Received: 28 October 2021 Revised: 24 January 2022 Accepted: 27 February 2022

DOI: 10.1111/pbr.13011

ORIGINAL ARTICLE

A multi-season analysis of barnyard millet (Echinochloa

frumentacea) germplasm lines for shoot fly resistance and
multi-trait stability

Poluru Gunnampati Padmaja | Andiappan Kalaisekar | Vilas Achutarao Tonapi |

Ragimasalawada Madhusudhana

ICAR-Indian Institute of Millets Research,

Hyderabad, India
Correspondence
Ragimasalawada Madhusudhana, Principal
Scientist (Plant Breeding), ICAR-Indian
Institute of Millets Research, Rajendranagar,
Hyderabad 500 030, Telangana, India.
Email: madhu@millets.res.in
Funding information
ICAR-Indian Institute of Millets Research;
Indian Council of Agricultural Research
Abstract
Barnyard millet is one of the most important minor millet crops grown in rainfed
conditions for its health benefits. A large germplasm collection is available at the
global level and needs to be characterized for biotic stress factors. In this study,
106 barnyard millet lines were characterized for their resistance to shoot fly resis-
tance during the 2016, 2017 and 2018 rainy seasons, and Additive Main effect and
Multiplicative Interaction (AMMI) analysis was carried out to understand the geno-
type x environment interaction. The study also investigated the multi-trait stability of
genotypes using deadhearts on the main plant (MPDH) and tillers (TDH). The analysis
revealed the genetic difference that exists in the barnyard genetic material for shoot
fly resistance. Seedling epicuticular wax was found related to shoot fly infestation.
The infected plants had substantially higher levels of peroxidase and polyphenol
oxidase and total protein content when contrasted with their control plants. MPDH
and TDH have high G
E interaction, and the multi-trait stability index (MTSI)
analysis has identified 16 lines with lower shoot fly damage and stability.

KEYWORDS
barnyard millet, deadhearts, G
E, multi-trait stability index, shoot fly

1 | I N T RO DU CT I O N et al., 2018). Consumption and production of millets in India have

Barnyard millet is a C4 cereal crop cultivated for nutritious food and
fodder in several parts of the globe. Of the 35 species available in
barnyard millet (Sood, Khulbe, Gupta, et al., 2015), Indian barnyard
millet (Echinochloa frumentacea) and Japanese barnyard millet
(Echinochloa utilis) are cultivated on a large scale in India, Pakistan,
Nepal, Japan, Korea and the north-eastern parts of China, respectively
(Yabuno, 1987). In India, barnyard millet is cultivated in two distinct
agroclimatic zones, the Himalayan region in the north and the Deccan
plateau in the south, particularly in Tamil Nadu (Sood, Khulbe, Kumar,
et al., 2015). Globally, India is the largest producer of barnyard millet,
in both area (0.146 m ha1) and production (0.147 mt) with an aver-
age yield of 1034 kg ha1 during the last three years (Madhusudhana
dropped over years in support of maize, rice and wheat (Kumar
et al., 2009). Unpredictable rainfall and labour related to processing
have also contributed to decreased production of millet crops
(Dwivedi et al., 2012).
Barnyard millet is suitable for cropping systems, particularly in
hilly areas in which it is highly appreciated for its grain as well as fod-
der (Gomashe, 2016). The crop is primarily grown on marginal lands
with minimal input in hill slopes and wavy fields of hilly, tribal or mar-
ginal areas, where there are few options for crop divergence. The
duration of early maturity and its climate-resilient qualities give it an
added benefit in supporting agricultural production and the suste-
nance of farmers in these areas. Recently, barnyard millet has
attracted attention for its high nutritive value and as an excellent

Plant Breed. 2022;141:399–407. wileyonlinelibrary.com/journal/pbr © 2022 Wiley-VCH GmbH 399

400
biomass crop. Barnyard millet is highly nutritious and is gaining accep-
tance as a health food, as it contains carbohydrate (65%), protein
(11%), fat (3.9%), crude fibre (13.6%) and an excellent source of iron
(Fe), zinc (Zn) and antioxidative compounds (Hadimani
Malleshi, 1993; Saleh et al., 2013; Upadhyaya & Vetriventhan, 2018;
Veena Bharati et al., 2010; Vinoth & Ravindhran, 2017;
Watanabe, 1999). Its nutritional content makes it suitable for the for-
mulation of commercial foods, especially for people with diabetes
(Ugare et al., 2014), celiac and for infants and pregnant women.
Around the world, about 8600 barnyard millet germplasm acces-
sions are available, the largest collection held by Japan (3700 acces-
sions), followed by India (3200 accessions), China (700 accessions)
and the United States (300) (Gomashe, 2016). Recently, barnyard
millet has received some attention from the scientific community in
developing genetic and genomic resources (Upadhyaya et al., 2014;
Wallace et al., 2015) and understanding diversity at morphological
(Mehta et al., 2005; Nirmalakumari, 2009; Sood, Khulbe, Kumar,
et al., 2015; Williams et al., 2019) and molecular levels (Jayakodi
et al., 2019; Manimekalai et al., 2018; Moharil et al., 2016). Systematic
characterization and evaluation of germplasm are necessary to facili-
tate effective utilization of available germplasm and identify lines with
significantly distinct traits as donors for crop improvement. Barnyard
millet germplasm lines have been characterized for disease (Gupta
et al., 2010; Joshi et al., 2015; Nagaraja & Jayarame, 2010; Nagaraja &
Mantur, 2008; Rawat et al., 2018) and insect pests (Rawat
et al., 2019). Studies have also been conducted to understand grain
yield components (Joshi et al., 2015; Sood et al., 2016) and character
associations (Arunachalam & Vanniarajan, 2012; Dhagat et al., 1978;
Prabu et al., 2020; Prakash & Vanniarajan, 2015).
In recent times, there have been consistent efforts to breed high
yielding barnyard genotypes to achieve higher yields (Madhusudhana
et al., 2018). In this scheme, one of the major biotic factors that have
been encountered is the insect pest, shoot fly. Genetic enhancement of
crop varieties as opposed to biotic stresses is an important adaptation
strategy to achieve higher yields. Shoot fly causes economic damage in
barnyard millet by causing deadhearts (DH) on the main plant, basal til-
lers and on the nodal tillers (Rawat et al., 2020). It has been observed
that among small millets, barnyard millet is preferred by shoot fly after
sorghum (Davies & Reddy, 1981; Kalaisekar et al., 2016). Complex of
fly species comprising Atherigona soccata and A. falcata attacks the
barnyard millet. Eggs are laid singly on the under surface of seedlings
and cause DH similar to sorghum. A recent study (Rawat et al., 2020)
characterized six elite breeding lines for shoot fly resistance. Given the
nature of damage caused by the shoot fly, it is necessary to character-
ize available germplasm for shoot fly reaction and find suitable donors
to breed for resistance. In addition to higher resistance, understanding
the interaction and stability of the genotype
environment is also a
key criterion to ensure consistent performance of a genotype in differ-
ent environments. The Additive Main effect and Multiplicative Interac-
tion (AMMI) model is the most widely used model to capture the
largest genotype (G) and environment (E) interaction.
Generally, the phenotypic stability of genotypes is measured
based on a single trait. Selection of a stable superior genotype for a
larger number of traits is needed in plant breeding. MTSI (Multi-Trait
PADMAJA ET AL.
Stability Index) identifies stable genotypes by considering all mea-
sured traits (Olivoto et al., 2019). The present study characterizes a
set of 106 diverse barnyard millet germplasm lines for their reaction
& to shoot fly and their phenotypic stability based on two traits besides
estimating the role of biochemicals in imparting resistance. The infor-
mation generated in the study is the first report that will be useful for
the breeding of shoot fly resistance in barnyard millet to achieve sus-
tainable agriculture, especially in the hilly and tribal tracts where barn-
yard millet is a staple crop.
2 | M A T E R I A L S A N D M ET H O D S
2.1 | Field evaluation for shoot fly resistance
A total of 106 barnyard millet (Echinochloa frumentacea L.) genotypes
(Table 1) were evaluated during the late kharif (August–November)
seasons of 2016, 2017 and 2018 for shoot fly resistance. All the
TABLE 1 List of barnyard genotypes included in the study
County of
Entry origin
IEC 52 (1), IEC 53 (2), IEC 54 (3), IEC 56 (4), IEC 57 India
(5), IEC 60 (6), IEC 71 (7), IEC 80 (8), IEC 100 (9),
IEC 133 (10), IEC 158 (11), IEC 159 (12), IEC 162
(13), IEC 178 (14), IEC 179 (15), IEC 183 (16), IEC
196 (17), IEC 208 (18), IEC 217 (19), IEC 229 (20),
IEC 239 (21), IEC 240 (22), IEC 265 (23), IEC 269
(24), IEC 285 (25), IEC 286 (26), IEC 350 (28), IEC
360 (29), IEC 365 (30), IEC 374 (31), IEC 381 (32),
IEC 383 (33), IEC 391 (34), IEC 395 (35), IEC 396
(36), IEC 573 (37), IEC 607 (38), IEC 613 (39), IEC
619 (40), IEC 647 (42), IEC 650 (43), IEC 651 (44),
IEC 672 (46), IEC 675 (47), IEC 688 (48), IEC 690
(49), Oodalu (56), KOPBM5 (57), KOPBM6-1 (58),
KOPBM7 (59), KOPBM8 (60), KOPBM9-1 (61),
KOPBM10-1 (62), KOPBM11 (63), KOPBM12 (64),
KOPBM15 (65), KOPBM16 (66), KOPBM17 (67),
KOPBM18 (68), KOPBM25 (69), KOPBM27 (70),
KOPBM28 (71), KOPBM29 (72), KOPBM30 (73),
KOPBM31 (74), KOPBM32 (75), KOPBM33 (76),
KOPBM34 (77), KOPBM35 (78), KOPBM39 (79),
KOPBM43 (80), KOPBM44 (81), KOPBM45 (82),
KOPBM46 (83), KOPBM47 (84), KOPBM51 (85),
VL-129 (86), GECH-33 (87), GECH-38 (88), GECH-
43 (89), GECH-57 (90), GECH-91 (91), GECH-111
(92), GECH-231 (93), GECH-244 (94), GECH-266
(95), GECH-310 (96), GECH-397 (97), GECH-407
(98), GECH-468 (99), GECH-476 (100), GECH-514
(101), Local (102), VL 172 (103), VL 207 (104), ERP
93 (105), ERP 100 (106)
IEC 348 (27) Malawi
IEC 624 (41) Cameroon
IEC 661 (45) Pakistan
IEC 701 (50), IEC 706 (51), IEC 731 (52), IEC 747 (53), Unknown
IEC 758 (54), IEC 786 (55)
Note: Figures in parenthesis indicate the genotype number used in the
study.
PADMAJA ET AL.
experiments were carried out with three replications in a randomized
complete block design at ICAR-Indian Institute of Millets Research
(IIMR) experimental farms located at an altitude of 523 m above mean
sea level in Rajendranagar, Hyderabad, Telangana, India. During 2016,
the experiment was planted at A3 block (17.1931 N and 78.2310 E),
whereas during 2017 and 2018, it was planted at A6 block
 
(17.1926 N and 78.2413 E) with sorghum as the previous crop. Dur-
ing the cropping season of three years, mean rainfall was 513 mm, the
maximum temperature was 30.4 C and the minimum temperature
was 18.8 C. Each entry was planted in a 4-m single row plot, and the
rows were 60 cm apart. The plants were thinned seven days after
seedling emergence (DAE) to maintain a 10-cm spacing between the
plants. Field evaluations were conducted under high stress conditions
using the Interland fish meal technique (Soto, 1974). DH of shoot fly
(%) were estimated in the main plant (MPDH) and in the basal tillers
(TDH). Plants with DH were recorded in all plots at 28 DAE in the
main plant and the number of DH in tillers later in tiller formation at
60 DAE. The DH% (ratio of the number of DH to the total number of
plants
100) was used for evaluating overall shoot fly resistance.
During Kharif 2018, data on additional traits such as seedling
height (SH), epicuticular wax (EW), and biochemical parameters (pro-
tein, peroxidase, and polyphenol oxidase) were recorded using leaf
samples collected from the field experiment.
2.2 | SH
SH (cm) was determined with a scale and measured from the bottom
to the tip of the fully expanded leaf at 21 DAE on each entry from
three replications.
2.3 | EW
Wax was estimated from the samples taken from all three replications.
The determination of EW was done using the method of Ebercon
et al. (1977). The development of the method was based on the colour
change that is generated due to the reaction of wax with acidic
K2Cr2O7 (Bragdon, 1951). The individual sample consisted of leaf
discs having a total surface area of 32 cm2. Each sample was dipped in
15 ml of double-distilled chloroform for 15 s, and the extract was fil-
tered and evaporated in a boiling water bath until the smell of chloro-
form was not detected. After adding 5 ml of reagent, the samples
were placed in boiling water for 30 min. After cooling, 12 ml of
deionized water was added and several minutes for colour develop-
ment and cooling. The absorbance of the sample was read at 590 nm.
2.4 | Biochemical analysis
Data on biochemical parameters (protein, peroxidase and polyphenol
oxidase) were recorded from a single replication of healthy and DH
plants.
401
2.5 | Preparation of samples
One gram of ground tissue from three seedlings of each of the
106 genotypes, fly-infested and uninfected shoots, was collected from
the field for biochemical analyses at 21 DAE. Seedlings without DH
symptoms served as control.
2.6 | Protein
The sample preparation and protein assay followed the method pro-
posed by Hildebrand et al. (1986). Briefly, soluble proteins were
obtained by crushing seedlings in a chilled mortar with 3.0 ml of
20-mM 4–2-hydroxymethyl-1-piperazineethane sulfonic acid (HEPES)
buffer (pH 7.2) comprising a protease inhibitor concoction (0.3/1 g of
tissue contains 4-(2-aminoethyl) benzene sulfonyl fluoride, bestatin,
pepstatin A, E-64, leupeptin and 1,10-phenanthroline) and 1% poly-
vinylpyrrolidone. The homogenate was centrifuged at 10,000
g for
10 min at 4 C. The supernatant was collected and stored (<3 h) at 4 C
until protein analysis.
2.7 | Assays
The result of the shoot fly feeding on protein content and enzyme
(peroxidase and polyphenol oxidase) activities was assessed using a
spectrophotometer. The total protein content was evaluated with
bovine serum albumin as a control (Lowry et al., 1951). Peroxidase
activity was computed by examining the increase in absorbance at
470 nm for 1 min (Hildebrand et al., 1986; Hori et al., 1997). The
enzymatic reaction was initiated by adding 10 ml of 30% hydrogen
peroxide to a cuvette that contains 300 μl of 18-mM guaiacol, 100 μl
of 200-mM HEPES (pH 7.0), 585 μl of distilled water and 5 μl of sor-
ghum extract. The specific activity of peroxidase was established
using the molar absorptivity of guaiacol at 470 nm (26.6
103/
M/cm).
Polyphenol oxidase activity was calculated following a protocol
modified from Hori et al. (1997). Enzyme activity was tracked at
470 nm for 1 min after the onset of the reaction. The reaction was
started by adding 20 μl of barnyard extract to a cuvette comprising
500 μl of 1.6% catechol in HEPES buffer, 380 μl of distilled water and
100 μl of 200 mM HEPES buffer (pH 6.0). Polyphenol oxidase activity
was determined as a change in absorbance at A470 per minute per
milligram of protein.
2.8 | Statistical analysis
All statistical calculations were performed using GenStat software ver-
sion 21 (VSN International Limited, 2021). AMMI analysis was carried
out using data on DH (MPDH and TDH) as per the statistical model
given by Zobel et al. (1988). The Multi-Trait Stability Index (MTSI) is
based on the genotype–ideotype distance (Euclidian) using the scores
402 PADMAJA ET AL.

achieved in factor analysis (Olivoto et al., 2019). MTSI was calculated mean of 28.70%. For TDH, the range recorded was from 32.42%
from both MPDH and TDH trait values. A Varimax rotation principle (KOPBM17) to 51.67% (IEC 53) with an overall mean of 42.50%. The
was utilized for analysing the final loadings. Genotype scores were mean difference between MPDH (28.70%) and TDH (42.50%) was
achieved using standardized WAASBY means. The ideotype scores significant (P < .001). During 2018, the data on seedling height (SH),
were achieved considering that an ideotype has the lowest WAASBY epicuticular wax (EW) and biochemical parameters were recorded. A
values (100) for all the observed traits. The MTSI was determined as wide genotypic variation was also observed for SH, EW (Tables 2 and
follows: 4) and biochemical parameters (Table 2). The height of the seedling
varied from 18.30 cm (IEC 80) to 37.10 cm (IEC 661) with a mean of
" #0:5
X
f  2 28 cm. The range in EW was from 5.40 (KOPBM9-1) to 18.00
MTSIi ¼ F ij  F j ,
j¼1
(KOPBM43).

where MTSI is the MTSI for the ith genotype, Fij is the jth score of the
ith genotype and Fj is the jth score of the ideotype. The genotype with
the lowest MTSI is then nearer to the ideotype and therefore exhibits
a high mean performance and stability for all investigated variables
(Olivoto et al., 2019).
3 | RESULTS
3.1 | Summary statistics
Large variation was observed for all traits, as evidenced by the results
of the variance analysis (Tables 2–4). The genotypes with low and
high trait values in each year and across years are presented in
Table 2. Considerable variation was observed for both MPDH and
TDH during individual years and across years. Across years, the range
for MPDH ranged from 20.63% (IEC 285) to 37.63% (IEC 53) with a
3.2 | Biochemical differences in the genotypes due
to shoot fly infestation
The affected seedlings (seedlings with DH) and healthy (seedlings
without DH) were analysed for biochemical components during 2018.
Biochemical expression levels, that is, protein, peroxidase and poly-
phenol oxidase, showed significant differences between healthy and
DH seedlings (Table 2). A large variation in biochemical parameters
was observed within healthy and infected plants. In healthy seedlings,
the protein ranged from 2.63 (GECH-514) to 7.10 (IEC 229), whereas
in shoot fly-infested seedlings, it ranged from 4.00 (IEC 56) to 8.20
(KOPBM6-1). The difference in mean protein levels between healthy
seedlings (4.85) and shoot fly infested seedlings (6.24) was significant
(P < .001). Similarly, the difference in peroxidase expression levels
between healthy and infected seedlings was significantly different
(P < .001). Among healthy seedlings, the peroxidase value varied from
16.84 (IEC 240) to 76.98 (GECH-46), whereas in infested seedlings, it

TABLE 2 Genetic variability parameters for target traits

Trait Year Minimum Maximum Mean# SEM h2b (%)

b
MPDH (%) 2016 13.57 (KOPBM6-1) 53.23 (IEC 239) 31.01 0.52 0.72
a
2017 14.97 (KOPBM32) 40.17 (IEC 53) 25.40 0.37 0.35
2018 21.87 (KOPBM16) 41.23 (KOPBM35) 29.70b 0.32 0.65
Across 20.63 (IEC 285) 37.63 (IEC 53) 28.70
TDH (%) 2016 41.40 (IEC 265) 57.47 (IEC 183) 49.20c 0.27 0.20
a
2017 15.50 (GECH-231) 53.03 (KOPBM25) 35.10 0.65 0.66
2018 31.43 (KOPBM18) 59.27 (IEC 53) 43.10b 0.46 0.72
Across 32.42 (KOPBM17) 51.67 (IEC 53) 42.50
Seedling height (cm) 2018 18.30 (IEC 80) 37.10 (IEC 661) 27.89
Wax (μg/g) 2018 5.40 (KOPBM9-1) 18.00 (KOPBM43) 11.09
Protein—healthy plants 2018 2.63 (GECH-514) 7.10 (IEC 229) 4.85a
Protein—infected plants 2018 4.00 (IEC 56) 8.20 (KOPBM6-1) 6.24b
Peroxidase—healthy plants 2018 16.84 (IEC 240) 76.98 (GECH-46) 40.25a
Peroxidase—infected plants 2018 24.41 (IEC 269) 80.66 (KOPBM10-1) 55.71b
Polyphenol—healthy plants 2018 0.15 (IEC 80) 2.91 (IEC 381) 1.44b
Polyphenol—infected plants 2018 0.32 (IEC 178) 3.25 (IEC 381) 1.78a

Note: Names in parenthesis indicate the genotype.

Abbreviations: MPDH, main plant deadheart; TDH, tiller deadheart.
#
Class means with different letters are statistically different.
PADMAJA ET AL. 403

T A B L E 3 Additive main effects and multiplicative interaction T A B L E 5 Mean of genotypes selected by MTSI applied for MPDH
analysis of variance for MPDH and TDH, of the genotypes across and TDH traits
years
S. no Genotype MPDH TDH
Source df MPDH %SS TDH %SS
1 KOPBM17 (67) 26.01 32.42
Environments 2 2693* 15.98 15760** 47.40 2 IEC 285 (25) 20.63 39.98
Genotypes 105 98.6** 30.73 145** 22.83 3 IEC 391 (34) 27.66 32.64
G
E 210 85.5** 53.28 94** 29.75 4 IEC 758 (54) 22.04 40.24
IPCA 1 106 125.4** 130** 5 KOPBM45 (82) 23.11 38.38
IPCA 2 104 44.8* 57** 6 IEC 162 (13) 25.83 35.61
Error 630 32.9 39 7 IEC 360 (29) 23.37 38.80
Total 953 61.1 109 8 IEC 706 (51) 23.41 39.48
Var (%) Var (%) 9 IEC 613 (39) 25.77 36.81
IPCA 1 74.02 69.78 10 KOPBM9–1 (61) 23.98 37.82
IPCA 2 25.98 30.22 11 IEC 265 (23) 26.77 33.90
Total 100 100 12 KOPBM6–1 (58) 21.42 42.41

Abbreviations: MPDH, main plant deadheart; TDH, tiller deadheart; SS, 13 IEC 647 (42) 25.03 39.21
sum of squares. 14 IEC 786 (55) 27.97 34.73
*Significant at 5% level.
15 GECH-231 (93) 26.71 37.53
**Significant at 1% level.
16 IEC 286 (26) 25.11 39.81
Mean 24.68 37.49
TABLE 4 ANOVA for seedling height and epicuticular wax (year
Note: Values in parenthesis gives the genotype number.
2018) Abbreviations: MPDH, main plant deadheart; TDH, tiller deadheart.
Source df Seedling height-MSS Epicuticular wax-MSS
Replication 2 20.23 0.59
Genotypes 101 53.94*** 27.87*** 3.4 | Multi-trait stability
Error 202 17.67 0.61
Total 305 The MTSI was calculated involving both MPDH and TDH. Table 5
provides the mean performance of selected genotypes based on the
Abbreviation: ANOVA, analysis of variance. MSS, Mean Sum of Squares.
***Significant at .1% level. MTSI, and Table 6 gives the selection differential and selection gains
of characters founded on MTSI. Figure 1 shows the MTSI values of
the selected (red) and unselected (black) genotypes. KOPBM17 (67),
varied from 24.41 (IEC 269) to 80.66 (KOPBM10-1). For polyphenols, IEC 285 (25), IEC 391 (34), IEC 758 (54), KOPBM45 (82), IEC
the levels were high (1.78) in infested seedlings compared with 162 (13), IEC 360 (29), IEC 706 (51), IEC 613 (39), KOPBM9-1 (61),
healthy seedlings (1.44), and the mean difference was also statistically IEC 265 (23), KOPBM6-1 (58), IEC 647 (42), IEC 786 (55), GECH-231
significant (P < .001). The protein content and the activity of two (93) and IEC 286 (26) were the genotypes, which were selected with a
enzymes, peroxidase and polyphenol oxidase, were significantly mean of 24.70% MPDH and 37.50% TDH. The selection differential
higher in shoot fly-infested seedlings than in unaffected healthy was >4 with a reduction in the trait value of 11–14%. The % selection
plants. gain was also 2–4%.

3.3 | G
E interaction 4 | DI SCU SSION

AMMI analysis of variance (ANOVA) showed significant differences
for genotypes, environment and their interaction for MPDH and TDH
(Table 3). Effect of the year was higher for TDH (47.4% of the total
sum of squares) compared with MPDH (15.98%). The genotypic effect
was greater for MPDH (30.73%) than for TDH (22.83%). G
E was
higher in MPDH (53.28%) as compared with 29.75% of TDH. Further-
more, G
E was divided into the principal component axes of interac-
tion (IPCA) and the residuals. Both IPCA were highly significant for
both traits and cumulatively explained 100% variation.
Barnyard millet has been one of the fastest growing minor cereal
crops cultivated by tribal and poor farmers in marginal soils in rainfed
situations (Joshi et al., 2015). It is becoming increasingly important as
a good source of nutrition and seen as an ideal crop for subsistence
farmers. It is cultivated as an alternative to rice and other major crops
during the failure of monsoons in India (Gupta et al., 2009). Under-
standing trait diversity in germplasm collection is fundamental and
useful for genetic enhancement programmes. One of the major limita-
tions in the successful cultivation of Barnyard millet is the damage
404 PADMAJA ET AL.

T A B L E 6 Selection differential and

Traits Xo Xs SD SD (%) h2 SG SG (%) Sense Goal
selection gains of characters based on
MPDH 28.70 24.7 4.03 14.0 0.13 0.53 1.87 Decrease 100 multi-trait stability index
TDH 42.50 37.5 4.98 11.7 0.34 1.73 4.08 Decrease 100

Note: Xo = mean of genotypes; Xs = mean of selected genotypes; SD = selection differential;

SG = selection gain; h2 = heritability.
Abbreviations: MPDH, main plant deadheart; TDH, tiller deadheart.

F I G U R E 1 Genotypes selected by
multi-trait stability index applied on
MPDH (main plant deadheart) and TDH
(tiller deadheart) [Color figure can be
viewed at wileyonlinelibrary.com]

caused by the insect shoot fly. The insect attacks the crop on the main
plant and tillers, thereby reducing the formation of effective panicles.
In this study, large variability was observed for shoot fly resistance
among the 106 germplasm lines evaluated. The range seen for DH in
both MPDH and TDH (Table 2) indicated moderate levels of resis-
tance. None of the genotypes was found to be highly resistant (with
<10 DH%). In a recent study with limited barnyard accessions (6) eval-
uated for shoot fly resistance, Rawat et al. (2019) found DHBM
996 and TNEF-204 as resistant to shoot fly. The current study
involved a larger collection of germplasm lines and identified IEC
285 and KOPBM17 for the resistance of the shoot fly over seasons.
IEC 53 was found to be highly susceptible for both MPDH and TDH
over seasons. Identified sources of resistance can be employed in
resistance breeding. The resistant and susceptible sources are useful
in the development of biparental mapping populations to identify the
genes/QTL (Quantitative Trait Loci) involved in shoot fly resistance as
done in sorghum (Satish et al., 2009).
EW was found to be strongly and positively associated with
MPDH and TDH (r = .39**, P > .01). This finding is similar to that of
sorghum, where genotypes with a higher EW content are associated
with a high susceptibility to shoot fly (Padmaja et al., 2013). Further
qualitative analysis of wax is required to understand the structural dif-
ference in the wax crystals as observed in sorghum (Nwanze
et al., 1992; Padmaja et al., 2009). The glossiness of leaf surface in
sorghum is an important component of sorghum shoot fly resistance
and reflects the quantitative and qualitative difference in EW. QTL
mapping for glossiness traits in sorghum has identified several wax
biosynthesis genes (Satish et al., 2009, 2012). Such studies involving
biparental mapping populations in barnyard millet may also reveal the
role of wax in the resistance of the shoot fly. The height of the seed-
ling was not associated with MPDH and TDH. The correlations also
did not reveal a significant association between MPDH and TDH
(r = .15, P > .08), which indicated differential stage susceptibility of
genotypes.
PADMAJA ET AL.
Plant resistance to biotic and abiotic stresses is often governed
through the metabolism of phenolic compounds (Dicko
et al., 2005). Increased activities of phenolic-related enzymes and
the accumulation of phenolic compounds have been correlated
with the resistance of cereals to biotic stresses (Mohammadi &
Kazemi, 2002; Padmaja et al., 2014). In this study, we looked at
shoot fly feeding-induced damage on barnyard plants and the
resulting effects on plant biochemical and enzymatic changes. In
insect-injured plants due to herbivory, there has been a quantita-
tive increase in the levels of biochemical substances such as pro-
teins, phenols and enzymes activities. The results suggest that
barnyard millet genotypes may be able to withstand the feeding of
the shoot fly by improving their activities of protein, peroxidase
and polyphenol oxidase as reported in sorghum against the shoot
fly (Padmaja et al., 2014). Increase in the total protein in wheat
infested with the Russian wheat aphid and the corn leaf aphid (Ni
et al., 2001), peroxidases in barley against aphids (Chaman
et al., 2001) and polyphenols in tomato (Thipyapong et al., 2004)
were reported.
AMMI ANOVA suggested significant differences between
genotypes, seasons and their interactions, affirming the impact of
environment on the expression of MPDH and TDH phenotypic
traits. The high environmental effect on TDH signifies that TDH
expression is greatly influenced by the environment. For MPDH, the
largest portion of the trait variation was due to the G
E
interaction rather than the main effects. As G
E was significant
for both MPDH and TDH, the genotype x environment interaction
(GEI) was further partitioned using PCA. The result of ANOVA has
shown that the first two IPCA were very significant at (P < .01)
suggesting the incorporation of the first two interactions of the
PCA axes into the model. Hence, the best matching AMMI model
for this multi-location yield trial data was AMMI-2 (Table 3). Olivoto
et al. (2019) have put forth a multi-trait stability index (MTSI) for
the selection of stable genotypes based on several traits and mean
performance. MTSI is based on the genotype–ideotype Euclidian
distance using scores received in factor analysis. The ideotype has
the highest WAASBY values for each observed trait. The lower the
value of MTSI, the better the stability and performance of the
genotype. Figure 1 shows the stable genotypes chosen based on
MTSI with a 15% selection differential. The top 5 selected stable
genotypes in the order were KOPBM17, IEC 285, IEC 391, IEC
758 and KOPBM45 (Table 5). The use of these genotypes would be
highly beneficial in improving the resistance of the shoot fly, as
reflected in the negative genetic gain of 1.87% (for MPDH) and
4.08% (for TDH) (Table 6).
The study brought out the genetic difference that exists in the
barnyard germplasm lines for shoot fly resistance, and the genotypes
have shown differential stage susceptibility. EW plays a major role in
the resistance, and more studies are needed to understand the effect
of wax structure in imparting resistance. MPDH and TDH have high
G
E interaction, and the MTSI used in the study has identified a few
lines, which can be employed in breeding to achieve better shoot fly
resistance.
405
AC KNOW LEDG EME NT S
The authors sincerely acknowledge the Indian Council of Agricultural
Research (ICAR), New Delhi, and the ICAR-Indian Institute of Millets
Research (ICAR-IIMR), Hyderabad, for providing funds to carry out
this study. We are also thankful to ICAR-AICRP on Small millets,
Bengaluru, for providing the genotypes used in the study.
CONFLIC T OF INT ER E ST
The authors declare that they have no known competing financial
interests or personal connections that could have seemed to influence
the work described in this paper.
AUTHOR CONTRIBU TIONS
Author PG Padmaja and author R Madhusudhana conceived the
research. Author PG Padmaja conducted experiments. Authors PG
Padmaja, A Kalaisekar and Vilas A Tonapi contributed materials.
Author R Madhusudhana analysed the data and conducted statistical
analyses. Authors PG Padmaja and R Madhusudhana wrote the manu-
script. Authors PG Padmaja and R Madhusudhana secured funding. All
authors read and approved the manuscript.
DATA AVAILABILITY STAT EMEN T
Research data are not shared.
OR CID
Ragimasalawada Madhusudhana https://orcid.org/0000-0001-
7316-0986
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How to cite this article: Padmaja, P. G., Kalaisekar, A., Tonapi,
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barnyard millet (Echinochloa frumentacea) germplasm lines for
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