The Scientific World Journal
Volume 2014 (2014), Article ID 308042, 14 pages
http://dx.doi.org/10.1155/2014/308042

Research Article

Heritability and Genetic Advance among Chili Pepper Genotypes for Heat Tolerance and Morphophysiological Characteristics

Magaji G. Usman,¹ M. Y. Rafii,1,2 M. R. Ismail,1,2 M. A. Malek,2,3 and Mohammad Abdul Latif1,4

¹Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia
²Institute of Tropical Agriculture, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia
³Bangladesh Institute of Nuclear Agriculture, Mymensingh 2202, Bangladesh
⁴Bangladesh Rice Research Institute, Gazipur 1701, Bangladesh

Received 10 March 2014; Revised 14 July 2014; Accepted 14 July 2014; Published 16 November 2014

Academic Editor: Antonio Ferrante

Copyright © 2014 Magaji G. Usman et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

High temperature tolerance is an important component of adaptation to arid and semiarid cropping environment in chili pepper. Two experiments were carried out to study the genetic variability among chili pepper for heat tolerance and morphophysiological traits and to estimate heritability and genetic advance expected from selection. There was a highly significant variation among the genotypes in response to high temperature (CMT), photosynthesis rate, plant height, disease incidence, fruit length, fruit weight, number of fruits, and yield per plant. At 5% selection intensity, high genetic advance as percent of the mean (>20%) was observed for CMT, photosynthesis rate, fruit length, fruit weight, number of fruits, and yield per plant. Similarly, high heritability (>60%) was also observed indicating the substantial effect of additive gene more than the environmental effect. Yield per plant showed strong to moderately positive correlations () at phenotypic level while at genotypic level correlation coefficient ranged from 0.16 to 0.72 for CMT, plant height, fruit length, and number of fruits. Cluster analysis revealed eight groups and Group VIII recorded the highest CMT and yield. Group IV recorded 13 genotypes while Groups II, VII, and VIII recorded one each. The results showed that the availability of genetic variance could be useful for exploitation through selection for further breeding purposes.

1. Introduction

Chili pepper (Capsicum annuum L. and Capsicum frutescence L.) is widely cultivated, primarily as a spice crop [1]. The optimum day temperatures for chili pepper growth range from 20 to 30°C [2], and day temperatures rise above 30°C year round in Malaysia [3]. The domestic production of chili pepper in Malaysia can hardly meet 70% of demand due to poor performance of local varieties under high temperature. Furthermore, high temperature is one of the major problems for chili cultivation in Malaysia. Such conditions are the important factors limiting the production of chili pepper. Therefore, understanding the effect and mechanism of high temperature on chili pepper are the important factors for the improvement of the quality of the crop. However, chili pepper, as well as other crops such as groundnut [4] and heat tolerant genotypes will be needed to sustain their production under high temperature environments.

Yield is a determining factor for crop improvement [5]. Chili pepper, as in other crops, yield is a quantitative trait that is influenced by a number of yield contributing parameters. The selection of desirable genotypes is usually based on yield and yield components. It is therefore necessary to study the mutual relationship between yield and yield components for efficient utilization of the genetic stock in crop improvement program of chili pepper. Variability in plants is the first step in understanding how to improve or produce new plants. Heritability is the degree of genetic control associated to some important traits [6]. It indicates how much of the genetic variability has a genetic origin and gives necessary information for the genetic selection process [7]. To improve grain yield potentials of crops in any breeding programs, it is necessary to obtain adequate information on the magnitude and type of genetic variability and their corresponding heritability. This is because selection of superior genotypes is proportional to the amount of genetic variability present and the extent to which the characters are inherited. Heritability is used to indicate the relative degree to which a character is transmitted from parent to offspring. The magnitude of such estimates also suggests the extent to which improvement is possible through selection [5].

Temperature and other abiotic stresses are clearly limiting factors for the growth and development of crop. Indeed stresses due to high temperature can be harmful to all phases of plant development, and global climate change is thought to cause extreme environmental fluctuations in most agricultural regions [8]. Temperature increment due to changing climatic condition is a serious threat [9] which affects crop production. So, understanding how the plants respond to stress is a challenging area of research [10]. Cell membrane thermostability (CMT) is a phenotypic parameter used in measuring electrolyte leakage from leafs of plants at different temperatures. CMT is a very sensitive and rapid method to identify heat tolerance in plants [11]. Several studies have indicated that CMT is effective in detecting genetic difference among several crops for heat tolerance [12, 13]. However, the mechanism for heat tolerance using the electrolyte leakage of crops under heat stress need to be more exploited in order to identify heat tolerant lines for the development of high yielding heat tolerant hybrid varieties, which will contribute to achieve self-sufficiency in chili production in Malaysia. Genotypes within several crop species are found to differ with respect to heat tolerance, where heat tolerant genotypes are referred to as giving the highest yield under high temperature condition [14–16]. Heat tolerance can be referred to as performance of a plant with respect to its yield or physiological processes under elevated temperature as compared with its performance under optimal temperature [17]. Genotypic differences in tolerance/susceptibility have been reported in chili pepper for pollen tube length and pollen germination and membrane stability [18, 19]. However, only a very limited number of genotypes of chili pepper have being studied. Furthermore, association between heat tolerance and cell membrane thermostability with respect to selection criteria for breeding purposes has not been investigated.

The objectives of this research was to identify chili pepper genotypes tolerant to high temperature and study the genotypic variation among the genotypes for heat tolerance and other yield related traits, to determine whether genotypic differences in tolerance to high temperature were associated with membrane thermostability, to study the correlation between heat tolerance and morpho-physiological traits and determine whether heat tolerance can be used as selection criteria in chili pepper breeding program.

2. Materials and Methods

2.1. Site and Location

This research was conducted between May and August 2013 using the Rain Shelter facility of the Agro-technology experimental site, Institute of Tropical Agriculture, Universiti Putra Malaysia. It is located on 3°02′ N latitude and 101°42′ East longitude and altitude is 31 m above sea level. The average climatic conditions are represented in Table 1.

Table 1: The average temperature, solar radiation, and rainfall of the experimental site during the period of the experiment (2013/2014).

Months
Temperature (°C)
Solar radiation MJ/m²
Rainfall (mm)

Maximum
Minimum

May
38.5
24.5
22.70
—

June
36.3
24.2
21.2
4.2

July
36.3
23.8
20.20
100

August
39.3
23.8
—
—

Source: Malaysian Meteorological Department (http://www.accuweather.com/en/my/kuala-lumpur/233776/month/233776?monyr=7/01/2013#), 2013.

2.2. Plant Husbandry

Experiment was carried out in a prepared polythene bag under rain shelter condition. All genotypes used for this experiment were collected from Asian Vegetable Research and Development Centre, AVRDC, in Taiwan, except for Kulai, which is a local variety cultivated in Malaysia (Table 2). Thirty-six genotypes belonging to two species of Capsicum were sown in seed trays on 8th May, 2013, with 1-2 seeds per cell and the growing medium was peat moss. They were later transplanted to the pots filled with cocoa peat. The pots were 17 cm × 30 cm with small holes to drain excess water. The experiment was laid in a randomized complete block design (RCBD) in three replications. Two pots were assigned for each genotype in each replication (72 pots per replication). The pots were oriented east to west and spaced 75 cm × 150 cm. Seedling emerged within 7–10 days after sowing and were transplanted 4 weeks after sowing. All pots were irrigated and fertilized using fertigation system of cropping with drip system of irrigation.

Table 2: List of chili pepper genotypes and their pedigree, specie, and heat tolerance used in this experiment.

Genotype
Source
Specie
Pedigree
Heat tolerancea

AVPP9703
CCA 1410A
C. annuum
HDA210/Szechwan10/MC4
excellent

AVPP9901
CCA 3106
C. annuum
HM-19/155-42//G-4
good

AVPP9806
CCA 196-A
C. annuum
SlamChiliF1/HDA248
—b

AVPP9813
CCA 323
C. Frutescens
Kulim/HDA295 (Berke’s Joy)
excellent

AVPP0002
CCA 3331
C. annuum
Arunalu/Tumpang
excellent

AVPP0012
Jin’s Joy
C. annuum
Jin’s Joy
fair

AVPP0102
CCA 3636
C. annuum
IR/LongFruit
excellent

AVPP0004
Maor/Perennial
C. frutescens
Maor/Perennial
good

AVPP0104
CCA 3468
C. annuum
MI-2/Taiwan83-168-1-1//MI-2
good

AVPP0105
CCA 321
C. annuum
Kulim/HDA248
excellent

AVPP9905
Susan’s Joy
C. annuum
Susan’s Joy
excellent

AVPP9815
CCA226A-3
C. frutescens
Corbadzijiski/HDA248
—

AVPP0201
CCA 4283
C. annuum
(Szechwan9/PerennialHDV)/Jin’s Curlicue//F1 Hy Hot 3 F3 sel.
—

AVPP0116
CCA 3743
C. annuum
PSP-11/Jin’s Delight//Kulai
excellent

AVPP9805
CCA 2345
C. annuum
HDA248/5*LongFruitA

AVPP0109
MSH-1 seln
C. annuum
F1 MSH-1
excellent

AVPP0305
CCA 4824
C. frutescens
PBC325/PBC308

AVPP0101
CCA 3617
C. annuum
FriesdorferSelex/LongFruit
excellent

AVPP0805
CCA6306-1-3-3-3-1
C. annuum
Jatilaba/CCA321//Jatilaba
—

AVPP0307
CCA 4860
C. annuum
LongThick/9852-173
—

AVPP0506
CCA5213
C. frutescens
PBC 481 sel.//Kulium/HDA248
—

AVPP0803
CCA6875-1-1-3-1-3
C. annuum
Var F/UF8752-3
—

AVPP0907

C. annuum
Kulim/HDA248//Lorai
—

AVPP0306
CCA 4851
C. annuum
VC232/9945-1856
—

AVPP0804
CCA6875-8-1-3-1-1
C. annuum
Var F/UF8752-3
—

AVPP0512
CCA5217
C. frutescens
Jin’s Joy//Kulium/HDA248
—

AVPP0514
CCA5218
C. annuum
Jin’s Joy//Kulai*3/PBC932
—

AVPP0702
CCA6023-9-1-1-2-1
C. annuum
Jin’s Joy/4/Bangchang-selex///HDA210/Szechwan10//MC4
—

AVPP0103
CCA 260
C. annuum
Szechwan9/Perennial
excellent

AVPP0014
Mr. Lee No.3 seln
C. annuum
Mr. Lee No.3 selex
fair

AVPP0705
CCA5684-05-1-2-1-1
C. frutescens
SSK-1/0209-4//SSK-1/PBC495
—

AVPP0904

C. annuum
Jin’s Joy//Kulim/HDA248/Jin’s Joy//Kulim/HDA248
—

AVPP0513
CCA5218
C. frutescens
Jin’s Joy//Kulai*3/PBC932
—

AVPP9812
CCA 336-B
C. frutescens
PBC385/HDA248
fair

C05573
PBC 142
C. annuum
Pant C-1 (long term ck.)
good

Kulai
Local
C. annuum

—

AVRDC, unpublished data.

bData not available.

2.3. Procedure for Cell Membrane Thermostability

The membrane stability test was conducted at the Plant Physiology laboratory, Department of Crop Science, Universiti Putra Malaysia. The leaf cell membrane thermostability (CMT) in the chili pepper genotypes was assessed according to the procedure described [20]. A sample for assay consists of a paired set, namely, control () and treatment () set of 6 leaf disks each 1.3 cm². The disks were cut from five fully expanded 3rd or 4th leaves from the top of the stem axis from each genotype. Samples were replicated three times each. Prior to assay, the paired set of leaf disks were placed in two separate test tubes (50 mL) and washed thoroughly with four exchanges of de-ionized water, 10 mL each time, to remove electrolytes adhering to the cut surface of the leaf discs. After the final wash, both sets of test tubes were filled with 10 mL de-ionized water and sealed with aluminium foil to avoid evaporation. The set of the test tubes were incubated for 20 min at 50°C in a temperature controlled water bath, while the set of test tubes were kept at room temperature (approximately 25°C). Both sets of test tubes were then incubated at 4°C (kept in a refrigerator) for 24 hrs. Initial conductance reading of both sets (CEC 1 and TEC 1) was made using an electrical conductivity meter (Starter) after bringing test tubes to room temperature. Tubes were then sealed again with aluminium foil and autoclaved at 121°C and 0.15 MPa for 20 min. to completely kill the leaf tissue. Autoclaved tubes were cooled to room temperature; content was mixed thoroughly and final conductance (CEC 2 and TEC 2) reading was taken. The CMT was calculated using the following equation, where TEC and CEC are a measure of conductance in treated and control test tubes, respectively, at initial (CEC 1 and TEC 1) and final (CEC 2 and TEC 2) conductance measurements [19]:

2.4. Morpho-Physiological Parameters

Characters assessed include disease incidence (%), plant height (cm), days to flowering, fruit length (cm), fruit weight (g), number of fruits, yield per plant (g), chlorophyll content, photosynthesis rate, and degree of pungency.

2.5. Chlorophyll Determination

Measurement of chlorophyll content was done following [21] procedure. Fresh leaves were collected from each replication from each genotype. One cm² leaf disks were cut from the leaves using leaf puncher and were transferred immediately into scintillation vials containing 20 mL of 80% acetone. The vials were capped and covered with aluminium foil after which kept in the dark for 7 days. UV spectrophotometer was used to measure the absorbance at 647 and 664 nm wavelengths. Using the following formulas, total actual chlorophyll was calculated:

2.6. Photosynthesis Rate (µmol/m²/s) Measurement

Net photosynthetic for the 36 genotypes was measured from the leaves of 90-day-old seedlings. The uppermost expanded leaves were selected for the measurement using an LI-6400XT portable photosynthesis system (LiCOR Inc., Lincoln, Nebraska, USA).

2.7. Degree of Pungency

The extractions of capsaicin from the chili pepper samples was done using the method described [22] and were analyzed using Ultra Fast Liquid Chromatography (UFLC) as described [23]. Whole chili fruits from the 36 genotypes were collected for determining degree of pungency (spicy level). Scoville Heat Units was used to calculate the heat units for all samples. Scoville Heat Units are calculated in parts per million of heat (ppmH) based on sample dry weight according to the following formula from [24]:

Conversion to Scoville Heat Units was made by multiplying ppmH by a factor of 15. They are classified as follows:

(i)(0–700 SHU) nonpungent(ii)(700–3,000 SHU) mildly pungent(iii)(25,000–70,000 SHU) highly pungent(iv)(3,000–25,000 SHU) moderately pungent(v)(>80,000 SHU) very highly pungent [25].

2.8. Statistical Analysis

Results were analyzed using SAS software (version 9.1) for all traits and means were separated using Duncan’s Multiple Range Test (DMRT) and Least Significance Difference (LSD) at 5% level.

2.8.1. Phenotypic and Genotypic Coefficient of Variation

The estimates of phenotypic and genotypic coefficient of variation were calculated as described [26] as follows:

where PCV is phenotypic coefficient of Variance, VP is phenotypic variance, GCV is Genotypic Coefficient of Variance, and Vg is genotypic variance. GCV and PCV values were categorized as low (0–10%), moderate (10–20%), and high (20% and above) as indicated by [27, 28].

2.8.2. Heritability

It was estimated as the ratio of total genotypic variance to the phenotypic variance according to [7]:

where Broad sense heritability. The heritability percentage was categorized as low (0–30%), moderate (30–60%), and high ≥60% as given by [29].

2.8.3. Genetic Advance

The extent of genetic advance expected through selection for the character was calculated as in [29]:

where is heritability, is phenotypic standard deviation, and is selection deferential (2.06 at 5%).

Genetic Gain (%) = GA × 100; it is categorized as low (0–10%), moderate (10–20%) and high (20% and above) [29].

2.9. Multivariate Analysis

Cluster and Principal Component Analysis were carried out to assess the genetic diversity of cell membrane thermostability and yield characters using NTSYS-pc software (version 2.1). Data were analyzed based on Euclidian distance method, dissimilarity coefficient. In order to determine the genetic relationships among the chili pepper accessions the UPGMA algorithm and SAHN clustering were applied. The PCA of the 36 chili pepper genotypes were calculated by EIGEN module of NTSYS-pc software.

3. Results

3.1. Cell Membrane Thermostability (CMT)

Looking at the climatic condition at which the plants were grown, cell membrane thermostability would prove effective for screening the genotypes for heat tolerance. The result from this study showed that there was a highly significant () difference among the genotypes in the relative injury (RI) and cell membrane thermostability (CMT) (Table 3). The cell membrane thermostability values ranged from 11.70 (AVPP9901) to 89.27% (AVPP0702) with a mean value of 64.62%. Most of the genotypes were recorded with high CMT values (>60%). Genotypes AVPP0105, AVPP9905, AVPP0116, AVPP0506, AVPP0803, C05573, AVPP0014, AVPP0702, AVPP0513, and AVPP0904 were found to be the most heat tolerant with the highest CMT values, while genotypes AVPP9703, AVPP9901, and AVPP0002 recorded the lowest CMT values indicating sensitivity to heat at 50°C.

Table 3: Relative injury as determined by cell membrane thermostability test at 25 and 50°C for 20 min for 36 genotypes of Capsicum spp.

GENOTYPE
Relative injury (%)
CMT (%)v

25°Ca
50°Ca
Injury*

AVPP9703
31.00
89.00
84.13
15.87

AVPP9901
26.33
91.2
88.30
11.70

AVPP9806
18.57
59.07
50.00
50.00

AVPP9813
22.00
62.10
51.03
48.97

AVPP0002
28.67
86.83
81.57
18.43

AVPP0012
22.07
81.23
75.77
24.23

AVPP0102
31.70
84.10
76.33
23.67

AVPP0004
23.53
56.93
43.27
56.73

AVPP0104
23.40
71.30
62.40
37.60

AVPP0105
25.23
38.57
17.67
82.33

AVPP9905
29.63
40.20
14.90
85.10

AVPP9815
30.93
68.43
54.20
45.80

AVPP0201
32.83
47.97
22.20
77.80

AVPP0116
32.67
40.90
11.97
88.0

AVPP9805
27.07
56.90
41.03
58.97

AVPP0109
36.03
61.40
39.03
60.97

AVPP0305
31.93
45.23
18.97
81.03

AVPP0101
42.47
68.50
45.63
54.37

AVPP0805
21.67
36.40
18.67
81.33

AVPP0307
23.20
43.03
25.90
74.10

AVPP0506
34.63
42.23
11.90
88.10

AVPP0803
21.50
34.20
15.87
84.13

AVPP0907
32.33
32.33
29.97
69.73

AVPP0306
26.27
49.07
30.83
69.17

AVPP0804
26.73
46.67
27.30
72.70

AVPP0512
23.40
39.27
20.67
79.33

AVPP0514
28.43
49.20
29.03
70.97

AVPP0702
33.20
40.43
10.73
89.27

AVPP0103
31.83
44.97
18.80
81.20

AVPP0014
27.20
35.40
11.57
88.43

AVPP0705
28.70
48.10
27.27
72.73

AVPP0904
27.23
39.20
16.50
83.50

AVPP0513
37.17
46.27
13.43
86.57

AVPP9812
29.80
43.37
19.37
80.63

C05573
25.97
38.47
17.03
82.97

Kulai
21.37
60.93
50.13
49.87

Means
28.24
53.92
35.37
64.62

LSD ()
7.39
11.95
17.43
17.39

SEM
0.61
1.67
2.37
2.37

Mean values were separated using Least Significance Difference at 5% level of probability.

Calculated as (initial conductivity/final conductivity) 100.

Calculated as calibrated RI = {1 − [1 − (TEC1/TEC2)]/[1 − (CEC1/CEC2)]}   100.

vCalculated as [1 − (TEC1/TEC2)]/[1 − (CEC1/CEC2)] 100.

LSD = Least significance difference.

SEM = Standard error of mean.

3.2. Morphophysiological Characters

All the morphophysiological characters studied in this experiment showed highly significant () difference except days to flowering and chlorophyll content (Table 4). The means for the 9 traits are presented in Table 5. The yield per plant ranged from 108.69 (AVPP0804) to 1144.3 g (AVPP9905). The plant height at harvest ranged from 41.4 to 92.13 cm which were recorded by AVPP0804 and AVPP0116 genotypes, respectively (Table 5). Significant range of variations was also observed for plant height at harvest, disease incidence, fruit length, fruit weight, and number of fruits. Similarly, a highly significant () variation was observed among the 36 genotypes for photosynthesis rate and degree of pungency. The genotype Kulai recorded the highest rate of photosynthesis (19.32) while AVPP0705 gave the lowest (3.75). The average rate of photosynthesis in this investigation was to be 13.29 µmol/m²/s. Degree of pungency or spicy level of the chili were found to vary significantly () among the genotypes (Table 4). The genotype AVPP0705 showed the highest pungency (247245.28 SHU). The capsaicin and dihydrocapsaicin contents ranged from 175.26 to 133.1 mg/L, respectively (Table 6). However, no significant variation was observed for chlorophyll content and days to flowering.

Table 4: Analysis of variance for 11 characters for the 36 Capsicum spp.

Traits
Genotype (DF = 35)
Error (DF = 70)
CV (%)

PLH6WK
200.00*
113.50
24.09

PLHH
478.23**
102.28
14.55

Days to flowering
19.90ns
16.24
6.31

Disease incidence
524.02*
318.52
49.07

Chlorophyll content
5.56ns
5.99
37.23

Photosynthesis rate
37.97**
0.0016
0.305

Fruit length
18.64**
2.73
14.26

Fruit weight
161.95**
3.84
20.45

Number of fruits
6456.66**
1385.09
46.42

Yield
146873**
50594.56
49.59

Membrane stability
1622.85**
112.73
16.43

SHUs
4945258353.7**
7448153.84
7.30

Significant at 5%, highly significant at 1%, ns = not significant, PLHH: plant height at harvest, PLH6WK: plant height at 6 weeks after transplanting, SHUs = Scoville heat units.

Table 5: Means for 10 characters studied in 36 genotypes of Capsicum spp.

Genotype
PH6WK
PLHH
DF
DI (%)
CPL
Photo
FL (cm)
FW (g)
NF
(g/pl)

AVPP9703
45.07
60.60
63.00
31.25
5.64
14.28
10.79
9.08
49.33
338.08

AVPP9901
32.15
47.10
68.50
78.89
6.43
10.92
8.28
6.07
62.00
299.36

AVPP9806
41.25
77.87
60.67
31.59
6.30
15.24
13.41
10.93
47.00
332.37

AVPP9813
56.33
72.07
61.67
30.73
5.75
12.22
10.73
9.43
78.00
554.79

AVPP0002
37.62
60.23
62.83
40.79
6.30
10.92
9.09
3.29
208.33
490.44

AVPP0012
44.80
77.60
67.00
49.10
7.35
15.24
13.16
8.08
20.00
102.03

AVPP0102
56.73
74.23
61.75
39.53
9.65
12.22
12.37
10.30
61.67
574.83

AVPP0004
43.47
77.07
65.00
38.85
4.60
10.92
9.19
6.03
86.67
479.63

AVPP0104
40.85
59.13
60.50
9.11
7.01
15.24
8.72
2.34
150.33
385.10

AVPP0105
39.50
78.57
63.00
40.37
5.91
12.22
12.31
9.67
142.33
813.79

AVPP9905
43.97
68.27
62.67
41.24
5.57
12.74
16.43
22.94
63.00
1144.30

AVPP9815
27.43
50.93
62.33
36.71
7.43
14.58
9.76
6.03
61.67
329.39

AVPP0201
45.23
59.40
60.67
29.80
7.61
11.48
11.71
6.19
78.67
407.36

AVPP0116
60.75
92.13
64.50
28.14
5.60
17.62
10.55
11.65
46.33
411.35

AVPP9805
28.43
45.37
65.00
28.83
7.90
13.56
15.35
14.67
21.00
282.83

AVPP0109
40.07
66.67
63.83
41.86
7.07
16.93
11.29
4.27
130.33
487.72

AVPP0305
55.10
67.30
59.00
30.47
7.92
10.47
12.91
9.73
116.00
711.27

AVPP0101
54.20
65.03
64.50
61.19
7.00
15.71
9.67
8.69
27.33
126.65

AVPP0805
54.78
86.13
63.17
33.18
5.79
14.47
9.92
4.96
95.00
396.67

AVPP0307
49.30
88.67
65.50
26.91
11.93
11.69
14.45
10.71
71.33
659.66

AVPP0506
38.77
75.47
64.50
25.31
5.58
8.85
10.14
4.77
88.00
332.80

AVPP0803
33.70
61.90
64.00
22.82
5.81
16.04
15.45
44.13
8.00
379.74

AVPP0907
41.78
88.87
69.83
42.95
5.86
16.91
6.97
2.47
46.67
263.13

AVPP0306
45.43
71.90
66.00
55.85
6.11
11.13
11.33
9.69
81.67
479.70

AVPP0804
30.60
41.40
67.50
59.85
7.36
13.04
7.83
14.00
6.33
108.69

AVPP0512
49.08
55.07
61.33
41.27
7.29
13.12
12.86
9.66
61.33
270.53

AVPP0514
38.77
67.83
67.00
19.79
5.29
16.96
14.00
12.71
68.67
582.34

AVPP0702
39.13
56.47
63.67
39.14
6.72
10.04
12.66
8.61
73.00
398.99

AVPP0103
44.20
72.43
66.33
42.05
6.54
16.12
8.99
3.54
81.67
249.99

AVPP0014
48.32
68.07
62.33
29.74
6.00
13.09
13.83
10.79
113.00
821.47

AVPP0705
46.40
83.80
63.33
16.69
6.88
3.75
8.02
3.57
151.00
349.93

AVPP0904
55.62
79.97
65.00
38.00
4.54
7.60
12.33
8.13
106.33
643.22

AVPP0513
44.43
71.80
66.00
23.16
5.62
6.02
12.60
9.64
39.33
283.38

AVPP9812
44.50
76.00
62.00
29.05
5.87
13.07
11.57
7.01
100.67
581.27

C05573
42.88
85.53
67.25
35.22
6.04
18.41
10.87
3.51
180.33
436.92

Kulai
51.50
72.17
59.00
39.96
6.44
19.32
17.23
17.68
64.00
818.10

Mean
44.23
69.53
63.89
36.37
6.58
13.12
11.58
9.58
80.18
453.55

LSD ()
17.35
16.47
6.56
29.06
3.99
0.066
2.69
3.19
60.61
366.29

SEM
1.16
1.45
0.45
1.96
0.23
0.020
0.27
0.72
5.53
28.57

PLH6WK: Plant height at 6 weeks; PLHH: plant height at harvest; DF: days to flowering; DI: disease incidence; CPL: chlorophyll content; Photo: photosynthesis rate; FL: fruit length; FW: fruit weight; NF: number of fruits; : yield; LSD: least significance difference; SEM: standard error mean.

Table 6: The capsaicins and dihydrocapsaicin content of the thirty-six chili pepper genotype samples (mg/L).

Genotypes
Capsaicin (mg/L)
Dihydrocapsaicin (mg/L)
Scoville Heat Units
Pungency (SHUs’ scale)

AVPP9703
0.00
0.00
0.00
Non-pungent

AVPP9901
205.16
84.17
36809.30
Highly pungent

AVPP9806
167.81
95.91
32731.96
Highly pungent

AVPP9813
23.28
0.00
3250.48
Moderately pungent

AVPP0002
476.03
405.55
105796.29
Very highly pungent

AVPP0012
97.87
50.52
18564.11
Moderately pungent

AVPP0102
0.00
0.00
0.00
Non-pungent

AVPP0004
13.61
0.00
1899.62
Mildly pungent

AVPP0104
439.32
445.64
104557.81
Very highly pungent

AVPP0105
120.34
78.47
24413.04
Moderately pungent

AVPP9905
175.43
241.83
47945.70
Highly pungent

AVPP9815
25.57
25.98
6090.68
Moderately pungent

AVPP0201
18.04
0.00
2519.57
Mildly pungent

AVPP0116
32.78
21.57
6669.59
Moderately pungent

AVPP9805
129.17
164.79
34015.68
Highly pungent

AVPP0109
57.15
55.10
13323.25
Moderately pungent

AVPP0305
313.05
360.08
78628.18
Highly pungent

AVPP0101
39.65
63.45
11688.78
Moderately pungent

AVPP0805
411.98
299.98
86615.90
Very highly pungent

AVPP0307
0.00
0.00
0.00
Non-pungent

AVPP0506
582.78
355.88
115887.85
Very highly pungent

AVPP0803
0.00
0.00
0.00
Non-pungent

AVPP0907
141.97
69.03
26517.41
Highly pungent

AVPP0306
107.97
84.69
23288.70
Moderately pungent

AVPP0804
18.099
0.00
2527.43
Mildly pungent

AVPP0512
0.00
0.00
0.00
Non-pungent

AVPP0514
216.78
137.03
43558.53
Highly pungent

AVPP0702
102.37
82.00
22245.67
Moderately pungent

AVPP0103
323.04
231.16
67524.10
Highly pungent

AVPP0014
48.28
13.99
8099.05
Moderately pungent

AVPP0705
1277.56
710.02
247245.28
Very highly pungent

AVPP0904
191.15
181.29
44270.75
Highly pungent

AVPP0513
70.21
51.25
15030.29
Moderately pungent

AVPP9812
23.06
12.31
4742.27
Moderately pungent

C05573
376.42
408.03
92127.13
Very highly pungent

Kulai
83.61
61.77
17665.58
Moderately pungent

Mean
175.26
133.10
37395.83

LSD ()
19.67
36.88
5540.40

SEM
7.22
12.31
5828.29

LSD: Least significance difference, SEM: Standard error mean.

3.3. Genotypic Variation for Heat Tolerance and Yield Characters in Chili Pepper

The significant variation among the genotypes for heat tolerance is shown in Figure 1. The figure showed that the genotypes varied with respect to heat tolerance. Most of the genotypes were found to be above the mean. Figure 2 showed variation in the genotypes with respect to yield per plant. Similarly, significant variations were observed for plant height, number of fruits, fruit length, disease incidence, and fruit weight.

Figure 1: Variation among 36 chili pepper genotypes in cell membrane thermostability.

Figure 2: Variation in yield per plant for 36 genotypes of Capsicum spp.

3.4. Heritability and Genetic Advance as Indices for Heat Tolerance and Morpho-Physiological Characters Selection in Chili Pepper

3.4.1. Coefficient of Variation

The extent of variability in respect of phenotypic and genotypic variances and phenotypic and genotypic coefficients of variance (GCV) for CMT and morphophysiological characters is represented in Table 6. The GCV estimate ranged from 1.73 (DF) to 75.78 (FW) over all the characters.

Moreover, high GCV and PCV were observed (>20%) in the traits for cell membrane thermostability, photosynthesis rate, disease incidence, fruit weight, and number of fruits and yield. Moderate GCV were also recorded for plant height and fruit length. The lowest were recorded for days to flowering and fruit length (<10%) (Table 7).

Table 7: Estimate of broad sense heritability, genotypic, and phenotypic coefficients of variance, relative differences between GCV and PCV, and genetic advance for ten traits in 36 Capsicum spp.

Traits
(%)
(%)
(%)
GCV (%)
PCV (%)
RD
GA (%)

PLH6WK
20.25
28.83
142.34
12.14
26.99
55.02
11.24

PLHH
55.06
125.32
227.59
16.1
21.7
25.81
17.11

DF
6.99
1.22
17.46
1.73
6.54
73.55
0.94

DI
17.7
68.50
387.02
22.76
54.09
57.92
19.72

CPL
2.56
−0.15
5.85
5.89
36.56
83.98
1.94

PHOTO
100
12.66
12.66
26.77
26.77
0
55

FL
66
5.30
8.03
19.88
24.47
18.76
33.27

FW
93.21
52.70
56.54
75.78
78.49
28.29
150.71

NF
54.96
1690.50
3075.60
51.28
69.17
25.86
78.31

38.81
32092.80
82687.40
39.5
63.4
37.7
50.69

CMT
81.7
503.37
616.1
34.72
38.41
5.71
64.64

PLH6WK: plant height at 6 weeks; PLHH: plant height at harvest; DF: days to flowering; DI: disease incidence; CPL: chlorophyll content, PHOTO: photosynthesis rate; FL: fruit length; FW: fruit weight; NF: number of fruits; CMT: cell membrane thermostability; : heritability; : genotypic variance; : phenotypic variance; : yield; PCV: phenotypic coefficients of variance; GCV: genotypic coefficients of variance; RD: relative difference between GCV and PCV; GA: genetic advance.

3.4.2. Heritability and Genetic Advance

Broad sense heritability values for the ten traits are presented in Table 7. Traits such as membrane thermostability, photosynthesis rate, fruit weight, and fruit length showed a relatively high heritability values (>60%). The values estimated for plant height at harvest, number of fruits and yield per plant were moderate (30–60%). Fruit length, fruit weight, number of fruits, yield, and cell membrane thermostability exhibited the highest predicted genetic advance as compared to the other traits. High heritability (>60%) together with high genetic advance (>20%) was observed for CMT, photosynthesis rate, fruit weight, and fruit length traits. However, moderate heritability with high genetic advance was observed for number of fruits and yield per plant (Table 7).

3.4.3. Correlation Coefficient

Simple phenotypic correlation coefficients for the traits studied using SAS software (version 9.1) are shown in Table 8. Yield per plant showed strong to moderately positive correlations () at phenotypic level while at genotypic level correlation coefficient ranged from (0.16 to 0.72) for plant height at 6WAT and at harvest, days to flowering, fruit length, and number of fruits. However, negative correlation was observed for disease incidence with yield per plant. Similarly, cell membrane thermostability was found to correlate negatively with disease incidence. Positive phenotypic correlation between CMT and plant height and genotypic correlation between CMT and yield were also observed. Other correlation coefficients between pairs of traits that are of some interest to the breeders are shown in Table 8.

Table 8: Coefficient of phenotypic and genotypic (bold) correlations among the investigated traits in 36 Capsicum spp.

PLHH
DF
DI
Photo
CPL
FL
FW
NF

CMT

PLHH
1
0.70 **
−0.49 **
0.01 **
NA
−0.03 **
−0.21 **
0.22ns
0.19 **
0.39ns

DF
−0.11ns
1
1 **
1 **
NA
NA
−1 **
−0.23 *
−0.67 **
0.04ns

DI
−0.21*
0.28**
1
0.23 **
NA
−0.41 **
−0.19 **
−0.22 **
−0.21 **
−0.44 **

Photo
0.01ns
0.04ns
0.08ns
1
NA
1 **
NA
−0.02 **
NA
−0.25 **

CPL
−0.04ns
−0.04ns
0.03ns
−0.04ns
1
0.09 **
0.12 **
NA
NA
NA

FL
0.03ns
−0.27**
−0.18ns
0.08ns
0.08ns
1
0.67ns
−0.36 **
0.72 **
0.26 **

FW
−0.14ns
−0.08ns
−0.04ns
0.12ns
−0.04ns
0.60**
1
−0.59 **
0.31 **
0.20 **

NF
0.27**
−0.27**
−0.26**
−0.06ns
−0.07ns
−0.13ns
−0.40**
1
0.16 **
0.07ns

0.29**
−0.38**
−0.23**
−0.07ns
−0.07ns
0.39**
0.18ns
0.56**
1
0.35 **

CMT
0.20*
0.06ns
−0.24**
−0.22**
−0.16ns
0.13ns
0.17ns
−0.02ns
0.14ns
1

Significant at 5%, highly significant at 1%, PLHH: plant height at harvest; DF: days to flowering; DI: disease incidence; CPL: chlorophyll content; FL: fruit length; FW: fruit weight; NF: number of fruits; CMT: cell membrane thermostability; Photo: Photosynthesis rate; ns: not significant; NA: not available; : yield.

3.5. Genetic Distance-Based Analysis for Cell Membrane Thermostability and Yield

3.5.1. Cluster Analysis

The standardized data was employed to calculate the Euclidean distances among the 36 chili pepper genotypes and an UPGMA dendrogram was constructed (Figure 3). The dendrograms of the 36 chili pepper genotypes were grouped into 8 major groups based on the cell membrane thermostability and yield traits at 1.26 dissimilarity coefficients. The cutoff at this point was for the convenience of discussion. Group IV recorded the highest number with 13 genotypes followed by groups III and VI with 7 genotypes each. Group I and V recorded 4 and 2 genotypes, respectively, while groups II, VII, and VIII had 1 genotype each. Group VIII gave the highest CMT value and recorded the highest yield, while Group II gave the lowest yield and Group I gave the lowest CMT value (Table 9).

Table 9: Mean values of cell membrane thermostability and yield per plant characters for 8 groups revealed by cluster analysis on 36 genotypes of Capsicum spp.

Clusters
Genotypes
Yield
CMT

Group I
AVPP9703, AVPP9901, AVPP0002, AVPP0102
425.68
17.42

Group II
AVPP0012
102.03
24.23

Group III
AVPP9806, AVPP9815, AVPP0104, AVPP9813, AVPP0004, AVPP0109, AVPP0306
435.53
52.75

Group IV
AVPP0201, AVPP0805, AVPP0803, C05573, AVPP0116, AVPP0702, AVPP0506, AVPP0513, AVPP0512, AVPP0103, AVPP9805, AVPP0907, AVPP0705
343.35
80.01

Group V
AVPP0101, AVPP0804
117.67
63.53

Group VI
AVPP0105, AVPP0014, AVPP0305, AVPP0904, AVPP9812, AVPP0307, AVPP0514
687.57
80.14

Group VII
Kulai
818.10
49.87

Group VIII
AVPP9905
1144.30
85.10

CMT: Cell membrane thermostability.

Figure 3: Relationship among the 36 chilli pepper genotypes based on cell membrane thermostability and yield characters using SAHN clustering on UPGMA method.

3.5.2. Principal Component Analysis

Principal component analysis was carried out on the cell membrane thermostability and yield per plant characters on the 36 genotypes. The two-dimensional graphical illustration (Figure 4) showed that most of the genotypes were dispersed at close distance while few were dispersed at great distances as reflected by the Eigen vector. The farthest genotypes from the centroid were AVPP9905, Kulai, AVPP0102, AVPP0002, AVPP9901, AVPP9703, AVPP0012, AVPP0804, and AVPP0101, while other genotypes are more or less close to the centroid. The variation percentages of the PC1 and PC2 are 62.1 and 37.9%, respectively, with PC1 showing the highest of the total variation.

Figure 4: Two-dimensional graph principal component analysis showing relationship among the genotypes for cell membrane thermostability and yield.

4. Discussion

4.1. Cell Membrane Thermostability (CMT)

The membrane is the first line of defense with many heat-responsive sensors that help plants to activate defense mechanisms early in heat shock. The mean value of CMT from this investigation was 64.62% indicating in most of the genotypes that membrane integrity was not damaged by the high temperature treatment of 50°C for 20 min. This shows that most of the genotypes assessed are tolerant to heat having relative injury of <40%. Increased electrolyte leakage indicates decreased cell membrane thermostability (CMT), which has long been used as an indirect mechanism of heat-stress tolerance in several crop plant species, including tomato, potato [30], and wheat [31]. Therefore membrane leakage can be used as a means of screening vegetative plants for heat tolerance in chili pepper. This is in conformity with the works of [19] that measured CMT in 12 ornamental pepper genotypes and found most of the genotypes to be thermotolerant with a mean of 59.50%. Membrane thermostability was also measured by [4] in diverse groundnut genotypes and observed heat tolerant genotypes has low percentage of membrane leakage, with high CMT. He further reported that genotypes of diverse origin indicate different sources of tolerance to heat.

4.2. Morphophysiological Characters

The investigation revealed considerable amount of variation for the characters studied. Such wide variation indicated the scope for improving the genotypes studied for these characters with respect to heat tolerance and morphophysiological characters. High performance chili pepper genotypes, namely, AVPP9905, AVPP0105, and Kulai, in terms of growth and yield were observed in this investigation. Photosynthesis rate is the rate at which the plant can photosynthesize its own food. It measures how efficient the plant is in carbon assimilation. The variation observed in this investigation among the 36 genotypes gave room for possible selection and improvement. Similar results were observed by [32, 33] who reported significant variation in growth and yield characters of pepper genotypes. On the contrary, [34] reported significant variation among the genotypes studied for days to 50% flowering. This might be attributed to the differences in the genetic sources of the genotypes studied. In the case of degree of pungency, genotype AVPP0705 was found to record the highest capsaicin content and was the highest in pungency which was significantly () higher than all the genotypes tested. Genotypes AVPP9703, AVPP0512, AVPP0307, AVPP0803, and AVPP0102 were found to record no capsaicin and therefore be non-pungent. Similar variation in capsaicin content of different peppers has been previously reported [35–37]. In addition, capsaicin and dihydrocapsaicin have the same trend in contents of the capsaicinoids, and in particular capsaicin was found in higher contents than dihydrocapsaicin in all genotypes studied except C05573, AVPP9905, and AVPP9805.

4.3. Genetic Variation for Heat Tolerance and Yield Traits in Chili Pepper

The most tolerant genotypes seem to have high membrane stability (CMT > 60%) at high temperature of 50°C. Cell membrane thermostability therefore showed some potential for screening for heat tolerance in chili pepper. This is in line with the works of [38] who found large plant-to-plant variation in cell membrane thermostability measurements in Chrysanthemum cultivars. Most of the genotypes studied by [4] showed that membrane integrity was damaged by the high temperature treatment of 54°C for 15 min. On the contrary, [39] found no significant variation of CMT for ten lines of cabbage (Brassica oleracea L.). Heat tolerant genotypes having greater CMT at high temperature are said to have higher optimum and maximum temperatures for growth and development processes. This also indicates the effectiveness of cell membrane thermostability parameter as rapid and sensitive in determining heat tolerance among chili pepper genotypes. Similarly, variations were observed among the genotypes for yield indicating differences in their yield performance under the same condition. This might be due to the differences in their genetic make-up. This investigation is in harmony with the work of [32].

4.4. Heritability and Genetic Advance as Indices for Heat Tolerance and Morphophysiological Characters Selection in Chili Pepper

4.4.1. Coefficient of Variation

The extent of genetic variation in heat tolerance as well as other morphophysiological characters is better judged by the estimation of the genotypic coefficient of variation (GCV) in relation to its phenotypic coefficient of variation (PCV). A small difference between GCV and PCV was observed in plant height at harvest, fruit length, fruit weight, number of fruits, and heat tolerance characters indicating that variations among the genotypes were mostly due to genetic factors. This indicates a high significant effect of genotype on phenotypic expression with very little effect of environment. On the other hand, large differences between GCV and PCV were observed for the characters days to flowering, disease incidence, chlorophyll content, and yield per plant. This indicates the influence of the environment over these characters. High GCV observed for some traits such as CMT and yield per plant in this investigation indicates their high variability and that further selection could be used to improve the genotypes for the traits, while low GCV indicates limited improvement for the traits through selection. This is similar to the works of [40].

4.4.2. Heritability and Genetic Advance

Effective selection can be achieved only when additive effects are sufficiently higher than the environmental effect. Report from [29] showed that effectiveness of selection depends not only on heritability but also on genetic advance. High GCV together with high heritability and genetic advance provide more information than other parameters alone [41]. High heritability together with high genetic advance observed in this investigation indicates that these traits such as CMT are mainly controlled by additive type of genes. Therefore, selection may be effective for improving these traits in chili pepper. Works of [42] observed high heritability with genetic advance in several characters in chili. This investigation is in agreement with the work of [6] who observed high genetic advance accompanied with high heritability in drought tolerance in sorghum. In this study, cell membrane thermostability, fruit length, and fruit weight are found to be important characters to be taken into consideration for effective selection in pepper. Thus, selection in this population of chili pepper would prove successful once the fixed genetic component is freed from environmental influence.

4.4.3. Correlation between Traits

Relationship existing between cell membrane thermostability and disease incidence indicating the tendency of heat tolerant genotypes possesses disease resistant traits as compared to heat sensitive genotypes. As revealed from this investigation heat tolerant genotypes may be disease resistant genotypes. The likeliness of negative correlation between cell membrane thermostability and disease score as found in this study has also been reported by [43]. On the other hand, no significant association between cell membrane thermostability (heat tolerant trait) and yield per plant was observed.

4.5. Genetic Distance-Based Analysis for Cell Membrane Thermostability and Yield

Genetic divergence is one of the criteria of parent selection. Knowledge of genetic diversity among plant populations and its quantitative assessment usually helps a breeder in choosing desirable parents for breeding program as selection of parents on the basis of divergence analysis would be more effective [44].

4.5.1. Clustering Analysis

The 36 genotypes of Capsicum spp grouped into 8 clusters based on cell membrane thermostability and yield traits at distant coefficient of 1.26 indicates a level of diversity among the genotypes. Group VIII, which has only one genotype (AVPP9905), recorded the highest yield and CMT values and this proved that this genotype might have dissimilar genes as compared to the other genotypes for controlling these traits. To improve the heat tolerance of Kulai (an adaptable and susceptible genotype) that recorded the second highest yield it needs to be crossed with AVPP9905. Similarly, AVPP9905 can be crossed with AVPP0012 for high yield and heat tolerance. Similar results were obtained by [45] who studied significant variation in terms of morphological traits in distribution of chili pepper. Genetic diversity among 45 chili pepper genotypes studied by [46] observed six clusters and [16] reported eight distinct grouped in 50 accessions of chili.

4.5.2. Principal Component Analysis

The principal component analysis (PCA) yielded eigenvalues of each principal component axis of ordination of genotypes with the first axis totally accounting for the variation among the genotypes. The result of the PCA further justifies the clustering analysis. Genetically similar genotypes were grouped together. According to [7], genetically distant parents are able to exert high heterosis. Considering the variation and diversity analysis of the genotypes genotype AVPP9905 having high yield and more tolerant to heat (high CMT) from group VIII, 13 genotypes from group IV with high CMT but low yield and 7 genotypes from group VI with high CMT and Kulai with high yield and low CMT from cluster VII were found promising. Therefore, for hybridization program crosses among these genotypes for heat tolerance and yield could be effective. Similar results were observed by [46, 47].

5. Conclusion

Cell membrane thermostability is a useful parameter for thermotolerance in chili pepper. The present data indicated that most of the genotypes studied are consistently tolerant to high temperature measured based on membrane thermostability phenotyping parameter. Furthermore, these genotypes are from diverse origin indicating different sources of heat tolerance. However, the genetic parameters discussed above are function of environmental variability, so estimates may differ in other environments. High and moderate heritability and high genetic advance shown by the different characters, namely, plant height at harvest, fruit length, fruit weight and number of fruits, yield, and cell membrane thermostability determine the genetic effects of the phenotypic expression of these characters that they are fundamentally from the additive type. The prevalence of genetic variance for heat tolerance and morphophysiological traits studied can be exploited through selection as the estimate of high broad sense heritability and genetic advance allows doing so.

Considering the group distance, agronomic performance, and variability the crosses between AVPP9905 and Kulai; Kulai and AVPP0105, AVPP0014, AVPP0305, AVPP0904, AVPP9812, AVPP0307, AVPP0514; Kulai and AVPP0201, AVPP0805, AVPP0803, C05573, AVPP0116, AVPP0702, AVPP0506, AVPP0513, AVPP0512, AVPP0103, AVPP9805, AVPP0907, and AVPP0705 could be suggested for future hybridization program for heat tolerance and high yield.

Conflict of Interests

The authors declare no conflict of interests.

Authors’ Contribution

All authors contributed equally to this work.

Acknowledgments

The authors are grateful to Ministry of Education, Malaysia, for adequate funding of the research through the Fundamental Research Grant Scheme (FRGS/1/2012/STWN03/UPM/02/2:07-01-13-1240FR). The authors are grateful to Asian Vegetable Research and Development Centre, Taiwan, for the supply of seeds. Gratitude also goes to Yussuf Oladosu Adeniyi for his constructive criticism.

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