Genetic relatedness among indigenous rice varieties in the Eastern Himalayan region based on nucleotide sequences of the Waxy gene
© Choudhury et al.; licensee BioMed Central. 2014
Received: 2 June 2013
Accepted: 17 December 2014
Published: 29 December 2014
Indigenous rice varieties in the Eastern Himalayan region of Northeast India are traditionally classified into sali, boro and jum ecotypes based on geographical locality and the season of cultivation. In this study, we used DNA sequence data from the Waxy (Wx) gene to infer the genetic relatedness among indigenous rice varieties in Northeast India and to assess the genetic distinctiveness of ecotypes.
The results of all three analyses (Bayesian, Maximum Parsimony and Neighbor Joining) were congruent and revealed two genetically distinct clusters of rice varieties in the region. The large group comprised several varieties of sali and boro ecotypes, and all agronomically improved varieties. The small group consisted of only traditionally cultivated indigenous rice varieties, which included one boro, few sali and all jum varieties. The fixation index analysis revealed a very low level of differentiation between sali and boro (FST = 0.005), moderate differentiation between sali and jum (FST = 0.108) and high differentiation between jum and boro (FST = 0.230) ecotypes.
The genetic relatedness analyses revealed that sali, boro and jum ecotypes are genetically heterogeneous, and the current classification based on cultivation type is not congruent with the genetic background of rice varieties. Indigenous rice varieties chosen from genetically distinct clusters could be used in breeding programs to improve genetic gain through heterosis, while maintaining high genetic diversity.
The indigenous rice varieties in NE India show remarkable diversity in morphological and agronomic traits including high variability in size, shape, aroma and nutritional properties of grains , disease resistance  and abiotic stress tolerance . A recent study revealed high levels of genetic diversity in these rice varieties with the highest genetic diversity in the varieties of the sali ecotype, followed by the jum and boro ecotypes . These rice varieties with exceptional phenotypic and genetic diversity can serve as an important source of germplasm for the genetic improvement of cultivated rice. A thorough understanding of genetic relatedness among these rice varieties is crucial for designing breeding programs for the genetic improvement of rice, allowing us to capitalize on genetic gain through heterosis while maintaining high genetic diversity.
The objective of the present study is to infer the genetic relatedness among indigenous rice varieties of sali, boro and jum ecotypes cultivated in NE India using the nucleotide sequences of the Wx gene. As a single copy nuclear gene with high polymorphism, the nucleotide sequences of the Wx gene is an ideal genomic tool to assess the genetic relatedness of rice varieties. The Wx gene, which encodes granule-bound starch synthase [8, 9], determines the amylose content in the endosperm and influences the glutinous nature of the rice grain. The nucleotide sequences of three Wx genes (Wx-A1, Wx-B1 and Wx- D1) reported from wheat  have been used successfully to infer genetic relatedness among wheat cultivars , highlighting the Wx gene’s suitability for determining genetic relatedness in crop plants.
The variety name, cultivation type and collection sites of traditionally cultivated indigenous and agronomically improved rice varieties in Northeast India (AP, Arunachal Pradesh; AS, Assam; ML, Meghalaya; MZ, Mizoram)
N. Lakhimpur, (AS)
N. Lakhimpur, (AS)
N. Lakhimpur, (AS)
N. Lakhimpur, (AS)
Garo Hills (ML)
West Siang (AP)
PCR amplification and sequencing
Oligonucleotide primer sequences used for amplification of the Wx gene
The DNA sequences were analyzed using the computer program Geneious version 5.4.6 (http://www.geneious.com/). The resulting consensus sequences were aligned using the software program ClustalW v2 . We used Bayesian, maximum-parsimony (MP) and neighbor-joining (NJ) methods to infer genetic relatedness of rice varieties. The Bayesian analysis infers the phylogenetic relationships based on posterior probability distribution using evolutionary models , whereas the MP analysis infers the evolutionary tree(s) with the minimum number of nucleotide changes . The NJ method uses a pairwise distance matrix to infer the genetic relatedness among taxa . Thus, the use of a variety of approaches that differ in underlying assumptions provided a means to assess the robustness of resulting phylogenetic trees.
The best model of nucleotide substitution obtained through Modeltest analy ses based on Akaike Information Criterion (AIC)
HKY + I + G
A = 0.275
C = 0.239
G = 0.213
T = 0.273
Ti/Tv ratio = 3.489
Among-site rate variation
Proportion of invariable sites (I) = 0.937
Variable sites (G)
Gamma distribution shape parameter = 0.148
Using mixed χ2 distribution
P-value = < 0.00001
The phylogenetic trees based on NJ and MP methods were inferred using the PAUP*  software. Kimura 2-parameter distances  were used in the NJ analysis following Saitou and Nei . The MP analyses were performed with full heuristic search with tree bisection-reconnection branch swapping and random order of taxon addition option. The robustness of tree topologies was tested with 1000 bootstrap replicates. Nodes with greater than 50% bootstrap support were retained in the tree.
Genetic relatedness among rice varieties was further analyzed through haplotype networks. In this analysis, a series of nested clades based on haplotypic or allelic networks were reconstructed. The haplotype network analysis infers evolutionary relationships among intraspecific populations and closely related species . The median-joining algorithm  as implemented in the software package NETWORK 4.5.1 (Fluxus Technology) was used in this analysis. The level of differentiation between the ecotypes was estimated by calculating FST values between pairs of populations using the DnaSP software .
The MP analysis resulted in 141 equally parsimonious trees with total length of 140 steps, and the consensus tree topology was similar to the tree based on the Bayesian analysis. Most varieties of the sali ecotype clustered within Group-I along with the varieties of boro and jum ecotypes (Additional file 1: Figure S2). The other clade (Group-II) comprised only indigenous rice varieties. The varieties clustered within Group-III were identical to the group that clustered together in the Bayesian analysis. The sole difference between the Bayesian and MP-based trees was the placement of two varieties of the sali ecotype (Harinarayan and Kakiberoin), which occupied a basal position in Group-II in the former analysis and in Group-I in the latter. The NJ analysis also showed similar tree topology, except for the Group-III varieties, which formed a separate cluster and occupied a basal position in Group -I (Additional file 1: Figure S3).
The pairwise differentiation ( F ST ) of ecotypes
Sali and Boro
Sali and Jum
Jum and Boro
In the present study, we investigated genetic relatedness among three different rice ecotypes in the eastern Himalyan region of NE India. The Bayesian, MP and haplotype network analyses resulted in similar tree topologies consisting of two major groups. This clustering pattern was not congruent with three commonly cultivated ecotypes (sali, boro and jum) in NE India, and suggests a polyphyletic nature of rice ecotypes . This could be attributable to two possible reasons. First, exchange of seed material between regions mediated through human migration , often associated with migration of traditional farmers seeking better opportunities , could lead to cultivation of genetically different varieties within a given geographical locality. Second, large scale flooding during monsoon rainy seasons often damages crop plants, and farmers generally seek seeds from other regions leading to seed exchange between different agroclimatic regions. The polyphyletic nature of rice varieties in the region is in agreement with a previous study based on chloroplast DNA, which suggested polyphyletic maternal lineages for O. sativa ssp. indica. Similar results were also reported in other crop species, including sweet sorghum and grain sorghum lines of Sorghum bicolor ssp. bicolor. Based on the nucleotide sequences of the Wx gene, eight to ten genetically distinct indigenous rice varieties within Group-II are discernible. Similarly, rice varieties in the genetically distinct Group III may also contain unique genotypes. Thus, these indigenous rice varieties can serve as a valuable germplasm for genetic improvement of cultivated rice.
Cultivated rice has been subject to human mediated selection for various traits of agronomic and ecological importance. Adaptation to various agroclimatic conditions and human-mediated selection may have contributed to diversification of rice varieties in the NE Indian region . The jum and boro ecotypes showed a high level of population differentiation (FST = 0.230), indicating local adaptation to contrasting habitats leading to high level of population differentiation [35–37]. The cultivation of varieties of the jum ecotype in dry, upland habitats, and the cultivation of varieties of the boro ecotype in low-lying irrigated land during the winter season may have led to the genetic isolation and genetic differentiation of varieties of these two ecotypes. Very low FST value (0.005) between sali and boro ecotypes at the Wx gene reflects high levels of gene flow between rice varieties of these two ecotypes  or the latter ecotype may have originated from the sali ecotype. Since cultivated rice is mostly self-pollinating , gene flow among varieties is minimal. Thus, the observed low differentiation between these two ecotypes could be attributable to the fact that the boro ecotype may have been selected from the sali ecotype to grow in low-lying areas during the winter season.
The present study based on the nucleotide sequence data of the Wx gene revealed a) the polyphyletic nature of sali, boro and jum rice ecotypes and b) two genetically distinct groups of rice varieties in NE India. One group consisted of only traditionally cultivated varieties, while the other group comprised both agronomically improved and traditionally cultivated rice varieties. The occurrence of genetically distinct groups of rice varieties in the region highlights the importance of rice genetic resources in NE India as potential source of germplasm for genetic improvement of cultivated rice to maintain global food security under changing climatic conditions.
Availability of supporting data
The aligned DNA sequences and phylogeny trees were submitted to TreeBASE (Accession number S14972) which can be accessed from the URL http://purl.org/phylo/treebase/phylows/study/TB2:S14972.
The authors thank the farmers of Northeast India and International Rice Research Institute, Philippines for providing samples for the present study. This study was supported by fNSERC Discovery Grant. BIC received MELS merit scholarship from FRQNT and Faculty of Arts and Science Graduate Fellowship from Concordia University. The comments received from anonymous reviewers are gratefully acknowledged.
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