Assessment of Genetic Divergence in Long Day Onion (Allium cepa L.) through Principal Component and Single Linkage Cluster Analysis

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Onion is an important vegetable crop used by all the sections of people,round the year throughout the world for its distinct flavour and health healing properties. It is a photosensitive crop and forms bulbs at certain day length. Long day onion requires 14 hours or more day length to initiate the bulbing. In India, majority of growing area is under short day onion except in hillyregion. Long day onion is grown in temperate region of India with productivity ranging from 10 to 23 t/ha.Though it covers large temperate area from Jammu and Kashmir to Arunachal Pradesh but efforts on varietal improvement programme on long day onion are very limited. There is no commercial long day variety available for cultivation except some old introductions like Yellow Globe and Brown Spanish. Farmers use their own seed without caring isolation distance to maintain the purity which leads to a great variability in shape and size of bulb with inherent low yield.

The magnitude of genetic diversity in onion germplasm is a critical component in breeding for new cultivar. Selection of genetically diverse parents in breeding programme on the basis of divergence would be more promising to get the heterotic F1, and to create a broad spectrum of variability in segregating generation (Meena and Bahadur, 2015). Presence of genetic diversity play a vital role in plant breeding for getting higher yield, uniformly disired quality and resitance to biotic and abiotic stresses. A systematic understanding of gentic diversity in different traits is essential for targeted breeding programme. There are numerical taxonomic techniques available to classify the variation pattern at inter and intra specific level (Ario and Odulaza, 1999). Multivariate analysis is an effective tool for characterization and classification of plant genetic resources, when a large number of accessions are assessed for several traits (Peter and Matrinelli, 1989). Different type of multivariate analysis such as principal component analysis (PCA) and single linkage cluster analysis (SLCA) are used to identify groups of accessions that have desirable traits for breeding and assessing the pattern of variation in germplasm collection. PCA enables easier understanding of impact and relationship among the different traits. However PCA alone would not give an adequate character representation in term of relative importance when multiple characters are considered simultaneously (Shalini et al., 2003). To complement the results of such multivariate analysis, Single Linkage Cluster Analysis(SLCA) is employed to classify the variation. It is an agglomerative technique which shows the patterns of exact genotype position in population. (Ariyo and Odulaza, 1999) by sorting them in distinct group.Thus this study is aimed to identify the major characters responsible for variation among the onion genotypes with a view to group accessions and for identifying the potential parental stocks within the group of local germplasm by employing the multivariate analysis.

Thirty four long day onion accessions (Allium cepa L.) including two varieties collected from growing hot spot of Kashmir valley and conserved at active germplasm site of ICAR-Central Institute of Temperate Horticulture, Srinagar (J&K) were evaluated (Table 1). The seedlings of 45 day old were transplanted in main field during rabi season. Each accession was grown in ten rows of two metre length with a spacing of 10x15 cm. The experiment was conducted in randomized block design with three replications. Geographical position of the experimental site lies between latitude of 34005 N and longitude of 74050 E at an altitude of 1640 MSL. The average maximum and minimum temperature were 19. 630 C, 6. 520 C respectively with annual precipitation of 160.72 mm and relative humidity 58.35%, evaporation 2.45mm. The soil characteristics viz. pH= 6.81 and EC = 0.36 dSm-1 were recorded during the cropping season. Recommended uniform agronomic and cultural practices were adopted to obtain better expression of phenotypic characters. Data was recorded on nineteen quantitative traits. Disease severity rating was measured on 0-5 scale - (0 grade - No disease, 1 grade - 1-10%, 2 grade -11- 20%, 3 grade -21-30%, 4 grade -31-50% and 5 grade -51-100%). Whereas, pest (thrips) damage (1-5 scale) - (1 -1-20% foliage damage, 2- 21-40 foliage damage, 3-41-60% foliage damage, 4-61- 80% foliage damage and 5-81-100% foliage. The genotypes with <5% infestation was considered immune, 6-10% infestation resistant, 10-20 infestation moderately resistant, 21-40% infestation moderately suceptble, 41-60% infestation susceptible >60% infestation considered highly susceptible. Data collected on the quantitative characters were analyzed using SAS Microsoft Windows 9.2 (SAS Institute, 2011), employing the method outlined by Steel and Torrie (1980) using statistical XL STAT-2011. Principal Component Analysis and Single Linkage Cluster Analysis (SLCA) were used for the determination of genetic variation and percentage similarity within the genotypes. Eigen - Vectors and principal component score were used to assess the relative discriminatory power of its axis and their associated characters. The cluster procedure was used to produce distinct groups of 34 genotypes on the basis of genetic relationship while using the character variation. Average intra-cluster distance was calculated by the following formula as suggested by Singh and Choudhary (1985). SLCA summarized the position of accessions into a dendogramat an interval of 5% level of dissimilarity starting from 100 % level of dissimilarity (Kendall, 1980).

Table1
Accessions with their geographical information used in study

The genotypes evaluated for all horticultural traits varied significantly (Table 2). The phenotypic variability expressed by range, standard deviation, and coefficient of variation. The plant height ranges from 63.33 to 91.66 cm. Genotype CITH-O-9 recorded highestplant height, whereas CITH-O-32 recorded lowest plant height (63.33 cm). Number of leaves ranged from 6.33 (CITH-O-9 ) to 14.0 cm (CITH-O-19. Polar diameter of bulb ranged from 5.18cm (CITH-O-7) to 11.97cm (CITH-O-13). Equatorial diameterof bulb ranged from 5.87cm (CITH-O-5) to 11.08 cm (CITH-O-8). Polar and equatorial diameter ratio reflects the bulb shape which is an important parameter indirectly related to yield storage life and market preference. The bulbs of genotype having < 1 Polar and equatorial diameter ratio ( P: E ) considered as flat and genotypes having P: E ratio 1 considered globe and those having P: E ratio> 1 were considered as torpedo. Genotype CITH-O-11, had P: E ratio 1,whereas CITH-O-4, CITH-O-32 and CITH-O-13have < 1 P: Eratio and remaining genotypes were having >1 P: E ratio. Neck thickness of bulb affects the storage life. Neck thickness ranged from 0.42 to 2.46 cm. The minimum neck thickness (0.42 cm) was observed with genotype CITH-O-7 , whereas CITH-O-19 had maximum neck thickness (2.46 cm). Bulb grade determines the market price and quality. A grade bulb ranged from 12 to 86.11 per cent (Table 3). Genotype CITH-O-12 recorded highest A grade percentage of bulbs. B grade bulb ranged from 00 to 34.55 per cent. Double bulbs which are major drawback in onion production ranged from 00 to 34.28 percent. Total soluble solids important quality trait in onion ranged from 0.36 to 16 per cent. Genotype CITH-O-29 scored highest TSS (16 %) whereas minimum TSS(0.36%) was recorded with genotype CITH-O-5.Average bulb weight which is directly correlated with yield, ranged from 154. 51 t o 470. 3 0 g.Genotype CISH-O-9 recorded the highest average bulb weight (470.30 g) whereas smallest bulb size was recorded with CITH-O-29 (154.51 g). Foliar disease of onion is major problem in long day conditions. The incidence of purple blotch ranged from 7.00 to 30.71%. Genotype CITH-0-29 has recorded highest infestation (30.31%) whereas, least infestation was observed with variety Brown Spanish (7%). Downy mildew infestation ranged from 13 . 48 to 30. 50%. T he lowest ( 13. 48%)infesta tion of downy mildew wa s obser ved ingenotype CITH-0-9 whereas highest infestation of was recorded with CITH-0-2 (30.50%). Thrips ismajor damaging insect in long day onion. Number of thrips/plant ranged from 7.66 to 31.00 / plant.T he minimum infesta tion of thrips /plant was recorded with genotype CITH-0-6 (7.66/plant) whereas, maximum number of thrips / plant wasobserved with CITH -0-17 (31.00/plant). Prematurebolting a burning problem in onion ranged from 0 to 3.66 % among the genotypes evaluated. The highest percent of bolt ing was observed in genotype CITH-0-11 (3.66%), where as four teengeno types recorded 0% bolting. Marketable bulb percenta geis an important trait from economic point of view. The percentage of marketable bulbr anged from 48.26 to 100% . The lowes market table bulb percent age (48 . 26 %) was recorded with genotype CITH-09 where as CITH-0-28 had recorded 1 0 0 % market ablebulbs. Marketa blebulb yield ranged from 33 1. 28 to1212.56 q/ha. The lowest marketable bulb yield was recorded with genotype CITH-0-31 (331.28 q/ha ) where as highes market ablebulb wa srecorded with CITH-0-8 (1212.56 q/ha). Total yield ranged from 494.5 to 1505.06 q/ha. Among thegenotype evaluated, CITH-09 recorded highesttota l yield, where as minimum tota l yield was observed with CITH-0-31. The genotype having the highest desirable traits may be utilised for crop improvement programme for a particular trait. Coefficient of variation (%) reflected the extent of variationfor evaluated phenotypic traits was highestfor double bulb percentage and B grads of bulbs,T. S. S and market ablebulb yield (q/h) . High coefficient of variation a mongst udied traitsindica tedan ppreciable variability which isprerequisite of acrop improvement program. Similar type of variability was also reported by Arya et al. (2017). The observed variability foundamong the onion genotypes might be related to genetic makeup of genotype as per Kandil et al. (2010).

Table 2
Variation in quantitative traits of onion accessions

Table 3
The Principal component latent vector for Eigen values and proportion of variance accounted for different components with respect of different traits

Based on degree of divergence 34 genotypes were grouped into 7 principal component having Eigen value >1 and cumulatively accounted for 83.87% of total variability (Table 4a and 4b). The PC-I contributed for 24.73% of total variation was positively loaded with bulb weight, marketable bulb percentage, total and marketable bulb yield. It was negatively correlated with number of thrips per plant, downy mildew infestation, (%) bolters and doubles, B and C grades bulbs. The PC-II reflected 15.27% of total variability and was positively loaded with plant height, neck thickness and negatively with downy mildew infestation. The PC-III was positively loaded with T.S.S. (%), number of leaves/plant and contributed 12.69 % of total variation. The 4th principal component contributed 10.90% of total variability was, associated with A grade bulb (%) and negatively correlated with purple blotch disease.The PC-V accounted 8.33% of total variation was positively loaded with polar diameter, Polar: Equatorial. Diameter ratios, bolter percentage and negatively loaded with C grade bulb percentage. Principal Component-VI contributed 6.42% of total variation and was positively correlated with downy mildew (%) but it was negative associated with purple blotch. PC-VII accounted for 5.53% of total variation was positively loaded with bolter percentage, purple blotch percentage, and number of thrips/plant and was negativity correlated with neck thickness.

The positive and negative loading of quantitative traits reflects the positive and negative correlation trend between the components and variables suggesting that these principle components may be used to summarise the variables. The traits with largest absolute value closer to unit within first component influence the cluster more than those to lower absolute value closer to zero. Thus in present study the differentiation of genotypes in different principal component was because of high contribution of few characters rather than small contribution of each character. The desirable characters loaded positively and undesirable characters loaded negatively in first seven PC’s could be in consideration while selecting the genotype for appropriate traits and yield potential. The principal component analysis has also been used for showing the genetic diversity in many species (Ravindra et al., 2018; Singh et al., 2017). The Bi-plot of PC-I & PC-II indicated that the some isolated genotype clearly define the diversity among the evaluated germplasm. The genotype CITH-0-13, CITH-0-4, CITH-0-22, CITH-0-19, CITH-0-9, CITH-06, CITH-O-2 and variety Coral Red were most divergent (Fig 1) usually is customary to select oneof the important variable from these identified groupsfor targeted improvement programme. Hence PC-Ifor higher total yield, PC-II for plant height, PC-IIIfor high T.S.S, PC-IV for maximum A Grade bulb,PC-V for wider polar diameter of bulb, PC-VI forresistance to purple blotch, PC-VII for thin Neckthickness of bulb were ideal for selection . Theresults of present study are useful as it furnish theinformation about the group where certain traits aremore important, allowing breeder to execute breedingfor specific target. Biological implication of principalcomponent analysis can be quantified by contributionof different variable in each PC as revealed by Eigenvector and cluster scor e at the component axissuggest that some relationship exist among theindividuals with the cluster but not provided the exactposition of genotypes in groups.

Table 4a
Cluster means for 19 characters in 34 onion genotypes based on agglomerative hierarchical clustering analysis

Based on single linkage cluster analysis genotypes were grouped into five clusters by quantifying their share and cluster means for all the traits (Table 4 a,b). The cluster-I and Cluster-III accommodated maximum number of genotype (9) followed by cluster II (8), Cluster-IV (7) and cluster -V(1) contributing 26.47, 23.53, 20.58 and 2.94%, of total population respectively. On the basis of cluster means, the cluster-I was important for high TSS (15.06%), marketable bulbs percentage (92.66%) powdery mildew (8.24%) and purple loch (16.08%) resistance. Cluster-II was important for plant height (83 cm) number of leaves/plant(12), polar diameter (8.54 cm) and P:E ratio (1.54) cluster -III was important A grade bulb percentage (69.21%)whereas cluster-IV was important equatorial diameter ( 9.48 cm),minimum neck thickness (0.42 cm) thrips resistance (9.33 thrips/ plant). Cluster -V was important for average bulb weight (377.76 g) marketable yield (1109.59 q/ha) and total yield (1208.83 q/ha). The genotype of cluster having high means value of particular traits would contribute more positively in their off springs if used as a parent. Arya et al. (2017) and Singh et al. (2017) also suggest that clusters having high meanvalue of the traits may be used for hybridizationprogram to get better segregates.

Table 4b
Grouping of 34 onion genotypes into five clusters based on agglomerative hierarchical clustering analysis

Fig. 1
Bi-plot for 1st and 2nd PC for genotypes of onion in relation horticultural traits

Fig. 2
Dendrogram depicting genetic relationship among 34 genotypes based on horticultural traits produced by complete linkage analysis (Scale-Euclidean distance at .05)

Proximity matrix obtained, suggest the resolution for 34 onion genotype distributed in five clusters with wide range of diversity for the traits (Table 5.) The highest inter cluster distance between cluster II and Cluster I (803.47%) followed by cluster-III and I (697.8) cluster V and cluster I (582.56). Based on convention that distantly related parents give better recombinants and hybrid. It could be expected that hybridization between genotype of these cluster will results high heterotic F., s and better recombinants in segregating generations. These genotypes of distant clustercould serve useful source of genes for different desirable traits in onion. The findings are in conformity with finding of Singh et al. (2017), Chattopadhay et al.(2015) and Ravindra et al. (2018) who reported that genotypes among the cluster having high distance when used in hybridization programme will obtain awide spectrum of variation in sergeants.

Table 5
Average Inter and intra cluster distance

Dendogram obtained from single linkage cluster analysis by using the Euclidian distance depicted the clear relationship and exact position of genotype in the clusters. All the genotype were distinct at 100 percent of dissimilarity and formed nine duster at 87% of dissimilarity, and formed five clusters at 57 of dissimilarity (Fig II). The dissimilarity ranged from 57 to 100% among the delineated genotypes enough to suggest the variability for crop improvement in onion. (Denton and Nwangburuka, 2011).

Genotypes CITH-0-29, CITH-0-26, CITH—8, CITH-0-24 and CITH-05 were divergent in cluster position on the basis of Euclidian distance which reflected higher distance among these genotypes and may be used for hybridization to get the better segregates. Singh et al., 2013 and Santara et al., 2017 also reported such variability by using the single linkage cluster analysis in Onion.This genetic diversity analysis would be useful to avoid the selecting parents from genetically homogenous cluster and maintain the broad genetic base for breeding programme in long day onion.