Genetic variance and heritability estimation of hybridized pepper plants (Capsicum annuum L.) F2 progeny for begomovirus resistance in growth stage

Pepper is widely cultivated as a condiment and cash crop in Indonesia. However, Pepper yellow leaf curl disease (PepYLCD) caused by begomovirus is currently seriously affect the domestic pepper production. Breeding for begomovirus resistance material by crossing is currently necessary to overcome the constraint. The present study is aimed to determine the resistance of pepper (C. annuum) plants F2 progenies to begomovirus infection in the growth stage. Two local C. annuum accessions, BaPep-5 as a resistance donor for pepy-1 begomovirus resistance gene (locally called Perintis) and BaPep-4 as a susceptible parent (locally called Kencana) were crossed to generate F2 progenies. The research was conducted in Agricultural Extension Training Centre (BLPP) Saree and Horticulture Laboratory of Syiah Kuala University from February to July 2020. 500 F2 progenies were transplanted to the field along with 15 plants of each parent as control. The result suggested that plant height and crown width had the highest broad sense heritability value, whereas the dichotomous height, stem diameter, secondary branch, and tertiary branch had the lowest broad sense heritability value. Coefficient of genetic variance and coefficient of phenotypic variance from overall characteristics were relatively low which suggest the narrow sense to slightly narrow sense heritability.


Introduction
Pepper (C. annuum) is an important versatile condiment and cash crop in Indonesia. In 2019, 2.7 M tonnes of fresh market pepper was produced to meet high demand of the domestic market [1]. Pepper production is occupying 155,000 hectares land and involving more than 500,000 local farmers [2]. Pepper is vulnerable to various abiotic and biotic stress [3]. Currently pepper yellow leaf curl disease (PepYLCD) is one of the most serious threat for pepper production in Indonesia that cause substantial yield loss up to 100% [4]. In 1999, PepYLCD was firstly noted in West Java and followed by subsequent incidence report from Central Java in 2003 [5]. PepYLCD has extensively spread to Sumatra in 2005  [6]. In addition, PepYLCD was also observed in five pepper cultivation field in Aceh Province, Indonesia with more than 81% plants exhibited the typical symptoms and symptoms reached 100% at four out of five field [7]. PepYLCD is caused by viruses belongs to the genus of begomovirus of the Geminiviridae family [8]. The genus comprises 445 species composed of a circular single-stranded DNA component and its transmission rely dependently on the insect vector Bemisia tabaci Genn. [9]. PepYLCD symptom is recognized by distinct vein yellowing, mosaic, severe leaf curl, failure of flower and fruit formation followed by significant yield-loss [10].
Current management for begomovirus caused disease in pepper rely dependently on vector population control which is less effective [11]. As the most efficient and effective method, breeding of resistance material is urgently required. One of the ways to breed for resistance is by crossing. Crossing is aimed to combine all attractive traits into a newly generated genotype with broad genetic diversity, increase the hybrid vigour, and examine the potential of parent or progeny test [12]. Essential information in breeding newly attractive cultivar includes the fulfilled assumption for determined inheritance pattern of a desired trait where genes between parent spread equally, thereby to determine the inheritance pattern of desired trait, groups of genotypes representing the tolerance response is chosen as parent [13]. Consequently, genotype with high potential can be used as parent in the breeding of PepYLCD resistance cultivars [14].
BaPep-5 is a locally grown C. annuum accession in Aceh Province, Indonesia. In our preliminary study, a pepper yellow leaf curl disease virus resistance-1 (pepy-1) gene has been identified from BaPep-5 [15] after the challenge inoculation of prevalent isolates in Aceh, pepper yellow leaf curl Indonesia virus (PepYLCIV) and Pepper yellow leaf curl Aceh virus (PepYLCAV) [16]. Therefore, BaPep-5 is currently essential germplasm for begomovirus resistance. Because estimation of genetic parameters is an essential process for plant breeding, notably in obtaining heritability and genetic variety information [17], in this present study we evaluate the begomovirus resistance response in BaPep-5 by estimating the genetic variance and heritability value related to growth stage.

Experiment place and time
The experiment was conducted at the Agricultural Extension Training Centre (BLPP) Saree, Lembah Seulawah, Aceh Besar (458 m asl approximate temperature ± 24ºC -28ºC, Soil type: Andisol). Analysis of growth was conducted at the Horticulture Laboratory of Agrotechnology Department, Faculty of Agriculture, Universitas Syiah Kuala, Darussalam Banda Aceh. The study was started from February to July 2020.

Plants materials
500 F2 progenies (C. annuum) derived from a cross between BaPep-5 and the begomovirus susceptible accession BaPep-4 was used as the plant material. 15 plants of the respective parent were included as control.

Cultivation
Pepper plants were sown in a 87 m x 1 m plot, a total of 6 plots were formed and covered with plastic mulch. A 30 cm depth of drainage was implemented with a 50 cm distance of each plot. Plants were transplanted with 40 cm x 50 cm plant-to-plant distance. Initial fertilizer application was conducted by applying 20 tons ha -1 of organic fertilizer, the application of dolomite lime 1.4 tonnes ha -1 and 50 kg ha -1 anorganic fertilizer NPK Mutiara (16:16:16). Irrigation was maintained for every morning. Pest and disease management was applied with chemical pesticides every 10 days from 7 days post transplanting (DPT) to 90 DPT.  [20]. Begomovirus which infect pepper plants usually caused the symptoms such as blanching of the leaf vein which continue to develop as yellowing, vein swelling, and curled leaves. Heavier infection of begomovirus lead to reduced size of leaves with bright yellowing, and dwarf phenotype of plant. In the field, symptom caused by begomovirus is various. Several plants will exhibit complete yellowing leaves, stripped green and yellow, and yellowing leaves with curled edges , this typical symptoms spread is generally occur evenly [6].

Begomovirus transmission
Virus transmission in the field is highly dependent on the vector. Begomovirus which originated from pepper plants is not capable of being transmitted mechanically by extracted liquid from the infected plants, yet it can only transmit by the insect vector B. tabaci and side grafting. The efficiency of infection by insect vector is higher compared to that of grafting, therefore to conduct experiment related to begomovirus infection, the insect vector transmission is commonly implemented [14].
Several terms are frequently used to highlight the relationship between plant, virus, and vector particularly from the insect group such acquisition access period (AAP), inoculation access period (IAP), latent period, and persistency. AAP is a period for insect vector to obtain liquid from plant cell. Whereas IAP is an important period for vector to absorb the plants cell liquid and transfer it to the healthy plants. Latent periods occur between AAP and IAP. Persistency is period for the insect vector to be sustainably fit to run further IAP after its previous infection. Three categories of persistency; nonpersistent, semi persistent, dan persistent [21]. Begomovirus is transmitted in a persistent or cicrculative manner. Virus will be able to maintain its life cycle inside vector body at least one week and the vector  [22]. Gaswanto et al., [23] stated that minimal AAP and IAP for B. tabaci is 15 minutes, respectively.

Genetic parameter estimation
Phenotypic expression of a plant is determined by genetic factor, environment, and interaction of both factors. The phenotypic variation in F2 population could be expressed due to the segregation of first filial (F1) which enables a crossing over at certain period allowing more quantity and more variation of gamete formation affected by multiple genes. Genetic factor become the most concerned since it is inherited from parents to its progeny. Heritability value is the principle in determining selection character.
Heritability will create precise visualization of characters affected by genetic or environment which can be further used for examining the genetic relationship between two parents and the derived progenies. Heritability is classified according to several criteria from low to high. Heritability is classified as high if the value is higher than 50%, quite high if the value is 20-50%, and low if the value is less than 20% [24]. The result of general mean, phenotypic variance, environmental variance, genotypic variance, and heritability are shown in Table 1.  [18] Variance in a population is affected by the value of genotypic variance and phenotypic variance observed in the evaluated trait. A higher genotypic variance suggesting that the trait is predominantly influenced by its genotypic factor, whereas a higher phenotypic variance suggesting that the trait is predominantly influenced by its environmental factor. In Table 1, all the observed traits had higher phenotypic variance compared to genotypic variance which demonstrated a high influence of environmental factor on phenotypic expression.
The broad sense heritability values shown in Table 1 indicates that plant height (230.84%) and crown width (224.71%) have high heritability values whereas dichotomous height (0%), stem diameter (0%), secondary branches (0.74%) and tertiary branches (2.92%) had low heritability values. Mahmud-Kramer equation for heritability analysis was established using P1 and P2 population data as a pure accession to determine the heritability and the results suggests that high heritability in several trait was contributed by the ability of genetic trait in particular individual to inherit the trait to its offspring.
The heritability value for the character of plant height is relatively high (230.84%) indicating that the expression of genes holds an important role in plant height which more influenced by genetic factors, whereas environmental factors slightly affect the character of plant height such as Begomoviral disease which causes plants to grow abnormally. The heritability value of the crown width character (224.71%) also had a high heritability value which indicated that the genes from the parents influenced or dominated the character of the width of the crown more than the experimental environmental conditions. According to [12], quantitative characters in plants are controlled by many genes, each of which has a small effect on quantitative characters, these characters are heavily influenced by environmental factors. Characters that have high heritability values indicate that genotype variety plays a more important role than environmental variety. The low heritability value suggests that environmental factors affect the character more. According to [25], the heritability value of quantitative characters which is relatively high indicates the phenotypic diversity in that generation is the diversity that can be passed on to their offspring.
The relationship between plant height and production is very close, it is suspected that there is a relationship with the formation of the number of productive branches, the taller the plant, the more opportunities for the emergence of productive branches, and with the number of productive branches, presumably the more flowers will be formed [26]. According to [27], in addition to having many productive branches, tall plants are more favoured by farmers because taller plants can prepare better vegetative organs, this affects the amount of photosynthesis so that many produce flowers which later become fruit. Generally, a reduction in plant height is common in plants that are infected by viruses because the the chlorophyll was hijacked by virus component and resulted in reduced efficiency of photosynthesis. Very high viral activity is thought to affect plant metabolic processes, thereby reducing primary metabolites and plant growth. The main metabolism is the process of photosynthesis which is connected with the pigment chlorophyll. Plants infected with the virus are thought to reduce their growth in both the vegetative and generative phases [4].
Diversity can be determined by the coefficient of genetic diversity (GCV), there are various coefficients of genetic diversity in Table 2 ranging from 0-21.28%. The value of the coefficient of genetic diversity is influenced by the experimental environment where the more heterogeneous the experimental environment, the greater the coefficient of diversity. The overall agronomic characters of plant height, dichotomous height, stem diameter, secondary branches, tertiary branches, and crown widths had criteria for GCV and PCV which are moderately narrow to narrow. The low GCV and PCV values indicate that environmental influences influence these characters more. Selection of characters with narrow coefficient of genetic diversity is difficult to increase their genetic potential. if the diversity is high then there is an opportunity to expect for genetic improvements and will presumably resulted in well-established selection [28].
According to [29], the GCV and PCV values which almost coincide with these characters indicate that the diversity of a character is caused by genetic factors. Stating the existence of a high criterion on