Production and characterization of inter and intraspecific hybridization eggplant

The eggplant is a highly valuable horticultural crop grown all over the world and it is of substantial economic importance in Asia. However, its production is severely threatened by several soil-borne and foliar diseases, insect-pests, drought, heat, and frost damage. Therefore, efforts to transfer useful resistance genes into eggplant from their wild relatives is important. In the present study, interspecific and intraspecific hybridization was carried out, that included three cultivated genotypes of eggplant ( Solanum melongena MEE, Solanum melongena MEP , Solanum melongena MEB) and one wild Solanum species ( Solanum incanum INC). The F 1 hybrids were made by inter and intraspecific hybridization. A total of 632 possible inter and intraspecific reciprocal crosses was performed where only three were successful. The minimum days to flowering were observed in parent MEP, and maximum plant height was measured in MEE×MEB. Maximum fruit length was observed in parent MEB. Furthermore, fruit diameter, leaf width, leaf length, and fruit yield per plant were found maximum in hybrid MEExINC. Our results suggest that these materials will be of great interest for the genetic improvement of eggplant; they may have a tremendous potential to increase tolerance to abiotic stresses, such as to drought and heat, as well as increased nutrient and herbal values. Findings of this study will be helpful for the human health, ultimately contributing to the development of a new generation of plants adapted to climate.

Eggplant wild species (Solanum incanum, also known as Sodom or thorn apple) is found growing naturally in both tropics and subtropics (Waweru et al., 2017) and has been regarded as wild progenitor of Solanum melongena (Prohens et al., 2013). It is herbaceous or a soft wooded shrub having spiny stem and leaves covered with velvety hairs. Its extract is used to control ticks in cattle (Mwaura et al., 2011). Moreover, its fruit has been used to cure mycotic infections in Africa. Compost is also prepared by this wild plant (Mwaura et al., 2011). Furthermore, extract of its fruit has been used to control nematode (Meloidogyne spp.) in Capsicum annuum (Waweru et al., 2017) and so it can be used as an environmentally safe nematicide. It is believed to contain high quantity of phenolic compounds and therefore can be used in improvement of eggplant quality through breeding (Prohens et al., 2013). However, Kaushik et al. (2016) reported small fruit size of the hybrids developed by crossing cultivated eggplant with Solanum incanum. Mangino et al. (2020) evaluated the performance of 16 introgression lines (ILs) containing chromosomal segments of S. incanum and reported improved vigour in the ILs but exhibited low value of various fruit related parameters. Availability of large number of accessions (167) of S. incanum (Taher et al., 2017) offers to select the best performing one for distant hybridization.
Inter and intraspecific hybridization transfers desired genes from one specie to the other or same species which play an important role to improve the crop yield and disease resistance (Ghani et al., 2020). Interspecific hybridization is an important approach in plant breeding to incorporate useful genes to crop improvement (Sekara et al., 2007). The wild species of eggplant are a rich source of genes for resistance to diseases or insects. For example, bacterial wilt resistance in S. aethiopicum. (Collonnier et al., 2001); resistance to little leaf, shoot and fruit borer in S. indicum (Bahgat et al., 2008); Verticillium and Fusarium wilt resistance in S. incanum (Prohens et al., 2013). However, in interspecific hybridization, the production of hybrid seed is greatly hampered due to certain fertilization barriers. The crossability and hybridization studies of different S. melongena accessions with its related species have been carried out with inconsistent results (Behera & Singh, 2002). Crossability of S. melongena with S. incanum has also been reported (Kumar et al., 2020). The breeding materials developed through this program will help in further improvement of eggplant particularly in resistance breeding, but the fruit weight is reduced, particularly in S. incanum (Kumar et al., 2020).
The identification of hybrids relies on both morphological traits and molecular markers. Different molecular markers, including RAPD, AFLPs, and SSR have been used in plant genetic analysis. In the present study, a marker system called sequence related amplified polymorphism (SRAP) was chosen because it is simple, reproducible and comparatively less expensive than other types of markers (Li & Quiros, 2001). Moreover, SRAP is also more informative than RAPD, ISSR, SSR, AFLP (Budak et al., 2004). With respect to true hybrid, SRAPs have provided a reliable tool for the estimation of the degree of genetic relatedness among number of species in the plant kingdom (Li & Quiros, 2001). Thus, SRAP markers are commonly used as they are rapid, simple, and cost effective to access the genetic purity of interspecific and intraspecific hybrids.
In the present study, we reported the successful interspecific hybridization of eggplant and its wild relative S. incanum. We tried to describe the characteristics of intraspecific and interspecific hybrids between S. melongena x S. melongena and S. melongena x S. incanum and found positive gain in fruit size and fruit yield per plant compared to the parents. Given the high quality (phenolic contents) of S. incanum, and tolerance to drought and resistance to a few diseases (Prohens et al., 2013: Mangino et al., 2020 as well as suitability as rootstock (Gisbert et al., 2011), these materials may be of great interest for developing a new generation of eggplant varieties adapted to climate change and tolerance to drought and heat stress.

Morphological parameters
Different morphological parameters were observed i.e. leaf length, leaf width, plant height, fruit length and fruit diameter. Leaf dimensions were recorded by taking measurements of three mature leaves from each plant at the vegetative phase. Plant height was measured after flowering while fruit length and fruit diameter were obtained from mature fruit. These parameters were taken by using meter scale. Fruits were harvested at maturity and yield per plant was determined.

Pollen viability
The mature flower buds were bagged with butter paper bag one day before anthesis for the pollen viability assessment. Three to four flowers were collected of each wild and cultivated species at 8 a.m. on the day of anthesis. Fresh pollen from anthers of each flower of parents and F 1 were shattered on microscope glass slide and stained with 1% (w/v) acetocarmine. After drying of acetocarmine, slides were examined under microscope (400 X) using the method described by Alexander (1969).

SRAP (Sequence related amplified polymorphism) analysis
Eggplant six SRAP primers were selected from sequence previously reported (Li & Quiros, 2001). For this purpose, these six pair of primers were used Forward primer (Table 1). A modified version of the CTAB method was used to extract genomic DNA. PCR amplification was performed under the following conditions: 5 min of denaturing at 94°C, and 5 cycles of three steps, including 1 min of denaturing at 94°C, 1 min of annealing at 35°C and 1 min of elongation at 72°C. This was followed by 35 cycles of 94°C/45 s, 50°C/45 s, 72°C/1 min, 1 cycle of 72°C for 10 min at 4°C. Each PCR product (6 µL) was resolved in 6% denaturing poly acryl-amide gel electrophoresis (PAGE) at 1500 W for 2.5 h. The amplified products were visualised by simplified silver staining. In order to obtain reproducible and clear banding patterns, each amplification was repeated three times, and only bands showing consistent amplifications were scored.

Statistical analysis
The statistical analyses were carried out by using Statistix 8.1 software. Standard deviation (SD) was also calculated. Complete randomize block design (RCBD) was used and also for the pair-wise comparisons least significant difference (LSD) test was used with 95% (p<0.05) confidence.

Inter and intraspecific crossing
Intraspecific hybrids were produced by using different cross combinations (Table 2). Highest hybridization r e s e m b l a n c e w a s o b s e r v e d i n MEE×MEB with a 14.81% successful cross ratio than other combinations. On the other hand, different cross combinations with S. incanum were also performed to produce F 1 interspecific hybrids. In this cross-success ratio 6.12% was observed in MEE×INC combination, but its reciprocal was unsuccessful. The MEE×INC hybrid had the maximum number of seeds per fruit (Table 2).

Morphological observation
The crossability among the S. melongena genotypes with their wild relatives showed wide range of variations in morphological characters. The interspecific hybrids were intermediate in most of the parental characteristics. This is a common phenomenon in interspecific hybrids in eggplant with wild relatives (Prohens et al., 2013: Kaushik et al., 2016  yield of plants. Moreover, Haydar et al. (2007) also found that the days taken for flower setting is directly related to the fruit yield in tomato. Parent MEP has minimum days to flowering about 61.6 days than the other parents and hybrids. The maximum sepal length was measured in parent MEB (2.16 cm) and minimum length was 1.66 cm in hybrid MEE×INC (Table 3) (Table  3). The maximum stem diameter was observed in hybrid MEPxMEE other than hybrids and parents (Table 3). Leaves are main sites for photosynthetic reaction and play a key role in fruit size and yield (Shakoor et al., 2010) Figure 1c). Interspecific hybridization between cultivated eggplant and its wild relatives is an important way of deploying desirable genes for eggplant improvement (Liu et al., 2015). In this study, the MEE x INC hybrid was successfully obtained by crossing cultivated eggplant and S. incanum. However, when S. incanum was used as female, no fruit setting was observed, whereas only cross combinations of cultivated eggplant and S. incanum showed fruit setting when S. incanum was used as male. Most of the attempts to produce interspecific hybrids have resulted in sterility between the eggplant S. melongena and its wild relatives (Nasrallah & Hopp, 1963). Latest literature described that the hybrids of S. melongena x S. incanum and S. incanum × S. melongena were fertile and produced seeds (Kaushik et al., 2016). The INC parent had highest number of seeds per fruit (1177.2) and the lowest numbers of seeds (185) was recorded in MEE×MEB as compared to parents and hybrids (Figure 1d). The maximum number of seeds per fruit was also found in S. melongena x S. incanum as compared to parents and other hybrids ( Figure 2a). Higher number of seeds per fruit was also reported in a cross between S. melongena x S. incanum (Plazas et al., 2016). But the other study showed that the number of seeds per fruit was low in S. melongena and their wild relatives (Devi et al., 2015).

Pollen viability estimation
Pollen viability of hybrids and their parents was found different. The INC, MEE and MEB were semi-fertile and showed pink color of pollen grains. The pollen grains of MEP gave dark red color and were highly fertile, i.e. 70.1% pollens were fertile, while 63.4% pollen were fertile in hybrid MEP x MEE. On the other hand, the least pollen viability (38.9%) was observed in MEE x MEB. However, the maximum (65.04%) semi-fertile pollens were observed in MEB parent and minimum semi-fertility  was observed in 35.07%. Pollen grain of MEExMEB gave pink color that indicated semi-fertile pollen while pollens of MEExINC and MEPxMEE were dark red colored indicating high fertility and so they can be used in further breeding program (Figure 2b). A previous study also reported wide range of variation for pollen viability in S. melongena, wild relatives and their hybrid progenies (Devi et al., 2015). Almost all the pollen was aborted and empty, only 1.2% appearing normal, and pollen fertility was also high with 77.7% stainable pollen which was also larger than that of the parents.

Molecular analysis by SRAP markers
SRAP analysis was also performed to confirm the hybridity of MEPxMEE, MEExINC and MEExMEB. For confirmation of hybridity, all plants were analyzed by SRAP markers, and the results showed that they had specific bands from the two parents ( Figure  2c). Thus, all plants were true hybrids. Similarly, all hybrids were found to be true hybrids between 'Clementine' mandarin and K1 blood orange (Oruç & Dalkiliç, 2017). Mishra et al. (2011) demonstrated the effectiveness of SRAP marker in genetic analysis and hybrid identification of intraspecific C. arabica coffee hybrids.
These results suggest that genotypic difference can result in variable results of heterosis observed for positive gain in fruit diameter and yield of S. melongena x S. incanum. In this regard, this material will be of great interest for the genetic improvement of eggplant; it may have a tremendous potential to increase tolerance to abiotic stresses, such as to drought and heat, as well as increased the nutrient values and herbal values which will be helpful in improving the human health. Finally, we hope that this study opens the way for the incorporation of the S. incanum in new World gene pool for eggplant breeding, ultimately contributing to the development of a new generation of plants adapted to climate change and with improved nutritional, herbal, and diseases and pest resistance properties.