EC418536, EC539050 & EC539057)

1-1-2 2 T. alexandrinum EC539048 and EC539047

1-1-3 3 T. alexandrinum (EC539054 & EC418539) and T. clypeatum EC528536

1-1-4 2 T. alexandrinum EC418538 and T. dasyurum EC528537

1-2 1 T. alexandrinum EC528530

1-3 1 T. clypeatum EC511807

1-4 2 T. hirtum EC429039 and EC425037

2 2-1 2 T. angustifolium EC539063 and EC425062

2-2 3 T. angustifolium (EC539058 & EC539064) and T. glomeratum EC401700

3 3-1 4 T. subterraneum (EC539068, EC401717 & EC539069) and T. pilulare EC528547

3-2 1 T. subterraneum EC401718

4 1 T. echinatum EC425075

5

5-1 5 T. apertum EC401712, T.fragiferum EC528539 and T. vesiculosum (EC401716, EC Ooty &

EC Palampur)

5-2 6 T. echinatum EC425078, T. lappaceum (EC528542 & EC402165), T. grandiflorum EC528540,

T. ligusticum EC528544 and T. balanse EC511775

5-3 1 T. diffusum EC528538

5-4 3 T. hybridum EC401702 , EC425029 and EC401701

6

6-1 1 T. nigrescence

6-2 5 T. repens EC400385, EC402155, EC400984, EC400379 and EC401705

6-3 1 T. repens EC401706

7 2 T. pratense EC400980 & EC400979

8

8-1 3 T. dasyurum EC511808 and T. echinatum (EC425076 & EC425077)

8-2 2 T. spumosum EC511781 and EC528549

8-3 6 T. glomeratum EC405033 and T. resupinatum (EC401715, EC425073, EC425074, EC425072

and EC425071)

9 3 T. compestre EC402155, EC425027 & EC425028

10 1 T. repens EC400987

11 1 T. alexandrinum EC511781

78

Compatibility between different species is an indication of

close affinity between two species.

In Trifolium, the incompatibility barriers are very strong.

Evans (1962 b) reported the existence of high degree of

incompatibility between species of Trifolium.

The present study is an attempt to know the genetic

similarity among 10 Trifolium species and their relationship

with T. alexandrinum based on compatibility studies. 79

80

S. No. Female Parent Male Parent Obs-1 Obs-2 Obs-3 Obs-4 P-I P-II P-III P-VI P-V Observations Seed Setting

Control cross

T.alexandrinum cv Wardan

T.alexandrinum cv Wardan

5hrs 10 1.8 1.7 1.9 1.7 2.4 2.4 1.4 Normal seed setting

Normal

1 T.alexandrinum cv Wardan

T.angustifolium EC539058

1hr 10 0.2 0.1 0 0 0 0 0 No withering No 5hrs 10 1.8 1.5 0.5 0.3 0.3 0.2 0 8hrs 5 0 0 0 0 0 0 0

2 T.alexandrinum EC511781

T.clypeatum EC528536

1hr 5 0.2 0.2 0 0 0 0 0 No withering No 3hrs 5 0 0 0 0 0 0 0 5hrs 5 0 0 0 0 0 0 0 8hrs 5 0 0 0 0 0 0 0


T.grandiflorum ECEC528540

3hrs 5 0.6 1.2 0 1 0.2 0 0 Withering 2 DAP & shrivel ovaries

No 5hrs 5 0.8 0.8 0 0.2 0.4 0.2 0

8hrs 5 1.2 2.2 1.6 1.2 1.2 1 0.2


T. compestre EC5425027

1hr 5 0.6 0.6 0.4 0 0 0 0 No withering up to 5 days

No 3hrs 5 1.2 0.8 0.8 0.2 0 0 0 5hrs 5 0.8 1.4 0.8 0.4 0.2 0 0


T .repens EC400385

1hr 10 0.9 0.6 0.5 0 0 0 0 No withering No 5hrs 11 0.82 0.82 0.6 0.18 0 0 0


T .ligusticum EC528544

1hr 10 0.5 0.6 0.5 0.1 0 0 0 Withering 2 DAP No 5hrs 10 1.6 0.5 0.2 0.2 0.4 0.1 0


T. balansae EC511775

1hr 10 1.1 0.6 0 0 0 0 0 No withering No 5hrs 10 0.5 0.4 0 0 0 0 0

8 T.alexandrinum cv BB-2

T. hirtum EC425039

1hr 10 0.3 0.2 0 0 0 0 0 No withering No 5hrs 10 0.2 0.4 0.5 0 0 0 0


T. pilulare EC528547


10 T.alexandrinum cv BB-2

T. hybridum EC41702


Obs-1=Flowers harvested after 5hrs Obs-2=Number of pistils observed.

Obs-3Number of pollen attached (Average). Obs-4=Hairy region of stigma with pollen tubes or pollen germination (Average).

81

Fig: Pollen tube growth in different cross combinations

Control cross (A-J)

(Wardan) x T. clypeatum EC528536 (K-L)

(Wardan) x T. hirtum EC425039 (M)

(Wardan) x T. angustifolium EC539058 (N-X)

82

Fig: Pollen tube growth in different cross combinations:-

(Wardan) x T. balansae EC511775 (A-D)

(Wardan) x T. compestre EC5425027 (E-H)

(Wardan) x T. ligusticum EC528544 (I-Q)

(Wardan) x T. repense EC400385 (R-T)

Good mitotic preparations with well spread

metaphase were studied.

5 cultivars of T. alexandrinum representing 3

ecotypes viz., Mescawi (Wardan, BB-2 & BB-3), Saidi

(Ex-8) and Fahli (FAO-5) were analyzed on the basis

of mitotic preparations.

The number of chromosomes were found to be

2n=16 in Wardan, BB-2, FAO-5 and Ex-8 while in BB-

3, the number of chromosome was 2n=32.

83

84

BB-3 Wardan BB-2 Ex-8 FAO-5

Mitotic preparations of different ecotypes of berseem (T. alexandrinum)

prethesis submission seminar on 9th may 2012

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