Variability Assessment of Aromatic and Fine Rice Germplasm in Bangladesh Based on Quantitative Traits

The study was conducted to investigate genetic variability among 113 aromatic and fine local rice genotypes of which five were exotic in origin. The test genotypes were evaluated for 19 growth traits, yield components, and yield. All the quantitative traits varied significantly among the test genotypes. High heritability along with high genetic advance was observed for flag leaf area, secondary branches per panicle, filled grains per panicle, grain length, grain breadth, grain length breadth ratio, and 1000 grain weight. Such findings suggested preponderance of additive gene action in gene expression for these characters. Grain yield was significantly and positively correlated with days to flowering, days to maturity, panicle length, filled grains per panicle, and 1000 grain weight. According to D 2 cluster analysis, 113 test genotypes formed 10 clusters. Selection of parents from the clusters V and X followed by hybridization would possibly result in desirable heterosis for the development of heterotic rice hybrids. Finally, molecular characterizations of the studied germplasm are required for high resolution QTL mapping and validating the presence of candidate genes responsible for valuable characters.


Introduction
Bangladesh is mainly a country of rice based cropping system, where thousands of local rice varieties are being cultivated from the time immemorial [1]. To date, farmers across the country are used to cultivate different local varieties or landraces particularly in the unfavourable ecosystems. Local variety including aromatic rice genotypes occupied about 12.16% of the rice growing areas in Bangladesh [2]. Many of these local varieties have some special characteristics such as aroma, better taste, and higher cooking quality which also provide additional value in socioeconomic aspects. Moreover, aromatic rice constitutes a special group of rice genotypes well known in many countries across the world for their aroma and/or super fine grain quality [3]. Bangladesh has a stock of above 8,000 rice germplasms of which nearly 100 are aromatic [1,4]. It is worthwhile to mention that aromatic rice is closely related to the social and cultural heritage in Bangladesh and it is consumed during weddings and other festivals [5]. Aromatic and fine rice germplasm native to Bangladesh generally have short bold and medium bold grain type with mild to strong aroma [6,7]. In Bangladesh, among the different aromatic rice varieties, Chinigura is the predominant one that covers more than 70% of rice farms in the northern districts of Naogaon and Dinajpur. Other important aromatic rice varieties are Kalijira (predominantly grown in Mymensingh) and Kataribhog (mainly cultivated in Dinajpur) [8]. Most of the aromatic rice varieties in Bangladesh are of locally adapted, photoperiod-sensitive, and grown during Aman season under rainfed lowland ecosystem. The production cost of aromatic and fine rice is low compared to that of coarse rice. Therefore, the income potential is higher with aromatic fine rice cultivation, since its cultivation does not usually require additional expenditures 2 The Scientific World Journal on fertilizer, pesticides, and irrigation. However, the average yield of high yielding rainfed lowland rice is 3.4 t/ha, whereas that of aromatic rice is 2.0-2.3 t/ha [9].
Knowledge on genetic diversity among crop populations and its quantitative assessment usually helps a breeder to select suitable parents to be utilized in breeding programmes [10][11][12][13][14][15]. Among the different cereal crops, rice is (Oryza sativa) one of the best models to undertake the study of genome structure and genetic diversity. Its diploid genome is relatively smaller in size (430 Mb) with a significant level of genetic polymorphism and a large amount of well-conserved genetically diverse material [16][17][18].
In a breeding programme, genetic improvement primarily depends upon the amount of genetic variability present in the population. In many cases, characters are mostly governed by poly genes which are highly influenced by the environment. Therefore, it is difficult to predict whether the existing variability is heritable or not. Furthermore, heritability of a genetic trait is very important in determining the response to selection because it implies the extent of transmissibility of that trait into next generations [19,20]. In addition, high genetic advance coupled with high heritability offers the most effective condition for selection for a specific character [21].
These days, plant breeders usually evaluate genetic diversity on the basis of morphological traits because they are more economic, faster, and easier to score compared to the molecular traits [22,23]. Investigation to these traits also does not require any sophisticated procedure or advanced equipment. In addition, these traits can be transmitted without adapting any special biochemical or molecular techniques. The rice plant is morphologically diverse, especially in terms of the vegetative traits such as plant height and leaf length. Our previous studies involving local aromatic and nonaromatic rice germplasm from Bangladesh using morphological, physicochemical, and molecular markers revealed high genetic diversity [6,[24][25][26][27][28]. However, such investigations on aromatic and fine rice genotypes are not yet to be conducted. Therefore, the present study was undertaken to assess the genetic diversity of aromatic and fine rice genotypes in Bangladesh.

Experimental
Site. The experiment was conducted at the research farm of Bangladesh Rice Research Institute (BRRI), Gazipur, during July to December (T. Aman season), 2011. Geographically, the place is located at about 24.00 ∘ N latitude and 90.25 ∘ E longitude with an elevation of 8.4 meters from the sea level and is characterized by subtropical climate. The soil of the experimental site was clay loam in texture.

Plant Materials.
A total of 113 aromatic and fine rice genotypes (Table 1) were selected from BRRI genebank. Pregerminated seeds were sown in the seed bed.

Experimental Design and
Setting the Experiment. The experiment was conducted following a randomized complete block design with three replicates for each treatment. Thirtyday-old seedlings of each test genotypes were transplanted on the 15th August, 2011 using single seedling per hill in 2.4 m 2 plot with 25 cm and 20 cm space between rows and plants, respectively.

Intercultural
Operations. Fertilizers were applied @ 80 : 60 : 40: 12 kg N : P : K : S per hectare. However, except N, the other fertilizers were applied at final land preparation. Nitrogen was applied in three equal splits, at 15 days after transplanting (DAT), at 35 DAT, and just before flowering. Intercultural operations and pest control measures were done as and when necessary.

Data
Collection. Data were collected on culm diameter (mm), flag leaf area (cm 2 ), plant height (cm), days to flowering, days to maturity, effective tiller number (ET No.), panicle length (cm), primary branch length (cm), secondary branch length (cm), primary braches per panicle, secondary branches per panicle, number of filled grains per panicle, number of unfilled grains per panicle, grain length (mm), grain breadth (mm), grain length breadth ratio, 1000grain weight (g), grain yield per hill (g), and harvest index (HI). Kernel quality was determined using dehusked grains. Kernels were classified on the basis of length (size) and for / ratio (shape) following classification described by Cruz and Khush [29] (Table 2).

Statistical
Analysis. Univariate analysis of the individual character (ANOVA) including the estimation of mean, range, and coefficient of variation (CV%) was conducted using a statistical software package MstatC. The test of significance was performed using Fisher's ( ) test. Multivariate analysis was conducted using another statistical software package GEN-STAT version 5.5. Genetic parameters were also estimated to understand genetic variations among the test genotypes and to determine genetic and environmental effects on different characters. These genetic parameters were calculated using the following formula [31][32][33]. These parameters include the following: where GMS is the genotypic mean sum of squares, EMS is the error mean sum of squares, and is number of replication.
where 2 is the genotypic variance and 2 is the mean squares of error. (c) Genotypic coefficient of variation (GCV)% = √ 2 / × 100, where 2 is the genotypic variance and is the mean of character.
The Scientific World Journal 3     where 2 is the phenotypic variance and is the mean of trait.
(e) Heritability (broad sense) where 2 is the genotypic variance and 2 is the phenotypic variance.

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The Scientific World Journal where GA is a percent of the mean assuming selection of the superior 5% of accession: where is a constant, √ 2 / is the phenotypic standard deviation, ℎ 2 is the heritability, and is the mean of traits.

Variation in Grain Diversity and Genetic
Parameters among Accessions. The grain morphology varied considerably in genotypes collected from BRRI genebank ( Figure 1) with respect to awning, colour and size of awns, lemma and palea with presence or absence of coloured furrows and spots, pubescence, and varied coloured apiculus and sterile lemma. Analysis of variance of 19 quantitative characters based on individual sample means showed highly significant ( ≤ 0.01) variations among the genotypes for all the characters outlined in Table 3. The range, mean, standard error, coefficients of variation, and value of 19 characters are presented in Table 4. The coefficient of variation ranged from 1.69 to 35.81% which indicates considerable variation among the character studied. Out of 19 traits, unfilled grains per panicle, harvest index, yield per plant, filled grains per panicle, primary branches per panicle, and secondary branches per panicle found with relatively higher coefficient of variation (35.81, 20.45, 18.70, 16.79, 11.61, and 11.03%, resp.) than the other traits. These possibly occurred because of sampling error and/or characters were more influenced by the environmental factors. In this study, most of the growth traits showed higher PCV compared to yield and yield component traits. However, lower PCV belonged to days to maturity  panicle (29.32%) were recorded with higher values of PCV. However, panicle length (6.31%), days to flowering (7.40%), and plant height (10.04%) were found with lower values. The higher GCV was associated with 1000-grain weight (33.18%) whereas the value was fairly low in case of panicle length (5.06%). Results also showed narrow differences between PCV and GCV for most of the traits. Heritability ranged from 29.03 to 97.44%. The highest and the lowest amount of heritability were recorded at grain length and yield per plant, respectively. Days to flowering, grain breadth, grain length breadth ratio, plant height, and days to maturity were highly heritable,  all with an estimated 2 > 0.90 whereas other characters showed relatively low heritability. GA ranged from 0.03% for harvest index to 48.19% for filled grains per panicle. The genetic advance as percent of mean (GA%) ranged from 6.41% in panicle length to 50.85% in 1000 grain weight. In this study, flag leaf area, secondary branches per panicle, filled grains per panicle, grain length, grain breadth, grain length breadth ratio, and 1000-grain weight showed high heritability and high genetic advance indicated the presence of additive genes controlling these characters (Table 5).

Association between Traits.
Pearson's correlation coefficient was computed between 19 quantitative traits among 113 accessions of aromatic and fine rice genotypes (Table 6). Culm diameter was significantly and positively correlated with flag leaf area, days to flowering, days to maturity, plan height, and primary branches per panicle. Plant height showed highly significant positive correlation with culm diameter, days to flowering, days to maturity, and panicle length. Grain yield was highly significant (

Principal Component Analysis (PCA).
Eigen values (latent roots) of 19 principal component axes and percentage of total variation accounted for them obtained from component analysis are presented in Table 7. The result revealed that the first axis largely accounted for the variations observed among the genotypes (48.8%) followed by the second axis (10.37%). The first nine axes accounted for about 90% of the total variations among the 19 characters describing 113 aromatic and fine rice genotypes where only 59.17% variation was accounted for the first two axes.

Cluster Analysis.
The pattern of distribution of 113 aromatic and fine rice genotypes were grouped into 10 clusters shown in Table 8. The number of genotypes ranged from 3 to 19 in different cluster. The distribution pattern indicated that the maximum number of test genotypes (19) was grouped into the cluster I followed by 18 in clusters VIII, 17 in clusters III, 13 in clusters IV, 11 in clusters II, 10 in clusters X, 8 in clusters V and IX, and 6 in cluster VII. Cluster VI contained the lowest (3) number of genotypes. Results of 10 higher and 10 lower intergenotypic distances estimated from distant matrix of Principal Coordinate Analysis are shown in Table 9. Highest intergenotypic distance was 2.274 observed between Gopalbhog and Kalobakri followed by the distance of 2.126 observed between Haitsail TAPL101 and Kalobakri. The 10th highest distance of 1.522 was observed between Jirabuti and Straw TAPL554 followed by 1.528 observed between Ranisaluit and Jirakatari. The lowest distance was calculated (0.299) between Doiagura and Jiradhan followed by the distance of 0.301 observed between Kamini soru and Jirabhog (bolder).

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The Scientific World Journal Intra-and intercluster distances value are presented in Table 10. There were marked variations in intracluster distances which ranged from 0.61 in cluster VI to 1.27 in cluster II indicating homogeneous nature of the genotypes within the cluster. The highest intracluster distance was computed for cluster II (1.27) which was comprised of eleven genotypes followed by cluster IV (1.01) with thirteen genotypes. The genotypes under cluster II (with the highest intracluster mean) were most heterogeneous and genotypes under cluster VI (with the lowest intracluster mean) were comparatively homogenous.
The intercluster distances ranged from 3.710 to 16.116. Regarding the intercluster distance, the highest value was found between clusters V and X (16.116) followed by clusters II and X (15.791) and so on. On the other hand, the lowest intercluster distance was observed between clusters I and III (3.710) followed by clusters III and IX (3.775) indicating that genotypes of these clusters were genetically closed.
The mean values for all of 19 characters along with the marking of the highest ( ) and lowest ( ) for each of the cluster are presented in Table 11. Differences in cluster means existed for almost all the characters. Genotypes of cluster VI produced the highest mean for days to flowering (DF), days to maturity (DM), plant height (PH), and yield per plant (Y/P). Genotypes in cluster II had higher mean values for flag leaf area (FLA), secondary branch length (SBL), grain length (GL), grain length breadth ratio (GLBR), and 1000grain weight (1000). Higher mean values for panicle length (PL), secondary branches per panicle (SB No.), filled grains per panicle (FG/P), and harvest index (HI) were recorded in cluster X whereas those for effective tiller (ET No.) per plant and grain breadth (GB) were recorded in cluster IV.

Discussion
During the current study, all traits showed highly significant ( < 0.01) variations among 113 accessions, which originated in Bangladesh except Khazar, Basmati 37, Basmati 370, Basmati Sufaid 106, and Basmati Sufaid 187 genotypes. Our results are in close agreement with those of Pandey et al. [34] who recorded highly significant variability among the different rice genotypes. Similarly the finding of Wang et al. [35] also gives support to the current findings. The findings of Chandra et al. [36] and Abarshahr et al. [37] further strengthen the current findings, who also found valuable and highly significant and positive variability among their studied genotypes.
The dependence of grain yield on other traits has been reported for many crops [38]. As mentioned, in this study, yield of plant had positive correlation with 8 quantitative traits. Lasalita-Zapico et al. [39] studied correlation coefficient of 10 quantitative traits for 32 upland rice varieties. In this distinguished significant positive correlation the majority of the morphological traits was recorded except flag leaf angle that had negative correlation with most of characters such as panicle length, leaf length, leaf width, ligule length, leaf area, and culm length. In our studies, grain yield positively correlated with panicle length. The findings indicate that plants with high panicles have high number of filled grains thereby increasing rice yield. Similar correlations were reported by Zafar et al. [40].
The calculation of heritability and genetic advance are used to help the breeder to select traits that are highly heritable as compared to a trait which is less heritable [33]. Both high heritability and genetic advance value obtained in this study, flag leaf area, secondary branches per panicle, filled grains per panicle, grain length, grain breadth, length breadth ratio, and 1000-grain weight indicated reasonable variation for this traits. This suggests that selection can be easily practiced by using these traits to improve grain yield in aromatic rice genotypes. The results support the findings of Sedeek et al. [41], Laxuman et al. [42], and Pandey et al. [34] who reported such type of heritability in rice.
In the present study, 113 aromatic and fine rice genotypes were clustered into ten groups based on 19 quantitative traits. This result supports the findings of Singh et al. [43] and Rao et al. [44] who reported ten clusters in rice genotypes. Ahmadikhah et al. [38] clustered 58 rice varieties into four groups based on 18 morphological traits and genetic distance was around 0.75. Group A was comprised of only one genotype and groups B, C, and D contained 14, 20, and 23 genotypes, respectively. Veasey et al. [45] computed clustering for 23 populations of rice by 20 morphological characteristics. So the varieties were clustered into 10 groups; the last group was the biggest group with seven members and groups 1, 2, 7, and 8 were the smallest groups including only one variety. So, genotypes having distant clusters could be hybridized to get the higher heterotic responses. The similar  Principal component analysis indicated diversity among 113 aromatic and fine rice genotypes. "Proper values" measure the importance and contribution of each component to total variance, whereas each coefficient of proper vectors indicates the degree of contribution of every original variable with which each principal component is associated. The higher the coefficients are, regardless of the direction (positive or negative), the more effective they will be in discriminating between accessions. In the present study, the first three axes accounted for about 66% of the total variations. Lasalita-Zapico et al. [39] computed approximately 82.7% of total variation among 32 upland rice varieties, 66.9% variation for PC1, and 15.87% for PC2. Rajiv et al. [49] reported the first two principal components accounting for 82.1% of total variation in control and 68.6% in the stress induced genotypes. To obtain greater heterosis, genotypes having distant clusters could be used as parents for hybridization program. In Bangladesh, most of the aromatic rice genotypes are traditional, photoperiod-sensitive, tall stature, and lower yields with mild to strong aroma and also they showed high variability (6,28). In the present study, it was observed that the genotypes in clusters V and X (16.116) were more diverse than the genotypes of clusters I and III (3.710). Considering cluster distance and cluster mean, the highest mean value for panicle length (cm), secondary branch length (cm), filled grains per panicle, and harvest index was observed in cluster X, which means that those traits might be selected for their high heterosis. Therefore, selection of parents for hybridization program from clusters V and X may result in the desirable heterosis for heterotic rice hybrids. Genotypes under cluster II may also give higher heterosis, if crossing is done within the genotypes of this cluster due to high value of intracluster distance.

Conclusion
In the present study, flag leaf area, secondary branches per panicle, filled grains per panicle, grain length, grain breadth, grain length breadth ratio, and 1000-grain weight showed high heritability and high genetic advance in percent of mean had high heritability and high genetic advance. Yield of plant had positively correlated with days to flowering, days to maturity, panicle length, filled grains per panicle, and 1000grain weight. The cluster analysis placed 113 aromatic and fine rice genotypes into ten groups. The highest intercluster distance was observed between clusters V and X followed by clusters II and X. The maximum value of intercluster distance indicated that the genotypes belonging to cluster V were far diverged from those of cluster X. So, it is expected in our results that parent's selection for hybridization from the clusters V and X may give the desirable heterosis for heterotic rice hybrids. Finally, molecular characterizations of the studied germplasm are required for high resolution QTL mapping and validating the presence of candidate genes responsible for valuable characters.