Brassica juncea L. commonly known as Indian mustard is globally used as oilseed, vegetable and condiments. The crop species is said to be one of the earliest domesticated species. B. juncea annually grows as wild and cultivated species over a large geographical range, across the Asia, Africa, Australia, America and Europe  . In the genus Brassicaceae, Indian mustard is one of the highly significant crop species. In Pakistan after cotton and rape seed, Indian mustard is the third highly important source of oil   .
So for the prosperity of the country, it is essential to increase the production of these crops and for maximum production, exploitation of genetic diversity of these crops is essential. Pakistan being an agricultural country but unfortunately it is deficient in the oilseed production and a large amount ($ 1054.7 million) is spent on the import about 77% of edible oil. After petroleum, Pakistan pays the second largest import bill for edible oil among food items  .
Diversity can be determined by three different levels, first one is genetic diversity, in which the variation observed at gene and germplasm level, the second one is diversity of species, in which richness of species at a specific location and the third one is the Eco-system diversity, in which crop species interact with their environment. Diversity is potent for different crops to fulfill the gap between demand and production across the world  . Various morpho-biochem- ical and molecular methods are used to study genetic variability among local and exotic plant germplasm. The proper evaluation of important crop species helps in the identification and utilization of improved genotypes  -  . Therefore, the present study was performed to estimate and characterize the genetic variation and relationships among Indian mustard genotypes through agro-mor- phological traits to identify and select promising germplasm for traits of economic significance.
2. Materials and Methods
Plant material comprises 167 germplasm including 1 check cultivar of B. juncea L. for estimation of agro-morphological variation. The germplasm were provided by PGRI gene-bank, NARC, Islamabad, Pakistan. The present research work was conducted in the experimental area of NARC (33˚43'N and 73˚06'E) during the year 2012-2013 using augmented design. The average annual rainfall in this area ranges from 500 - 900 mm with 70% in summer and 30% in winter. Row to row distance of 70 cm was maintained with one accession per row and the length of the row was 2.5 meter for each and every accession and genotype. During seed bed preparation the field was irrigated before sowing to provide conditions of optimum moisture while afterword no irrigations were given and the crop was grown under rain-fed conditions. The experimental germplasm were planted at a depth of 3 - 4 cm using hand drill. After germination to maintain proper plant population thinning was carried out by hand. After 30 days of germination weeding was carried out and because of no serious attack of insect pest no insecticides and pesticides were sprayed throughout the plant growth stage. The crop was harvested when almost more than 75% of plants were turned yellowish in color. Data were recorded for twenty quantitative traits such as Days to 50% flowering, Days to 100% flowering, Days to 50% maturity, Days to 100% maturity, Leaf length, Leaf width, Leaf width/leaf length ratio, Leaves per plant, Plant height, Primary branches per plant, Main raceme length, Silique per main raceme, Silique length, Silique width, Silique length and silique width ratio, Stem thickness, Seed per silique, 1000-seed weight, Seed yield per plant, Shattering percentage (%). The description of calculating different parameters is given in Table 1. The recorded morphological traits data were averaged and analyzed for simple statistics i.e. mean, variance, range, frequency distribution, coefficient of variance and standard deviation using computer software (MS-Excel 2007). Cluster and principal component analysis (PCA) was performed on the recorded data for quantitative traits. Before to cluster and PCA, mean of each parameter was standardized so that to avoiding scaling differences effects. For all the pairs of accessions Euclidean distance co-efficient were calculated. The Euclidean dissimilarity co-efficient matrices were utilized to estimate the association among the B. juncea germplasm with cluster analysis through complete linkage method (Statistica version 7.0 and NTSYS pc v 2.1).
3.1. Correlation Study Based on Agronomic and Morphological Traits
During the present research work days to 50% flowering exhibited highly significant positive correlation with the parameter i.e. days to 100% flowering (0.86**), leaf length (0.28**) and leaves per plant (0.21**), similarly days to 50% flowering, had significant positive correlation with days to maturity 100% and leaf length/leaf width ratio (0.18*), plant height (0.17*), days to maturity 50%, leaf width and primary branches per plant (0.16*). While days to flowering 50% had significant negative correlation was observed with silique width (−0.18*). Days to 100% flowering had highly positive significant correlation with leaf length (0.37**), days to maturity 100% (0.29**), days to maturity 50%, leaves per plant and stem thickness (0.27**) and primary branches per plant (0.24**). While the trait like plant height (0.19*) and leaf width (0.18*) had found significant positive correlation with days to 100% flowering (Table 2).
Table 1. Detailed information for calculating 20 different quantitative traits of B. juncea L. genotypes.
Table 2. Correlation coefficient among agro-morphological characters in B. juncea L. accessions.
Note: *significant at 0.05 (0.15 to 0.19 value); **highly significant at 0.20 & above probability level (more than 0.00 level); The green, yellow, white, red colors shows strong positive, weak positive, moderate positive, and strong negative correlations among different traits respectively.
3.2. Principal Component Analysis Based on Agronomic and Morphological Traits
Principal component analysis (PCA) was carried out based on twenty quantitative morphological characters. The 7 principal components account for 73.92% of the overall variability among the studied B. juncea L. accessions for the total phenotypic variations (Table 3, Figures 1-6). Among these seven principal components (PCs), the PCA-I was found to have 23.35% out of the total variability. Silique length (0.073), silique length/width ratio (0.224) and shattering percentage (0.506) contributed positively to first principal component. In contrast, days to flowering 50% and 100% (−0.385 and −0.494), days to maturity 50% and 100% (−0.680 and −0.679), leaf length (−0.711), leaf width (−0.569), leaf length/leaf width ratio (−0.112), leaves per plant (−0.616), stem thickness (−0.606), plant height (−0.513), primary branches per plant (−0.645), main raceme length (−0.391), siliqua per main raceme (−0.653), silique width (−0.193), seeds per silique (−0.230), 1000-seed weight (−0.203) and seed yield per plant (−0.251) contributed negatively.
The contribution of PC-II in total differences was 12.87% and was positively associated with days to maturity 50 and 100% (0.520 and 0.540), leaf length/ width ratio (0.540), silique on main raceme (0.034), silique length (0.234), silique width (0.435), seed per silique (0.530), 1000-seed weight (0.270) and seed yield per plant (0.394), whereas days to flowering 50 and 100% (−0.176 and −0.133), leaf length (−0.149), leaf width (−0.506), leaves per plant (−0.187), stem thickness (−0.153), plant height (−0.580), primary branches per plant (−0.280), main raceme length (−0.243), silique length/width ratio (−0.245) and shattering percentage (−0.215) were negative associated with PC-II.
PC-III showed 11.24% of the total agro-morphological variation and positively associated with leaf width (0.161), leaves per plant (0.033), plant height (0.144), primary branches per plant (−0.015), main raceme length (0.365), siliqua per main raceme (0.266), seeds per silique (0.205), 1000-seed weight (0.232) and silique width (0.578), while negatively associated with days to flowering 50%
Table 3. Principal components (PCs) of agronomic traits among B. juncea L. genotypes.
Figure 1. Scatter diagram og PC-I and II for agro-morphological traits.
Figure 2. Scatter diagram of PC-I and III for agro-morphological traits.
Figure 3. Scatter diagram of PC-II and III for agro-morphological traits.
Figure 4. Contribution of morphological traits in the first two principal components.
Figure 5. Contribution of morphological traits in the second and third principal components.
Figure 6. Contribution of morphological traits in the first and third principal components.
and 100% (−0.610 and −0.587), days to maturity 50% and 100% (−0.183 and −0.207), leaf length (−0.066), leaf length/leaf width ratio (−0.353), stem thickness (−0.029), silique length (−0.290), silique length/width ratio (−0.696), seed yield per plant (−0.248) and shattering percentage (−0.119).
The PC-IV contributed 8.07% in the total variability and were positively contributed to days to maturity 50% and 100% (0.273 and 0.252), leaf width (0.093), stem thickness (0.193), plant height (0.133), primary branches per plant (0.243), main raceme length (0.095), silique per main raceme (0.040), silique length (0.592), silique length/width ratio (0.490) and seed yield per plant (0.236). Similarly the traits i.e. days to flowering 50% and 100% (−0.463 and −0.443), leaf length (−0.186), leaf length/width ratio (−0.423), leaves per plant (−0.180), silique width (−0.108), seed per silique (−0.103), 1000-seed weight (−0.116) and shattering percentage (−0.069) contributed negatively.
In the total variability the contribution of PC-V was observed 6.89% and the parameter i.e. leaf length (0.329), leaf width (0.103), leaf length/width ratio (0.274), main raceme length (0.246), siliqua per main raceme (0.319), silique length (0.228), silique length/width ratio (0.349), seeds per silique (0.332) were positively contributed. Whereas the characters i.e. days to flowering 50 and 100% (−0.054 and −0.135), days to maturity 50% and 100% (−0.151 and −0.147), leaves per plant (−0.026), stem thickness (−0.492), plant height (−0.118), primary branches per plant (−0.033), silique width (−0.253), 1000-seed weight (−0.007), seed yield per plant and shattering percentage (−0.388 and −0.445) contributed negatively.
The PC-VI depicted proportion of variability as 6.35% and were positively associated with days to flowering 50% and 100% (0.138 and 0.095), days to maturity 50 and 100% (0.063 and 0.037), plant height (0.155), main raceme length (0.563), siliqua per main raceme (0.394), silique length/width ratio (0.090), 1000-seed weight (0.378), and seed yield per plant (0.125). Similarly the traits i.e. leaf length (−0.396), leaf width (−0.417), leaf length/width ratio (−0.021), leaves per plant (−0.196), stem thickness (−0.025), primary branches per plant (−0.160), silique length (−0.224), silique width (−0.259), seeds per silique (−0.238) and shattering percentage (−0.069) were negatively associated with PC-VI.
PC-VII accounted 5.15% of the total variation among agro-morphological traits. Days to maturity 50% and 100% (0.022 and 0.016), leaf length (0.004), leaf length/width ratio (0.192), leaves per plant (0.228), stem thickness (0.165), plant height (0.106), silique length/width ratio (0.022), seeds per silique (0.059) and seed yield per plant (0.248) were contributed positively to PC-VII. Whereas days to flowering 50% and 100% (−0.309 and −0.251), leaf width (−0.107), primary branches per plant (−0.116), main raceme length (−0.143), siliqua per main raceme length (−0.030), silique length (−0.509), silique width (−0.414), 1000-seed weight (−0.338) and shattering percentage (−0.295) depicted negative contribution.
Maximum variation was calculated for shattering (427.2) followed by plant height (345.6), days to flowering 100% (336.2), main raceme length (210.0), days to flowering 50% (185.1), days to maturity 100% (122.9), days to maturity 50% (83.8). The results obtained from the present investigation were in closed agreement with that of     .
Our results were strengthened by many earlier plant breeders and scientists. The highly significant and positive correlation was observed between the parameters of days to flowering 50% and 100% and days to maturity 50% and 100% physiological maturity. These results were supported by the results of Nasim et al.  , Alemayehu and Becker  , by reporting highly significant and positive correlation among days to 50% and 100% flowering and days to 50% and 100% maturity. The results obtained by Zare  were also strongly supported our finding, who reported positive and highly significant correlation among important traits like days to flowering, days to maturity and plant height of B. napus L. accessions. Similarly Azadgoleh et al.  also reported positive and highly significant association among days to physiological maturity and plant height. Similarly the results of Rabbani et al.  also strengthen our findings. In the current investigation main raceme length was highly significant positive correlated with the trait of number of silique per main raceme. The results obtained by Rabbani et al.  and Nasim et al.  had also confirmed our findings. Plant height was also found positive and highly significant correlated to primary branches per plant, main raceme length and silique per main raceme which was also close agreement by previous research of Yasir et al.  who studied strong positive and significant association of plant height with primary branches per plant, main raceme length, pod on main raceme, pod length and seeds per pod.
Numerous plant breeders and plant scientist in the earlier era had practiced and got remarkable results of diversity in agro-morphological parameters for various Brassica individuals through two complementary methods i.e. cluster analysis and principal component analysis (PCA). These techniques were successfully observed in Indian mustard (Brassica juncea L.) germplasm by Gupta et al.  , in Ethiopian mustard by Alemayehu and Becker  and in white head cabbage Balkaya et al.  . All the above mentioned plant breeders and plant scientist’s results gives support to our present investigation that these 2 techniques are very supportive in estimated associations among individuals originated from different environments in a more clear approach. The classification of present studied Brassica juncea L. genotypes into various groups was not due to of its origination from diverse ecological zones of the country and world. Amurrio et al.  , who investigated qualitative and quantitative traits of Iberian pea genotypes, were agreement with our results. During the present investigation among different accessions of Brassica juncea L. considerable level of genetic differences was observed for different agronomic and morphological characters. These studied parameters can play significant and interesting role in Brassica juncea L. cultivars and varieties development programs in future. In present study the seven groups were recorded and PC1 showed highest level of variability. The same findings were also recorded by Dias et al.  in diverse germplasm of Brassica oleracea L. originated from Portugal.
Among all the characters, valuable genetic diversity was observed through different agro-morphological traits i.e. days to maturity 50% and days to maturity 100%, days to 50% flowering and days to flowering 100%, leaf length and leaf width, main raceme length siliquae per main raceme, leaf width, primary branches per plant, silique length. Most of the traits contributed highly significant association for the said trait during the present investigation. These genotypes show that there is great potential in the studied accessions, which can be used in future breeding program as a productive genotypes.
We acknowledge cooperation of Gene bank of PGRI, NARC, Islamabad, Pakistan for providing B. juncea germplasm.
Conflict of Interest
The author’s declares that there is no conflict of interests regarding the publication of this article.
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