Marker aided screening for Ty, Ph and Mi genes
The best lines were selected from their field/artificial reaction against multiple diseases (ToLCD+LB+RKN) analyzed with functional/linked molecular markers (Table 2). A total 5 lines out of 132 lines were found to be positive for different combination of targeted genes (Ty, Ph and Mi). From these 5 multiple disease resistant lines, 2 lines (PDRT-104 and PDRT-122) were found to be carrying all the six genes, PDRT-104 was heterozygous resistance (+/-) for Ty-1, Ty-2 and Ty-3 while homozygous resistant (+/+) for Ph-2, Ph-3 and Mi-1.2 and PDRT-122 was homozygous (+/+) for Ty-1, Ty-2, Ty-3, Ph-2 and Ph-3 genes while heterozygous resistance (+/-) to Mi-1.2, similarly, Kumar et al. (2019c) reported single line (TW-4-5-7) harboured all the six genes. The line PDRT-118 was homozygous resistance (+/+) for Ty-3, Ph-2 and heterozygous (+/-) for Mi-1.2 while there was no amplification for Ty-1 and Ty-2 genes, PDRT-124 homozygous for Ty-1, Ty-2, Ty-3, Ph-2 genes and heterozygous (+/-) for Mi-1.2 gene while PDRT-125 showed homozygous resistance (+/+) for Ty-1, Ty-2, Ty-3, Ph-2 and Mi-1.2 genes.
Table 2 Details of molecular markers used for selection of tomato lines
Sr.
no.
|
Marker name
|
Type of DNA marker
|
Gene
|
Restriction endonuclease
|
Expected Amplicon size (S, R)
~bp
|
Forward primer sequence
|
Reverse primer sequence
|
Reference
|
1.
|
JB-1
|
CAPS
|
Ty-1
|
TaqI
|
400, 500
|
AACCATTATCCGGTTC ACTC
|
TTTCCATTCCTTGTTTC TCTG
|
De Castro et al. (2007)
|
2.
|
TG105
|
CAPS
|
Ty-2
|
TaqI
|
200, 350
|
CTTCAGAATTCTGTTT TAGT
|
ATGTCACATTTGTTGC TTGGACCATCC
|
Garcia et al. (2007)
|
3.
|
FLUW-25
|
SCAR
|
Ty-3
|
-
|
450, 600
|
CAAGTGTGCATATAC TTCATA(T/G)TCACC
|
CCATAATATAACCTCT GTTTCTATTTCGAC
|
Ji et al. (2007a)
|
4.
|
dTG422
|
CAPS
|
Ph-2
|
HinfI
|
245, 275
|
Dr. Martha Mutschler personal communication, College of Agriculture and Life Sciences, Cornell University, USA
|
5.
|
TG328
|
CAPS
|
Ph-3
|
ApoI
|
274, 243
|
6.
|
Mi23
|
SCAR
|
Mi-1.2
|
-
|
420, 380
|
TGGAAAAATGTGAAT TTCTTTTG
|
GCATACTATAGGCTTG TTTACCC
|
Seah et al. (2007)
|
CAPS= Cleaved Amplified Polymorphic Sequence, SCAR= Sequence Characterized Amplified Region.
Phenotyping for Multiple disease resistance
Evaluation for Leaf Curl Disease resistance
Natural screening during kharif season against ToLCD deciphered out of 132 lines, 28 lines were highly resistant, 31 were resistant and rest of the lines were categorized from moderately resistant to highly susceptible. During the vector mediated transmission under insect proof net house, 24 lines were highly resistant, 14 were resistant and remaining were grouped into moderately resistant to highly susceptible. In vector mediated screening against ToLCD, amid these 5 lines (Table 3 and 4) namely, PDRT-122, PDRT-124 and PDRT-125 were found to be highly resistant with coefficient of infection (CI) 2.55, 4.21 and 3.10, respectively. One line namely, PDRT-104 (CI=5.12) exhibited as resistant while line PDRT-118 noted CI value of 15.22 grouped as moderately resistant. Both the resistant and susceptible checks viz., PVB-4 and Punjab Chuhhara respectively, were reacted well as anticipated in this inspection.
Evaluation against Late blight resistance
Appraisal of LB resistance was executed through field screening from February to March, 2020 and deciphered that, 41 lines revealed as highly resistant, 36 were resistant and rest of the lines out of 132 lines grouped under moderately resistant to highly susceptible. The artificial screening of LB was carried out via “detached leaf assay” by spraying inoculum concentration (5×104 sporangia/ml) with sustaining high relative humidity (RH) with cool temperature in the month of January, 2020. No one of the line was revealed to be immune to pathogen signifying that, there was presence of highly uniformed isolates of P. infestans with opportune environment for growth. Owing to one week after spraying of inocula, disease severity index (DSI) was observed and deciphered that, out of 132 lines screened, 9 lines were characterized to highly resistant (HR) and 20 were resistant (R). Amid them, 5 lines (Table 3 and 4) were characterized to multiple disease resistance two lines viz., PDRT-104, PDRT-122, showed lowest DSI= 24.01±2.34, 7.50±0.83 grouped under resistant (R) and highly resistant (HR), respectively. Moreover, three lines viz. PDRT-124, PDRT-125 and PDRT-118 exhibited DSI of 25.66±0.66, 35.84±2.51 and 35±1.67 respectively, categorized as moderately resistance (MR). Both the Resistant and susceptible check viz., LBR-10 and Punjab Chuhhara reacted well as supposed with DSI of 2.05±0.02 and 87.50±0.83 respectively.
Evaluation for Root knot nematodes resistance
Field appraisal exhibited the 13 lines, out of 132, were resistant and 27 lines were moderately resistant (MR) against RKN malady. Artificial appraisal was directed through sick plot technique and found that only 10 lines, out of 132 were resistant (R) and 15 were moderately resistant for RKN disease. Among them, 5 lines (Table 3 and 4) were resistant against multiple targeted disease and pest, four lines i.e., PDRT-104, PDRT-122, PDRT-124 and PDRT-125) exhibited minute nematode infection with root gall index (RGI) 0.55, 0.99, 0.65 and 0.4 respectively, grouped as resistant (R) against RKN. Besides, single lines viz. PDRT-118 (RGI=1.95) was found moderately resistant against RKN. While, Resistant (NR-14) and susceptible (Punjab Chuhhara) checks performed well as supposed with RGI of 0.00 and 5.0 respectively.
Table 3 Reaction of multiple disease resistant tomato lines in field appraisal against ToLCD, LB and RKN
Name of lines
|
Tomato leaf curl disease
|
Late blight
|
Root knot nematodes
|
DI
|
PDI
|
CI
|
Reaction
90 DAT
|
DSI
|
Reaction
|
RGI
|
Reaction
|
PDRT-104
|
0.00±0.00
|
0.00±0.00
|
0
|
HR
|
33.67±1.53
|
MR
|
0.1±0.1
|
R
|
PDRT-118
|
83.92±1.8
|
34.01±1.36
|
17.84
|
MR
|
24.69±1.44
|
R
|
2.4±0.1
|
MR
|
PDRT-122
|
13.2±1.09
|
2.85±0.81
|
1.86
|
HR
|
0.00±0.00
|
HR
|
1.1±0.1
|
R
|
PDRT-124
|
26.85±1.72
|
5.2±1.12
|
2.64
|
HR
|
0.00±0.00
|
HR
|
0.00±0.00
|
R
|
PDRT-125
|
13.21±1.09
|
3.13±1.09
|
1.81
|
HR
|
0.00±0.00
|
HR
|
0.00±0.00
|
R
|
PVB-4(RC)
|
0.00±0.00
|
0.00±0.00
|
0
|
HR
|
32.14±1.02
|
HS
|
5.35±0.15
|
MS
|
LBR-10(RC)
|
100.00±0.00
|
47.79±0.86
|
24.33
|
MS
|
0.00±0.00
|
HR
|
7.4±0.1
|
S
|
NR-14(RC)
|
96.56±3.44
|
50.19±0.83
|
40.81
|
S
|
43.72±1.4
|
MS
|
0.00±0.00
|
R
|
Punjab Chuhhara (SC)
|
100.00±0.00
|
99.1±0.9
|
97.68
|
HS
|
96.5±1.5
|
HS
|
10±0.00
|
HS
|
DI=disease incidence, PDI=percent disease index, CI=coefficient of infection, HR=highly resistant, R-resistant, HS-highly susceptible, DAT-days after transplanting, RC-resistant check, SC-susceptible check, DSI-disease severity index, RGI-root gall index.
Table 4 Reaction of multiple disease resistant tomato lines in artificial and molecular appraisal against ToLCD, LB and RKN
Name of Line
|
Tomato leaf curl disease
|
Late blight
|
Root knot nematodes
|
DI (%)
|
PDI (%)
|
CI
|
45 DAI
|
Ty-1
|
Ty-2
|
Ty-3
|
DSI
|
7 DAI
|
Ph-2
|
Ph-3
|
RGI
|
60 DAS
|
Mi-1.2
|
PDRT-104
|
41.49±1.37
|
20.47±2.10
|
5.12
|
R
|
+/-
|
+/-
|
+/-
|
24.01±2.34
|
R
|
+/+
|
+/+
|
0.55±0.05
|
R
|
+/+
|
PDRT-118
|
54.64±2.51
|
30.44±1.87
|
15.22
|
MR
|
-
|
-
|
+/+
|
35.00±1.67
|
MR
|
+/+
|
-/-
|
1.95±0.05
|
MR
|
+/-
|
PDRT-122
|
30.88±2.31
|
10.19±2.03
|
2.55
|
HR
|
+/+
|
+/+
|
+/+
|
7.50±0.83
|
HR
|
+/+
|
+/+
|
0.99±0.01
|
R
|
+/-
|
PDRT-124
|
71.94±0.51
|
16.85±1.53
|
4.21
|
HR
|
+/+
|
+/+
|
+/+
|
25.66±0.66
|
MR
|
+/+
|
-/-
|
0.65±0.15
|
R
|
+/-
|
PDRT-125
|
56.15±1.00
|
12.39±0.15
|
3.10
|
HR
|
+/+
|
+/+
|
+/+
|
35.84±2.51
|
MR
|
+/+
|
-/-
|
0.40±0.10
|
R
|
+/+
|
PVB-4(RC)
|
0.00±0.00
|
0.00±0.00
|
0.00
|
HR
|
+/+
|
+/+
|
+/+
|
55.00±1.67
|
S
|
-/-
|
-/-
|
2.45±0.05
|
MS
|
-/-
|
LBR-10(RC)
|
84.92±0.80
|
81.07±1.48
|
81.07
|
HS
|
-/-
|
-/-
|
-/-
|
2.05±0.02
|
HR
|
+/+
|
+/+
|
3.25±0.05
|
S
|
-/-
|
NR-14(RC)
|
99.06±0.94
|
77.83±2.32
|
77.83
|
HS
|
-/-
|
-/-
|
-/-
|
56.34±1.34
|
S
|
-/-
|
-/-
|
0.00±0.00
|
R
|
+/+
|
Punjab Chuhhara (SC)
|
100.00±0.00
|
99.47±0.53
|
99.47
|
HS
|
-/-
|
-/-
|
-/-
|
87.50±0.83
|
HS
|
-/-
|
-/-
|
5.00±0.00
|
HS
|
-/-
|
DI=disease incidence, PDI=percent disease index, CI=coefficient of infection, HR=highly resistant, R=resistant, MR=moderately resistant, HS=highly susceptible, DAT=days after transplanting, DAS=days after sowing, DAI-days after inoculation, RC=resistant check, SC=susceptible check, DSI=disease severity index, RGI=root gall index, +/+ (homozygous resistant), +/- (heterozygous resistant), –/– (homozygous susceptible), - (not amplified).
Evaluation for horticultural traits:
The nine horticultural attributes were evaluated during this study. The 5 best lines (Table 5) possessing multiple disease resistance was compared with resistant and susceptible checks. The lines namely, PDRT-104 (3.07), PDRT-122 (3.44), PDRT-124 (3.04) and PDRT-125 (2.78) gave higher yield kg/plant over all the checks investigated. The line PDRT-104 (6.34%) contributed maximum amount of dry matter and TSS (4.68°B) than all the checks. While, PDRT-122 contributed higher for lycopene content about 5.43 mg/100g. The lines namely, PDRT-104 (0.38), PDRT-118 (0.23) and PDRT-125(0.48) identified for lesser amount of titratable acidity (g/100ml). Likewise, Kumar et al, (2019a) reported the lines namely, TW-4-5G-12, TW-5-1E-7, PR-DH-15-7-11 and PR-DH-28-11G-13 were also more yielder than all the three checks along with other horticultural traits.
Table 5 Mean performance of multiple disease resistant tomato lines for horticultural traits
Name of line
|
Average fruit weight (g)
|
Total fruit yield (kg plant-1)
|
Number of locules fruit-1
|
Pericarp thickness (mm)
|
P/E ratio
|
Dry matter
(%)
|
Total soluble solids (°Brix)
|
Lycopene content (mg/100g)
|
Titratable acidity (g/100ml)
|
PDRT-104
|
58.08±1.45
|
3.07±0.09
|
3.34±0.13
|
3.38±0.03
|
0.94±0.04
|
6.34±0.20
|
4.68±0.30
|
4.28±0.04
|
0.38±0.02
|
PDRT-118
|
82.37±4.19
|
2.48±0.08
|
2.50±0.25
|
4.00±0.55
|
0.85±0.09
|
4.23±0.10
|
3.56±0.56
|
4.48±0.10
|
0.23±0.04
|
PDRT-122
|
79.16±0.97
|
3.44±0.23
|
3.11±0.11
|
3.29±0.06
|
0.96±0.03
|
4.75±0.05
|
4.51±0.15
|
5.43±0.06
|
0.60±0.02
|
PDRT-124
|
59.87±0.67
|
3.04±0.09
|
3.65±0.11
|
3.41±0.07
|
0.91±0.02
|
4.66±0.05
|
3.96±0.05
|
4.70±0.32
|
0.52±0.02
|
PDRT-125
|
92.96±1.12
|
2.78±0.19
|
3.00±0.16
|
5.26±0.25
|
0.87±0.18
|
5.18±0.04
|
3.95±0.20
|
4.46±0.30
|
0.48±0.03
|
Punjab Varkha Bahar-4 (Check)
|
87.82±1.35
|
2.52±0.03
|
3.18±0.08
|
4.59±0.02
|
1.04±0.01
|
4.28±0.03
|
4.00±0.02
|
3.52±0.03
|
0.47±0.03
|
LBR-10 (Check)
|
102.64±2.49
|
2.32±0.20
|
3.06±0.11
|
4.72±0.04
|
0.89±0.01
|
5.28±0.04
|
3.49±0.03
|
3.52±0.03
|
0.56±0.03
|
NR-14 (Check)
|
86.59±0.95
|
2.22±0.03
|
3.02±0.10
|
3.70±0.03
|
1.32±0.09
|
5.11±0.01
|
4.06±0.07
|
3.77±0.02
|
0.52±0.01
|
Punjab Chuhhara (Check)
|
81.15±1.01
|
2.40±0.08
|
3.59±0.09
|
5.90±0.03
|
1.73±0.02
|
4.10±0.01
|
4.53±0.02
|
2.87±0.02
|
0.82±0.01
|
C.D. @ 5%
|
10.27
|
0.44
|
0.57
|
0.76
|
0.21
|
0.42
|
0.56
|
1.20
|
0.08
|
S.E. (m)
|
3.67
|
0.16
|
0.20
|
0.27
|
0.08
|
0.15
|
0.20
|
0.43
|
0.03
|
S.E. (d)
|
5.19
|
0.22
|
0.29
|
0.38
|
0.11
|
0.21
|
0.28
|
0.60
|
0.04
|
C.V.
|
6.95
|
11.33
|
10.55
|
8.87
|
9.20
|
4.54
|
7.88
|
15.18
|
7.56
|