Genetic divergence for seedling and qualitative traits of tomato (Solanum lycopersicum) germplasm

One hundred and eighty-five tomato genotypes were explored for seedling and qualitative characters of tomato fruits and plants. Significant variations were detected for all studied traits. Hypocotyl length (HL) ranged from 13.2 to 71.4 mm with a mean value of 40.86±0.86. Primary leaf length (PLL) showed the range from 12.1 to 42.3 mm. Primary leaf width (PLW) ranged from 3.2 to 12.1 mm and the mean value was 6.46±0.12. Among qualitative analysis, eighty percent of genotypes showed good health. Various qualitative traits (fruit shape, color and size, foliage density and leaf type) for vegetative and reproductive stages were sufficient to differentiate the tomato germplasm in addition to seedlings. Dendrogram based on qualitative traits (fruit and plant) of 185 genotypes of tomato germplasm comprising five main clusters. Germplasm comprised almost all the classes reported somewhere else demonstrating prime diversity for diverse qualitative traits of seedlings in the germplasm of tomato preserved in the gene bank excluding hypocotyl pubescence. The present study of the seedling will be helpful with qualitative data of fruit and plant for establishment of the core set in providing information about the study of morphological evaluation, genetic relationships and representation of genetic diversity.


Introduction
Tomato (Solanum lycopersicum L.) belongs to a Solanaceae family that comprises more than three thousand species of tomato. It is a significant vegetable crop grown in the fields or greenhouses have distinctive economic position around the world in the horticultural industry [1, 2]. is a diploid species and has 2n=2x=24 chromosomes. The haploid genome of tomato species has 12 chromosomes and genome size equivalent to 950 Mb about 77% of which is heterochromatin and 23% euchromatin [3]. Tomato ranks second most important vegetable crop, and as a result of the robust breeding program, it has wider adaptation and is being cultivated all over the world after potato [1]. Tomato has globally achieved the position of the most widely used vegetables because of its multipurpose uses, better adaptability and extensively studied crop with respect to genomics, genetics and breeding [4]. Lycopersicon esculentum var. cerasiforme (cherry tomato) is thought to be an ancestor of cultivated tomato based on its wide presence in tropical America [5]. As a domesticated crop, it was first accepted in America though the innovative places of domestication are unclear [6]. Mexico has accepted a most likely site of domestication with Peru as a center of diversity for wild relatives [7]. It is self-pollinated fruit berry represent low to moderate genetic diversity due to bottlenecks in the germplasm of cultivated tomato, particularly in Europe [8,9]. Tomatoes are grown on 3.7 million hectares in the world with a total production of around 100 million tones [10]. China being the top producer (34 million tons), followed by USA (12.7

million tons) and
Pakistan is at the 34th position in fresh tomato production with annual production is 575,923 tons [11]. Tomatoes and their products are popular nutritive food and continue to increase because they are a good source of minerals, vitamins and antioxidants [12]. Tomato is a short-lived perennial but usually grown as annuals throughout the world. It is a member of the Solanaceae family and a highly self-pollinated plant. The cultivated tomato has gained popularity very rapidly in world food crops and attained the status of most consumed vegetables in the world [13]. It is understood that tomato is native to South America, the Incas and Aztecs began cultivating tomato plants as early as 700AD [14]. Genetic evidence shows that the progenitors of tomatoes were herbaceous green plants with small green fruit and a center of diversity in the highlands of Peru [15]. It is believed that tomato is introduced by British colonists at the beginning of the 19th century in the region of Indo-Pak [16].
The lumpy tomato, a larger mutation from a smaller fruit, smoother, originated in Mesoamerica and may be the direct ancestor of some modern cultivated tomatoes [17]. Assessment of the intra-population genetic variability in tomato has been reported important by Mazzucato et al. [18]. These authors analyzed morphological and molecular descriptors in Italian landraces of tomato. Seedling traits give an indication for plant growth and could also be used as plant descriptors as well as markers. Monogenic traits were the first to be employed for varietal identification and for markers which are still important for most of the crops [19,20]. he seedling qualitative descriptors have been utilized for the identification of various crop varieties and as genetic markers for investigation of quantitative traits loci [21,22]. The present study was initiated to characterize tomato germplasm for seedling traits and qualitative traits of tomato fruit and plants to investigate the magnitude of diversity among genotypes and clusters. This data will serve the purpose of genotypic identification and later will be utilized for establishing core collection to have the maximum diversity in less number of genotypes.

Materials and methods
The study was conducted to assess the genetic diversity of tomato germplasm on the basis of seedlings and qualitative traits of fruit (Fig. 1). One hundred and eightyfive genotypes of tomato germplasm included 150 genotypes from Solanum lycopersicum (L.) and 33 genotypes from Solanum lycopersicum var. cerasiforme and two check varieties Sahil and Roma. A research study conducted in green and shade house of Bio-resources Conservation Institute (BCI) NARC, Islamabad, Pakistan One hundred and eighty-five accessions of tomato were obtained from the National Gene Bank, BCI (NARC) Islamabad, Pakistan. Field experiments were carried out at NARC and nursery was raised in the green-house. The seeds were sown in plastic pots on 15 February during 2016 and the pots were kept in the greenhouse at 25 o C and irrigated with the help of sprinkler at alternate days, or when required. Onemonth-old seedlings were transplanted with the layout of Augmented Design in the shade-house. Each genotype was planted in two rows keeping 75 cm row spacing and 50 cm plant spacing in the shade-house (130 feet length, 30 feet width) at Crop Science Institute (CSI), NARC, Islamabad. Sahil and Roma (check varieties) were repeated after every 20 rows of genotypes. Morphophysiological level All the genotypes were evaluated at the seedling stage for 5 qualitative (Hypocotyl color, hypocotyl color intensity, pubescence on hypocotyl, overall leaf color, seedling vigor) and 3 quantitative (Primary leaf length, leaf width, Hypocotyl length) characters (Table 1). Data were recorded for seedling traits by following the descriptor of tomato established by the International Board for Plant Genetic Resources [23]. For the seedling stage, qualitative data (hypocotyl color, the intensity of color and pubescence density) were recorded after the development of the first two leaves. Other discrete traits including seedling vigor and overall color of leaf were also noted on genotype basis, hence represent single value for each genotype however quantitative traits, viz., hypocotyls length (mm), primary leaf length (mm) and primary leaf width (mm) were recorded on average of ten seedlings selected randomly from each genotype. Plant characters were recorded in the shadehouse after seven weeks of transplantation. The qualitative traits (Table 2) included stem pubescence density, foliage density, number of leaves under the first inflorescence, leaf type, leaf attitude, exterior color of immature fruit and stem internode length were recorded whereas, data for exterior color of mature fruit, fruit size and fruit shape were recorded at fully ripened stage.

Statistical analysis
Descriptive statistics (mean, standard error, standard deviation and range) were computed for all seedling traits and frequency distribution for qualitative traits to estimate the genetic diversity present in the germplasm of tomato. The dendrogram was constructed for morphological qualitative traits based on the dissimilarity matrix by the un-weighted pair group of arithmetic means analysis (UPGMA) [24]. Analyses were carried out using the R software.

Results and discussion
Tomato germplasm comprised of a total of 185 accessions along two check varieties (Roma and Sahil) were investigated for genetic variations. The genetic base of tomato varieties is narrow, which makes it difficult to identify different varieties using molecular markers. Even though morphological analysis is the first step, however, better presentation is expected when coupled agronomic traits with molecular markers in various crop analysis [25]. Quantitative and qualitative parameters were studied for seedlings, plants and tomato fruits evaluation. Quantitative traits comprised of hypocotyl length, primary leaf length and primary leaf width at the seedling stage were presented in the (Table  3) Data for hypocotyl pubescence, hypocotyl color, color intensity, leaf color and seedling vigor at the seedling stage were presented in (Table 4) according to data descriptors presented in (Table 1).

Table 1. Descriptors key (IBPGR 1996) used in estimation of diversity at seedling stage of tomato germplasm
Data for seedling recorded after development of first two leaves Exterior color of mature fruit ECMF 2-Black red, 5-orange, 7-pink, 9-red, 10-Verigated red, 11-yellow, Fruit shape* FS 1-Ellipsoid, 2-cylindrical, 3-flattened, 4heart-shaped, 5-rounded Legends: Data for plant descriptors* were noted when the fruits of 50% were ripened. Data for fruit descriptors* were noted at the fully mature stage of fruits. Data for the exterior color of immature fruit*were noted before maturity. Fruit shape* were noted after the fruits turn color

Seedling stage
The data for seedling traits were recorded in the greenhouse. The results for quantitative traits are presented in (Table  3) Keeping in view the importance of tomato in daily consumers' life, and the constraints of less research work conducted in Pakistan, the present study was initiated to evaluate a broad-based genetic stock for morphological evaluation with economic importance. Previously genetic variation was detected in all characters except stem pubescence studied by Rizvi et al. [31] who investigated the genetic diversity of three hundred and eighty accessions for nine seedling traits of tomato and concluded that spectrum of exploitation will be improved by the characterization/ evaluation of germplasm for various characters. The present study will be helpful in providing information about the study of morphological evaluation, genetic relationships and representation of genetic diversity. Germplasm comprised almost all the classes reported somewhere else demonstrating prime diversity for diverse qualitative traits of seedlings in the germplasm of tomato preserved in the gene bank excluding hypocotyl pubescence. Characterization of tomato genotypes, their wild relative's presence or absence of pubescence on fruits and vegetative parts has been used as a morphological trait [32]. The morphological evaluation of tomato hypocotyl has been used to detect the homozygous or heterozygous nature of tomato germplasm for mutant [33]. Monogenic characters were the first to be employed and they are still important for markers and varietal identification for most of the crops [19,20]. Six traits were of discrete nature out of nine seedling characters and may likely to be unified as genotype identification in single or in combination [34].To predict plant health and growth, diversity for seedling vigor traits is also important. Qualitative traits of seedling have been employed as a genetic marker and for the identification of various crop varieties for the examination of quantitative traits loci [21,35]. The application of a seedling marker for the identification of hybrids resulted to lower down the expense of hybrid seeds [36].

Qualitative traits of tomato plants and fruits
Qualitative parameters were studied for morphological evaluation. The plant descriptors including stem pubescence density, number of leaves underneath the first inflorescence, stem internode length, foliage density, leaf type and leaf attitude were recorded and presented in (Table 5). Fruit descriptors included the exterior color of immature fruit, exterior color of mature fruit, fruit shape, size were displayed in (Table 6).

Frequency distribution
Stem pubescence density was a common characteristic of plants of tomato among various qualitative traits ( Table 5). The frequency distribution of qualitative traits showed that germplasm was distributed into two classes based on pubescence density. In the first-class frequency of genotypes with the sparse pubescent stem (19 accessions) was 10%. Highly dense pubescent stems (89%) were observed in 166 accessions. Stem internode length exposed that short length of internode was characteristic of 42% of (79) accessions of tomato germplasm. Long internodes were 7% in 16 accessions however, 48% of the genotypes (90) were exhibited medium internodes. Sparse foliage density was observed in 26 accessions with a frequency of 14 percent. Genotypes having dense foliage were 77 i.e. 41% of germplasm while 44% of the germplasm (82 accessions) represented medium foliage density. Two categories of accessions were observed one with few and second with many leaves under the first inflorescence. The majority of the accessions (155) had many leaves with a frequency of 84% whereas 34 accessions had few leaves with a frequency of 18% of germplasm. It was observed that different categories for leaf attitude were horizontal, semi-erect and drooping. The majority of genotypes (86) were horizontal with the frequency of 46% population followed by drooping (82 genotypes; 42%) and semi-erect leaf attitude with 9% population of 17 genotypes approximately. Leaf type trait was divided into 4 categories i.e. Standard leaf type was observed in the majority of accessions (147) with a total population of 79%, followed by hirsutum in 22 accession (12%) and 10 genotypes exhibited potato leaf type i.e. merely of 6% frequency of a total population. However, pimnellifolium leaf type was observed in 4 genotypes having a lower frequency of 2% of the total population. Various degrees for the exterior color of immature fruits showed a wide range of variation with different levels of frequencies ( Table 6). The majority of the genotypes (95) with a frequency of 51% had light green fruits while green fruits were observed with less frequency of 38% in 71 genotypes. Greenish white immature fruits were counted in 28 accessions with 15% frequency while dark green fruits were noted in 18 genotypes with a lower frequency of 10% population. Moreover, the germplasm revealed adequate divergence for fruit shape. Different fruit shapes were detected with different frequencies. The variation displayed in the size and shape of the fruit is presented in (Fig. 2). Fruit size is an important trait in tomato that depends upon consumers' preference, in the genotypes evaluated, diverse fruit size was observed. Out of four classes as projected by IBPGR for descriptors of tomato only three were noted. Small-sized fruits (3.0-5.0 cm) were produced by 38 genotypes with a frequency of 21%. Large-sized fruits were produced by the majority of the genotypes (89) with a frequency of 48%. Intermediate sized fruits (5.1-8.0 cm) were observed in 58 genotypes having a frequency of 31%. For the exterior color of mature fruits, the germplasm was found uniform equally. Majority of genotypes (148) produced red fruits with highest frequency (79%) of population while, orange color fruit was Fruit shape also showed wide diversity in studied germplasm. Flattened shape fruits were recorded 11 % of the germplasm (21 accessions). Round fruit shape was noted in 150 genotypes having a frequency of 81% of total population. The genotypes with cylindrical, ellipsoid and heart-shaped fruits were less frequent, i.e. 11, 2 and 1 accessions with frequencies of 6, 1 and 0.5% percent, respectively.    Stuffing, 6233, 17904, LA-1969A, LA-4285A, TH-15-095, TH-15-103, TH-15-114, TH-15-117, TH-10-5-0011, TH-10-5-0015, TH-10-5-0012, TH-10-5-0026, TH-

Cluster analysis
Cluster analysis on the basis of 10 qualitative traits classified all the germplasm into five main clusters at linkage distance 1.75. The dendrogram was constructed using the complete linkage method and presented in the (Fig. 3). Cluster I grouped 62 genotypes and was the second largest group having nearly 33 % of the total population. Cluster II was the largest comprising of 87 genotypes which were 47% of the total population. The cluster III consisted of five genotypes (TH-10-5-0021, TH-10-5-0043, Tom 10, Burnely Fortune and Santa) which were 2.7% of the population and this was followed by cluster IV with 9 genotypes (

Conclusion
The morphological diversity observed among different tomato genotypes could be associated with variation of factors. The morphological variations observed in tomato genotypes could be described as differences in environmental and genetic conditions. The nutrient status of the soil is a basic factor of climatic conditions that significantly contribute to the variances in the performances of the cultivar. Characterization of tomato germplasm at the seedling stage and qualitative traits of tomato plants is suggested for broadening the use of core collections so as to use more efficiently crop germplasm in minimum time.