Chapter-IV

RESULT AND DISCUSSION

The present investigation on “Morphological, molecular and biochemical characterization of elite germplasm lines of

wheat (TriticumaestivumL.)”was planned to meet the objectives as under:

1. Phenotypic and molecular characterization of LTN among wheat genotypes.

2. To estimate the variability for each trait.

3. Diversity analysis

4. To estimate correlation coefficient among the traits studied.

5. Molecular characterization of leaf tip necrosis genes using SSR markers.

6. Histo-chemical study of leaf tip necrosis by DAB solution.

4.1 Characterization of wheat lines for leaf tip necrosis under field conditions.

Figure.4.1 Leaves of wheat with (ltn)

4.1.1: Scoring of leaf tip necrosis trait:

Expression of LTN in the leaves was measured only in terms of presence or absence.

Table 4.1: Characterisation of genotypes on the basis of presence and absence of LTN

Genotype Ltn Genotype Ltn Genotype Ltn Genotype Ltn Genotyp

e

Ltn

1 P 18 P 35 P 52 A 69 P

2 P 19 P 36 P 53 P 70 A

3 P 20 P 37 P 54 P 71 P

4 P 21 P 38 A 55 P 72 P

5 P 22 A 39 P 56 P 73 P

6 P 23 P 40 P 57 P 74 P

7 P 24 P 41 A 58 P 75 P

8 P 25 P 42 P 59 A 76 A

9 A 26 P 43 P 60 A 77 P

10 P 27 P 44 P 61 P 78 A

11 P 28 P 45 A 60 P 79 P

12 P 29 P 46 P 63 P

13 P 30 P 47 A 64 A

14 P 31 P 48 P 65 P

15 P 32 A 49 P 66 P

16 A 33 P 50 P 67 P

17 P 34 A 51 P 68 A

Note: P indicating the presence of LTN and A indicating the absence of LTN.

4.2:Molecular biology result:

MarkersCsLV34, STS1BL2 and cfd71 were used to identify the Lr34, Lr46and Lr67respectively. These are leaf rust resistant

genes which is closely related to leaf tip necrosis in the given wheat genotypes.

Table 4.2: Distribution of the markers for Lrgenes among 24 wheat cultivars

Genotype Lr34 Lr46 Lr67

Baaz + + +

Bavi-9 - + +

Bavi-3 + + +

K 68 + + +

Bavi -6 - - +

Bavi– 10 + + +

Bavi– Cora - + +

Bavi– 12 + + +

Veery -42

- - -

Tia + + +

Tinik - + +

K 8027 + + +

Bw 362 - + +

UVL 404 - + +

UVL 404 - + +

Lok 1 + + +

Ning 8027 - + +

Mons -Mz-21-31Cmsz 72476- + +

CAPAN 4128 - + +

HUWJ 13 - - +

Dharwar Dry - + -

IA 814-467 - + +

BP8-477 - - +

HD 2206/nork + + +

RAJ 3814 - + +

HW 2005 - + +

Monsukan&Zar + + +

Sanghai 4 –X + + +

Sonalika - + +

M 3104 - + +

C 306 + + -

LDSG 54/3247 + + +

BH 1146 - 470 + + +

Atila - + +

CHD-14-473 - + +

Bonly + - -

CPAN 4119 - + -

Chiria– 7 + + +

Kaz + + +

9M-16-468 - - -

BL-997 + + +

MONSALDS - + -

Chiriya 1 - + +

NW-1012 + + +

NW-1014 - + -

DBW 14 + - -

K 0307 + + -

PBW 343 - + +

HP 1716 - - +

HD 2733 + + -

K 9107 + + +

HD 2824- - +

UP 2338 + - +

NW 1012 - + +

DBW 17 + - +

Chirya -3 + + +

Yangmai -6 - + +

Ning 8119 + + -

Francolin - + +

BL 3063 - - -

Aditya + + -

VL 829 + + +

LABH - - +

LAHAR + + +

HUW 609 - - -

KIRITATI/2*WBLL1 + + +

HUW234+LR34/PRINIA//PFAU/WEAVER

- - +

ELVIRA/5/CNDO/R143//ENTE/MEXI75/3/AE.SQ/4/2*OCI/6/VEE/PJN//KAUZ/3/PASTOR

- + -

PFAU/WEAVER*2//KIRITATI - + -

KIRITATI//SERI/RAYON + - -

WAXWING*2/KIRITATI + - -

WAXWING*2/4/SNI/TRAP#1/3/KAUZ*2/TRAP//KAUZ

+ + +

WAXWING*2/KRONSTAD F2004 - - -

WAXWING*2/CIRCUS - + +

CROC_1/AE.SQUARROSA (205)//KAUZ/3/SASIA/4/TROST

- - -

PRL/2*PASTOR//PBW343*2/KUKUNA/3/TACUPETO F2001*2/KUKUNA

+ + +

WHEAR/3/URES/PRL//BAV92/4/TIMBA

+ - +

Yangmai -6 - - +

Note: The sign + and– ,indicates that the marker is present or absent, respectively

4.2.1:Amplification of CsLV34 marker (Lr 34) for LTN:

Lr34 gives durable resistance against leaf rust (Ezzahiri and Roelfs, 1989). The Lr34 is the APR leaf rust resistance gene and it is an

important part of the HexaploidT. Aestivum L. genome. Dyck (1987) was first described Lr34. Cytogenetic analysis was earlier used to

locate the Lr34 gene on thearm of chromosome 7DS (Dyck, 1994). This gene is currently found in many wheat cultivars. The marker

for this gene, developed by SSR, RFLP and more recently RFLP was converted to the sequence tagged site (STS) [Lagudahet al,.

2006].The size of amplification product with this marker was 150 bp.The + sign indicating the presence of Lr34 gene and –

sign indicating the absence of Lr34 gene among the wheat genotypes.

sample of gel pictures:

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

200 bp

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

Fig no- 4.3 Amplicon of Lr 67 in twenty four genotypes of wheat

600 bp

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

Fig no- 4.2 Amplicon of Lr 46 in twenty four genotypes of wheat

4.2.2:Amplification of SSR primer for Lr46 associated withLTN:

In present study, 24 genotypes were used for testing Lr46 genes presence/absence.STS1BL2amplified bands were in the size

of 600bp and all the genotypes showed clear DNA bands.The Lr46 is also slow rusting gene same as like Lr34 and give better

result along with the combination of Lr34 (Singh et al., 1998). The Lr46 locus, conferring resistance to both leaf rust and stripe rust,

is located in the terminal portion of the long arm of wheat chromosome 1BL (Mateos-Hernandez et al., 2005) this positively

associated with spot blotch resistance gene Sb1 (Lillemoet al., 2013).

4.2.3:Amplification of SSR markerfor Lr67associated withLTN:

SSR marker (cfd71) was tested and found that amplified in the size of 200 bp in all the 24 wheat genotypes tested.

APR gene for leaf rust (Lr67) and stripe rust located in the centromeric region of chromosome 4DL and however Lr67 phenotypically

resembled Lr34, the degree of resistance conferred by Lr67 was less than that conferred by Lr34(Herrera-Foessel et al., 2011).

4.3: Biometrical Analysis

The result and discussion are given in as follows.

4.3.1) Analysis of variance

The analysis of variance for 12 traits including grain yield and AUDPC with its related traits in present set of wheat genotypes is given in table 4.2.It is evident from the analysis of the variance among the genotypes all the 12 traits showed highly significant variance indicated the presence of genetic variability among this set of germplasm.

4.3.2.: Variability, Heritability, Genetic advance

The mean value of all the 12 traits is given in Apendix 1. The estimates of range, mean, phenotypic coefficient of variation (PCV), genotypic coefficient of variation (GCV), Heritability (H) and Genetic advance (GA) are presented in table 4.3

4.3.2.1:Mean performance and range for important traits:

Auricle pigmentation- Auricle pigmentation ranged from 1 to 3 with mean value 2.78(Tabel 4.4). Most of the genotypes have green (3) auricle pigmentation.

Flag Leaf attitude- Leaf angle ranged from 1 to 4 with mean 2.05 (Table4.4). Leaves of most of thegenotype were semi-erect.

Flag leaf length(cm) - Data in the Table4.4 explain that flag leaf length ranged from 10.37cm to 24.13cm with mean value 19.29cm. Genotype Bavi-6(24.13) had longer Flag leaf length as compare to genotype Chiriya-7 (10.37) which had lowest Flag leaf length.

Flag leaf breadth (cm)- Flag leaf breadth ranged from 1cm to 2.3cm with mean 1.55cm. Genotype K0307 had longer FLB compare to genotype CROC_1/AE.SQUARROSA(205)/KAUZ/3/SASIA/4TROST and PRL/2*PASTOR/PBW343*2/KUKUNA/3/TACUPETOF2001*2/KUKUNA had short length of 1cm.

Table 4.3: ANOVA for 12 characters in 79 genotypes of wheat.

Source of variance

Entry Replication Error

DF 78 2 156

Auricle pigmentation

0.845** 0.004 0.004

Flag leafattitude

1.302** 0.004 0.004

Flag leaf length 24.312** 12.678** 1.294

Flag leaf breadth

0.108** 12.678** 0.029

Daystoheading 25.871** 32.33** 0.777

Spike length 2.976** 17.8849** 0.685

No ofseeds per year

85.89** 134.91** 13.25

Plant height 358.75** 0.118 9.63

Pedunclelength 149.68** 7.016 6.303

TGW 50.88** 15.309 16.37

Plot Yeild 1053** 197.1** 359.33

AUDPC 19922** 11315** 1551.6

*Significance at 5% level of significance**Significance at 1% level of significance

Table 4.4 Range, mean, components of variation and genetic parameters for 12 traits in 79 wheat germplasm.

ParametersAuricle

pigmentationFlag leaf attitude

Flag leaf

lengthFlag leaf breadth

Days to heading

spike length

No.of Seed/ear

Plant height

Peduncle length TGW

Plot Yield AUDPC

Min 1 1 10.37 1 64 5.8 25 62.67 15.67 24.8 78.56 187.24Max 3 4 24.13 2.3 79.33 10.87 55.33 120.3 48.67 41.2 166.55 654.32Mean 2.78 2.05 19.29 1.55 70.68 8.41 37.76 83.64 34 33.4 120.99 351.915

σ2g 0.28 0.43 7.67 0.03 8.36 0.76 24.21 116.37 47.79 11.50 231.22 6123.47

σ2p 0.28 0.44 8.97 0.06 9.14 1.45 37.46 126.01 54.10 27.87 590.55 7675.07

H 0.99 0.99 0.86 0.48 0.92 0.53 0.65 0.92 0.88 0.41 0.39 0.80

GA 1.08 1.35 5.28 0.23 5.70 1.31 8.15 21.36 13.39 4.49 19.60 143.99GA as % of Mean 38.96 65.79 27.36 14.88 8.06 15.54 21.58 25.53 39.37 13.44 16.20 40.92

GCV(%) 19.05 32.09 14.36 10.47 4.09 10.39 13.03 12.90 20.33 10.15 12.57 22.24

PCV(%) 19.18 32.23 15.52 15.18 4.28 14.31 16.21 13.42 21.63 15.81 20.09 24.89LSD 0.105 0.105 1.8352 0.28 1.422 1.335 5.872 5.007 4.049 6.83 30.6 63.529

σ2g = Genotypic variance σ2p = Phenotypic variance

GCV = Genotypic coefficient of variation PCV =Phenotypic coefficient of variation

Days to heading-The data in the Table4.4revealed that it varied from 63 to 79 days with mean value of 70.68 days. The genotype Lok-1 showed lesser days to flowering(63) while genotype Atila showed highest days to 50% flowering (79).The wide range in variability indicated the better scope of improvement in terms of this character. Dwivediet al. (2003) also observed variation in this trait.

Spike length(cm)- Spike length varied from 5.8cm to 10.87cm with mean value of 8.41cm. the genotype Bavi-3,Bavi-9,NW-1012,NW-1014, UVL404 (11cm) had longer spike as compared to genotype Bonly (5cm) which showed short spike. Lalet al.(2009).

Number of Seeds per ear- Number of seeds varied from 25 to55 with mean value 37.76. The genotype UVL404 had more number of seeds while genotype BP8-477 had only 25 seeds.

Plant height(cm)-Plant height exhibited a range of 62.67cm to 120.3 cm with mean value 83.64cm. The RAJ 3814 genotype 120.3 was recorded as the tallest variety while genotype BAVI-3 had height of 62.67cm. Due to problem of lodging and less responsiveness to inputs, by tall type, Dwarf genotype are ideal in wheat for good yield. Pawaret al.(2002) and Asifet al. (2004) got variation with plan height.

Peduncle length(cm)- The data in table revealed that peduncle length varied from 15.67cm to 48.67cm with mean value 34 cm. The genotype RAJ 3814(48.67 cm) had long peduncle as compared to genotype BAAZ had 15.67 length. Lalet al.(2009) got variation for peduncle length in wheat.

1000-grain weight (g)-.The thousand grain weight in the present study ranged from 24.8gm to 41.2gm with mean value of 33.4gm .genotype WAXWING*2/KIRITATIhave high grain weight while genotype BAVI-9 have very low grain weight.

Plot Yield (g)-Grain yield data in the present study ranged from 78.56gm to 166.55gm with a mean value of 120.99gm. The highest value of grain yield was observed in genotype BAVI-10 while genotype CPAN 4119 have low value of grain yield. Phadnawiset al.(2002) and Gupta and Verma (2000) observed variation for this trait.

Area Under Disease Progress Curve -Data for this trait revealed it varied from 187.24 to 654.32 with mean value 351.91. The genotype RAJ 3814 have highest AUDPC while genotype BAVI-3 have lowest AUDPC. Sharma et al.(1998) has used variation for this trait.

4.3.2.2: Genetic Variability- The genetic variability parameters can be measured through the phenotypic and genotypic coefficient of variation, heritability, and genetic advance for all the traits taken. These parameters play an important role in selection for improvement in the concerned characters. Table 4.4 gives the estimates of phenotypic and genotypic coefficient variation for the 12 traits studied.

In the present investigation, a perusal of variance and its component revealed that the genotypic and phenotypic variances were found to be highest for AUDPC (6123.47 and 7675.07) followed by Plot yield (231.22 and 590.55) while low value of genotypic and

phenotypic variances were observed for Flag leaf breadth (0.03 and 0.06) followed by auricle pigmentation (0.28 and 0.28) respectively. In this study phenotypic variances was found to be higher than genotypic variance for all 12 traits under study, indicating the presence of influence of environment on the external manifestation of these characters.

Coefficients of variations truly provide a relative measure of variance, among the different traits. The value of genotype coefficient of variation (GCV) and phenotypic coefficient of variation (PCV) had been presented in table 4.4. Genotypic coefficient of variation was found to be highest for Flag leaf attitude (32.09), followed by AUDPC (22.24) and peduncle length(20.33) ,while low value for Days to heading (4.09).

The Phenotypic coefficient of variation ranged from 32.23(Flag leaf attitude) to 4.28(days to heading) and have been found to be highest for flag leaf attitude (32.23). Therefore estimation of degree of genotype and phenotype correlation of grain yield with yield components is very important to utilize the available genetic variability through selection. (Singhet al.,1998).

PCV and GCV are the indicators of presence of the degree of genetic variation.The amount of heritable portion of variation can only be determined with the help of the estimates of heritability and genetic advance.

4.3.2.3: Heritability and Genetic advance

Heritability is a measure of the extent of phenotypic variation caused by the action of genes. Heritability is a parameter which is widely used in the establishment of breeding programs and formation of selection indexes (Falconer,1985). High genetic advance coupled with heritability estimates offers the most effective condition for selection (Lariket al.2000). Heritability in broad sense according to Lush (1949) is the ratio of total genotype variance to phenotype variance, expressed in percentage. There is a direct relationship between heritability and response to selection, which is referred to as genetic progress. The expected response to selection is also called genetic advance (GA). Johnson et al., (1995) suggested that without genetic advance the estimates of heritability will not be of practical value and emphasized the concurrent use of genetic advance along with heritability.

The estimate of heritability for different characters under study ranged between 99% to 39%. The highest heritability was observed for auricle pigmentation and flag leaf attitude (99%) followed by Days to heading and Plant height (92%). Lowest value of heritability was observed in case of Plot yield (39%). Genetic advance as percent of mean indicates predominance of additive gene action. Johnson et al.(1995) showed that high heritability should be accompanied by high genetic advance as percent of mean to arrive at more reliable conclusion.

4.3.3 Correlation coefficient:

The correlation coefficient measures the extent and direction (positive/negative) of relationship between two variables. Correlationcoefficient of 12 traits have been calculated and presented in Table 4.5.

In the present investigation,days to heading(0.176**), plant height (0.975**) and peduncle length (0.748**) found highly significant and positive correlation with AUDPC.While plot yield (-0.498**) and 1000 grain weight (-0.387**) highly significant and negative correlation with AUDPC. Spike length (-0.147*) showing negatively significant with AUDPC. Khan et al.(2009) observed similar result.

Plot yield showed highly significant and positive correlation with 1000 grain weight (0.520**), number of seeds per ear (0.260**) while negative and highly significant with AUDPC (-0.498**). Phadnawiset al.(2009) showed similar results.

Aurclepigmentation(0.219**) showed highly significant and positive correlation with Flag leaf attitude while Days to heading(-0.208**) and Number of seed per year (-0.124*) exhibited highly significant andnegative correlation with Flag leaf attitude respectively.

Table 4.5 :Estimates of correlation coefficient between yield and its related traits in 79wheat germplasm.

Trait APIG FLA FLL FLB DH SL NOS PH PL TGW PY

FLA 0.219** 1

FLL -0.049 0.000 1

FLB -0.012 0.083 0.003 1

DH 0.003 -0.208** -0.060 -0.061 1

SL -0.011 0.107 0.038 -0.078 -0.112 1

NOS -0.015 -0.124* 0.037 0.025 0.044 0.0373 1

PH -0.066 -0.011 -0.060 -0.18 0.198** -0.141* -0.047 1

PL -0.034 -0.054 0.168** -0.129 0.079 -0.111 0.095 0.584** 1

TGW -0.086 0.047 0.013 0.025 -0.055 0.091 -0.079 0.146* 0.100 1

PY -0.041 -0.037 0.041 0.044 0.013 0.089 0.260** 0.086 0.067 0.520** 1

AUDPC -0.068 -0.024 -0.003 -0.107 0.176** -0.147* -0.013 0.975** 0.748** -0.387** -0.498**

Table 4.6: Grouping of Wheat genotypes into 12 clusters (Ward’s method).No. of cluster

No. of genotypes Cluster Members

1 12 1, 3, 20, 34, 49, 50, 53, 55, 71, 76, 77, 782 8 6, 7, 8, 16, 18, 25, 41, 653 10 5, 14, 29, 42, 46, 57, 69, 70, 73, 794 14 9, 10, 21, 31, 44, 45, 48, 58, 62, 64, 67, 68, 72, 745 3 17, 30, 516 5 11, 12, 27, 43, 637 11 2, 4, 28, 36, 47, 52, 56, 59, 60, 61, 668 3 13, 24, 759 1 5410 5 15, 22, 23, 33, 3911 3 19, 26, 35

12 4 32, 37, 38, 40

Table 4.6: Details of all the 12 clusters indicating the contribution of individual traitsin each clusters.SN No.

of cluster APIG FLA FLL FLB DH SL NOS PH PL TGW PY AUDPC

112 2.92 2.08 16.39 1.54 71.67 8.46 38.44 78.28 28.72 34.49 129.54 495.28

28 3.00 2.63 19.07 1.57 68.00 9.63 39.21 82.17 32.92 32.22 125.04 529.48

310 2.90 1.80 21.62 1.56 69.60 7.89 44.50 81.87 36.27 31.99 130.96 534.25

414 2.86 2.17 20.19 1.53 68.93 9.12 37.43 87.64 37.60 38.46 129.25 449.64 Resistant

53 3.00 2.67 19.86 1.71 65.67 7.31 32.67 74.44 27.22 37.66 130.26 458.06

65 1.40 1.60 20.15 1.72 69.60 8.14 37.93 89.27 34.87 37.85 137.60 453.83

711 2.82 1.73 19.60 1.58 70.09 8.34 37.21 70.97 27.42 29.44 103.47 554.62

83 2.00 2.67 18.44 1.61 67.67 8.32 30.56 82.78 32.00 25.44 80.30 599.72 Susceptible

91 3.00 1.00 17.03 4.93 74.00 8.00 35.67 74.33 26.67 33.70 121.11 500.83

105 3.00 2.20 15.97 1.39 68.60 7.35 34.53 100.93 41.87 33.14 115.11 515.50

113 3.00 2.33 20.94 1.41 79.00 7.42 34.67 104.56 42.11 31.74 108.15 526.67

124 3.00 1.50 21.24 1.44 71.00 8.23 34.58 93.42 43.33 28.50 103.61 581.88

Genetic Divergence:

In order to study the genetic divergence, 79 wheat lines were subjected to D2 analysis in the present investigation. Analysis employed WARD’S method so as to group the genotype into different clusters. Through Ward’s method all the genotypes were grouped into 12 clusters which are presented in Table 4.6.

Genetic divergence study revealed that Cluster 4 have highest number of genotype (14) which include genotype number9, 10, 21, 31, 44, 45, 48, 58, 62, 64, 67, 68, 72, 74, while Cluster 9 have lowest number of genotype (1) i.e. genotype number 54.The table 4.7 presented that out of the 12 cluster, most of clusters had approximately same mean for most of the traits except 1000-grain weight, plot yield and AUDPC. Cluster no. 4 showed low AUDPC(449.64), high yield(129.25 g) and high 1000-grain weight(38.46 g), which may be considered as a group of resistance lines. While all the lines of cluster 8had shown high AUDPC(599.72), low plot yield(80.30 g) and low 1000 grain weight(25.44 g). So cluster no.4 and 8 are diverse for Spot blotch resistance hence, the lines from these clusters may be used for developing mapping population for spot blotch resistance in wheat.

Genotype

38403732263519393322231554247513665647364595260612826327124311513017484421676245311068725874649695742702914467379525186516784167177537678505549342031

R-Squared

1.0 0.8 0.6 0.4 0.2 0.0

Dendogram using 79 gemplasm of wheat with 12 phenotypic traits

4 .1.4.Histochemical staining:

More H2O2is produced in resistant genotypes resulting hypersensitive response and dense staining with 3, 3-

Diaminobenzidine. The brown staining on the leaves confirmed the production of hydrogen peroxide.The browning on

leaves was denser in resistant genotypes than susceptible genotypes.

Susceptible genotypes Resistant genotypes

Fig No: 4 DAB staining in resistant and susceptible wheat Genotypes.

Necrosis Necrosis

Auricle

pigmen

tation

Flag le

af atti

tude

Flag le

af len

gth

Flag le

af brea

dth

Days to

heading

spike le

ngth(cm

)

No.of Seed

/ear

Plant h

eight(c

m)

Peduncle

length

TGW(gm

)

Plot Yield

AUDPC0

5

10

15

20

25

30

35

GCV(%)PCV(%)

Characters studied

Figure 6- Histogram depicting estimates of GCV (%) and PCV(%)for 12characters in wheat.