Genetic Diversity of Seed Storage Proteins in Pigmented and Non-pigmented Indigenous Paddy (Oryza sativa L.) Landraces of Assam, India

Indigenous landraces of major crops are the product of natural evolution and selection pressure and hence gift of nature, which sustained the food security of mankind for thousands of years. Red rice is a group of paddy where the kernel colour has characteristics of red to purple red colour of varied intensity. Red rice is relatively unfamiliar to general public since it is seldom marketed but it has attracted the attention of researchers because anthocyanin is known to be a powerful antioxidant. Pigmented (red, brown, purple and black) rice in particular has gained a lot of attention as raw materials for the production of commercial health food supplements due to its high phenolic, anthocyanin and antioxidant contents. Based on cultivation practice and season in Assam paddy cultivars are classified into four major groups, viz., Ahu (autumn paddy), Sali (winter paddy), Boro (summer paddy) and Bao (deep water paddy). Red rice landraces are found among all the four groups, particularly in Bao group which are rich in nutritive and nutraceutical values. Loying et al., (2010) studied such lesser-known landraces, particularly their genetic diversity is very limited. Protein polymorphism serves as genetic marker as they are direct products of active genes and are quite polymorphic and generally heritable (Gepts, 1986). Both DNA based and seed protein profile-based markers are used for molecular characterization by a number of workers. However, DNA based markers generate variable designate results since different primers generate different profiles, which are often not reproducible and consistent. By contrast seed protein profile is consistent, stable and reproducible since seed proteins are storage protein and only variation is due to genetic factors. The high stability of seed protein profile and its additive nature make it a promising tool for distinguishing genotypes of particular plant species. Therefore, in the present study SDS-PAGE technique was employed for analysis of seed protein diversity in different red and white indigenous landraces collected from different districts of Assam. That apart even far and many landraces of conventional white rice are also endangered to various degrees of continuous negligence. This loss of agrobiodiversity is a matter of serious concern and threat to food security (Swaminathan, 2011). The present study was undertaken in the backdrop of such scenario. The study involves molecular characterization and diversity analysis of pigmented (red) and non-pigmented (white) by seed protein profile with SDS-PAGE technique. Seed protein electrophoretic profile is gaining acceptance and International Seed Testing Association (ISTA, 1996) has recognised it as a tool for identification of cultivar species and to study intraspecific variability.

Altogether 20 indigenous landraces of paddy were collected for evaluation. Among them eleven were pigmented red rice and other nine landraces were non pigmented. All these landraces were collected from the different districts of Assam, viz., Nagaon, Sonitpur, Darrang, etc. The pigmented red paddy landraces used in this study were Amona (AM1), Biroi (BI2), Kabra (KA3), Kablam (KA4), Kabrabadam (KA5), Bil Bao (BB6), Godasali (GS7), Lalkartisali (LK8), Ronga Kurmi (RK9), Kura Binni (KB10), Kokowa (KK11) (Fig 1) and the non pigmented white paddy landraces were Agnisali (AS12), Lotasali (LS13), Suhagmani (SM14), Kalizira (KZ15), Laki (LK16), Tengeri (TG17), Ranjit Amon (RA18), Boro (BR19), Bismuthi (BS20) (Fig 1). Seed protein profiles were worked out by SDS-PAGE method outlined by Laemmli (1970). Manually dehusked grains (300 mg) were extracted with ice-cold Tris-buffer 0.2 M (PH: 7.5). Seed proteins were resolved in two phase acrylamide gel, stacking gel 4%, separating gel 13.5%. Protein bands were visualized by staining with Coomassie Brilliant Blue R-250 stains. Protein molecular weight marker (PMW-M, Bangalore Gennie) was co-electrophoresed to determine the molecular weight of the individual protein. After gel scoring the data were analysed to prepare similarity index (SI) matrix using Nei and Lis coefficient. From this, the dendrogram was prepared by unweighted pair group method with arithmetic average (UPGMA) using NTSYS PC V2, 02j Software.

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A number of workers working with different paddy groups show that genetic diversity in paddy can be effectively analysed and documented based on seed protein profile. Analysis of seed protein profile resolved by SDS-PAGE revealed a total of 15 protein bands among the landraces in the present study (Table 1). Considerable variation has been observed, in protein profile which is evident from the fact that seed protein varied from 12.1 Kd to > 122 Kd (Table 1). Among the landraces, the highest number of 15 protein bands was observed in Ranjit Amon, Boro and Bismuthi. On the other hand, lowest number of protein bands was observed in Amona with 9 protein bands (Table 1). Three other cultivar is Lalkartisali, Kura Binni and Laki exhibited second highest number of protein bands with 14 protein bands each (Fig 4, Table 1). However, their profile was different. Among the protein bands, some were consistently found to be present in all the landraces. Among them two have molecular weight >122 Kd, while another had molecular weight 112 Kd (Fig 4). Among low molecular weight protein only the one with molecular weight 15.0 Kd was found in all the 20 landraces (Table 1). On the other hand, the protein with molecular weight 74 Kd occurred in lowest frequency since it was found in only 11 landraces (Table 1). Loying et al., (2010) worked with 10 deep water paddy (Bao) cultivars of Assam and reported a 22 protein bands ranging in size from 97.40 Kd to -~13.2 Kd. The authors also reported three protein bands of 26.7, 17.0 and 15 .7 Kd as molecular marker for deep water paddy. Tiwari (2010) worked with 12 indigenous landraces of Boro rice or spring rice reported a total 14 protein bands in the size range of 16.2 to 9.0 Kd. Like Joha paddy, Boro landraces also exhibited considerable genetic diversity and three proteins with molecular weight 16.2, 24.75 and 31.4 Kd were found to be marker for Boro group landraces, since they were consistently found in all the landraces (Dutta Roy et al., 2010).



Analysis of similarity index (SI) matrix revealed considerable variability among the landraces as revealed by SI values. Among the landraces, SI values varied from 47% to 100% (Table 2). On the higher size 100% SI value was observed between a number of landraces e.g., between Agnisali and Tengeri as well as between Tengeri and Suhagmani (Table 2). On the other hand, Amona exhibited the least similarity with any other landrace which is reveal from the fact that the SI value of Amona with other landraces varied from 60 to only 73 (Table 2). The variability in SI matrix was reflected in the dendrogram which reveal the phylogenetic relationship among the landraces. Dendrogram analysis reveals a total 4 cluster (Fig 2). The first and biggest cluster contains a total of 7 landraces with similarity coefficient among them ranging from 85 to 100 (Fig 2). The second cluster had 5 landraces viz., Lalkartisali, Kura Binni, Ranjit Amon, Boro and Bismuthi. The fourth cluster was the smallest with two landraces, viz., Godasali and Ronga Kurmi with similarity coefficient of 85 between them (Fig 2). The most notable aspect of the dendrogram is that red rice and white rice did not form separate clusters; rather both red and white rice cultivars were found to belong to same cluster. The first cluster had seven landraces and out of them, five were red rice while the remaining two were white. Likewise, in cluster 2 there were five landraces and out of them, three were white while the rest two were red (Fig 2). However, those landraces which exhibited 100% similarity were either red or white e.g. Agnisali, Suhagmani and Tengeri exhibited 100% SI value and all three were white (Fig 2). Similarly, Biroi, Kabalam exhibited 100% similarity and both are red (Fig 2). Apart from qualitative difference, there was also some quantitative difference also as revealed by pixel intensity analysis (Fig 3). Pixel intensity spectra reveal that most of the high molecular weight proteins have low concentration of protein and in the gel, they appeared as very thin band and corresponding pixel intensity are also lowest. By contrast most of the low molecular weight protein had relatively high concentration of protein. In the gel they appeared as thick and deeply stain bands. Among them the protein with molecular weight 15 Kd was found to be thickest and most prominent in the gel and its corresponding pixel intensity was also found to be highest. Another low molecular weight protein with size 22.2 Kd was found to exhibit second highest pixel intensity implying that protein concentration for this band is second highest (Fig 3).

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Among the landraces in the present study, two namely Amona and Ronga kurmi appears to be unique and very distantly related to the remaining landraces. Because they did not belong to any cluster in the dendrogram (Fig 2). The landrace Amona exhibited genetic distance of about 0.84 and SI value in the range of 57.14 to 69.23 implying that genetically it is most distantly related to other landraces in the present study. The other landrace Ronga kurmi also exhibited similar genetic dissimilarity with others. By contrast, some landraces are morphologically different with aspect to seed morphology, but at molecular level they were found to be identical. For instance, Tengeri, Suhagmani and Agnisali were morphologically distinct but at molecular level they were, found to be identical with genetic distance 0.0 between them (Fig 2).

Dendrogram analysis showed that within the same clusters there is both red rice as well as white rice landraces in random and there was no separate cluster for red or white rice. Quantitative study by pixel intensity analysis showed that mostly low molecular weight proteins particularly the one with 15 Kd have relatively high concentration of protein while the high molecular weight proteins occur in relatively low concentration. However, in the present study two high molecular weight protein (97.4 and 90.0 Kd) occurred in relatively high amount. Santos et al., (2013) predicted three proteins in wild rice Oryza glumaepatula as glutelin (34-36 Kd), albumin (15-25 Kd) and prolamin (15-18 Kd).

Several rice protein bands have been detected only on specific rice both of SDS PAGE. This study suggested the specific protein bands can be used as biochemical markers for rice and further more study to predict their functional protein. No crop has as much genetic variability as paddy and paddy represents the largest gene pool with a recorded number of 75,0000 to 10,0000(IRRI). Many workers, corroborates the views that molecular analysis is a powerful and proven tool to find out genetic similarity and dissimilarity among closely related landraces and it can be used to identify a particular landrace or cultivar as recognized by International Seed Tenting Association (ISTA).

The authors are thankful to village headmen and village farmers for sharing their knowledge, help and support during the field study. We are also thankful to the Director, IASST, Guwahati, Assam for instrumental and laboratory facilities.