Journal of Genetics (2019) 98:92 © Indian Academy of Sciences https://doi.org/10.1007/s12041-019-1131-0

RESEARCH ARTICLE

Genetic diversity and population structure analysis of Asian and African aromatic ( L.) genotypes

ANURAG MISHRA1,2, PUSHPENDRA KUMAR2∗, MD. SHAMIM3, KAPIL K. TIWARI4, PARVEEN FATIMA2, DEEPTI SRIVASTAVA5, RAJENDRA SINGH6 and PRASHANT YADAV7

1Division of Genetics, Indian Agricultural Research Institute, New Delhi 110 012, India 2Department of Agricultural Biotechnology, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut 250 110, India 3Department of Molecular Biology and Genetic Engineering, Dr.Kalam Agricultural College, Bihar Agricultural University, Sabour, Bhagalpur 813 210, India 4Department of Plant Molecular Biology and Biotechnology, C. P. College of Agriculture, S. D. Agricultural University, S. K. Nagar 385 506, India 5Integral Institute of Agricultural Science and Technology, Integral University, Lucknow 226 026, India 6Zonal Research Station, Nagina, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut 250 110, India 7Directorate of Rapeseed Mustard Research, Bharatpur 321 304, India *For correspondence. E-mail: [email protected].

Received 18 October 2018; revised 2 April 2019; accepted 14 June 2019; published online 6 September 2019

Abstract. Rice germplasms collected from different regions could be used as valuable resources for the future breeding programme. For the utilization of such collections, knowledge about the level and distribution of genetic diversity among these collections will facilitate the breeder. In this study, we report the phenotypic correlation, biochemical quality parameters and population genetic analysis of 35 rice accessions including 34 aromatic rice from different countries and a nonaromatic, Nagina 22, a well-known drought resistance variety. Further biochemical quality analysis, gel consistency test, molecular diversity analysis with 55 simple sequence repeat markers, population structure analysis and pair wise FST analysis were also conducted to assess the genetic diversity. The collected rice genotypes showed significant variability in different agronomic traits, i.e. spikelet per panicle, branches per panicle etc. Results obtained from the above tests demonstrated the importance of regional genetic studies for understanding the diversification of aromatic rice in Asian and African rice.

Keywords. rice; aroma; alkali spreading value; gel consistency; amylose; diversity; simple sequence repeat primers.

Introduction chemical compound 2-acetyl-1-pyrroline (2AP). Due to the presence of aroma in aromatic rice, they are in higher Rice (Oryza sativa L.) is one of the most widely demand than nonaromatic rice and also plays a vital role cultivated crops in the world. It is a diverse crop, due to in global rice trading (Blakeney et al. 1992; Weber et al. its adaptation to different geographical, ecological and 2000; Shobha et al. 2006). Aromatic rice can be further climatic regions. Most of the world’s rice is cultivated grouped into long and short grain type. Among the long and consumed in Asia, home for more than half of the grain aromatic rice, Basmati rice is more valuable because global population (Chakravarthi and Naravaneni 2006). of its cooking quality and are at higher demand. Basmati On the basis of aroma, rice can be grouped into aromatic rice elongates at least twice its original size after cook- and nonaromatic. Aromatic rice (scented rice or fragrance ing and has a delicious taste, superior aroma as well as rice) is very popular in Asia, United States, Europe and distinct flavour. India and Pakistan are the traditional in Middle East. Aroma in rice is due to the presence of producers and exporters of Basmati rice. In international

1 92 Page 2 of 19 Anurag Mishra et al. rice market, Basmati rice fetches three times higher price in cases where the morphological markers are inaccurate. than high-quality nonBasmati rice. Quality parameters Genetic diversity, relationship and population structure like grain dimensions, rice colour, texture of cooked rice, studies among the rice genotypes are valuable for different intensity of aroma and cooking quality analysis revealed purposes (Ndjiondjop et al. 2018). These purpose include large variation in rice under different agro-climatic con- the selection of parental combinations for the creation of ditions. Cooking quality traits of rice also varied despite different progenies that are phenotypically superior and having similar amylose content (AC), gel consistency (GC) with significantly higher yield potential compared to their and alkali spreading value (ASV). However, any aromatic parents (Mohammadi and Prasanna 2003). In the present rice is not accepted as Basmati in the trade (Sharma and study, we conducted morphological, biochemical, molec- Goel 2011). The molecular or DNA markers are preferred ular characterization along with diversity analysis of these over the traditional morphological and protein markers, aromatic and nonaromatic rice genotypes. The informa- because molecular markers are stable at different environ- tion generated in the present study can be further used in mental conditions as well as various developmental stages the aromatic rice breeding programme. (Yong-Jin et al. 2009). Among the molecular markers, sim- ple sequence repeat (SSR) is widely used in genetic diversity and gene pool study. SSR or microsatellite markers are tandem repeats of few nucleotides interspersed through- out the genome and can be amplified using primers that Materials and methods flank these regions (Grist et al. 1993). Various SSR mark- ers that varied in the degree of polymorphism have been Plant materials developed and their position is mapped in rice (Temnykh et al. (2000); McCouch et al. (2002)). Diversity assessment Thirty-five diverse rice germplasms collected from differ- of Indian rice germplasms have been conducted by the ent sources were sown in the randomized block design help of various researchers using molecular markers (Saini (RBD) manner with three replication at the field of the et al. 2004; Ram et al. 2007; Sundaram et al. 2008; Kumar Crop Research Centre and Laboratory, Sardar Vallab- et al. 2010; Sivaranjani et al. 2010; Vanniarajan et al. 2012). hbhai Patel University of Agriculture and Technology, Taxonomic classification using molecular marker is a nec- Meerut. Data of different parameters were recorded in two essary step to determine population structure especially consecutive years (figure 1; table 1).

Figure 1. Different location of countries from where genotypes were taken for the study of diversity. Asian and African aromatic rice genotypes Page 3 of 19 92

Table 1. Detailed information of aromatic rice genotypes used in the study.

No. of ancestor Release Genes for Genotype Pedigree used year Origin important traits

1 Basmati 370 Pure line selection for – 1962 Pakistan Traditional basmati export Dehradun traditional quality, long duration, LS basmati 2 PusaSugandh 4 P614-1-2/P 614-2-4-3 2005 India Evolved basmati irrigated, super fine, semidwarf, long duration, LS 3 Taraori basmati Pure line selection from 1996 Taroarikernal, Traditional basmati, Karnal local India semidwarf, long duration 4 Type 3 basmati Pure line selection for 1990 Dehradun, Traditional basmati, basmati Dehradun basmati India tall long duration, export quality, LS 5 Pusa basmati 1 Pusa 150/Karnal local 3 1996 India Evolved basmati high yield, irrigated, super fine, semidwarf, short duration, export quality, LS 6 Basmati 386 Pure line selection from – 1994 Punjab, India Traditional basmati export Karnal local quality, super fine and long duration, LS 7 PusaSugandh 5 Pusa 3A× Haryana India Medium duration irrigated, basmati super fine, semidwarf, LS 8 Vallabh basmati 22 Pusa 1121 × Type 3 2009 India Export quality, medium duration, super fine, LS 9 Vallabh basmati 21 Khalasa 7/Pusa 2013 SVPU&T, Evolved basmati 1121/Type-3 Meerut, India 10 Haryana basmati Sona/Basmati 370 7 1991 Pau, India Basmati 11 Basmati CSR 30 BR4-10/Basmati 370 – 2001, 2012 CSSRI, Evolved basmati, salinity KernaL tolerant. 12 Ranbir basmati Single plant selection 1996 Jammu Basmati &Kashmir, India 13 Vallabh basmati 24 Pusa 1121×Type 3 2015 SVPU&T, Export quality, medium Meerut, India duration, super fine, LS 14 Kalanamak Land race Sidharthnagar, Aromatic, short grain India 15 IR7734-93-2-3-2 NSIC RC 148/PSB RC 7 2002 IRRI Aromatic 18//NSIC RC 148 16 IR60080-46A IR47686-08-4-3/CT 12 1996 IRRI Aromatic 6516-21-4-4 17 IR78554-145-1-3-2 IR72861-13-2-1-2/IR- 14 IRRI Aromatic 68450-36-3-2-3 18 IR78006-55-2-3-3 IR67406-6-2-3/IR72860- 13 2005 IRRI Aromatic 80-3-3-3 19 IR77736-54-3-1-2 NSIC RC 148/PSB RC 7 2002 IRRI Aromatic 64//NSIC RC 148 20 IR77512-2-1-22 IR68726-3-3-1- 2002 IRRI Aromatic, semidwarf 2/IR71730-51-2 21 IR78537-32-1-2-1 IR65610-38-2-4-2-6- 14 2005 IRRI Aromatic 3/IR60912-93-3-2-3-3 22 WAB99-84 ITA 257/WABUKA 10 1995 Warda Aromatic 23 TOX3440-17-1-1-1-1-1 TOX891-212-1-201- 6 1998 Warda Aromatic, resistant to shield 105/TOX3056-5-1 bug 24 WAS515-B-10A-1-4 Unknown – 2000 Warda Aromatic 25 TOX3867-19-1-2-3-3 TOX3118-78-2-1/ITA 8 2000 Warda Aromatic 234 26 WAS272-B-B-5-H5 3290/WABC 165 6 2005 Warda Aromatic 92 Page 4 of 19 Anurag Mishra et al.

Table 1 (contd)

No. of ancestor Genes for Genotype Pedigree used Release year Origin important traits

27 TOX3226-5-2-2-2 ITA 235/ 8 1995 IITA Aromatic, resistant to IR9828-91-2-3/CT 19 blast and motile virus 28 WAS197-B-6-3-12 IR31851-96-2-3-2- 14 2005 Senegal Aromatic 1/IR66231-37-1-2 29 WAS197-B-5-2-5 IR31851-96-2-3-2- 14 2005 Senegal Aromatic 1/IR66231-37-1-2 30 WAS-197-B-5-2-16 IR31851-96-2-3-2- 14 2005 Senegal Aromatic 1/IR66231-37-1-2 31 WAS-197-B-4-1-25 IR31851-96-2-3-2- 14 2005 Senegal Aromatic 1/IR66231-37-1-2 32 WAS197-B-4-1-22 IR31851-96-2-3-2- 14 2005 Senegal Aromatic 1/IR66231-37-1-2 33 WAS197-B-6-3-16 IR31851-96-2-3-2- 14 2005 Senegal Aromatic 1/IR66231-37-1-2 34 WAS197-B-6-3-4 IR31851-96-2-3-2- 14 2005 Senegal Aromatic 1/IR66231-37-1-2 35 Nagina 22* – – Nagina, India Drought and heat stress resistance

◦ Evaluation of morphological traits 27–30 C. The spreading value of the grain was measured at a seven-point scale as follow: 1, when grain is not affected; Data were recorded on five competitive plants from the 2, grain is swollen; 3, swollen grain with incomplete narrow middle row of the each genotype for yield and yield com- collar; 4, swollen grain with complete wide collar; 5, split ponent traits such as days to 50% flowering (DF), days or segmented grain with complete wide collar; 6, dispersed to maturity (DM), plant height (PH), number of tillers and merging grain with collar; 7, completely dispersed and (NT), panicle length (PL), branches per panicle (BPP), intermingled grain (Perez and Juliano 1978). panicle bearing tillers (PBT), spikelet per panicle (SPP), filled grains per panicle (FGPP), unfilled grains per pan- icle (UGPP), spikelet fertility percentage (SFP), leaf area AC (LA), length breadth ratio (LB), test weight (TW), biolog- ical yield (BY), grain yield per plant (GYPP) and harvest Rice flour, 100 mg was taken in 1 mL of 95% ethanol and index (HI). 9 mL of 1.0 N NaOH. This was mixed well and heated in a boiling water bath for 10 min. Samples were diluted in 100 mL distilled water. Five mL of sample was taken Evaluation of grain quality traits in 1-mL acetic acid (57.75 mL in 1-L H2O) to acidify the sample along with 1.5 mL of iodine solution (0.2% iodine Aroma test: Five grams of rice grains were taken in 15 mL + 2% potassium iodide) and the volume was made 100 of water and soaked for 10 min. Soaked seeds were cooked mL with distilled water. The samples were incubated at for 15 min, transferred into a Petri dish and placed in the room temperature for 20 min. The absorbance was mea- refrigerator for 20 min. Then the cooked rice was sniffed by sured at 620 nm using a spectrophotometer (Perkin Elmer, a random panel of five experts and classified as SS, strongly Lambda 25). NaOH solution was used as a control. The scented rice; MS, mildly scented rice; NS, nonscented rice AC of different varieties was calculated in comparison category as per the scoring (Anonymous 2004). with a standard graph as described by Perez and Juliano (1978).

ASV and clearing test GC Six-milled rice grains were taken in Petri plates and 10 mL of potassium hydroxide (19.54 g of potassium hydroxide Rice flour, 100 mg was taken in a test tube (2 × 19.5 cm), dissolved in 500 mL H2O and final volume was adjusted 0.2 mL of ethanol containing 0.25% thymol blue and 2.0 to 1 L) was added to the samples. The samples were kept mL of 0.2 N of KOH were added and kept in boiling water undisturbed for 23 h in an incubator set at temperature of bath for 8 min, cooled, mixed well and kept in an ice bath Asian and African aromatic rice genotypes Page 5 of 19 92

Table 2. Variability observed for phenotypic traits.

Trait Mean ± SE Range CV (%)

DF 96.44 ± 1.40 53.20 (Nagina 22)–124.85 (Kalanamak) 1.76 DM 126.55 ± 0.95 84.37 (Nagina 22)–156.01 (Kalanamak) 1.30 PH in cm 114.87 ± 2.32 85.60 (TOX3440-17-1-1-1-1-1)–158.90 (Kalanamak) 3.51 PL in cm 30.17 ± 1.69 22.83 (Nagina 22)–43.24 (Taraori Basmati) 9.73 BPP 11.80 ± 0.38 8.93 (Nagina 22)–14.50 (IR78554-145-1-3-2) 5.61 NT 13.57 ± 1.05 9.47 (WAS197-B-5-2-5)–19.00 (Basmati 370) 13.40 PBT 11.86 ± 0.87 7.90 (WAS197-B-5-2-5)–17.97 (Basmati 370) 12.85 SPP 163.70 ± 2.41 90.00 (TOX3440-17-1-1-1-1-1)–253.00 (Vallabh Basmati 24) 2.56 FGPP 153.79 ± 2.50 103.63 (Taraori basmati)–238.25 (Vallabh basmati 24) 2.82 UGPP 11.07 ± 1.39 4.60 (PusaSugandh 4)–17.40 (IR78006-55-2-3-3) 20.99 SFP 93.08± 1.06 89.34 (Ranbir Basmati)–96.45 (Basmati 370) 1.98 LA 27.16 ± 1.63 20.13 (TOX3440-17-1-1-1-1-1)–41.64 (Basmati 370) 10.55 LB 3.83 ± 0.18 2.38 (WAS515-B-10A-1-4)–5.38 (Vallabh basmati 22) 8.19 BY in g 32.27 ± 0.73 2.58 (WAS272-B-B-5-15)–67.35 (TOX3440-17-1-1-1-1-1) 3.88 TW in g 23.28 ± 0.25 19.32 (TOX3867-19-1-2-3-3)–29.07 (WAS515-B-10A-1-4) 1.84 GYPP in g 11.34 ± 0.18 0.72 (WAS272-B-B-5-15)–23.81 (TOX3440-17-1-1-1-1-1) 2.72 HI 34.83 ± 1.98 28.01 (IR78554-145-1-3-2)–42.88 (Nagina 22) 9.87

for 20 min. Later the test tubes were laid horizontally for Data analysis 1 h and measurements were taken using graph paper. The degree of disintegration of the kernel was evaluated using The phenotypic data were analysed for the descriptive pur- a 7-point scale as described by Bhattacharya (1979). pose, as well as for character association. Cluster analysis was performed using a pair group distance with Euclidean similarity measures (Ward’s analysis) using XLSTAT soft- Selection of SSR markers ware for a genotypic factor that scores corresponding to significant principal components (PCs). Analysis of Fifty-five microsatellites (SSR) markers were selected ran- the population structure of the germplasms was done domly from the set developed by the Cornell University, using Structure 2.3.4 software based on SSR genotyping Ithaca (McCouch et al. 2002) for the molecular character- data. The dendrogram was constructed using the software ization of collected rice genotypes (table 2). package MEGA7. The polymorphism of markers were determined by calculating the polymorphism information content (PIC) for each SSR locus, according to the formula Marker assay (Botstein et al. 1980),  2 Total genomic DNA isolation was done by the CTAB PIC = 1 − pi method (Murray and Tompson 1980) and assayed with Where pi is the frequency of the Ith allele (Smith et al. a total of 55 SSR markers. The PCR reactions were 1997). carried out in sterile 0.2 mL thin-walled PCR tubes. Amplification was carried out in 20 μL reaction volume (DNA template 25–50 ng 1 μL, 10 mM dNTP mix- Results ture 0.6 μL, 1 U/μL Taq DNA polymerase 0.5 μL). PCR reaction buffer 2.0 μL, forward (F) primer 1.0 μL, The morphological trait of rice germplasm shows a wide reverse (R) primer 1.0 μL, double distilled water 13.5 μL. range of variation among the genotypes. The morpholog- Amplification was done in a thermo-cycler (LongGene) ical trait such as PL, NT, PBT, UGPP, LA, LB and HI which was programmed as follows: pre denaturation at showed the high coefficient of variation (table 2) whereas, 94◦C for 3 min, 35 cycles of denaturation at 94◦Cfor DF, DM, PH, BPP, SPP, FGPP, SFP, BY,TGW and GYP 1 min, annealing at 55◦C for 1 min and primer exten- showed the lowest coefficient of variation. Days to 50% sion at 72◦C for 1 min) followed by final extension at flowering were recorded minimum for Nagina 22 (53.20 72◦C for 7 min. Amplified products were separated on days) while maximum for Kalanamak (124.85 days). The 3.5% agarose gels and run for 2 h in 1× TAE buffer. Ampli- days to maturity (DM) were early in Nagina 22 (84.37 days) fied alleles were visualized under UV transillumination by while late in Kalanamk (156.0 days). The PH ranged from using Alpha Imager gel documentation unit (UVITEC, 85.60 (TOX3440-17-1-1-1-1-1) to 158.90 cm (Kalanamak). Cambridge). The PL ranged from 22.83 cm (Nagina 22) to 43.24 cm 92 Page 6 of 19 Anurag Mishra et al.

Table 3. Phenotypic correlation coefficients of 17 traits among 35 rice genotypes.

Variables DF DM PH PL BPP NT PBT LA SPP FGPP UGPP SFP LB BY TW GYPP HI

DF 1 DM 0.978 1 PH 0.307 0.379 1 PL 0.419 0.430 0.452 1 BPP 0.204 0.217−0.023 0.164 1 NT 0.411 0.366 0.303 0.119−0.275 1 PBT 0.399 0.354 0.357 0.107−0.288 0.972 1 LA 0.391 0.387 0.303 0.242 0.082 0.166 0.113 1 SPP −0.130−0.047 0.070−0.019 0.297−0.288−0.316 0.193 1 FGPP −0.125−0.066 0.039−0.022 0.292−0.242−0.265 0.183 0.986 1 UGPP −0.055−0.034−0.097 0.125 0.483−0.322−0.346−0.100 0.498 0.468 1 SFP −0.029−0.033 0.110−0.135−0.337 0.162 0.184 0.282 0.098 0.162−0.762 1 LB 0.235 0.187−0.141 0.194 0.022 0.120 0.092 0.043−0.092−0.092−0.128 0.037 1 BY 0.225 0.114−0.204 0.053−0.243 0.059 0.075−0.126−0.242−0.195−0.086 0.002 0.326 1 TW −0.273−0.276−0.143 0.213−0.006−0.128−0.151−0.007 0.011−0.023 0.140−0.176 0.140 0.058 1 GYPP 0.123 0.007−0.192 0.018−0.252 0.028 0.057−0.183−0.247−0.193−0.105 0.018 0.293 0.971 0.021 1 HI −0.276−0.283 0.007−0.111 0.135−0.125−0.098−0.314−0.025−0.022−0.003−0.101−0.075−0.014−0.117 0.196 1

Critical values of Pearson’s correlation coefficient at df = 34, 0.368 (P = 0.05) and 6.36 (P = 0.01).

(Taroari Basmati). The BPP ranged from 8.93 branches HI (–0.314). SFP (0.282) showed a positive correlation (Nagina 22) to 14.5 branches (TOX3226-5-2-2-2-2). The with LA (0.193) and negative correlation with PBT NT was lowest in TOX3440-17-1-1-1-1-1 (9.47 tillers) (–0.316). UGPP showed a high positive correlation with while highest in Taraori Basmati (19.0 tillers). The PBT SPP (0.498) and negative correlation with SFP (–0.762). ranged from 7.90 tillers (WAS197-B-5-2-5) to 17.97 (Bas- SFP exhibited high positive correlation with LA (0.282) mati 370). The SPP ranged from 113.22 spikelets (Taraori and high negative correlation with UGPP (–0762). Length– Basmati) to 253.33 (Vallabh Basmati 24) in number. The breadth ratio showed a higher correlation with DF (0.235), FGPP ranged from 103.63 spikelets (Taraori Basmati) to and negative correlation with PH (–0.141), biological yield 238.25 (Vallabh Basmati 24). The UGPP ranged from 4.60 showed a highly positive correlation with the length– (Pusa Sugandh 4) to 17.40 (IR78006-55-2-3-3) spikelets. breadth ratio (0.326) and high negative correlated found The average SFP is lowest for 89.34% (Ranbir Basmati) with BPP (–0.243). Test weight showed a highly positive and highest 96.45% (Basmati 370). LA ranged from 20.13 correlation with PL (0.213) and highly negative correlation cm2 (TOX3440-17-1-1-1-1-1) to 41.64 cm2 (Basmati 370). with DM (–0.276). GYPP was highly positively correlated Average of LB ranged from 2.38 (WAS515-B-10A-1-4) with biological yield (0.971) while negatively correlated to 5.38 (Vallabh Basmati 22). The average BY varied with a BPP (–0.252). HI exhibited a high positive corre- from 2.58 g/plant (WAS272-B-B-5-15) to 67.35 g/plant lation with GYPP (0.196), while negative correlation with (TOX3440-17-1-1-1-1-1). The TW ranged from 19.32 g LA (–0.314) (table 3). (TOX3867-19-1-2-3-3) to 29.07 g (WAS515-B-10A-1-4). An independent estimate of the morphological variabil- The GYPP ranged from 0.72 g/plant (WAS272-B-B-5- ity present in the germplasm was performed by PCA. 15) 23.81 g/plant (TOX3440-17-1-1-1-1-1). The HI ranged Eigen values were derived from the PCA (table 4) of from 28.01% (IR78554-145-1-3-2) to 42.88% (Nagina 22) the correlation matrix and two major PC were counted (table 2). representing the significant part (40.5%) of total varia- tion. Two-dimensional scaling of genotype by the first two PCs (figure 2) showed three distinct group of genotypes. Phenotypic correlation On the basis of Ward’s dissimilarity matrix, genotypes were divided into three distinct groups (I, II and III). Pearson’s correlation coefficient between the phenotypic Cluster I contained four genotypes: Basmati 370, Taraori traits were analysed (table 3) and found that FGPP showed Basmati, Type 3 Basmati and Kalanamak. Cluster II high positive correlation with SPP (0.986) and a high neg- had 16 genotypes which are Pusa Sugandh 4, Pusa Bas- ative correlation with biological yield (–0.195), DF and mati 1, Basmati 386, Vallabh Basmati 22, Nagina 22, DM showed high positive correlation (0.978). PBT showed TOX3440-17-1-1-1-1-1, TOX3226-5-2-2-2, IR60080-46A, a high positive correlation with NT (0.972) and negative WAS197-B-5-2-16, WAS197-B-4-1-25, WAS197-B-4-1-22, correlation with FGPP (–0.265). LA showed positive cor- IR77736-54-3-1-12, Haryana Basmati, Basmati CSR 30, relation with SPP (0.193), while negative correlation with Ranbir Basmati and WAS197-B-6-3-4. Cluster III consists Asian and African aromatic rice genotypes Page 7 of 19 92

Table 4. Eigen values of correlation matrix and related statistics for the agronomic trait.

Eigen value Cumulative Eigen value Variability (%) Cumulative variability (%)

3.857 3.857 22.687 22.687 3.025 6.882 17.796 40.483 2.246 9.128 13.212 53.695 1.694 10.822 9.963 63.658 1.339 12.161 7.875 71.533 1.152 13.313 6.776 78.309 1.054 14.367 6.199 84.509 0.886 15.253 5.213 89.722 0.590 15.843 3.472 93.194 0.458 16.301 2.695 95.888 0.322 16.623 1.896 97.785 0.313 16.936 1.840 99.624 0.034 16.97 0.202 99.827 0.018 16.988 0.104 99.931 0.008 16.996 0.047 99.978 0.003 16.999 0.016 99.994 0.001 17 0.006 100.000

of 15 genotypes which are Pusa Sugandh 5, Vallabh and WAS197-B-6-3-4.Group II consisted of 18 genotypes: Basmati 24, IR7734-93-2-3-2, WAB99-84, WAS197-B- Pusa Basmati 1, Basmati 386, Nagina 22, IR7734-93-2-3-2, 6-3-12, WAS197-B-5-2-5, IR78554-145-1-3-2, WAS515- TOX3226-5-2-2-2, WAS197-B-6-3-12, WAS197-B-5-2-5, B-10A-1-4, TOX3867-19-1-2-3-3, IR78006-55-2-3-3, IR60080-46A, WAS515-B-10A-1-4, TOX3867-19-1-2-3-3, IR77512-2-1-22, Vallabh Basmati 21, IR78537-32-1-2-1, IR78006-55-2-3-3, WAS197-B-4-1-2-2, IR77736-54-3-1-2, WAS272-B-B-5-H5 and WAS197-B-6-3-16 (figures 2 & 3; IR77512-2-1-22, Vallabh Basmati 21, Basmati CSR 30, table 4). Ranbir Basmati and WAS197-B-6-3-16. Group III had only four genotypes that are WAS197-B-5-2-16, WAS197- B-4-1-2-5, Haryana Basmati and WAS272-B-B-5-H5 (fig- Grain (quality parameters) ure 4; table 5). Biochemical quality tests were conducted to assess the grain quality variations among all genotypes. Sensory test Genotyping and data analysis for aroma revealed a wide range of variations among the varieties. Basmati 370, Taraori Basmati, Pusa Basmati1 Considering the ability of molecular markers to differen- and Pusa Sugandh had a strong aroma, while other vari- tiate the genotypes based on the difference in the genomic eties showed the presence of mild aroma except Nagina regions, 55 SSR primer pairs were used in which 48 primers 22 (no aroma). According to ASV and gelatinication test were found to be polymorphic. PIC ranged from 0.055 (GT), studied rice varieties were divided into two groups, (RM4472) to 0.99 (RM1080) with an average of 0.36 i.e. intermediate and high. Basmati 386, Vallabh Basmati (table 6). Forty-eight loci led to the amplification of a 22, Vallabh Basmati 24, Nagina 22, IR78006-55-2-3-3 and total of 116 alleles with an average of 2.11 alleles per Vallabh Basmati 21 showed intermediate ASV,while all the locus. Primer RM7448 amplified maximum four alleles, other genotype showed high ASV.GC was measured which while RM1017, RM4469, RM4098, RM1067, RM3332, ranged from 35 mm (TOX3440-17-1-1-1-1) to 155 mm RM4472, RM4128, RM3759 and RM5635 amplified three (WAS272-B-B-5-H5)and according to gel consistency rice alleles. Bootstrap (1000 times booting) method was also varieties were further divided into soft, medium and hard. used for the dendrogram (figure 5) by MEGA 7 software Similarly, the level of amylose content ranged from 7.23% and all the 35 genotypes were grouped into three clusters as (WAS272-B-B-5-H5) to 23.01% (CSR30) (table 5). On the indicated by population structure analysis (P1, P2, P3 and basis of biochemical quality, parameters of 35 rice (i.e. P4). Cluster I consists of nine genotypes indicated by red amylose percentage, gel consistency, alkali spreading value colour as Pusa Sugandh 4, Vallabh Basmati 22, WAS197- and aroma) genotypes were divided into three distinct B-5-2-16, Basmati 370, IR77734-93-2-3-2, Taraori Bas- groups using Ward’s analysis (figure 4). Group I consist of mati, Nagina 22, Pusa Sugandh 5 and WAS197-B-5-2-5. 13 genotypes: Basmati 370, Pusa Sugandh 4, Taraori Bas- Cluster II consists of six genotypes indicated by blue colour mati, Type 3 Basmati, Pusa Sugandh 5, Vallabh Basmati as Type 3 Basmati, IR60080-46A, TOX3867-19-1-2-3-3, 22, Vallabh Basmati 24, Kalanamak, TOX3440-17-1-1- IR78006-55-2-3-3, Pusa Basmati 1 and WAS272-B-B-5- 1-1-1, WAB99-84, IR78554-145-1-3-2, IR 78537-32-1-2-1 H5. Cluster III consists of six genotypes indicated by 92 Page 8 of 19 Anurag Mishra et al.

Figure 2. Two-dimensional scaling of 35 genotypes with 17 parameters.

green colour TOX3440-17-1-1-1-1-1, TOX3226-5-2-2-2, Population structure and phylogenetic study Basmati 386, WAS197-B-6-3-1, WAB99-84 and Haryana Basmati. Other 14 remaining genotypes, namely Vallabh Population structure analysis of the collected rice geno- Basmati 21, Basmati CSR 30, Vallabh Basmati 24, Kalana- types provide support for characterization of genotypes mak, IR78554-145-1-3-2, IR77736-54-3-1-2, IR77512-2- and classified into three distinct groups; indica (nonaro- 1-22, IR78537-32-1-2-1, WAB99-84, WAS515-B-10A-1-4, matic), aus (Nagina 22) and aromatic. Structure analysis WAS197-B-4-1-25, WAS197-B-4-1-22, WAS197-B-6-3-16 was performed at K2 to K4 (figure 6). K3 represented and WAS197-B-6-3-4 did not fall in any group and are to dendrogram and selected for further analysis. The indicated by yellow in colour as population 4 (figure 6; maximum admixture was observed in Pusa Sugandh 5 table 6). from the Nagina 22 group. Of the 35 genotypes eight Asian and African aromatic rice genotypes Page 9 of 19 92

Figure 3. NJ tree on the basis of C. S. chord genetic distance.

showed admixture of more than 15%. This admixture Discussion may be due to the presence of improved genotypes. PCoA with SSR markers were used to determine the genetic In this study, we analysed the diversity at morphological, relatedness among the genotypes. The first and second biochemical and molecular level along with population PCoA explained 29.40% of the total variation (figures 6 structure analysis of 35 rice accessions from different &7). agroclimatic zones of Africa, Philippines and India, to 92 Page 10 of 19 Anurag Mishra et al.

Figure 4. Dendrogram of rice genotypes based on biochemical traits by Ward’s analysis. Asian and African aromatic rice genotypes Page 11 of 19 92

Table 5. Biochemical quality (aroma, ASV, GC and amylose percentage) analysis of 35 rice germplasms for diversity study.

Aroma Alkali spreading value Gel consistency Amylose percentage Genotype Range Aroma ASV GT Category GC (mm) Category Amylose % Category

1 Basmati 370 2 Strong aroma 2 75–79 High 48 Medium 21.89 Intermediate 2 PusaSugandh 4 1 Mild aroma 2 75–79 High 57 Medium 19.43 Low 3 Taraori basmati 2 Strong aroma 3 75–79 High 45 Medium 17.21 Low 4 Type 3 basmati 1 Mild aroma 1 75–79 High 54 Medium 18.04 Low 5 Pusa basmati 1 2 Strong aroma 3 75–79 High 85 Soft 20.36 Intermediate 6 Basmati 386 1 Mild aroma 5 70–74 Intermediate 91 Soft 21.56 Intermediate 7 PusaSugandh 5 2 Mild aroma 2 75–79 High 48 Medium 17.59 Low 8 Vallabh basmati 22 1 Mild aroma 4 70–74 Intermediate 60 Medium 19.08 Low 9 Vallabh basmati 21 1 Mild aroma 4 70–74 Intermediate 112 Soft 16.63 Low 10 Haryana basmati 2 Mild aroma 1 75–79 High 150 Soft 19.08 Low 11 Basmati CSR 30 2 Mild aroma 1 75–79 High 122 Soft 23.01 Intermediate 12 Ranbir basmati 2 Mild aroma 1 75–79 High 75 Soft 15.31 Low 13 Vallabh basmati 24 1 Mild aroma 5 70–74 Intermediate 47 Medium 21.42 Intermediate 14 Kalanamak 1 Mild aroma 2 75–79 High 47 Medium 17.05 Low 15 IR7734-93-2-3-2 1 Mild aroma 1 75–79 High 111 Soft 17.36 Low 16 IR60080-46A 1 Mild aroma 2 75–79 High 114 Soft 18.60 Low 17 IR78554-145-1-3-2 1 Mild aroma 2 75–79 High 44 Medium 18.60 Low 18 IR78006-55-2-3-3 1 Mild aroma 5 70–74 Intermediate 110 Soft 15.04 Low 19 IR77736-54-3-1-2 1 Mild aroma 1 75–79 High 83 Soft 19.55 Intermediate 20 IR77512-2-1-22 1 Mild aroma 1 75–79 High 122 Soft 18.75 Low 21 IR78537-32-1-2-1 1 Mild aroma 1 75–79 High 58 Medium 17.83 Low 22 WAB99-84 1 Mild aroma 3 75–79 High 36 Hard 21.96 Intermediate 23 TOX3440-17-1-1-1-1-1 1 Mild aroma 1 75–79 High 35 Hard 15.93 Low 24 WAS515-B-10A-1-4 1 Mild aroma 2 75–79 High 131 Soft 21.07 Intermediate 25 TOX3867-19-1-2-3-3 1 Mild aroma 2 75–79 High 120 soft 15.10 Low 26 WAS272-B-B-5-H5 1 Mild aroma 1 75–79 High 155 Soft 7.23 Low 27 TOX3226-5-2-2-2 1 Mild aroma 1 75–79 High 102 Soft 10.94 Low 28 WAS197-B-6-3-12 1 Mild aroma 1 75–79 High 118 Soft 18.10 Low 29 WAS197-B-5-2-5 1 Mild aroma 2 75–79 High 104 Soft 22.16 Intermediate 30 WAS-197-B-5-2-16 1 Mild aroma 2 75–79 High 148 Soft 13.26 Low 31 WAS-197-B-4-1-25 1 Mild aroma 2 75–79 High 149 soft 13.73 Low 32 WAS197-B-4-1-22 1 Mild aroma 2 75–79 High 103 Soft 15.97 Low 33 WAS197-B-6-3-16 1 Mild aroma 3 75–79 High 131 Soft 16.53 Low 34 WAS197-B-6-3-4 1 Mild aroma 2 75–79 High 60 Medium 16.14 Low 35 Nagina 22 0 No aroma 3 75–79 Intermediate 115 Soft 19.70 Low

form a base for upcoming rice improvement breeding thereby grain yield increases (Rehman et al. 2013). Positive programmes. At morphological level different trait such correlation between above mentioned phenotypic traits as DF, DM, PH, PL, BPP, NT, PBT, SPP, FGPP, UGPP, may bias the estimates of the diversity pattern due to multi- SFP, LA, LB, BY, TW, GYPP and HI showed high coeffi- collinearity. Two-dimensional scaling of genotypes by the cient of variations. Early flowering was observed in Nagina two first PCs showed three distinct group of genotypes. 22 and maximum plant height was observed in Basmati Two major PCs counted for the significant part of total 370 followed by Kalanamak. Variability in plant height variation (40.48%). Different characters distance matrix and other morphological parameters were also reported was used to generate the dendrogram showing the dis- in many previous studies (Ibrahim et al. 1990; Tahir et al. similarity among all the varieties (Ward 1963). All the 2002; Sarvestani et al. 2008; Golam et al. 2011). Pear- 35 genotypes were divided into three different clusters in son’s correlation coefficient between the phenotypic traits which traditional Basmati genotypes Basmati 370, Type showed a highly positive correlation between FGPP and 3 Basmati, Taraori Basmati and Kalanamk were closely SPP, LA and SPP, DF and DM, GYPP and BY. Positive related and were found in the same group (Sarhadi et al. correlation between FGPP and SPP may be due to the 2011; Khare et al. 2014). The third group had only a sin- direct effect of FGPP on SPP (Zhou et al. 2017). Simi- gle genotype Nagina 22. Sensory analysis for aroma also larly, LA and SPP were positively correlated because of showed a wide range of variation such as strong aroma the larger leaf area. It means plants have high amount was found in Basmati 370 and Taraori, while no aroma of photosynthesis energy which helps in grain filling was observed in Nagina 22. Scented or aromatic rice is 92 Page 12 of 19 Anurag Mishra et al. 3009 4523 0000 1197 4892 1132 9996 9666 2412 2506 5320 0000 5715 1719 2135 1703 6634 4671 0000 8032 1164 1711 7191 0559 2774 6211 0000 5928 5178 ...... PIC size Product temp. Annealing SSR motif no. Chromosome List of SSR primer observed product size and their PIC during diversity study. Primer Forward Reverse 2 RM3894 TATGCTCTCTCCTTCAGGCC CTTACCAACTCCGCACTTGC 3 gt 55 201 0 Table 6. 1 RM3759 GAATGAGCTAAGAACACGCC CTGATGGCCCAAGACTTTTG NA ga 55 124 0 17 RM3700 AAATGCCCCATGCACAAC TTGTCAGATTGTCACCAGGG 9 ga 55 143 0 16 RM1867 TACTTGGGTATCAGTCCTTG CGATCACCTGAATTTAAGAA 7 at 55 187 0 15 RM1367 GCATCGTTCATGTACACTGG CTGCTACGCTGCTACTCCTAG 2 ag 55 122 0 14 RM1093 AGGTTGATGAACCCGATGAG CTAGCTGCAGAACGGAGGAG 7 ag 50 150 0 13 RM1080 AGAGCCCTCGTAAGCCAAAG GGTCGTGAATCTCCTCCAAG 12 ac 50 206 0 12 RM1075 CCAGTTCAGTAGTTCACACACC GTTGGGTTGCTGTGTTGTTC 2 ac 50 200 0 11 RM1067 CGATGGAGAGAGAATGTCTAGC TAATACGCAAGGCAGAAGGG 1 ac 50 149 0 10 RM1037 CAAGGACTATGAGCCCCATC TGGCACCCCAGTCATGGTAG NA ac 55 219 0 9 RM7400 TGCAGCAGAAACACGAAGAG AACTCGCCATCATCTCCAAG 8 gatg 55 127 0 8 RM7252 GGAGGAGGAGAAGGGTTTTG ACGCGCTGTCAAGTTAAAGG NA atct 50 165 0 7 RM6329 GTGCATTTCTCTTTTAATAG AACGACACATTAGTAGATGA 3 ctt 55 151 0 6 RM6042 TCCAGCGAGAGAACAGCG GAGGGATGGAGGAAGGAAAG NA ccg 61 164 0 5 RM5654 TGCAACTCGCGTATACAATA CCAAGTTCGTTACAGCAGAG 2 aag 55 156 0 4 RM4098 CGTTTGGATGAAGAAGAAGA AGTGTTCGTTTCGGATTAGA 7 ta 55 110 0 3 RM5545 CAGCACTCCTCCCCTACCAG GGCTAAGTCAGCGTGAGACC 8 tg 61 156 0 29 RM1019 GTTTGAACAGTAGGACTTGT AGAACATCTCACACTTCTCT 8 ac 55 147 0 28 RM1018 ATCTTGTCCCACTGCACCAC TGTGACTGCTTTTCTGTCGC 4 ac 55 160 0 27 RM1017 TCACATCGATCGATCTCGAG GTGTACACGTGTGAGCGAGC NA ac 55 186 0 26 RM1015 TGTATGACTTTTTAGCATTG CCACATTCATTTAGATGTTA 6 ac 55 145 0 25 RM1003 GATTCTTCCTCCCCTTCGTG TTCCTGTCAGAACAGGGAGC 1 ac 55 128 0 24 RM1002 GAACCAGACAAGCAAAACGG AGCATGGGGATTTAGGAACC 3 ac 55 140 0 23 RM4472 CACCAGGATACATAATCATC TAGACAATATTTGAAAGGGA 2 ta 55 183 0 22 RM4128 AGTAACTCGATCAAACTAAC AGAGTCCATATAGAATTTCA 6 ta 55 141 0 21 RM4321 AGAAGGAAAGGTGATGATGA CCAACGTGACGTTTATAACA 3 ta 55 119 0 20 RM5700 ATTTTTCAGTGCATGTCTTC CAAAGGAAAACTCATGAAAG 12 aat 55 228 0 19 RM5432 GTTTCCCCACTTATCTCCCC AAGCGAGGAGGGGTTTAGAG 8 tc 55 216 0 18 RM3759 GCCACCCACTTTGAGCTTAG GATGCTGGTGCGGATCTG NA ga 55 124 0 Asian and African aromatic rice genotypes Page 13 of 19 92 0000 2431 8400 6392 0000 583 2773 3587 1164 3999 5475 3528 1164 2224 7007 7084 0000 1247 1164 3382 3368 2703 3204 7439 059 9039 ...... PIC size Product temp. Annealing SSR motif no. Chromosome ) contd ( Primer Forward Reverse 36 RM3700 AAATGCCCCATGCACAAC TTGTCAGATTGTCACCAGGG 9 ga 55 143 0 35 RM3333 AAGCTATCGACACCGTGACC GCACCTTACAATTTGGCACC 4 ct 55 185 0 34 RM3894 TATGCTCTCTCCTTCAGGCC CTTACCAACTCCGCACTTGC 3 gt 55 201 0 33 RM2811 AGCCTCCTACCTCTAAACCT GCGGAGAGAGTAAGAAGTTC 4 at 55 158 0 32 RM1031 GTGAAGGCACACCAACCG GACGAGGATCGAATTCGAAG 6 ac 55 127 0 31 RM1026 GCCTCTGGCAGAATAGCATC TATCACTTTGCTGCCTAGGC 9 ac 55 164 0 Table 6 30 RM1022 CATGGGATGAGGGAGTAATG CTTTGATAGCGGCTTTGTCC 3 ac 55 150 0 40 RM4355 GGGATGAGAGTAGAAGGCA TATATGGCAAGCCTAGCG 2 ta 55 142 0 39 RM4352 GTTGTTGCACCATAGTCAGA ATACATTCATGAAACCTGCC 3 ta 55 98 0 38 RM1024 GCATATACCATGGGGATTGG GGGATTGGGATAATGGTGTG 5 ac 55 141 0 37 RM3332 ATTCTCCTCGCTTCCTCCTC AAAGAGAGAGCCGAGCACTG 4 ct 55 115 0 54 RM804655 AGTACGATTTCTGTCAGCGTTGCTTAGT RM8045 GGATGAAAGTTGATGGATGATCTACTTGTT 1 TCGCGGTTAATGTCATCT taac GACTGACCCTAAAACCATACA 55 195 1 0 ag 55 150 0 53 RM7452 GAGGCCATGAACGGTCAC ACCCAATTATGGTAGCGTGC 5 taat 55 94 0 52 RM7451 TAATACGAGCAGCGATCGTG GCTAATTGCAGCTTGTGTCG 2 taat 55 154 0 51 RM7448 GATTCTGTGTTTCGCTGCTG TAGCCCGCTGCTCTTCTCTC 12 taat 55 198 0 50 RM5503 CTCTGGGTACACTTCACGAG GGGAAGAAGATAGGAGATGG 4 tc 50 185 0 49 RM7252 GGAGGAGGAGAAGGGTTTTG ACGCGCTGTGAAGTTAAAGG NA atct 55 180 0 48 RM6864 TACCTTCTGCTGCTGCTGTC GCTGGAGGCATCATTTTCAG NA tgc 50 225 0 47 RM5637 CAACTCCAACGACGATGAAC TGGTGAAGTGGAGTGGAGTG 8 aag 55 87 0 46 RM5635 AGCTGAACACTGCGTTTTAC GCTAGCTTAGCTTGCTCTCC 4 aag 55 167 0 45 RM5633 GTGTAGCTGCTAGGCCGAAC TTCCTTTCGCTACGTTGGAC 4 aag 50 211 0 44 RM3295 TCGTGTCATGCGATCGAC GCTTCGACTCGACCAAGATC 12 ct 50 450 0 43 RM5270 ACAACTACATGGGCTAATAA GAAATCCTCTGTATCATCAA 4 ta 55 162 0 42 RM4469 AATTTCTCATGTTTTCTTCC AGTTATTCTAAGGGAGGGAC 11 ta 55 173 0 41 RM4405 TGAAGCAATTTGATTTTCAG GAGCTGGCCTTTATTAACTG 9 ta 55 151 0 92 Page 14 of 19 Anurag Mishra et al.

Figure 5. Molecular dendrogram of 35 genotypes by SSR markers.

preferred in areas of Asia and Gulf countries which draws Della of the United States, Dulhabhog of Bangladesh, a premium price in certain specialty markets. In the Mid- Azucena and Milfor of the Philippines, Sadri varieties of dle East regions consumer prefer rice with strong aroma, Iran and Leuang Hawn of Thailand, Rojolele of Indone- however it is vice versa in Europe, as they consider scent sia, and Barah of Afghanistan (Cruz and Khush 2000). as the indication of spoilage and contamination (Efferson According to ASV and gelatinization temperature, geno- 1985). Among the different rice varieties; Basmati con- types are again divided into two categories (i.e. high and tains more aroma than the traditionally cultivated scented intermediate) depending on the coarseness of the grain. rice varieties and are widely grown in the different part of Basmati 386, Vallabh Basmati 22, Vallabh Basmati 24, India (Nadaf et al. 2006). Most of the high quality pre- Nagina 22, IR78006-55-2-3-3 and Vallabh Basmati 21 ferred varieties in the major rice-growing countries are the showed intermediate gelatinization temperature which is Basmati rice of India and Pakistan, KhaoDawk of Mali, highly desirable for the grain quality (Bansal et al. 2006). Asian and African aromatic rice genotypes Page 15 of 19 92

Figure 6. Structure analysis of SSR primers.

During the process of gelatinization, intermolecular bonds up of linear structure of amylose and chain structure of of starch molecules are broken down in the presence of amylopectin. Amylose and amylopectin play an impor- water and heat, thereby dissolving the starch granules in tant role in the determination of quality parameters of the endosperm. The gelatinization temperature determines rice (Cagampang et al. 1973; He and Suzuki 1987; Sowb- the time required for cooking. Gel consistency is another hagya et al. 1987; Rani and Bhattacharay 1989; Ong and parameter to index the tendency of cooked rice to harden Blanshard 1995; Singh et al. 2003; Cameron and Wang on cooling and is normally classified as hard, medium and 2005; Allahgholipour et al. 2006). AC and the chain struc- soft (Chen et al. 2012). On the basis of AC, rice grain is ture of amylopectin are found to be significantly correlated again classified into hard, medium and soft texture. The with hardiness and stickiness of the rice after cooking lowest amylose percentage is observed in WAS272-B-B- (Ramesh et al. 1999; Mizukami and Takeda 2000; Kibanda 5-H5 and the highest amylose percentage was recorded and Luzi-kihupi 2007). The amylose content in all grades in Basmati CSR 30. However, low AC was observed in of rice range from 7.23 to 23.01%. After cooking, rice majority of the traditional aromatic varieties in compari- becomes moist and sticky due to low amylose content son to Basmati rice varieties. Cooking quality is mainly (Nakamura et al. 2006). On the basis of GC, rice grains determined by the content and composition of starch of different genotypes were further divided into three and storage protein in the endosperm. Starch is made category: hard, medium and soft genotype. WAS272-B- 92 Page 16 of 19 Anurag Mishra et al.

Figure 7. Principle co-ordinate analysis of SSR primers.

B-5-H5, Pusa Basmati 1, Basmati 386, Basmati CSR 30 Fifty-five microsatellite markers were used for assessing Haryana basmati and Vallabh Basmati 21 categorized in the genetic diversity of 35 aromatic Basmati and other rice soft GC. GC is commonly measured by determining the genotypes of Asian and African origin, which showed sig- length of a cooled gel, and it reflects the firmness of the nificant variation among the genotypes. With the help of cooked rice. GC is performed to classify the rice varieties DNA markers, variation in the genomic region of differ- within the same amylose group. Varieties having a softer ent genotypes can be easily observed. Other scientists have GC are preferred and the soft tendency of cooked rice has successfully conducted the genetic diversity studies of both a higher degree of tenderness. All the genotypes studied cultivated as well as wild genotype accessions with help of for biochemical traits of grain quality showed a varia- the DNA-based markers (Virk et al. 1995; Rai 1999; Qian tion among the genotypes and are divided into three main et al. 2001; Samarajeewa et al. 2004; Wong et al. 2009; groups on the basis of dissimilarity matrix (Ward 1963) Huang et al. 2010; Marie et al. 2010; Malik et al. 2010; each one having nonBasmati and Basmati rice cultivars. Umamaheswari et al. 2010; Ram et al. 2010). Of the 55, 48 Asian and African aromatic rice genotypes Page 17 of 19 92

SSR markers were found to be polymorphic. In this study, germplasms to develop biotic and abiotic stress resistant PIC ranged from 0.05 to 0.99 with an average of 0.36. The cultivars. range and average PIC of SSR markers also reported by In conclusion, genetic diversity and population struc- other researchers was 0 to 0.57 and 0.33; 0 to 0.74 and ture of Asian and African rice genotypes have been studied 0.083 to 0.86 and 0.53; 0.20 to 0.90 and 0.60; 0.146 to on the basis of their morphological traits, grain quality 0.756 and 0.416 (Jain et al. 2004; Meti et al. 2013; Tiwari parameters and SSR-based molecular markers. Pheno- et al. 2015; Nachimuthu et al. 2015). The average number typic traits have been evaluated on the basis of Ward’s of polymorphic alleles is 42.20 and 2.11 alleles per locus, analysis and all traditional Basmati rice varieties were while some other workers found an average of 4.5, 14.6, grouped into a same cluster. Quality parameters showed 7.7, 13.0, 4.5 and 4.4 alleles per locus (Nagaraju et al. 2002; intermediate aroma in the Basmati 370, Taraori Basmati Siwach et al. 2004; Brondani et al. 2006; Jalaluddin et al. and Pusa Basmati 1. Finally, the molecular markers anal- 2007; Oliveira et al. 2007; Thomson et al. 2007; Herrera ysis showed that traditional varieties were clustered in et al. 2008; Mishra et al. 2016). Dendrogram generated the same group including the nonaromatic Nagina 22. by MEGA 7 software showed four main groups, and 16 Drought and heat resistance rice var. Nagina 22 can be subgroups. Variations between Basmati and nonBasmati used as a donor in the crop improvement programme varieties showed deviation as well as independent evolu- to transfer the desirable traits into basmati rice varieties tion of the basmati through artificial selection (Oliveira for drought and heat tolerance through marker-assisted et al. 2007). Traditional Basmati cultivar, i.e. Basmati 370, breeding method. Type 3 Basmati, Basmati 386 and Taraori Basmati were grouped into cluster I. However, evolved Basmati and aro- Acknowledgements matic rice cultivars were grouped together irrespective of their geographical origin. In earlier studies, high genetic Authors thank Dr H. S. Gaur, Former Vice Chancellor for pro- similarity among landraces were also observed (Kumar viding research facilities to conduct the present work in the et al. 2010; Sivaranjani et al. 2010; Pervaiz et al. 2011; Department of Agricultural Biotechnology, Sardar Vallabhbhai Vanniarajan et al. 2012). Patel University of Agriculture and Technology, India. When structure analysis was performed at K2, one group emerged with indica types of genotypes whereas other group formed by aus and aromatic rice genotypes. Further, when subclustering was performed at K3, all 35 References genotypes were divided into one additional cluster. In this additional cluster, aus and aromatic genotypes were Allahgholipour M. A. J. A., Alinia F., Nagamine T. and Kojima separated. Traditional genotypes (Basmati 370, Basmati Y.2006 Rice grain amylase and pasting properties for breeding 386, Taraori Basmati and Type 3 Basmati) are found better quality rice varieties. Plant Breed. 125, 357–362. Anonymous 2004 Laboratory manual on rice grain quality proce- in a same cluster. Also, three independent clusters were dure directorate of rice research, pp. 1–20. Directorate of Rice reported in previous studies (Ram et al. 2007). Nonaro- Research, Hyderabad. matic aus type genotype (Nagina 22) grown in rainfed Bansal U. K., Kaur H. and Saini R. G. 2006 Donors for quality conditions, released by rice research station Nagina, Bijnor characteristics in aromatic rice. Oryza 43, 197–202. India and used as a source of drought and heat resis- Bhattacharya K. R. 1979 Tests for . In Proceedings workshop on Chemical aspects of rice grain quality, pp. 363. tance allele/gene in rice improvement programme (Patra International Rice Research Institute, Los Bahos,. et al. 2016). In addition to the subgroup identified by Blakeney A. B., Welsh, L. A. and Martin M. 1992 In Proceedings the analysis, 15% individual showed genetic admixture, of the 42nd RACI Cereal Chemistry Conference, pp. 342–346. the maximum admixture was observed in Nagina 22. The Royal Australian Chemical Institute, Parkville. possible reason for this may be the cross-hybridization Botstein D., White R. L., Skolnick M., and Davis R. W. 1980 Construction of a genetic linkage map in man using restriction or gene flow through conscious breeding efforts made by fragment length polymorphisms. Am. J. Hum. Genet. 32, 314– humans for crop improvement programme. It is reported 331. that the aus type rice variety has 80% admixture of Indica Brondani C., Borba T. C. O., Rangel, Paulo H. N. and Brondani followed by evolved rice genotype 40% and minimum R. P.V.2006 Determination of genetic variability of traditional 30% for nonaromatic rice genotype (Garris et al. 2005; varieties of Brazilian rice using microsatellite markers. Genet. Mol. Biol. 29, 676–684. Parida et al. 2012). The present study suggests that Cagampang G. B., Perez C. M. and Juliano B. O. 1973 A gel the traditional and evolved basmati genotypes contain consistency test for eating quality of rice. J. Sci. Food Agric. their background, i.e. the ancestral genome and it is 24, 1589–1594. preserved by the genotypes. The genotypes of differ- Cameron D. K. and Wang Y. 2005 A better understanding of ent subgroups may carry diverse genes for different factors that affect the hardness and stickiness of long grain rice. Cerl. Chem. 82, 113–119. traits. Strategic use of diverse genotypes in the breed- Chakravarthi B. K. and Naravaneni R. 2006 SSR marker based ing programme would allow judicious explanation of DNA fingerprinting and diversity study in rice (Oryza sativa. these genes complexes for improvement of existing rice L). Afr. J. Biotecnol. 5, 684–688. 92 Page 18 of 19 Anurag Mishra et al.

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