Analysis of variance (ANOVA) for parents and backcross populations
The ANOVA for experimental design for 19 plant growth, yield, seed and biochemical traits is presented in Table 1 and 2. The ANOVA showed greater significant mean squares (MS) due to parents for all the studied 19 traits, it indicating that the presence of sufficient amount of inherent variability among the parents which could be exploited via selection. The MS due to developed BC2F1 populations were non-significant for plant spread, 1000 seed fresh weight and chlorophyll-a content, and significant for rest of the traits studied, which depicted the differential response of different BC2F1 population to studied traits. This is in agreement with the finding of Usman et al. (2014).
Recovery of recurrent parent traits in BC2F1 populations
To validate the successful revival of the recurrent parent genome (RPG) in the BC2F1 populations, the horticultural and morphological performances of the improved lines carrying the male sterility (ms10) gene together with the adaptable recurrent parent were compared by paired ‘t’ test analysis. The results are furnished in the Table 3-7.
Evaluation of BC2F1 population for plant growth traits
Plant height is considered as an important yield attributing trait, because it leads to more number of branches and prolonged harvest duration, ultimately results in higher productivity. The taller plants have less disease incidence than the dwarf ones (Sood and Kumar 2013). The mean performance of six recipient parental lines for plant height ranged from 67.67 cm (PAU SM-21) to 103.70 cm (PAU SM-17). On the other hand, plant height of the BC2F1 population, varied from 55.25 cm to 106.20 cm. The generation MS-12/PAU SM-17//PAU SM-17 (106.20 cm) showed maximum plant height followed by MS-12/PAU SM-3//PAU SM-3 (86.51 cm), and the minimum was recorded in MS-12/PAU SM-21//PAU SM-21 (55.25 cm) and MS-12/PAU SM-9//PAU SM-9 (63.47 cm). Plant spread for six parental lines ranged from 44.71 cm to 58.01 cm, and the maximum plant spread was recorded for PAU SM-1 followed by PAU SM-3, while, the minimum was observed for PAU SM-9 followed by PAU SM-17. The BC2F1 population means for plant spread varied from 48.94 cm in MS-12/PAU SM-9//PAU SM-9 to 61.93 cm in MS-12/PAU SM-3//PAU SM-3. Number of primary branches plant-1 is an essential yield contributing trait, having a direct effect on yield. The plant having more number of branches results increased productivity. Number of primary branches plant-1 of parental lines ranged from 3.04 to 4.53. The maximum number of primary branches plant-1 was recorded by PAU SM-3 followed by PAU SM-1, while, the minimum was observed in PAU SM-9 and PAU SM-21. Among the six developed BC2F1 populations, the maximum number of primary branches plant-1 was recorded by MS-12/PAU SM-3//PAU SM-3 (5.21) followed by MS-12/PAU SM-2//PAU SM-2 (4.72) and MS-12/PAU SM-1//PAU SM-1 (4.39), whereas, the minimum was recorded by MS-12/PAU SM-21//PAU SM-21 (3.24), MS-12/PAU SM-9//PAU SM-9 (3.73) and MS-12/PAU SM-17//PAU SM-17 (3.97). From the six BC2F1 populations, the paired t test was non-significant for mean values of five developed populations and their recurrent parent for plant growth traits except MS-12/PAU SM-9//PAU SM-9 for plant height and MS-12/PAU SM-17//PAU SM-17 for plant spread and number of primary branches plant-1 which showed significant differences with their respective recurrent parent.
Evaluation of BC2F1 population for fruit and yield traits
Development of high yielding cultivars along-with good fruit quality is important to enhance the crop productivity per unit area as well as the farmers’ income. Therefore, the total fruit yield and fruit traits are the most important economic characters concerned by most breeders. Fruit weight is one of the most important traits which have a direct effect on fruit yield plant-1. It is vital to clarify that the average weight of fruit can be altered according to the fruit yield plant-1. Under Indian marketing system, medium sized fruits (50-80 g) are generally preferred. The per se performance for fruit weight among the developed BC2F1 populations ranged from 33.33 g (MS-12/PAU SM-9//PAU SM-9) to 71.83 g (MS-12/PAU SM-3//PAU SM-3) in comparison to 43.98 g (PAU SM-21) to 84.88 g (PAU SM-3) of parental lines. For fruit length, the lowest and the highest mean values were recorded by the backcross generation MS-12/ PAU SM-9// PAU SM-9 and MS-12/PAU SM-3//PAU SM-3 in BC2F1 populations, while by PAU SM-21 and PAU SM-3 in parental lines. Fruit length of backcrossed populations ranged from 5.66 cm to 7.50 cm, on the contrary, fruit length of recurrent parents to be 4.82 cm to 7.38 cm. Per se performance for fruit width of parental lines ranged from 4.56 cm to 7.34 cm, on the other hand, of the BC2F1 population varied from 3.58 cm to 6.74 cm. Among the recurrent parents, the maximum fruit width was recorded by PAU SM-3 followed by PAU SM-1, while, the minimum fruit width was observed by PAU SM-21 followed by PAU SM-17. In the backcrossed populations, the maximum fruit width was recorded by MS-12/PAU SM-3//PAU SM-3 and MS-12/PAU SM-2//PAU SM-2, while, the minimum fruit width was recorded by MS-12/PAU SM-17//PAU SM-17 and MS-12/PAU SM-21//PAU SM-21. Pericarp thickness is a very important character which decides the firmness of fruit. Thick fleshed bell pepper can endure long shipping distance. Pericarp thickness of backcrossed population ranged from 3.14 mm in MS-12/PAU SM-17//PAU SM-17 to 3.93 mm in MS-12/PAU SM-3//PAU SM-3 whereas pericarp thickness of recurrent parents was varied from 3.29 mm in PAU SM-17 to 4.83 mm in PAU SM-3.
Number of lobes fruit-1 affects the shape of fruit in bell pepper. Generally, a medium sized, blocky and four lobed fruit are acceptable in the market. The backcross populations means for number of lobes fruit-1 ranged from 1.83 to 3.17, and that of recurrent parents varied from 2.90 to 3.50. Based on per se performance, the population namely MS-12/PAU SM-2//PAU SM-2, MS-12/PAU SM-1//PAU SM-1 and MS-12/PAU SM-3//PAU SM-3, and the parental lines namely PAU SM-3, PAU SM-2 and PAU SM-1 were identified with higher number of lobes fruit-1. Number of fruits plant-1 is the most important component trait, which is directly related with increased fruit yield plant-1. Number of fruit plant-1 of backcrossed populations varied from 28.59 to 39.18, which was higher than their recurrent parents i.e., 25.14 to 33.02. Among the parental lines, the maximum number of fruit plant-1 was recorded by PAU SM-17 followed by PAU SM-3 and PAU SM-21, while, the minimum number of fruit plant-1 was observed by PAU SM-2 followed by PAU SM-1. Among the backcrossed populations, the maximum number of fruit plant-1 was recorded by MS-12/PAU SM-9//PAU SM-9 followed by MS-12/PAU SM-3//PAU SM-3, while, the minimum was observed by MS-12/PAU SM-21//PAU SM-21 followed by MS-12/PAU SM-17//PAU SM-17. The main focus of cultivating a crop is to have the maximum yield per unit area for better returns. Moreover, high fruit yield is the ultimate goal of any breeding program; hence, it requires the highest consideration. It is the key factor in adaptation of variety by farmers. In the parental lines, the mean values of total fruit yield plant-1 varied from 1109.50 g to 2028.40 g. The maximum total fruit yield plant-1 was recorded by PAU SM-3 followed by PAU SM-1, while, the minimum was depicted by PAU SM-9 followed by PAU SM-21. On the contrary, total fruit yield plant-1 of the BC2F1 population was ranged from 1024.32 g to 2060.27 g. The maximum total fruit yield plant-1 was recorded by MS-12/PAU SM-1//PAU SM-1 followed by MS-12/PAU SM-3//PAU SM-3, while, the minimum was recorded by MS-12/PAU SM-17//PAU SM-17 followed by MS-12/PAU SM-9//PAU SM-9.
For studied fruit and yield traits all the developed populations depicted non-significant differences with their respective recurrent parents based on paired t-test analysis, except MS-12/PAU SM-21//PAU SM-21 for fruit length and number of lobes fruit-1, and MS-12/PAU SM-9//PAU SM-9 for number of lobes fruit-1 revealed significant differences with their recurrent parent. This suggested that fruit traits of individuals of the developed BC2F1 populations were in the range of the recurrent parent value. It indicated that the complete phenome of recurrent parent has been transfer or recovered in respective backcrossed population or genetic background of the backcrossed populations and their respective recurrent parent became identical for these traits. Into the genetic background of widely cultivated Malaysian variety of rice (MR219), Miah et al. (2015) successfully introgressed the blast resistant Pi gene (putative Piz) and newly developed improved line depicted similar phenotypic traits when compared with recurrent parent MR219, which proves it was similar to the MR219 genetic backgrounds.
To the significant t test populations, continued backcross with recurrent parent will be performed up-to the degree of similarity of the backcrossing progeny is greatly close to the recurrent parent. The final last backcross population will be self-pollinated to develop new GMS lines of bell pepper, which is almost isogenic to their recurrent parent in the future.
Evaluation of BC2F1 population for seed traits
Apart from the male sterility, GMS lines should be possessed the normal female fertility and higher number of seed fruit-1 (Meena et al. 2018). Number of seed fruit-1 ranged from 43.15 to 69.28 in parental lines. The maximum number of seed fruit-1 was recorded by PAU SM-3 (69.28) followed by PAU SM-21 (67.11), whereas, the minimum was recorded by PAU SM-17 (43.15) followed by PAU SM-1 (59.41). Among the BC2F1 populations, number of seed fruit-1 ranged from 40.93 to 60.47. The backcross population, MS-12/PAU SM-3//PAU SM-3 (60.47) showed maximum number of seed fruit-1 followed by MS-12/PAU SM-1//PAU SM-1 (58.57), and the minimum was recorded by MS-12/PAU SM-17//PAU SM-17 (40.93) and MS-12/PAU SM-2//PAU SM-2 (48.87). Test weight of fresh seed among the developed BC2F1 populations ranged from 8.00 g (MS-12/PAU SM-9//PAU SM-9) to 12.31 g (MS-12/PAU SM-3//PAU SM-3) in comparison to 9.20 g (PAU SM-9) to 13.48 g (PAU SM-3) in parental lines. Therefore, the performance of tested populations was statistically on par with that recorded for recurrent parents. Based on paired t-test analysis, it was observed that the developed breeding backcross populations had similar test (1000) weight of dry seed that of recurrent parent. Mean performance of 1000 seed dry weight for parental lines ranged from 5.94 g to 9.35 g. The maximum weight were recorded by PAU SM-2 followed by PAU SM-21 and PAU SM-3, whereas, weight were recorded by PAU SM-17 followed by PAU SM-9. Among the BC2F1 populations, it ranged from 6.74 to 8.18. The progeny MS-12/PAU SM-2//PAU SM-2 showed maximum weight followed by MS-12/PAU SM-17//PAU SM-17, and the minimum was recorded by MS-12/PAU SM-9//PAU SM-9 followed by MS-12/PAU SM-1//PAU SM-1. For the studied seed traits, the mean values of developed backcrossed population possessing ms10 gene was depicted statistically non-significant differences to their recurrent parents, on the basis of paired t-test analysis, it indicated that with the male sterility traits the developed progenies have good seed setting ability as their parental lines of bell pepper.
Evaluation of BC2F1 population for quality traits
Higher content of dry matter means higher processed product yield. Therefore, fruit dry matter is very important trait for breeding pepper for processing industry, since a large proportion of the pepper produced in the world is used in the form of powder (Lannes et al. 2007). The parental lines mean for dry matter content ranged from 5.55 to 8.87%. The parent line PAU SM-3 (8.87%) expressed the highest mean value followed by PAU SM-21 (7.78%), while, the minimum by PAU SM-17 (5.55%) and PAU SM-2 (6.68%). Per se performance of BC2F1 population for dry matter content ranged from 6.99 to 10.91%. In BC2F1 populations, the progeny MS-12/PAU SM-3//PAU SM-3 (9.56%) had maximum dry matter content followed by MS-12/PAU SM-1//PAU SM-1 (8.91%), and the minimum was recorded by MS-12/PAU SM-17//PAU SM-17 and MS-12/PAU SM-2//PAU SM-2. Ascorbic acid is the major component of the nutritional quality in bell pepper. Ascorbic acid content (mg.100g-1) for all the six developed backcross populations ranged from 80.56 mg.100g-1 to 115.01 mg.100g-1, that of the recurrent parents varied from 61.53 mg.100g-1 to 104.73 mg.100g-1. Among the parental lines, the maximum ascorbic acid content was observed by PAU SM-9 and PAU SM-3, whereas, the minimum was recorded by PAU SM-17 and PAU SM-2. In BC2F1 populations, the progeny MS-12/PAU SM-9//PAU SM-9 showed maximum ascorbic acid content followed by MS-12/PAU SM-3//PAU SM-3, and the minimum was recorded by MS-12/PAU SM-17//PAU SM-17 followed by MS-12/PAU SM-2//PAU SM-2. For the traits, dry matter and ascorbic acid, all the developed populations were highly similar to their recurrent parent, confirmed by non-significant values of the paired t-test analysis; this suggested that the developed populations had maximum recurrent parent phenome recovery for mentioned traits.
The backcrossed populations mean for capsaicin content ranged from 0.25 to 0.41%, on the other hand, the parental lines mean varied from 0.18 to 0.30%. The progeny MS-12/PAU SM-9//PAU SM-9 depicted the highest mean value for capsaicin content followed by MS-12/PAU SM-17//PAU SM-17, while, the lowest capsaicin content was recorded by MS-12/PAU SM-2//PAU SM-2 and MS-12/PAU SM-3//PAU SM-3. This variation for pungency and dry matter can be used in Capsicum breeding programs for the production of spice market oriented cultivars. Therefore, the developed GMS lines of bell pepper from this project could be utilized as maternal parent in hybrid breeding program to identify best cross-combination(s) with non-pungent high powder yield for industrial purposes. Among the parental lines, the maximum capsaicin content was recorded by PAU SM-21 followed by PAU SM-9, although, the minimum was observed by PAU SM-1 followed by PAU SM-3. Out of six BC2F1 populations, four populations showed non-significance difference with their respective recurrent parents which signified that developed populations had maximum recurrent parent genome recovery. Two populations namely MS-12/PAU SM-9//PAU SM-9 and MS-12/PAU SM-21//PAU SM-21 expressed significant differences with their recipient parent for capsaicin content.
The maximum mean values for chlorophyll ‘a’ (mg.100g-1) was expressed by PAU SM-21 followed by PAU SM-9, though, the minimum by PAU SM-17 followed by PAU SM-3, among the parental lines. On the other hand, among the BC2F1 population, the progeny MS-12/PAU SM-2//PAU SM-2 exhibited the highest mean values followed by MS-12/PAU SM-1//PAU SM-1, while, the minimum was recorded by MS-12/PAU SM-9//PAU SM-9 and MS-12/PAU SM-21//PAU SM-21. Chlorophyll ‘a’ of BC2F1 populations ranged from 0.17 mg to 0.29 mg.100g-1 whereas chlorophyll ‘a’ of recurrent parent was varied from 0.16 mg to 0.37 mg.100g-1. Among the parental lines, chlorophyll ‘b’ (mg.100g-1) content was ranged from 0.11 to 0.32 mg.100g-1. The maximum chlorophyll ‘b’ was recorded by PAU SM-2 followed by PAU SM-1, however, the minimum chlorophyll ‘b’ was observed in PAU SM-21 followed by PAU SM-17. The mean values of chlorophyll ‘b’ for BC2F1 populations ranged from 0.10 to 0.30 mg.100g-1. In the BC2F1 populations, the progeny MS-12/PAU SM-2//PAU SM-2 showed maximum chlorophyll ‘b’ followed by MS-12/PAU SM-1//PAU SM-1, and minimum was recorded by MS-12/PAU SM-17//PAU SM-17 followed by MS-12/PAU SM-21//PAU SM-21. For this trait, one population namely MS-12/PAU SM-9//PAU SM-9 revealed significant differences to the recurrent parent, while rest of the populations showed non-significance differences with their recurrent parents. The paired t-test analysis demonstrated similar mean values of total chlorophyll content (mg.100g-1) between the developed backcrossed population and their parental lines except for MS-12/PAU SM-9//PAU SM-9 and MS-12/PAU SM-21//PAU SM-21, this suggested that total chlorophyll content of individuals of four BC2F1 populations was in the range of chlorophyll content of recurrent parent, and it depicted that developed populations had maximum RPG recovery. For total chlorophyll content (mg.100g-1), the parental means varied from 0.15 to 0.32, on the contrary, the backcross population means ranged from 0.14 to 0.29 mg.100g-1. Among the six BC2F1 populations, MS-12/PAU SM-2//PAU SM-2 registered the highest per se performance followed by MS-12/PAU SM-1//PAU SM-1, while, the lowest was observed by MS-12/PAU SM-21//PAU SM-21 and MS-12/PAU SM-17//PAU SM-17.
Evaluation of BC2F1 populations for morphological traits
The color of the fruit determines the marketable acceptability of bell pepper. Generally uniform, non-blotchy fruits are desirable in the market. The green color of the fruit is principally due to the presence of chlorophyll which includes chlorophyll ‘a’, chlorophyll ‘b’ and total chlorophyll. It is evident from the Table 8, all developed BC2F1 populations had green fruit color as similar as parents. This indicated that all the populations had utmost recovered recurrent parent phenome. All the parental lines showed pendent fruit orientation except PAU SM-9 which showed erect fruit orientation. Likewise, all BC2F1 populations had pendent fruit orientation as their respective parents except MS-12/PAU SM-9//PAU SM-9 which had erect fruiting as that of recurrent parent. Fruit shape of bell pepper is a vital trait which determines the acceptability in the market. The standard shape for bell pepper is blocky, which is mostly preferred by the consumers. The fruit shape of all BC2F1 populations was blocky which is quite similar to their recurrent parents.
Assessment of fruit traits across the generations
The morphological traits of the donor parent (DP), hot pepper line namely MS-12, recurrent parent (RP), bell pepper lines namely PAU SM-1, PAU SM-2, PAU SM-3, PAU SM-9, PAU SM-17 and PAU SM-21, their F1 hybrids, BC1F1 and BC2F1 generations were evaluated to know the trend of recovery of recurrent parent phenome (RPP). The mean trait values of recurrent parents, reference F1, BC1F1 and BC2F1 populations were examined by LSD 5% and presented in Figure 1 (a-h). In PAU SM-1 generation, for number of lobes fruit-1, the perusal of mean values of donor parent, recurrent parent, F1 cross, BC1F1 and BC2F1 were 0, 3.2, 0, 1.14 and 3.16, respectively. Assessment of RPP content among BC1F1 and BC2F1 population and recurrent parent showed that average RPP content in BC1F1 was 35.63 and in BC2F1 was 98.75%. In PAU SM-3 generation, for fruit width (cm) the per se performance of F1 hybrid was 2.53 cm, BC1F1 and BC2F1 was 4.10 cm and 6.74 cm, respectively. Evaluation of RPP content between BC1F1, BC2F1 population and recurrent parent depicted that average RPP content was 55.86 and 91.83%. In PAU SM-17 generation, for pericarp thickness (mm), F1 cross performance was 1.57 mm while, presentation of BC1F1 and BC2F1 were 2.06 mm and 3.14 mm, respectively. The average RPP content was 62.61 and 95.44% in BC1F1 and BC2F1, respectively. In case of PAU SM-21 generation, the BC2F1 (1044.46 g) progeny was gave close resemblance to the recurrent parent (1117.18 g) in contrast donor parent (445.50 g) for total fruit yield plant-1. In PAU SM-2 generation, the findings revealed that total fruit yield plant-1 of F1 hybrid was 1573.83 g, whereas BC1F1 and BC2F1 progeny was 1731.73 g and 1871.28 g. In other studies conducted by different research group, the RPG recovery was found up to 91.6% in BC2F1 generation for pyramiding blast resistance genes Piz5 and Pi54 into an elite Basmati rice restorer line ‘PRR78’ using MAS (Singh et al. 2013). In BC2F1 populations, the genetic background recovery was observed 95.0% in case of Swarna and Samba Mashuri (Prigge et al. 2008). Our findings were similar to the findings of Ahmed et al. (2016), who achieved a recovery of 65.55 to 77.8 and 78.79 to 95.5 % in BC1F1 and BC2F1 populations, respectively. In BC5F1 generation, RPG recovery from 96.6% to 98.8% was recorded among CMS-A lines by Meena et al. (2019), which is similar to present findings.