Genetic Diversity Analysis of “Murungakayan” Rice Accessions based on Seed Morphology and Molecular Characterization

Genetic diversity of germplasm among species and within the species is important for systematic conservation of genetic resources. This study was conducted to assess the genetic diversity and to identify similar groups among the twenty-seven Murungakayan accessions conserved at the gene bank of Plant Genetic Resource Centre, Gannoruwa. Seed morphological characterization was done according to the standard descriptors for rice and data were analysed using Minitab 15.1. Molecular assessment was done using 31 SSR markers and PCR amplified products were resolved using 8% polyacrylamide gel electrophoresis. Bands were manually scored and analyzed using PowerMarker version 3.25. Morphological analysis grouped these accessions into ten clusters. The molecular analysis based on SSR markers generated 108 alleles across 27 populations. Polymorphism Information Content values ranged from 0.00 (RM 518 and RM 237) to 0.72 (RM 515) which revealed high genetic diversity. Nei’s genetic distance among accessions ranged from 0.00 to 0.9 and a dendogram grouped the accessions into eight clusters. Four sets of duplicates were identified with high genetic similarities. According to the molecular and morphological analyses, accessions 3490, 5378, 5614, 6279, 12824, 3329, 3491 and 5611 can be identified as the representative set of Murungakayan with high similarity. The results suggested that microsatellite markers along with morphological data could be used for identification of the representative set of Murungakayan accessions for systematic conservation and utilization.


INTRODUCTION
Landraces of rice play a very important role as the main staple food, in addition to their significance as a genetic resource for rice genetic improvement (Tang et al., 2002). Presently most of the rice lands in Sri Lanka (99%) are cultivated with New Improved Varieties (NIVs). At a minor extent, some traditional and old improved varieties are cultivated in the country (Rajapakse et al., 2000). The Plant Genetic Resource Centre (PGRC) explored and conserved the genetic resources of rice including traditional cultivars, wild relatives, old improved and exotic germplasm. Out of catalog of 14310 conserved accessions at PGRC, 4924 accessions were Oryza spp. Among them 684 were traditional varieties which include 2407 accessions (Gene bank information PGRC, 2017). Traditional rice cultivars continue to have a good and increasing market value (Wickramasinghe and Noda, 2008;Suriyagoda et al., 2011) due to their antioxidant properties and high nutritional value (Premakumara et al., 2013). Murungakayan is one of the popular traditional rice varieties with high nutritional value. Based on the Ayurvedic literature it was extensively recommended for pregnant mothers. Its nutritional values were considered conducive for the growth of the fetus and relieving the mother at her confinement (Withanawasam, 2017). There are 27 Murungakayan accessions available at PGRC. Accessions used in this study have been collected from different parts of the country hence there is a significant variation observed among them. Majority of them are spindle-shaped with red seed coat color.
The seed bank of PGRC, accessions are conserved by the vernacular name given by farmers. Therefore, the same cultivar may have been conserved under different names or conversely different cultivars under the same name. Study of genetic diversity among accessions appearing with the same cultivar name is critically important for systemic conservation and utilization of plant genetic resources. These genetic variabilities can be analyzed based on morphological and molecular data. Morphological data are the basis for the early and preliminary level of diversity analysis. Since molecular markers are independent of environment effects they can assist in the analysis of genetic relatedness and the identification and selection of desirable genotypes for crosses as well as for systematic germplasm conservation in gene banks. On the other hand, due to the high demand for traditional rice cultivars, Department of Agriculture plans to certify popular traditional cultivars with minimum standards for seed production. For that correct identification of a representative set of accessions for the cultivar is a prerequisite. Accordingly, the aim of this study is to identify the diversity among 27 Murungakayan accessions and to determine a representative set of accessions for seed production.

Experimental site and plant materials
This study was conducted at the Biotechnology division of Plant Genetic Resource Centre (PGRC), Gannoruwa, Peradeniya. Seeds of 27 Murungakayan rice accessions were obtained from the gene bank of PGRC. Accession number, accession name of the material used and its origin and organization collected from are given in Table 1.

Seed morphological and molecular characterization
Seven qualitative and quantitative data were recorded. Seeds were evaluated based on the seed shape, color and pubescence of lemma and palea, seed coat color, sterile lemma color, grain length and grain width. Characterization was done according to the descriptors of rice (IPGRI Database, 2017). DNA was extracted using modified CTAB (Cetyltrimethylammonium bromide) method optimized at PGRC, Gannoruwa (Wasala et al., 2017). Two grams of three-week old immature leaves from 27 plants were sampled to represent the whole population. Raw DNA was confirmed in 0.8% agarose gel and quantified by comparing with the band intensity of 25 ng λ DNA and concentration of DNA was adjusted to 20 ng/ µl.
Based on the grouping of accessions by seed morphology data, different bulks were prepared by pooling equal amount of DNA from each accessions for the convenience and for the cost effectiveness. A set of optimised 31 SSR markers (Temnykh et al.,2000) were selected for the molecular characterization. Polymerase chain reaction was performed according to the protocol optimized at PGRC (Wasala et al., 2012). A 15 μl of PCR reaction mixture was prepared for each DNA bulk and for BG 360 which was used as the standard accession. Amplification profile consisted of five touchdown cycles where annealing temperature was reduced by 2°C at each cycle using Takara Thermal Cycler Dice Touch (Code.TP350). PCR products were confirmed using 1.5 % agarose gel electrophoresis and resolved using 8% (Acrylamide: Bis of ratio 29:1) non denaturing polyacrylamide gel electrophoresis in 1X TBE buffer. Finally, bands were visualized under UV light using a gel documentation system (BIO-RAD) with the help of Quantity One software. If there were more than one band available within the bulk, they were expanded and rerun.

Data analysis
Cluster analysis was done using MINITAB 15.1. Gel images were scored manually by visual observation as presence (1) and absence (0) of every allele for all SSR markers. Scored data were analyzed using PowerMarker version 3.25 and MEGA 6.06. Major allele frequency, allele number, PIC value and gene diversity, were calculated across the population to identify the prevailing genetic diversity. Nei's genetic distance (1983) was calculated among the accessions. Phylogenetic tree was constructed by using Nei's genetic distance and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithm. Finally, representative set was selected placing more emphasize on molecular data.

Morphological analysis
A majority of morphological traits showed variation among accessions. Seed shape ranged from half spindle shape (6-7mm), spindle shape (7-8mm) to very spindle shape (>8mm). Most of the Murungakayan accessions displayed short pubescence on lemma and palea. One accession (AC: 3900) showed pubescence only on the upper portion. Four accessions (AC:6283, 5610,3855 and 3329) showed long pubescence. AC 3900, 3328, 3331 have brownish black furrows on the straw background while AC 3921, 5611, 3492 and 6280 have yellowish brown colored lemma and palea. In seed coat color, AC 3328 is having off white seed coat while rest of them have red color. AC 3331 and 3495 have both red and off white color seed coat but the majority are red in color (Table 2). Based on the morphological analysis 27 accessions were clustered into ten groups at more than 75% similarity level. According to the dendrogram most distantly related accession was AC 3328 which has white seed coat. Most closely related accessions were AC 5611 and AC 6280 followed by AC 3921 and AC 6281.

Genetic distance
Based on Nei's genetic distance (Nei's,1983), genetic distance ranged from 0.9 to 0.0. Maximum distance (0.9) was recorded between two sets. (Bg 360 -AC 6283 and Bg 360 -AC 3331). Since Bg 360 is an improved variety its genetic distance showed higher values from the rest of the Murungakayan accessions. Four sets of accessions were identified with high genetic similarities. During this study, a significant degree of genetic diversity was observed among the accessions. Compared to the genetic diversity analysis of traditional rice varieties in Sri Lanka such as Pokkali, Suwandal, Kuruluthuda and Kaluheenati, Murungakayan shows a number of similarity groups. There were no such similarity groups observed in Suwandal (Wasala et al., 2017), four pairs of similar groups were observed in Pokkali and Kaluheenati (Thotagodawatta et al., 2017;Siriwardhana et al., 2016), and one pair of genetically similar accession was identified in Kuruluthuda. (Karunadasa et al., 2017) However, when comparing genetic diversity, Murungakayan is having higher diversity (0.00-0.76) than Kaluheenati (0.110-0.70) but similar genetic diversity in Kuruluthuda (0.30-0.79) and Pokkali (0.33-0.77). Therefore, they have been subjected to selections when compared to other traditional rice varieties as they have been collected from farmers from all over the country. Based on the summary statistics higher major allele frequency was recorded in the SSR markers RM 237 and RM 518 as 1.0 which shows that they are monomorphic.
Murungakayan 302 was the parental line of H-4, which was an old improved verity. H-4 is the first bred variety in Sri Lanka with wide adaptability and resistant to bacterial leaf blight (RRDI database). During this study four accessions (AC 5613,5378,5614,3329) of Murungakayan 302 line has been studied. Based on molecular analysis three of them (AC 5378, 5614, 3329) are grouped into cluster 7 and the other accession (AC 5613) grouped separately into the cluster 6 showing that there is genetic variation present in the same line. Considering morphological and molecular analysis accessions: 3490, 5378, 5614, 6279, 12824, 3329, 3491 and 5611 are identified as the representative set of Murungakayan for systematic utilization.

CONCLUSIONS
The present study exhibited that there is a significant variation among 27 Murungakayan accessions. Giving more emphasize to molecular analyses eight accessions namely 3490, 5378, 5614, 6279, 12824, 3329, 3491 and 5611 having closer relatedness to each other were selected as the representative set of Murungakayan which can be used as donor parents in breeding programs, seed production and certification.