Inability of flowering plants to produce functional pollen. Male sterility is agronomically important for the hybrid seed production. Onion crop provides one of the rare examples of very early recognition of male sterility cultivar Italian Red (Jones and Emsweller 1936) Its inheritance and use in hybrid seed production (Jones and Clarke 1943). Since then male sterility is reported in a fairly large number of crops including vegetables.

1. Male sterility and its utilization in vegetable
improvement
Name of the Student : Mr. HEMANT GHEMERAY
ID No. : UHS14PGM537
Degree Programme and Subject : M.Sc. (Hort.) Dept. of Vegetable Sciences
College : College of Horticulture, Bengaluru.

2. What is Male Sterility ?
03-02-2017
COH Bengaluru
2
 Definition : Inability of flowering plants to
produce functional pollen.
 Male sterility is agronomically important for the hybrid seed
production.
•Onion crop provides one of the rare examples of very early
recognition of male sterility cultivar Italian Red (Jones and
Emsweller 1936)
•Its inheritance and use in hybrid seed production (Jones
and Clarke 1943).
•Since then male sterility is reported in fairly large number of crops
including vegetables.

3. 03-02-2017 COH Bengaluru 3
1. Mitochondrial mutation : mutation in mitochondrial bodies (mt DNA) (Shrivastva
& Sarkssian, 1969)
2. Barrier of tapetal layer : delayed degeneration of tapetal cells that block the
availability of nutrient to microspore (Polowick & Sawhney,1995)
3. Improper timing of callase activity : callase is an enzyme required for breakdown
of the callose that surrounds the pollen mother cells, helps in release of pollen ;
early or delayed callase activity lead to sterility.(Pritchard & Hutton, 1972;
Gottshalk & Kaul, 1974)
4. Role of Esterase : Esterase isozymes play role in the hydrolysis of Sporopollenin,
the polymer required for pollen formation. Decreased activity of esterase in male
sterile plant has been obeserved in tomato (Bhadula and Sawhney,1987) and in
radish (Zhon and Zhang, 1994)
5. Absence or malformation of male organs (stamens) in bisexual flowers: Failure to
develop normal microsporogenous tissue- anther.
6. Abnormal microsporogenesis--deformed or inviable pollen.

4. Significance of male sterility
• Genetic emasculation of plants.
• Economic & quality hybrid seed production.
• Larger quantity of hybrid seeds.
• As tester genotypes for assessing the combining ability.
03-02-2017 COH Bengaluru 4
Flower of male-fertile onion
Flower of male-sterile onion

5. Classification of Male sterility
On phenotypic basis
1. Sporogenous male sterility (eg dry/sticky pollen)
2. Structural male sterility (eg exerted stigma, stamenless filower in L.
hirsutum)
3. Functional male sterility (failure of anther dehiscence; eg tomato & brinjal)
On non genetic basis
1. Chemical male sterility
2. Physiological male sterility
3. Ecological male sterility
On genetic basis (spontaneous or induced)
1. Genetic male sterility
i) Temperature sensitive genic male sterilty
ii) Photoperiod sensitive genic male sterilty
iii) Transgenic male sterilty
2. Cytoplasmic male sterility
3. Cytoplasmic genetic male sterility
03-02-2017 COH Bengaluru 5

6. Genic Male Sterility (GMS) or Nuclear Male Sterility
 GMS has been reported in about 175 plant species (Kaul 1988) including important
vegetable crops
 Salient points
• Usually recessive & monogenic
• GMS does not have any undesirable agronomic characters
 Limitations of GMS
• Less Stable to temperature and photoperiod
• 50% of the fertile plants to be removed from the field
 Availability of marker gene- closely linked with ms gene
 Origin of GMS
 Spontaneous mutation
 Mutation by ionizing radiation. etc
03-02-2017 COH Bengaluru 6

7. 03-02-2017 COH Bengaluru 7

8. LINE A X LINE C
Male sterile Male fertile
msms MsMs
COMMERCIAL HYBRID
Msms (AXC)
(All male fertile)
03-02-2017 COH Bengaluru 8
Crops Gene
Commercially
utilized
Variety
Tomato Single recessive gene ps-2 gene -
Chilli Single recessive gene ms-12 & ms-3 gene CH-1, CH-3
Muskmelon Single recessive gene ms-1 gene
Punjab hybrid-
1

9. Many transgenes
produce genetic male
sterility observed in
tobacco, brassica spp.
tomato, cauliflower, etc.
 If transgenes are utilized
for hybrid seed production
required effective fertility
restorer system. eg.
Barnase/Barstar
- Barnase/Barstar –
transgenic tobacco
complete male sterile
Vegetable crops-
tomato, Cauliflower.
The barnase-barstar male sterility-fertility
restoration system was identified in Cauliflower and
Tomato ( Banga and Raman 1998)Cell cytotoxicity

10.  Determined by the cytoplasm
 It is the result of mutation in the mitochondrial genome (mt-DNA)
 CMS easily transferable trait n
 Most CMS associated genes are chimeric mitochondrial sequences
(Schnable and Wise 1998)
 Advantages of CMS
• Highly stable and not influenced by environmental conditions
 Limitations of CMS
• Not use where seed is the economic product
• CMS line has inferior agronomic performance
03-02-2017 COH Bengaluru 10
Cytoplasmic male sterility (CMS) systems

11. 03-02-2017 COH Bengaluru 11

12. Cytoplasmic genetic male sterility
Male sterility arises due to interaction of nuclear gene(s) conditioning sterility
with sterile cytoplasm
03-02-2017 COH Bengaluru 12
RR gene with F cytoplasm
(Fertile; R – line)
Genotypes of CGMS line
RR gene with S cytoplasm
(Fertile; R – line)
Rr gene with F cytoplasm
(Fertile)
msms F
RR
msms F
Rr
msms S
RR
msms S
Rr
msms F
rr
msms S
rr
Rr gene with S cytoplasm
(Fertile)
rr gene with F cytoplasm
(Fertile; B – line)
rr gene with S cytoplasm
(Sterile; A – line)

13. 03-02-2017 COH Bengaluru 13
Breeding Strategies Using CGMS lines In Hybridization
100% sterile and stable
under diverse condition
Unstable during
growing season
Mixture of sterile and
fertile plants
100% sterile in some
environ. & fertile in other
100% fertile and stable
under all environment
If line x tester
Indicates B line
(Maintainer line)
Indicates temperature
and humidity effect
Indicates R line
(Restorer line)
Related to climate
(temp. and humidity)
Indicates line is hetero-
genous at rf1 locus
Outcome
Conversion
programme
Benefit of
Seed production
Purification via
single plant selection
Hybrid development
programme
Should be Rejected
Breeding Strategy

14. Cytoplasmic genetic male sterility(CGMS) in vegetables crops
Crops Gene
Commerciall
y utilized
Variety
Chilli
Single recessive
gene
ms-2
Arka Meghana,
Arka Swetha, Arka
Harita, Kashi Surkh
Onion
Single recessive
gene
-
Arka Kirtiman, Arka
Lalima
Carrot
Single recessive
gene
-
Pusa Nayanjyothi,
Pusa Vasuda
03-02-2017 COH Bengaluru 14

15. Detection of MS System
By progeny performance or crossing with a few
normal (fertile) genotype
03-02-2017 COH Bengaluru 15
Trend I – All the progenies in all the rows may be sterile : CMS
Trend II – Some rows may consist of all fertile plants and some rows
sterile & fertile plants may occur in 1 : 1 ratio : GMS
Trend III – Some rows may have all fertile plants, some all sterile plants
& some have fertile and sterile plants in 1 : 1 ratio : CGMS

16. 03-02-2017 COH Bengaluru 16
GMS based hybrids:
CH-1 (MS 12 x Ludhiana long sel.)
CH-3 (MS 12 x S-2530)
Hybrids Identified through AICRP-
VC
CCH-2 (A1 x Pusa Jwala) and
CCH-3 (KA-2 x RPBC-473)
ARCH-228, Meghna
TOMATO
Ludhiana
ms33 IPA
ms2 IPA
ps2 L 3841
ps2 NS 101
ps2 San Pedro
ps2 UC 82-B
ms 10
36
ms 45
ms 15
47
CHILLI
Anand
CCA 4261
CCA 4759
CCA 4758
Ludhiana
CCA 4261
ONION
Bangalore
Ms1
Ms2
ms3
Male sterile genotypes available in India
S. No. Name of chilli hybrid Source
1 CH 1 (GMS based) PAU, Ludhiana
2 CH 3 (GMS based) PAU, Ludhiana
3 Arka Meghana (CGMS based) IIHR, Bangalore
4 Arka Sweta (CGMS based) IIHR, Bangalore
5 Arka Harita (CGMS based) IIHR, Bangalore
6 Arka Khyati (CGMS based) IIHR, Bangalore
7 CCH 2 (CGMS based) IIVR, Varanasi

17. Steps in development of male sterile line
1.Morphological and molecular evaluation of male sterility system.
2. Studies on blossom biology on floral morphology of male sterile and fertile flowers.
3. Transfer of male sterility into different genetic backgrounds to identify
corresponding maintainer & restorer lines/ pollen parents.
4.Studies on microsporogenesis and megasporogenesis of male sterile and male fertile
lines.
5.Development mapping population (parents, F1, F2 ,BC1, BC2 progenies) for genetic
studies.
6.Identification of molecular markers
7. Studies on reproductive biology and identify causes of male sterility (CMS,CGMS&
GMS system)).
8.. Use of diagnostic PCR kit to identify sterile, maintainer and restorer lines.
9. Development of stable male sterile lines, maintainers, restorer and male parental lines
to develop uniform efficient and durable F1hybrids.

18. Male sterility in selected vegetables
03-02-2017 COH Bengaluru 18
Mutant Description Inheritance Governing by
single recessive
gene
Stamenless Stamens absent Monogenic
recessive
sl
Positional sterility Stigma exerted Monogenic
recessive
ps
Pollen sterility Pollen abortive Monogenic
recessive
ms series
Functional sterility Anthers do not
dehisce
Monogenic
recessive
ps-2
Table 6: Different male sterile mutants in tomato.

19.  First report of MS within progenies of an onion cultivar
Italian Red (Jones & Emeweller, 1936); Male sterility
controlled by male sterile cytoplasm & recessive nuclear
gene (Jones & Clarke, 1943)
 3 types of Cytoplasm
1. S – cytoplasm
- anther morphology is normal but at anthesis these are
green, small & indehiscent
2. T - cytoplasm (Berninger in Jaune Paille des
Vertus(1965))
- anther morphology is disrupted. (Kaul, 1988)
03-02-2017 COH Bengaluru 19
Onion
Bennekam, 1979

20. Emasculation avoided
(STEP I)
Male sterile flower
Covering entire stigma with pollen
(STEP II)
USE OF CGMS SYSTEM IN HYBRID SEED PRODUCTION
IN CHILLI
03-02-2017 COH Bengaluru 20
Type of male sterility: CMS & CGMS
Present status: AB& R lines have been developed
(CGMS); AB&C lines( CMS) to be developed and commercialized
Incorporation of Anthracnose, phytophthora blight resistance & pungency with male
sterility & development of F1 hybrids
CHILLI

21. GMS :
 Due to shrivelled, brown & non exerted
anther
CMS & CGMS : 3 types of CMS
1. Petaloid type
- anther transformed into petal or petal
like structure, unable to produce
functional pollen
2. Brown anther type
- present in all orange type cultivars
- deformed, brown coloured anther
without functional pollen
3. Gum type
- derived from cross with D. carota var
gumifera
- total reduction of anthers & petals
03-02-2017 COH Bengaluru 21
Carrot
a) Normal (N-cytoplasm,
restored CMS plants)
b) Brown anther CMS (Sa)
c) Petaloid CMS (Sp)

22. Type of male sterility: GMS (Inductive)
Present status: AC lines have been developed
and commercialized
Incorporation of YVMS resistance with male
sterility & development of F1 hybrids
03-02-2017 COH Bengaluru 22
Okra

23. GMS lines in okra
03-02-2017 COH Bengaluru 23
Geneic male SterileMale fertile
GMS line can be maintained by sibbing

24. Cole vegetables
Transfer of Orgura cytoplasm of Raphanus to:
• broccoli (McCollum, 1981);
• Cauliflower (Hoser, Kranse & Antosik, 1987);
• Brussels sprout (Bannerot et al,1974) and in Cabbage
(McCollum, 1988).
• But seedling of all these CMS line developed chlorosis in
seedling & young leaves lead to delayed maturity.
• Transfer of sterile ‘Anand cytoplasm’ from B. rapa (originally
derived from wild spp B. tounetortii) to - B. olearcea trough
protoplast fusion ( Cardi & Earle, 1997)
03-02-2017 COH Bengaluru 24

25. IIHR CFMS-1
ogura ms
03-02-2017 COH Bengaluru 25

26. IIHRRGMS-2
IIHRRGMS-2 medium long fruits
Fertile pollen
Rudimentary
male flowers
Fertile flower spikes
Male Sterile
Male Sterile buds
Male Fertile
Sterile pollen
Ridge gourd
Type of male sterility: CGMS & GMS

27. Development of male sterile lines of tomato
and assessment of their utility in hybrid
development
03-02-2017 COH Bengaluru 27
Case -1

28. Material and methods
• ms33 IPA
• ms2IPA
• ps2 L 3841
• ps2 NS 101
• ps2 SanPedro
• ps2 UC 82-B1.
•
03-02-2017 COH Bengaluru 28
OBJECTIVE:
1.Transfer of trait(male sterility) through back cross
2. Evaluation of new stocks
3. Assessment of labour needs for hybrid seed production using new stocks

29. Table 1. Fruit characteristics of tomato male sterile lines
S.
No.
Genotype Sterility
No. Of
Locules
Pericarp
thickness
(mm)
TSS (%)
Fruit
weight
(g)
1 ms33 IPA
Pollen
Abortive
2.33 6.33 4.60 59.00
2 ms2IPA
Pollen
Abortive
2.00 6.33 4.75 62.33
3 ps2 L 3841 Functional 4.66 6.00 4.16 61.33
4 ps2 NS 101 Functional 3.00 6.00 4.60 59.33
5
ps2
SanPedro
Functional 3.83 6.66 4.80 112.66
6 ps2 UC 82-B Functional 4.10 6.30 4.63 67.00
03-02-2017 COH Bengaluru
29
Dhaliwal and Cheema., 2008

30. 03-02-2017 COH Bengaluru 30

31. Table 2. Time (minutes) required for crossing 50 flower buds on
male fertile ‘Ms33IPA’ (MF) and male sterile ‘ms33IPA’ (MS) plants in tomato
Worker
Activity
Emasculation Pollination on MF
Emasculation &
Pollination on
MF
Pollination on MS
Time saved in MS
over MF %
1 22.0 44.0 66.0 37.7 42.9
2 26.1 34.9 61.0 26.3 56.5
3 38.7 45.1 83.8 33.3 60.3
4 37.3 43.8 81.1 24.2 54.7
5 32.8 41.1 73.9 32.7 55.8
Mean 30.7 41.7 71.9 32.8 54.4
03-02-2017 COH Bengaluru
31
Dhaliwal and Cheema, 20080
10
20
30
40
50
60
70
80
90
1 2 3 4 5 Mean
Activity Emasculation
Activity Pollination on
MF
Activity Emasculation &
Pollination on MF E
Activity Pollination on
MS
Activity Time saved in
MS over MF %

32. Development and utilization of one new cytoplasmic male
sterile line of Chinese leaf mustard (Brassica juncea var.
rugosa Bailey)
03-02-2017 COH Bengaluru 32
Case 2

33. Materials and methods
• hau CMS (donor parent 00-6-102A).
• Xuelihong 0912B ( receptor parent leaf mustard)
• Novel cytoplasmic male sterility (CMS) designated as hau CMS
(00-6-102A) was identified in Brassica juncea previously.
• In present study, the hau CMS was transferred to leaf mustard
(B. juncea var. rugosa Bailey) for harvesting vegetative mass by
hybridization
F1BC8
03-02-2017 COH Bengaluru 33
00-6-102A( hau CMS) x 0912B
sterile hybrid x 0912B
(backcrossed)
line 0912A was obtained

34. Table 1: Biological characteristics of hau CMS line 0912A and
maintainer line 0912B in leaf mustard
Traits 0912A 0912B 0912A-0912B
Plant weight (g) 959.81 965.35 −5.54
Plant height (cm) 34.17 37.70 −3.53*
Canopy area (cm2 )
1917.64 1950.84 −33.2
Leaf length (cm) 36.39 37.05 −0.66
Leaf width (cm) 12.14 12.00 0.14
Petiole length (cm)
4.04 4.38 −0.34
Petiole width (cm)
1.57 1.62 −0.05
Tiller number 16.28 16.33 −0.05
Rosette leaf
number
166.11 160.06 6.05
03-02-2017 COH Bengaluru
34
Wan et al., 2014

35. Table 2: Leaf morphology of Brassica juncea 00-6-102A, hau
CMS line 0912A and maintainer line 0912B in leaf mustard.
Material
Leaf
colour
Leaf
shape
Leaf
edge
Leaf crack
Leaf
surface
Shine
surface
00-6-
102A
Green Obovate
Shallow
saw
tooth
Pinnatified
Slight
shrinkage
No
0912A
Deep
green
Long
Obovate
Saw
tooth
Pinnatified Smooth Yes
0912B
Deep
green
Long
Obovate
Saw
tooth
Pinnatified smooth yes
03-02-2017 COH Bengaluru 35
Wan et al., 2014

36. Fig. 1. Morphological traits (A) Brassica juncea 00-6-102A; (B) leaf mustard CMS line
0912A. (C) Leaf mustard maintainer line 0912B; (D) leaf mustard CMS line 0912A; (E) leaf
mustard maintainer line 0912B.
03-02-2017 COH Bengaluru
36

37. Table 3: The identification of the ‘sterility degree’ and ‘sterility
rate’ of hau CMS line 0912A in leaf mustard
Time Place Plants
Sterile
plants
Flowers
Sterility
ratio
Seeds
Sterility
degree
(%)
2010.3 Wuhan 90 90 2700 100 0 100
2010.7 Lanzhou 90 90 2700 100 0 100
2011.3 Wuhan 90 90 2700 100 0 100
2011.7 Lanzhou 90 90 2700 100 0 100
03-02-2017 COH Bengaluru 37
Wan et al., 2014
0
500
1000
1500
2000
2500
3000
2010.3 Wuhan
2010.7 Lanzhou
2011.3 Wuhan
2011.7 Lanzhou

38. Table 4: The comparison of flowering and seeding of hau
CMS line 0912A and maintainer line 0912B in leaf mustard
Lines
Corolla
expansion
Petal
length
Petal
width
Style
diameter
Style
length
Stamen
length
Seeds
per
silique
0912A 7.47 0.63 024 0.09 0.63 0.47 13.13
0912B 12.19 0.84 0.37 0.09 0.74 0.81 14.20
t 16.61** 9.84** 13.79** 0.5 8.49** 15.08** 1.06
03-02-2017 COH Bengaluru
38
Wan et al., 2014

39. Fig. 2. Flower morphology and the pollen vitality.
(A) The flower of hau CMS;
(B) the flower of 0912A;
(C) the flower of 0912B;
(D) pollen vitality of 0912A; (E) and (F) pollen vitality of 0912B.
03-02-2017
COH Bengaluru 39

40. Conclusion
• A new CMS line of leaf mustard with high potential in heterosis utilization
was bred and characterized for anther and pollen development.
• Researches on cytology, the restorer and maintainer relationship, and
polymorphism of mitochondrial DNA indicated that hau CMS was different
from the pol CMS, Shan 2A, ogu CMS, tour CMS, and nap CMS systems.
• hau CMS had no anthers while all the other CMS lines mentioned above
formed anthers but were devoid of functional pollen
• Fertility identification for two consecutive years showed that stamen of
0912A aborted completely, with no pollen formed, indicating the successful
transfer of hau CMS to 0912A
03-02-2017 COH Bengaluru 40

41. Chemical induction of male sterility and
Histological studies in Okra (Abelmoschus
esculentus L.)
03-02-2017 COH Bengaluru 41
Case 3

42. Material and methods
Variety : Arka Anamika
Chemicals : GA3, Ehtrel, Maleic Hydrazide
Foliar spray : three. (20, 20+30, 20+30+40)
Concentration : GA3 – 200, 300, 400 ppm.
Ethrel – 750, 1000, 1250
ppm
MH – 50, 100, 200 ppm.
Plant material

43. Treatments detail
T1- GA3 @ 200 ppm at 20 DAS
T2- GA3 @ 200 ppm at 20+30 DAS
T3-GA3 @ 200 ppm at 20+30+40 DAS
T4- GA3 @ 300 ppm at 20 DAS
T5- GA3 @ 300 ppm at 20+30 DAS
T6-GA3 @ 300 ppm at 20+30+40 DAS
T7- GA3 @ 400 ppm at 20 DAS
T8- GA3 @ 400 ppm at 20+30 DAS
T9- GA3 @ 400 ppm at 20+30+40 DAS
T10- Ethrel @ 750 ppm at 20 DAS
T11- Ethrel @750 ppm at 20+30 DAS
T12- Ethrel @750 ppm at 20+30+40 DAS
T13- Ethrel @ 1000 ppm at 20 DAS
T14- Ethrel @ 1000 ppm at 20+30 DAS
T15- Ethrel @ 1000 ppm at 20+30+40 DAS
T16- Ethrel @ 1250 ppm at 20 DAS
T17- Ethrel @ 1250 ppm at 20+30 DAS
T18- Ethrel @ 1250 ppm at 20+30+40 DAST19- MH @
50 ppm at 20 DAS
T20- MH @ 50 ppm at 20+30 DAS
T21-MH @ 50 ppm at 20+30+40 DAS
T22- MH @ 100 ppm at 20 DAS
T23- MH @ 100 ppm at 20+30 DAS
T24-MH @ 100 ppm at 20+30+40 DAS
T25-MH @ 200 ppm at 20 DAS
T26-MH @ 200 ppm at 20+30 DAS
T27-MH @ 200 ppm at 20+30+40 DAS
T28 - Control (water spray)
03-02-2017 COH Bengaluru 43

44. 03-02-2017 COH Bengaluru 44
65.23 90.37
76.50 50.40
1546.2 1601.9
3.05
21.6764.0745.506.73
14.83
84.3347.37

45. Treat
ment
Plant
height
(cm)
Treat
ment
Leaf
area
(cm2)
Treat
ment
NO. Of
branch
es
Treat
ment
Days to
flower
initiation
Treat
ment
Days to
50%
Flowering
T9 90.37 T28
1601.
9
T19 3.05 T9 39.77 T9 42.63
T8 89.87 T1
1538.
2
T20 3.00 T8 39.87 T8 42.70
T27 76.50 T18
1382.
2
T18 1.45 T27 47.37 T27 50.40
03-02-2017 COH Bengaluru 45
Table 1. Effect of application of different gametocides on plant height (cm), leaf area
(cm2), number of branches, number of days to flower initiation and days to 50 per cent
flowering in okra variety Arka Anamika
Deepak et al., 2007

46. T
Pollen
sterility
(%)
T
Ovular
sterility
(%)
T
Number
of fruits
per
plant
T
Seed yield
per plant
T27 84.33 T27 8.87 T1 6.00 T1 19.60
T18 82.10 T18 14.83 T2 6.00 T2 19.48
T28 9.10 T28 0.00 T28 6.73 T27 21.67
03-02-2017 COH Bengaluru 46
Table 2: Effect of application of different gametocides on pollen sterility
(%), ovular sterility (%), number of fruits per plant and seed yield per
plant in okra var. Arka Anamika
Deepak et al., 2007

47. 03-02-2017 COH Bengaluru 47

48. Conclusion
• Spraying of maleic hydrazide (200 ppm) at 20, 30 and
40 DAS was found to be better for higher pollen
sterility (84.33%) and also lower ovular sterility
(8.87%) followed by GA3 spray.
• Hence, MH can be considered as a safe gemetocide at
200 ppm
03-02-2017 COH Bengaluru 48

49. 03-02-2017 COH Bengaluru 49
Development of a codominant CAPS marker linked to
the Ms locus controlling fertility restoration in onion
(Allium cepa L.)
Case 4

50. 03-02-2017 COH Bengaluru 50
Fig. 1. Morphological characteristics of
fertile flowers
(A) and fertile anther
before (B, left) and after (B, right)
dehiscence vs. sterile flowers (C) and anther
before (D, left) and after (D, right)
dehiscence.
The objective of this study :
To identify molecular markers more tightly linked to the Ms locus.
 In addition, it was designed to assess the efficiency of allelic discrimination
of newly identified markers and previously reported OPT and PSAO markers and the
genetic relationship among those markers was investigated.

51. 03-02-2017 COH Bengaluru 51
MATERIALS AND METHODS
Plant material
Male-fertile line ‘H6’Male sterile line ‘506L x’
red bulb color
 A total of 301 plants from F2 and F3 populations were used for a genotyping
analysis and a molecular marker assessment.
Isolation of genomic DNA
RAPD analysis
 680 random primers used to screen polymorphisms between the
parent lines ‘506L’ and ‘H6’ and between the two different DNA
bulks from male-fertile and male-sterile F2 plants
yellow bulb color

52. 03-02-2017 COH Bengaluru 52
Sequencing and genome walking
 The amplified polymorphic fragment excised and purified.
 The purified PCR fragment was cloned into pCR®4-TOPO vector in TOPO TA
Cloning Kit
 Plasmids were purified with QIAprep Spin Miniprep Kit (Qiagen, Valencia,
CA).
 The sequencing reaction was performed using a BigDye® Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems; Foster City, CA).
 Once a partial sequence was acquired, DNA walking was performed using a
Universal Genome WalkerTM Kit (BD Biosciences, Palo Alto, CA).
CAPS marker analysis
 To convert the dominant RAPD marker into a codominant marker, both
sequences of male-fertile and male-sterile alleles were aligned to identify
polymorphic regions.
 For genotyping of segregating populations, PCR was performed
 PCR products were digested using AvaII.
 Digested products were separated on a 1% agarose gel for individual
genotyping

53. 03-02-2017 COH Bengaluru 53
RAPD marker analysis
Fig. 2. (A). A polymorphic band identified from RAPD between male-fertile and
male-sterile F2 plants using a random primer OBC14
B). Genotype analysis using the CAPS marker ACms.1100 derived from the RAPD
marker co-segregated with the restorer-of-fertility locus Ms
 680 RAPD markers screened
 41 polymorphic bands were identified
 But only BC14 primer produced a polymorphic band co -segregating with the
fertility in the male fertile bulk DNA & the band was absent in male sterile bulk

54. 03-02-2017 COH Bengaluru 54
Conversion of a RAPD marker to a codominant marker
fig

55. 03-02-2017 COH Bengaluru 55
Conclusion
 In this study, the RAPD marker OBC14.1000 was identified .
 ACms.1100 marker was developed from the OBC14.1000 marker to convert a
dominant marker to a codominant marker.
 The genotyping analyses using the OBC14.1000 and ACms.1100 markers
demonstrated that both markers are more reliable than any other markers
currently available.
 Therefore, these markers would be ideal for the allelic discrimination in marker
assisted breeding of onion to predict the genotype of a restorer-of- fertility gene having
better efficiency and can be the first step to identify a restorer-of-fertility gene.
 Further, these markers will be useful in hybrid onion seed production using CMS.

56. 03-02-2017 COH Bengaluru 56
Development of Genic Male-sterile Watermelon Lines with
Delayed-green Seedling Marker
X.P. Zhang, B.B. Rhodes, and W.V. Baird
Department of Horticulture, Clemson University, Clemson, SC 29634
Case 5

57. Materials and Methods
Breeding Lines:
• G17AB (containing the ms gene.)
• Pale90 (line containing the dg gene, was selected for yellow
cotyledons and delayedgreen true leaf)
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58. 03-02-2017 COH Bengaluru 58
Crosses between G17AB male sterile plants and delayed-
green plants were made

59. 03-02-2017 COH Bengaluru 59
Results and Discussion
ms and dg loci are inherited independently and confirms that the newly
ms selected dg mutant is inherited as a single recessive nuclear gene.
• All lines (MSDG-1, MSDG-2, were fixed for the dg seedling marker,
and each segregated (1 sterile : 1 fertile) for male fertility.
• MSDG-1 produces round fruit ,similar to G17AB.
•MSDG-2 produces fruit, similar to that of ‘Sugar Baby’.
The two breeding lines, MSDG-1 and MSDG2, will provide valuable
germplasm for introducing the ms male sterility and dg delayed green
into various genetic backgrounds using backcrossing without labor-
intensive manual cross- and self-pollinations
• The ms and dg genes can be introduced from lines developed in this
study into various genetic backgrounds

60. • The two breeding lines, MSDG-1 and
MSDG2, will provide valuable germplasm for
introducing the ms male sterility and dg
delayed green into various genetic
backgrounds using backcrossing without
labor-intensive manual cross- and self-
pollinations
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Conclusion

61.  Identification of new ms line through exploitation of other domesticated and wild
species.
 Identification of potential restorers through molecular techniques and their use for
development of hybrids
 Pollination mechanisms of male sterility in different vegetable crops should be
further investigated for effective hybrids seed production.
 Development of hybrids with multiple resistance/ tolerance to biotic as well as
abiotic stresses by transfer of genes using conventional and biotechnological
approaches.
 To identify potential markers for genetic purity testing.
 The hybrid seed production technologies should be generated
 Potentiality of transgenic male sterility should be use in vegetable crops.
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62. • Despite the complex maintenance process and additional labour requirement
to remove fertile segregants in hybrid seed production field, production of
male sterile based hybrid seeds is more economical than the seeds produced
by manual emasculation
• The research on male sterility in vegetables is a never ending process due
to rapid advancement of molecular advancements
• Substantial progress has been made in understanding the mechanism of male
sterility in selected vegetable crops techniques and their implementation.
• In fruit bearing vegetables like tomato, brinjal, chilli, muskmelon etc., identification
and utilization of functional male sterility are more attractive.
• In India, research on transgenic male sterility system was initiated in selected
vegetables
• Our first priority should be utilization of existing and established but unexploited
male sterility systems especially in chilli, onion, tomato,
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