Genetic Variability in Germplasm Accessions of Capsicum annuum L

Capsicum annuum is the most widely cultivated species of peppers (chilies) in the world. For culinary purposes, its fruits are used for pungency (capsaicin) and also color (capsanthin). Capsaicin is also used for medicinal purposes particularly in anti-inflammatory formulations. Genetic divergence among 38 accessions collected from diverse locations in India (28 from Uttar Pradesh, 5 from Assam, 3 from Maharashtra and 2 from Uttaranchal), was estimated from the data pooled over 3 consecutive years for 15 morphological, growth and chemotypic characters that included days to first and second flowering, fruit onset, plant height, primary, secondary and tertiary branches, leaf surface area, fruit length and diameter, fruit surface area, fresh and dry fruit weight, capsaicin and capsanthin content. Based on this characterization the plants could be grouped into 7 clusters wherein substantial diversity among accessions was indicated by the wide range of 2 D values (752.901 1918683.00). Accessions with distinct identity were marked, which are likely to be quite suitable for breeding through hybridization combining desirable traits. The accessions labeled number 38, 27, 26, 14 and 24 to high capsaicin content (%); 35, 23, 3, 16, 29 and 11 for high capsanthin content (%) and 26 and 27 for dual purpose had characteristics desirable. Above accessions could be utilized in hybridization programme for C. annuum crop improvement.


Introduction
Capsicum is the unique genus of the family Solanaceae finding diverse uses from nutritional and culinary to pharmaceutical uses.In this genus, native to the Americas, more than 30 species have been described, but only five of them, Capsicum annuum var.annuum, C. chinense, C. frutescens, C. baccatum var.pendulum, and C. pubescens are considered to be domesticated (Pandey, Pozzobon et al., 2006;Moscone et al., 2007) [1][2][3].Pungent peppers commonly known as chilies in India belong to the species C. annuum L. which is the most widely cultivated species in the world.Both hot and sweet peppers have been reported to have originated from C. annuum [4].
The pungent chemical principle of Capsicum is capsaicin, which is synthesized and accumulated specifically in fruits.The capsaicin has found use in a wide range of pain problems, including post-mastectomy syndrome, urticaria, psoriasis, diabetic neuropathy, arthritis, pruritis, contact allergy, post-surgical neuromas etc. [5].The sweet pepper on the other hand is one of the richest sources of carotenoids.The ketoxanthophylls, capsanthin and capsorubin are the major pigments contributing to the red color of Capsicum, while β-carotene and zeaxanthin are responcible for the yellow-orange colors [6].
The characterization and the evaluation of the Capsicum domesticated species are particularly interesting for gene bank curators, since a wide variability, not yet fully known and exploited, is available in these species (Guzmán et al. [7], 2005; Sudré et al. [8], 2006; Ince et al. [9], 2009).In this study, genetic variability for diverse traits in available germplasm collections assembled from different places in India were evaluated as a prelude to crop improvement.

Genetic divergence among 38 indigenous accessions of
Capsicum annuum collected from various geographical locations (Table 1) in India was studied.The plants were grown in randomized block design at the research farm of CIMAP, Lucknow, India during 1998, 1999 and 2000 with two replicates in each year.Each treatment consisted of single row 45 cm long and 60 cm apart.The plant received normal intercultural operations, irrigations and fertilizer applications (20 kg N, 30 kg P 2 O 5 and 30 kg K 2 O per hectare).
Observations were recorded for the 15 characters for days to first flowering, days to second flowering, days to fruit initiation, plant height, primary branches/plant, secondary branches/primary branch, tertiary branches/ secondary branch, leaf surface area, fruit length, fruit diameter, fruit surface area, fruit weight fresh and dry, capsaicin and capsanthin content.Mean values for all the characters (pooled over three years) were subjected to D 2 and canonical analysis of genetic divergence based on the procedures outlined by Mahalanobis [10] and Rao [4].

Results
Highly significant differences (P < 0.01) among the collection of C. annuum for all the studied 15 characters indicated the presence of considerable genetic diversity.Tremendous variation in shape and size of fruits were observed (Table 2, for the mean, ranges and C.D.).Hence D 2 values for 703 pairs of genotypes were developed.Based on these values, all 38 accessions/genotypes could be grouped into seven clusters such that the genotype within the clusters had smaller 2  D -values among themselves than those belonging to different clusters (Table 3).Divergence for the pooled characters (in terms of D 2 values) ranged from 752.901 to 1918683 and cluster mean (for 164 to 241 days-first flowering, 000 -243 days to second flowering, 259 -336 days of fruit initiation, 50 -106 cm plant height, 5 -8 primary branches, 7 -10 secondary branches, 5 -9 tertiary branches, 20.06 -45.39 cm 2 leaf surface area, 2.332 -9.960 cm fruit length, 0.802 -1.830 cm fruit diameter, 4.312 -27.400 cm 2 fruit surface area, 0.76 -4.17 mg fruit weight fresh, 0.20 -0.75 mg fruit weight dry, 0.000% -0.0869% capsaicin content and 0.063% -0.517% capsanthin content).It is evident from the results, Table 2 and Figure 3).Considerable differences in economic traits, growth and other parameters were noted (Tables 2 and 4).The comparative characteristic features of Capcicum annuum plant collected from various places  that largest cluster grouping was comprised of accessions collected from Lucknow (Uttar Pradesh); Pantnagar and Ramnagar (Uttaranchal); Assam and Pune (Maharashtra) followed by cluster II comprised of accessions originated from Uttar Pradesh, Uttaranchal and Maharashtra, and cluster III comprised the accessions collected from Uttar Pradesh and Assam states of India.Notwithstanding the fact that, based on 2 D -values, all 38 accessions/genotypes could be grouped into seven clusters such that the genotype within the clusters had smaller 2 D -values among themselves than those belonging to different clusters (Table 3).It is evident from the results, Table 3 and Figure 3).The highest intra cluster  of India are presented in Table 1.Days to first flowering varies from 174 -175, days to 2nd flowering 237 -247, days to fruit initiation 265 -267, plant height 81 -82 cm, primary branches/plant 7 -8, secondary branches/primary branch 7 -8, tertiary branches/ secondary branch 5 -6, leaves surface area 23.11 -23.30 per sq.cm., fruit length 3.6 -3.63 cm , fruit diameter 0.998 -1.00 cm fruit surface area 6.642 -6.65/sq.cm,fruit weight fresh 1.37 -1.38 mg, fruit weight dry 0.309 -0.311 mg, capsaicin content 0.072% -0.077%, capsanthin content 0.140 -0.142% (Table 5).Other morphological characters were also exhibited tremendous variation such as color of leaf flower and fruits, plant type, shape and morphology of fruit etc. (Table 5).
Accession number 5, 7, 30, 35 and 36 from Lucknow (Uttar Pradesh) appeared highly divergent being represented within four clusters.The clusters V to VII were highly divergent and unique including only one accession each (35-V, 30-VI and 36-VII cluster).All belonging from Lucknow (Uttar Pradesh) represented enormous variation with respect to their morphology, growth behavior, ecological requirement and genetic diversity.The primary branches per plant (7.4035, rank I) followed by plant height (4.8078, rank II) and days to first flowering (1.7709, rank III) at the primary axis (Z1) and the primary branches per plant (5.6000, rank I) followed by fruit weight fresh (0.5715, rank II) and leaf surface area (0.5147, rank III) at the secondary axis (Z2), respectively were the largest contributor to genetic divergence.The least contributor to genetic divergence were secondary branches per primary branch (-6.4503) followed by capsaicin content (-5.3054) and days of fruit initiation (-4.1256) at primary axis, respectively.Least contributing traits fruit surface area (-8.660), capsanthin content (-3.776) and capsaicin content (-2.7722) at the secondary axis, respectively (Table 4).The distribution pattern

Discussion
In the Capsicum annuum accessions, 55.263% of the accessions could be grouped within one cluster I (21 accessions); in cluster II, 18.421% (7 accessions); cluster III 13.158% (5 accessions), followed by cluster IV 5.263% (2 accessions).The cluster V, VI and VII were found unique included only one accession in each namely, 35, 30 and 36, which were in 7.896% of the total accessions (Table 3).An analysis of cluster composition revealed of accessions of diverse origin in the clusters indicates that genetic diversity observed within C. annuum was not related to geographic origin.Noted difference in plant characters probably occurred over time with free movement of plant material from location to location, mutation, hybridization, gene recombination and selection.Our findings were also in consonance of results of Pandey and Dobhal [1] and Varalakshmi and Haribabu [11] in C. annuum crop.Selection of promising accessions based on genetic divergence and capsaicin and capsanthin content quality are useful in chili crop improvement.The accessions labeled number 38, 27, 26, 14 and 24 to high capsaicin content (%); 35, 23, 3, 16, 29 and 11 for high capsanthin content (%) and 26 and 27 for dual purpose had characteristics desirable.Among the above, the accession no.35 (0.000% capsaicin content) should be utilized for the study of genetics of capsaicin content, making hybrids between the parents 0.000 X high capsaicin content.Mainly due to the versatility of application, C. annuum plants have become important from an economic point of view in several countries since their fruits can be used for different purposes, such as in cooking, in industry (e.g., production of "pepper spray") and for medicinal and ornamental purposes (Pickersgill,[12] 1971; [13] Moscone et al. [3], 2007).In Brazil this vegetable is of great importance, ranking second in the generation of foreign currency among exported vegetables (Embrapa,[6,14] 2009).Therefore, the above listed accessions of the C. annuum could be utilized, as parents for hybridization in recombination breeding programme followed by repeated selection to obtain maximum gain.

1 ( 2 D
), That the mean inter-cluster 2 Dvalues and the cluster mean indicated the highest divergence between clusters designated III and VII ( 2 D = 1665293.0),corroborating the proposed variation of the germplasm into different gene complexes based on cytogenetic aspects and botanical characters.Divergence was also noted between clusters designated I and VII ( 2 D = 1661982.0).II and VII ( 2 D = 1167484.0)and between IV and VII ( 2 D = 1119514.0).The lowest inter-cluster distance was noticed in between cluster I and cluster III ( 2 D = 76941.01)(Table

1 ,
that the mean inter-cluster 2 D -values and the cluster mean indicated the highest divergence between clusters designated III and VII ( 2 D = 1665293.0),corroborating the proposed variation of the germplasm into different gene complexes by Moscone et al. (2007) [3] in investigation based on cytogenetic aspects and botanical characters.Divergence was also noted between clusters designated I and VII ( 2 D = 1661982.0).II and VII ( 2 D = 1167484.0)and between IV and VII ( 2 D = 1119514.0).The lowest intercluster distance was noticed in between cluster I and cluster III ( 2 D = 76941.01)(Table

2 D
values depicted by clster III ( 2 D = 60444.35)having 5 accessions followed by cluster II ( 2 D = 55333.340having 7 and cluster I ( 2 D = 43480.89)having 21 accessions.The 0.00 intra-cluster value depicted by clusters V to VII having unique individual and highly diverse accession (Figure 1 in terms of D

Table 5 . Average intra and inter cluster D value in Capsicum annuum accessions.
2D .