Aromatic rice is superior-quality rice having fragrance along with other grain and cooking quality characteristics. Owing to these properties, they are popular among the consumers realizing a higher market value. In the rice gene pool, aromatic rice cultivars form a distinct group (Group V) as revealed by the isozyme analysis (Glaszmann, 1987; Khush, 2000). At the global level, most popular aromatic rices include Basmati rice from the Indo-Gangetic plains of the Indian subcontinent, Jasmine rice from Thailand, and Sadri rice from Iran.

North-eastern India is one of the major agro-biodiversity hotspots in the world, enriched with more than 10,000 diverse indigenous diverse rice cultivars including both aromatic and non-aromatic rice (Mao et al., 2009). Special among these are distinctly scented landraces such as Joha cultivars of Assam (Talukdar et al., 2017), Chakhao cultivars of Manipur, Tai cultivars of Mizoram, and Kampti cultivars of Arunachal Pradesh, which are grown and conserved by farmers over ages and distributed across different ecological niches (Durai et al., 2015; Roy et al., 2015). The Manipur state of North-eastern India is an isolated hilly region encircled by nine hill ranges and a central valley having climate varying from tropical to subalpine (GoM, 2018). Chakhao, meaning “delicious rice” in Manipuri, is the most popular aromatic rice of Manipur which also includes several lesser-known landraces. Chakhao cultivars have either pigmented (black, amubi) or non-pigmented (white, angouba) rice kernels. The cultivars with colored pericarp are distinct from other rice varieties originating from different parts of India (Gayacharan et al., 2018; Tulsiram et al., 2018). Particularly for sociocultural uses, farmers grow several of the Chakhao landraces such as Chakhao Poireiton, Chakhao Amubi, Chakhao Sempak, Ching Chakhao, Chakhao Angouba in local farm holds in smaller areas, covering less than 10% of the holdings. Historical accounts describe that black rice was restricted to the Royals, and the local Meitei community used it only during religious festivals and special occasions (Borah et al., 2018). Among the Chakhao rice, those with deep-pigmented kernels are popularly called “Manipur black rice.” They possess a high anthocyanin content in the pericarp, conferring antioxidant properties. Recognizing their exquisite nutraceutical quality, geographical indication (GI) status has been conferred to Manipur black rice in 2019 by the Government of India, registering it under GI No. 602 in the Geographical Indication Registry¹ (GoI, 2019).

There are several landraces of aromatic rice of Manipur that share the common epithet Chakhao but remain seldom characterized. Earlier explorations during different periods conducted in Manipur have collected several such landraces that are conserved in the National Gene Bank (NGB) at the ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR), New Delhi (Hore, 2005). In one study, the genetic diversity of 37 Chakhao landraces was assessed using 40 microsatellite (SSR) markers to reveal significant gene diversity (0.673) with markers having a PIC value of 0.63. These landraces were found grouped into six classes, having close correlation with farmers’ classification (Roy et al., 2014). Significant variation was also reported for yield-related traits in ten black rice genotypes of Manipur (Asem et al., 2019). By biochemical analyses, Asem et al. (2015) showed that the major anthocyanin fraction of black rice genotypes, Chakhao Poireiton and Chakhao Amubi, was delphinidin 3-galactoside, with Chakhao Poireiton having an average anthocyanin content of 740 mg/kg, and total phenolic content ranging from 5 to 6 g/kg of dried flour. Later, the 26 aromatic compounds were reported from Chakhao Poireiton, while 11 were reported from Chakhao Amubi (Asem et al., 2017). Another study by Chanu et al. (2016) reaffirmed the presence of high levels of anthocyanins, polyphenols, and zinc content having significant antioxidant activity in Chakhao landraces. However, the earlier studies suffered from one or other shortcomings, either having been carried out on a limited number of genotypes or having been characterized only for morphological, biochemical, or molecular variation. Therefore, a comprehensive study was felt necessary to assess the variation among several of the Chakhao rice including representative landraces, for agro-morphological, biochemical, and molecular diversity. There is no report of anthocyanin profiling and their variation across different black scented landraces cultivated in Manipur. Accordingly, the present study characterizes one of the comprehensive germplasm sets of aromatic rice landraces originating from Manipur including black rice, for phytochemical properties such as pigmentation, anthocyanin content, and antioxidant activities together with agro-morphological, molecular, and grain qualities such as cooking and aroma.

A total of 93 aromatic rice germplasm accessions collected from different parts of Manipur covering both hill and valley ecosystems were used in the study (Supplementary Table S1). Among these, 79 genotypes were sourced from NGB, ICAR-NBPGR, New Delhi; seven were collected from farmers’ field in Manipur and seven were sourced from ICAR-Regional Center for North-Eastern Hill Region (ICAR-RC-NEH), Manipur center. The genotypes were initially grouped based on a priori information on pericarp pigmentation, aroma, and adaptation ecologies (Table 1). The most contrasting feature of the study material was their diversity for spikelet and pericarp pigmentation (Figure 1). All the genotypes were initially multiplied at ICAR-Indian Agricultural Research Institute (ICAR-IARI), New Delhi, during Kharif 2017. During Kharif 2018, the genotypes were grown at the ICAR-RC-NEH Region, Manipur center, in lowland rainfed conditions. Each genotype was grown in three rows of 2.7 m length with a spacing of 20 cm between rows and 15 cm between plants. The field experiment was laid out in augmented design with four blocks and five non-aromatic checks, viz., RC Maniphou 7, RC Maniphou 10, RC Maniphou 11, RC Maniphou 12, and RC Maniphou 13. The experimental crop was raised with standard agronomic practices to maintain and harvest a good crop. The postharvest grain quality analysis, estimation of anthocyanin compounds, and molecular work were carried out at the Division of Genetics, and Division of Agricultural Chemicals, ICAR-IARI, New Delhi.

Aromatic rice genotypes from north-eastern India, particularly from Manipur, are relatively lesser known cultivars because of their confined cultivation within their geographical adaptation. Recent interest on their culinary and nutraceutical properties, particularly of Manipur black rice, turned the attention of rice scientists in understanding the genetics of these genotypes. Known by their vernacular names, cultivars carrying the epithet Chakhao—chak means “rice” and ahaoba meaning “delicious” (Dayanidhi et al., 2017)—are a conglomeration of local landraces that share similarity in grain aroma and cooking and taste properties but genetically different. The scented glutinous Chakhao with black pericarp are predominantly grown in valley districts of Manipur. The black rice of Manipur has a long history of exclusive adornment of royal cuisine and highly restricted use outside the aristocracy, and their culinary properties were lesser known until recently. Recent studies on black pigmented rice in general, have established their dietary significance especially on nutraceutical properties (Goufo and Trindade, 2014; Samyor et al., 2017). Notwithstanding, the genetic identity of aromatic rices of Manipur, particularly of black rice, needs to be established to realize their potential use. Therefore, there is an immediate need to characterize their genetic diversity and to utilize them in genetic improvement, as well as to conserve them for the posterity of future generations.

Agro-morphological characterization for assessing the genetic diversity in crops draws the foundation for genetic improvement. In rice, plant height, crop duration, and grain yield are three major agronomic parameters used for determining productivity, while secondary characteristics such as panicle length, grain number, grain weight, grain color, and shape are used for classification and identification (Bhandari et al., 2017). Although limited, past research on Chakhao rice landraces of Manipur was carried out using fewer genotypes using fewer traits (Roy et al., 2014; Asem et al., 2015; Chanu et al., 2016). Our analyses attempt to address this lacuna, by characterizing a larger set of aromatic landraces for agro-morphological, grain cooking quality, pigmentation, and antioxidant properties as well as at the molecular level. We found no distinct association of any trait(s) with the landrace names. On the contrary, we could find that landraces sharing the same epithets differed significantly for most of the traits assessed. However, the aromatic genotypes with a deep pericarp color differed significantly for plant height, panicle length, and grain yield per plant. In general, the most widely used epithet was Chakhao among Manipur rice. In our study, there were 69 landraces that carried the name Chakhao, or its close dialectical variants such as Chakhao and Chahou. Among these, there were 12 landraces that shared the name Chakhao Poireiton or its close resembling name Chakhao Poireitol. Twenty-four genotypes, however, were only called as Chakhao. Further, there were ten cultivars that carried the suffix Amubi and seven that had Angouba suffixed. Prominent cultivars such as Chakhao Poireiton generally had strongly scented deep purple/black kernel, with tall stature, long panicles, and low grain yield. Chakhao Angouba cultivars were white colored with moderate scent, while Chakhao Amubi included all categories of pericarp color, from light brown, variegated brown, to deep purple. Buhman was the second prominent name among the landraces in the panel, with 16 cultivars carrying this name. While the landraces from valleys mostly carried the prefix “Chakhao,” the landraces from the hill districts carried the name such as “Buhman” (Churachandpur district), “Maklei” (Ukhrul district), and “The Vumnu” (Chandel district). Majority of the light brown pericarp color genotypes originated from hill districts. Genotypes from hill districts varied significantly from valley genotypes for their plant height and panicle length. Irrespective of the collection ecology and pericarp color, grains of the landraces were of low amylose type, with majority of them having long slender kernels. The traditional preference of the local community of Manipur is for glutinous rice with soft cooking quality. They are exclusively used in kheer preparation called Chakhao Thongba. Chanu et al. (2016) also observed low amylose in two major cultivars, namely, Chakhao Poireiton and Chakhao Amubi. They also found these two cultivars to be nutritionally rich with high dietary fiber, protein, and minerals as compared to a popular rice cultivar, Sona Mashuri, grown in southern India. Another study that evaluated 10 aromatic rice together with three non-aromatic indigenous cultivars of Manipur for grain quality revealed that most of them possessed long bold chalky grains with high ASV and low to intermediate amylose content (Thongbam et al., 2010). Our study revealed that the landraces were low yielding, exhibited high spikelet sterility, were grain shattering, and were photo-sensitive (data not presented). Since most of these landraces are conserved and grown at their respective collection sites, in order to meet the necessities of social rituals rather than for subsistence farming, there has been no serious attempt made for their systematic genetic improvement. Besides, over the decades, there has been a decline in the area of aromatic rice of Manipur with farmers opting not to grow these landraces owing to their very low yield (Borah et al., 2018). If this trend continues, it might lead to genetic erosion of these valuable indigenous landraces. Alternatively, because of the benefits such as nutraceutical properties, pigmentation, glutinous endosperm, and aroma, pigmented rice is gaining popularity among the consumers across the world. Additionally, we found that some of the landraces also possessed desirable traits such as early maturity and relatively higher grain yield, which would provide ample scope for crop improvement. For systematic improvement of Manipur aromatic rice, for both yield and quality, it is desirable to generate a profile of desirable traits within this gene pool, such as kernel pigmentation and antioxidant properties as well as undesirable traits such as photosensitivity, seed shattering, low spikelet fertility, and poor yield.

Analysis of genome-wide variations using molecular markers is one of the means to delineate the evolutionary relationship between genotypes that are believed to share a common ancestry. SSRs are highly useful in assessing diversity among closely related rice cultivars (Singh et al., 2013; Yadav et al., 2013) as they provide better resolvability, are multi-allelic, provide genome-wide coverage, are highly reproducible, are easy to score, and are cost-effective (Akagi et al., 1997; Singh and Singh, 2015). There are several studies that used SSR markers for finding the genetic structure of the rice germplasm, as one of the most widely used molecular markers for genetic diversity studies (Nachimuthu et al., 2015; Singh et al., 2016; Aljumaili et al., 2018; Islam et al., 2018a, b; Pathaichindachote et al., 2019; Suvi et al., 2019; Verma et al., 2019). Further, SSR markers are also proven to be efficient in delineating major genetic groups of rice, namely, indica, temperate japonica, tropical japonica, aus, and aromatic (Roy et al., 2015, 2016; Wang et al., 2014). In the present study, the panel of 48 SSR markers used could establish the diversity pattern within the Manipur aromatic rice germplasm. Although the gene diversity (0.443) as well as the PIC value (0.394) was lower than those observed from previous studies (Das et al., 2013), this could be attributed to the narrow ecological range from which the lines were sourced, as well as to a greater number of genotypes tested in this study. Notwithstanding, a significant level of genetic variation observed within the lines could be due to their long history of evolution in specific ecologies. An attempt was made in the present study to classify the aromatic rice landraces into rice ecotypes indica, tropical and temperate japonica, and aus and their admixtures based on molecular data. The germplasm contained three subpopulations with the checks distributed in the first two subpopulations. As expected, indica genotypes (POP2) dominated japonica types (POP1) by number and possessed more admixtures. The hilly accessions were more of japonica type (POP1) and had very less admixtures. This indicated that even as there was population pressure coming from the majority of indica subtypes, the japonica types retained their genetic identity among the Manipur aromatic germplasm. Earlier, Roy et al. (2016) reported the predominance of japonica and their admixtures among the hill rice of northeast India. POP2 included genotypes such as MAR16, MAR51, MAR54, MAR57, MAR58, MAR68, and MAR98, which possessed most of the rare and unique alleles from both hill and valley regions. Pigmented rice formed a separate large subpopulation (POP3) among the genotypes tested. The origin of black rice of Manipur could be related to introduction of japonica rice from China, beginning from second century BC (Tensuba, 1993; Singh and Baghel, 2003; Lalit, 2007). Prominent among the members of POP3 were Chakhao landraces that included both pigmented and unpigmented types. Our study also revealed that the pigmented rice showed significant variation in the content of phytochemicals such as anthocyanin and phenolics, implicating that the Chakhao landraces had undergone isolated conservation in the local farm holds. As the RSA was found highly correlated with color parameters, these lines also possessed high antioxidant properties in the rice grain.

Earlier, genome analysis of 21 black rice landraces demonstrated that the origin of black rice gene occurred within tropical japonica, which later migrated to indica and then to temperate japonica (Oikawa et al., 2015). Signature of cross subspecific migration of black rice trait could also be seen in the present study, wherein one landrace Maklei (dark brown) from the hill district of Ukhrul was grouped along with japonica type, whereas the remaining black rice landraces were grouped within the indica subgroup cluster. However, this observation needs to be further confirmed by taking more reference entries of respective subgroups and evaluating the diversity with high-density genome-wide markers. Further, reinforcing the theory of cross subspecific migration, based on population structure, we could identify a subpopulation (POP3) with predominance in genotypes with deep purple pericarp as well as for hill and valley adapted genotypes. However, Roy et al. (2014) reported six subgroups such as Chakhao Poireiton, Chakhao Amubi, Chakhao, Maklei, Buhman, and Chakhao Angouba in 37 Chakhao landraces based on 47 random SSR markers, which could be due to a limited number of genotypes used in the study.

Rice pigmentation is attributed to the accumulation of anthocyanins in the pericarp of the grains. Anthocyanins are subgroups of flavonoids which are water-soluble pigments imparting different shades of red, blue, purple, to plant parts. They belong to a class of secondary metabolites of the polyphenol group. In the present study, C3G and P3G were found to be the predominant anthocyanin compounds, while the unidentified fraction was quantified as C3GE compounds. The identification was based on the mass spectral values depicted as m/z ratio. The aglycones, cyanidin and peonidin, have a m/z value of 287.05 and 301.07, respectively (Abdel-Aal et al., 2006; Kim et al., 2008; Lee, 2010), which, on addition of the sugar moiety attachment of glucose (m/z: 162.00), makes the total m/z to 449.1 and 463.1 corresponding to C3G and P3G. In our spectrum, corresponding signals were identified as the prominent peaks confirming the presence of C3G and P3G. The presence of both fractionated and unfractionated residues, served as a reconfirmation of the result. Identification of similar compounds was earlier reported from the black rice cultivars, Heugjinjubyeo (Lee, 2010) and Kilimheugmi (Ryu et al., 1998) from Korea and black rices of China (Sompong et al., 2011). Hou et al. (2013) identified the constituents of the C3GE fraction from the japonica black rice variety, Longjing No. 1 of China, as cyanidin 3,5-diglucoside and cyanidin-3-rutinoside. Among the Japanese black rice, Chen et al. (2012) identified four different anthocyanins such as C3G, P3G, malvidin, and petunidin-3-O-glucoside (Pt-3G). Asem et al. (2015) reported delphinidin 3-galactoside, delphinidin 3-arabinoside, cyanidin 3-galactoside, and C3G from Chakhao Poireiton. Further, they had also identified the first three of these compounds in Chakhao Amubi. However, the identification of these compounds in their study was based on the comparison of retention time (RT) reported in earlier publications, which is very subjective in nature leading to a possibility of errors due to changes in the solvent system and the instrumentation conditions of the assessment.

The total anthocyanins among the rice landraces in the present study varied widely, imparting different color shades to the kernels. It is interesting to note that genotypes sharing the same epithet(s) as a part of their names like Chakhao Poireiton, Chakhao Amubi, and Ching Chakhao but which were collected from different localities within Manipur significantly varied for total anthocyanin content and its constituents. There was no significant difference in patterns between genotypes from hill districts and valley districts. Although anthocyanin content is reported to be influenced by the environment (Somsana et al., 2013), in the present study, the genotypes were grown uniformly in the experimental plots at a valley region (Imphal), and hence, the variation among the different lines could be purely genetical. Further, pigmentation had no influence on the genotype adaptation to different ecologies. However, any influence of pericarp development, seed coat thickness, grain shape, and weight on the anthocyanin accumulation and color intensity among the pigmented rice of Manipur needs further detailed investigations. Shen et al. (2009) have reported that flavonoid and phenolic contents were positively related to grain shape and negatively related to grain weight. Likewise, we could also observe that the intensity of the pigmentation depended on the proportion and content of pigment compounds, starting from light color to dark. Deeply pigmented rice kernels rather appear black and are popularly traded as black or purple rice. The nutraceutical properties of the pigmented rice and their potential health benefits have been established using cell lines, animal models, and human clinical trials (Pojer et al., 2013; Das et al., 2014; Samyor et al., 2017; Khoo et al., 2017; Seechamnanturakit et al., 2018; Thanuja and Parimalavalli, 2018; Callcott et al., 2019; Limtrakul et al., 2019).

Traditionally, purple or black rice is grown and consumed in many Asian countries like China, Thailand, Sri Lanka, Republic of Korea, Vietnam, Indonesia, India, Philippines, and Japan. In India, popular black rice varieties include Chakhao rice from Manipur and Kalabhat from West Bengal. The anthocyanin content of black rice from Korea was as high as 493 mg.100g^–1, having a large fraction of C3G varying between 80 and 95.3% (Ryu et al., 1998), while Lee (2010) reported C3G content ranging from 52.1 ± 6.3 to 1601.0 ± 8.5 μg.g^–1 and P3G content from 0.0 to 82.6 ± 1.2 μg.g^–1. Anthocyanins being the constituent of polyphenols in plants, the total phenolic content show a significant correlation with anthocyanin content, particularly in pigmented rice (Dai and Mumper, 2010; Deng et al., 2013).

The radical scavenging property of antioxidant compounds provides immense health benefits by scavenging reactive oxygen species (ROS) and other harmful free radicals when oxidative stress occurs (Dröge, 2002). When cellular machinery fails to contain the stress, antioxidants such as anthocyanins can prevent damage either by delaying the oxidative process or by scavenging the excess free radicals. However, antioxidant property differs in different anthocyanins depending upon their molecular structure. The activity increases with the increased number of free hydroxyl group around the pyrone ring (Miguel, 2011). Among the most common anthocyanins, cyanidin is the most active against superoxide after delphinidin. In the present study, C3G was found to be the predominant anthocyanin pigment in Chakhao landraces, which could play an important role in scavenging the superoxides. The RSA of the pigmented rice was significantly higher than that of unpigmented genotypes, ranging from more than two to eight times. Chanu et al. (2016) reported an RSA of 72.5% in Chakhao Poireiton and 59.0% in Chakhao Amubi. Although RSA identified in this study was slightly less than these values, we could observe a maximum activity of 65.7% in our panel. However, the presence of a large variation in free RSA vis-à-vis total anthocyanin observed in different deep purple genotypes with similar epithets necessitates defining the baseline values for grain quality and nutraceutical standards aiding improvement and promotion of this speciality rice.

In the present study, we elucidated a large variation for the agro-morphological, grain quality, pigmentation, and phytochemical characteristics in the hitherto uncharacterized aromatic rice landraces of Manipur. Although several of them shared common epithets, we found that they are significantly diverse and shared distinct subpopulation memberships based on genetic data. Despite the commonness in names, the average gene diversity of the landraces was 0.443, implying their genetic uniqueness. This necessitates efforts for conservation, documentation, and utilization of this diversity for further improvement. The striking feature of Manipur aromatic rice is the variation for pericarp pigmentation ranging from white to dark purple/black color. Several of the pigmented genotypes are low in amylose with soft cooking quality and high anthocyanin content. Two growing niches in the region viz., valley and hill, also show a distinct pattern of genotype characteristics. A detailed investigation with high-throughput markers such as single nucleotide polymorphisms (SNPs) providing high-density genome-wide coverage can help in assessing the genetic relatedness of these genotypes with indica and japonica subtypes, as well as for mining novel allelic variants that may be present in this gene pool. There is also a need to assess grain quality through metabolic profiling, quantification of aroma, and micronutrient content, which can lay a strong foundation for in situ conservation and improvement of these unique landraces from Manipur.

All datasets presented in this study are included in the article/Supplementary Material.

AS and SG conceptualized the idea and supervised the experiments. SB, SG, and NS: carried molecular work. SB, HB, and SS carried and monitored quality, phytochemical estimations. RE, KV, and SB carried out the statistical analysis. SB, IS, NP, PB, and MN carried and monitored field experiments. SB, KV, and SG prepared manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.