Comparative analysis of agro-morphology, grain quality and aroma traits of

traditional and Basmati-type genotypes of rice, Oryza sativa L.

SOMNATH ROY1 , 2 , 6 , AMRITA BANERJEE

1 , 3 , B I JOY K. SENAPATI1 , 4 and GURUPADA SARKAR

1 , 5

1Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal 741252, India; 2Present address: NBPGR RegionalStation – Shillong, Umiam, Meghalaya 793 103, India; 3Present address: ICAR Research Complex for NEH Region, Umaim,Meghalaya 793 103, India; 4Present address: Regional Research Station, New Alluvial Zone, BCKV, SC: Chakdaha, Nadia,West Bengal 741222, India; 5Present address: Regional Research Station, New Alluvial Zone, BCKV, Gayeshpur, Nadia,West Bengal, India; 6Corresponding author, E-mail: sroypbr@gmail.com

With 3 figures and 4 tables

Received December 5, 2011|Accepted February 22, 2012Communicated by R. Singh

Abstract

The purposes of this study were to assess the variation in agro-

morphological and grain quality traits among traditional and Basmati-

type aromatic/quality rices and to investigate plausible relationships

between the traits. A set of 12 cultivars, comprising ten traditional and

two Basmati types, were studied. Highest variation was observed for

grains/panicle followed by grain yield/plant. Cluster analysis grouped

all traditional cultivars except �Tulaipanji�, which clustered with

Basmati varieties. Selection for long grain with slender shape will

simultaneously increase amylose content and alkali spreading value or

gelatinization temperature. Aroma score categorized rice varieties as

mild and strongly aromatic, which also was similar to aroma

genotyping with gene-based marker for betaine aldehyde dehydrogenase

2 (BADH2). Sequence analysis of BADH2 revealed that all strongly

aromatic and two mild aromatic rice varieties contain characteristic

8-bp deletion and three SNPs in exon 7 of BADH2 gene. Multiple

alignment of the DNA sequences revealed the addition of AT in

�Gobindabhog� and a T/A SNP in �Gobindabhog� and �Tulsibhog�exon 8.

Key words: rice — aroma — grain quality — correlation —sequence analysis — BADH2

Introduction

Improving the rice grain quality has been a major concern in

rice breeding programme to meet the market demand. Ricequality is based on a combination of subjective and objectivefactors, the relative importance of which depends upon theparticular end-use. The quality components, common to all

users, include grain appearance, milling quality, cooking andprocessing quality and nutritional quality, of which cookingand eating qualities are the most important components for

Asian rice consumers. Cooking and processing qualities thatare important across the users include texture and stickiness.The two most important quality indicators for these charac-

teristics are amylose content (AC) and gelatinization temper-ature. AC is controlled by one major gene with severalmodifiers (McKenzie and Rutger 1983, Kumar and Khush1986, 1987). Gelatinization temperature, measured by alkali

spreading value (ASV), seemed to have complex nature ofinheritance (Heda and Reddy 1986). Genetic variability hasbeen the major driving force used by man to meet not only its

food needs but also to produce high-quality cultivars. Know-ledge on variability and association between the grain yield

components helps breeders in tailoring the hybridization

programme.Fragrance in rice is one of the important grain quality traits

in rice. It is a key factor in determining market price along withgrain shape and size (Fitzgerald et al. 2009). Aromatic rice

genotypes constitute a special group well known for theiraroma (Kumar et al. 1996, Singh et al. 2000a). There do manylocally adapted aromatic and quality rice genotypes (Richharia

et al. 1965, Richharia and Govindaswamy 1966, Siddiq andShobharani 1998, Khush 2000) comprise small-, medium- andlong-grain types with mild-to-strong aroma (Singh et al.

2000a,b). Khush (2000) classified Indian aromatic rice germ-plasm as indicas among which Basmati types have beenidentified as genetically distinct cluster based on both isozyme

and microsatellite data (Glaszmann 1987, Aggarwal et al.2002, Nagaraju et al. 2002, Garris et al. 2003, Jain et al. 2004).The aromatic rice cultivars are generally poor yielders becauseof their disease susceptibility and limited adaptation outside

their original geographical distribution. Aromatic rice acces-sions have been indentified within at least three geneticsubpopulation of rice, including Group V (i.e. �Basmati� and�Sadri� varieties), indica (i.e. �Jasmine� varieties) and tropicaljaponica.Many studies reported the genetic control of aroma in rice

(reviewed by Sakthivel et al. 2009a). The inheritance offragrance was reported to be controlled by one or two orthree dominant or recessive genes or by QTLs that found to be

cross-specific/genotype-specific. Reports indicated that at leastsix of twelve rice chromosomes (4, 5, 8, 9, 11 and 12) wereimplicated to harbour the gene/genes (Sun et al. 2008). Thishas further complicated the genetic basis of fragrance in rice.

Availability of high-density molecular marker maps andgenome sequences in rice has allowed mapping, fine mappingand positional cloning of gene for fragrance. An eight-base

pair deletion and three SNP in exon 7 of the gene encodingbetaine aldehyde dehydrogenase 2 (BADH2) on chromosome8 of Oryza sativa have been identified as the primary cause of

fragrance in Jasmine- and Basmati-like rice (Bradbury et al.2005a). Functional BADH2 is either responsible for metabo-lizing 2-acetyl-1-pyrroline (2-AP), which has been identified asthe main compound among over 100 volatile compounds

reported to be responsible for characteristic aroma in the aerialparts of Basmati- and Jasmine-like rice lines (Buttery et al.

Plant Breeding doi:10.1111/j.1439-0523.2012.01967.x� 2012 Blackwell Verlag GmbH

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1983, Paule and Powers 1989), or for activating the fragrancepathway that competes for substrate which would otherwise beused in the production of 2-AP and so a non-functionalBADH2 enzyme results in increased flux of substrate down the

pathway of 2-AP production. In contrast, non-fragrant ricevarieties possess a full functional copy of BADH2 gene whilefragrant varieties possess BADH2 containing 8-bp deletion

and three SNPs, resulting in a frame-shift that generates apremature stop codon which disables BADH2 enzyme. RNAi-mediated down-regulation of BADH2 gene reported to

enhance the accumulation of 2-AP in non-aromatic rice cv.�Nipponbare� (Niu et al. 2008). Recently, Kovach et al. (2009)reported that despite the multiple origin of fragrance trait,BADH2 is the predominant allele in virtually all fragrant rice

varieties.Identification of the gene for fragrance and development of

stable marker system for aroma genotyping have created

worldwide interest to look for allelic variants at this locus inthe rice gene pool. In addition to 8-bp deletion in exon 7,several variations including a 7-bp insertion in exon 8

(Amarawathi et al. 2008), a 7-bp deletion in exon 2 (Shi et al.2008), absence of miniature interspersed transposable element(MITE) in promoter (Bourgis et al. 2008), two new SNPs in

the central section of intron 8 (Sun et al. 2008), a TT deletionin intron 2 and a repeated (AT)n insert in intron 4 (Chen et al.2008) of BADH2 were reported in various fragrant varieties.Although Bradbury et al. (2005a) have identified the gene

and Chen et al. (2008) and Bradbury et al. (2008) elucidatedthe role of BADH2 in 2-AP biosynthesis, the molecular basisof fragrance remained unsolved. The presence of rice varieties

exhibiting elevated levels of 2-AP, but lacking any knownfunctional allele of BADH2, raised the possibility of existenceof additional fragrance-causing alleles of BADH2. Further, the

involvement of several other aromatic compounds (Widjajaet al. 1996) indicates that many other genes might play a rolein effecting unique or modified fragrance in rice.

In this study, 12 aromatic/quality rice cultivars includingtraditional rice and improved Basmati cultivars have beenstudied for agro-morphological and grain quality traits tounderstand the association between grain yield and its com-

ponents. In addition, a search has been made about thevariation in the region spanning the 8-bp mutation in exon 7through sequence analysis of part of BADH2 gene in the tested

rice cultivars.

Materials and Methods

This study was conducted at the Regional Research Station, New

Alluvial Zone, Bidhan Chandra Krishi Viswavidyalaya, Sub-Centre:

Chakdah, Nadia, West Bengal (23�04¢N latitude, 88�31¢E longitude,

6 m altitude). A set of 12 rice cultivars were taken for this study from

the germplasm evaluation trial in 2006 and 2007. These rices

represented 10 traditional aromatic/quality rice and two Basmati

varieties (Table 1). Thirty-day-old seedlings were transplanted in

randomized block design with two replications. Each entry consisted

of five rows of 6.0 m length with a plant spacing of 0.20 m in either

direction. The crop was rainfed, and other normal agronomic practices

were followed during crop growth period. The soil is a clay loam with

pH 7.7. The field was fertilized with 40 kg N/ha in three increments,

20 kg P2O5/ha and 20 kg K2O/ha applied by hand broadcasting. One-

third of N and full amount of phosphorus and potassium were applied

during final land preparation. The second and third increments of N

were applied at the tillering stage and prior to panicle initiation,

respectively.

The days required from sowing to 50% flowering (DF) and maturity

(DM) were recorded on plot basis. Panicles/plant (PP), harvest index

(HI) and grain yield/plant (GY) were measured as the average of

randomly selected five plants. Panicle length (PL), panicle weight (PW)

and grains/panicle (GP) were determined based on five individual

measurements of main stems in each entry. After harvesting, the grains

were dried in open sun until moisture content reached 140 g/kg and

used for recording grain quality traits. Grain length (GL), grain

breadth (GB) and grain length-to-breadth ratio (GLB) for brown rice

were determined from randomly sampled (twice) 10 grains. About

1000-grain weight (GW) was determined twice for each entry. Rice

grains (100 g) were de-husked and milled in Satake TM-05 grain

testing mill. The method of Little et al. (1958) was used to find out

ASV. For AC determination, milled rice grains were ground in mortar

and pestle and screened through 60 mesh sieve. AC was determined as

described in Juliano et al. (1981). Aroma was detected by sniffing of by

a panel of ten experts and was scored as mild, medium and strong

following KOH-based method (Nagaraju et al. 1991).

Data analysis The 2-year data on various traits were subjected to

analysis of variance, and the mean values, standard deviation (SD),

standard error (SE), minimum and maximum values (summary

statistics) were determined. Ward�s hierarchical clustering was used

to assess the phenotypic diversity in rice cultivars. Clustering was

performed using SPSS statistical software (version 16 for Windows;

SPSS Inc., Chicago, IL, USA). The Z values were calculated from

mean values and used for cluster analysis. Pearson�s correlation

coefficients (r) for the agro-morphological and grain quality traits were

calculated using SPSS software.

Genotyping for aroma Genomic DNA was extracted from leaf samples

using a SDS-based micro-Prep method, and the polymerase chain

reactions (PCR) were performed in a thermal cycler (Eppendorf,

Hamburg, Germany). The genotyping for aroma in twelve rice lines

was carried out following Bradbury et al. (2005b) using external sense

primer (ESP), internal fragrant antisense primer (IFAP), internal non-

fragrant sense primer (INSP) and external antisense primer (EAP).

PCR products were analysed by electrophoresis in 1.0% agarose gel

stained by ethidium bromide (0.5 ug/ml).

Sequencing of BADH2 gene fragment We amplified �580-bp fragment

spanning the 8-bp mutation described by Bradbury et al. (2005a) from

12 rice cultivars. Among the four primers reported by Bradbury et al.

(2005b), ESP and EAP were used. The PCR was performed using 1 ll(�20 ng) of genomic DNA, 1 ll of each primer (10 lM), 2.5 ll of 10·buffer containing 15 mM MgCl2, 1 ll of 2.5 mM dNTPs, 1 U of Taq

DNA polymerase in a total volume of 25 ll by adding dd H2O.

Cycling conditions were an initial denaturation (94�C) for 5 min, 35

cycles of 94�C for 30 s, 55�C for 30 s and 72�C for 1 min, followed by

final extension of 72�C for 7 min.

Table 1: List of the rice varieties used for analysis

Sampleno.

Name ofvariety Type

Aromatest

PCRanalysis

1 Basmati 385 Traditional Basmati Strong Fragrant2 Gobindabhog Aromatic/quality Strong Fragrant3 Kalikhasa Aromatic/quality Strong Fragrant4 Khaskani Aromatic/quality Strong Fragrant5 Madhuri Aromatic/quality Mild Fragrant6 Radhunipagal Aromatic/quality Strong Fragrant7 Taroari Basmati Premium Basmati Strong Fragrant8 Tulaipanji Aromatic/quality Mild Fragrant9 Tulsibhog Aromatic/quality Mild Fragrant10 Dudheswar Quality Mild Non-fragrant11 Masino Selection Mild Non-fragrant12 Shantibhog Quality Nil Non-fragrant

PCR, polymerase chain reaction.

2 S . Roy , A . Baner j e e , B . K . Senapat i e t a l .

PCR amplifications of expected fragments were confirmed by

electrophoresis in ethidium bromide–stained (0.5 lg/ml) 1.0% agarose

gels. The PCR products of the 12 rice varieties were commercially

sequenced from Bangalore Genei Ltd., Bangalore, India. The PCR

fragments were sequenced from both 5¢ and 3¢ ends by primer walking.

The final sequence for each entry was deduced from forward and

reverse sequences. The identity and homology of the sequences were

first checked using the BLAST N programme from the NCBI website.

The annotated sequences were registered under GenBank accession

numbers JN599151–JN599162. The BADH2 nucleotide sequence data

of twelve rice varieties were subjected to pair-wise multiple alignments

using the CLUSTAL W algorithm in MegAlignTM programme of

Lasergene at its default settings.

Results

The mean values for all the agro-morphological and grain

quality traits of tested rice cultivars are depicted in Tables 2and 3, respectively. There was significant variation amongthem for all traits except GB. DF varied from 107 days(�Dudheswar�) to 115 days (�Tulaipanji�). The average number

of panicles/plant ranged from 6.6 (�Basmati 385�) to 14.0(�Kalikhasa�). Maximum PL was recorded in �Tulsibhog�(30.0 cm) and minimum in �Gobindabhog� (19.0 cm). The

average number of filled grains/panicle showed wide variationand ranged from 50 (�Taroari Basmati�) to 210 (�Randhunipa-gal�). GL ranged from 4.2 (�Gobindabhog�) to 7.3 mm

(�Basmati 385�), GB from 1.7 (�Taroari Basmati�) to 2.1 mm(�Khaskani�) and GLB from 2.1 (�Khaskani�) to 4.12 (�TaroariBasmati�).

GW ranged from 15.3 (�Randhunipagal�) to 19.5 g (�Basmati385�) and HI from 0.30 (�Gobindabhog�) to 0.38 (�Kalikhasa�).GY was highest in �Tulsibhog� (29.45 g) and lowest in �TaroariBasmati� (7.08 g). ASV ranged from 2.35 to 3.35 with a mean

value of 3.1. All the rice varieties had low-to-intermediate ACand it ranged from 15.46 (�Randhunipagal�) to 20.2 (�Madhuri�).The aroma score categorized the genotypes as mild (�Madhuri�,�Tulaipanji�, �Tulsibhog�, �Dudheswar� and �Masino�) andstrongly aromatic (�Basmati 385�, �Gobindabhog�, �Kalikhasa�,�Khaskani�, �Randhunipagal� and �Taroari Basmati�). �Shantibhog�was recorded as non-aromatic based on aroma test (Table 1).Among the characteristics studied in this experiment, highest

variation was recorded for GP (%CV = 38.5) and GY(%CV = 33.6). Rest of the traits showed low-to-intermediatevariation.The cluster analysis basedon agro-morphological andgrain quality traits placed the genotypes into two major clusters

(Fig. 1). Cluster one included nine traditional cultivars, whichfurther formed three subclusters. The second major clustergrouped two basmati lines (�Basmati 385� and �Taroari Basmati�)and one traditional aromatic cultivar �Tulaipanji�. These geno-types recorded higher values for GL, GLB, GW and AC.The Pearson�s correlation coefficients among the 15 traits

measured in this study are shown in Table 4. GY had significantpositive association with PP, PW, GP and GB. GL, GLB andAC had significant negative association with GY. PW and HIrecorded significant negative correlation with DF. The GW had

significant negative correlation with DM and HI. PL showedsignificant negative correlation with PP and positive associationwith GB. GP was significantly correlated in positive direction

with PW andGB, and in negative direction with GL, GLB, GWand AC. GL recorded negative correlation with GB and GY.The AC exhibited significant positive association with GLB,

GW and ASV and negative correlation with GP. ASV, anindicator of gelatinization temperature, showed significantlypositive association with GW and AC.

The PCR analysis of twelve rice genotypes with four BADH2gene-based primers (Bradbury et al. 2005b) revealed amplifi-cation of 577/585-bp fragments that serves as positive controlin all rice lines. In addition, nine varieties amplified a 257-bp

fragment resulting from primer pair ESP/IFAP and threevarieties (�Dudheswar�, �Masino� and �Shantibhog�) revealed thepresence of a 355-bp fragment specific to non-aromatic rice

varieties (Fig. 2). The results of aroma test by sensory methodwere also at par with the PCR assay. Though, the rice varietiespossessed mild aroma they showed variable results in PCR

assay (Table 1).We sequenced the �580-bp PCR fragment amplified by ESP

and EAP primer pair from 12 rice cultivars for more precise

genotyping of the BADH2 alleles. This region of BADH2alleles contains three exons (exon 6–8) and three introns (6–8).The pair-wise alignment of the sequences revealed the presenceof 8-bp deletion and three SNPs in exon 7 (Fig. 3). These

characteristics were not observed in three non-aromatic rices

Table 2: Mean values of eight agro-morphological traits for 12 rice varieties

Variety DF DM PP PL PW GP HI GY

Basmati 385 108 138 7.4 27.0 2.18 135 0.33 15.90Gobindabhog 113.5 145.5 11.1 20.1 1.97 170.5 0.31 23.49Kalikhasa 108 138.5 13.0 24.0 2.10 78.5 0.37 17.38Khaskani 107.5 139 11.0 25.0 2.54 190 0.36 26.01Madhuri 111 139 12.0 22.5 1.93 136.5 0.32 24.83Randhunipagal 110 143 8.5 27.0 1.95 204 0.32 24.42Taroari Basmati 112 142 8.0 25.0 2.15 54.5 0.34 8.37Tulaipanji 114 143 9.5 26.0 1.11 66.5 0.34 10.82Tulsibhog 107.5 139.5 10.7 29.0 2.37 185 0.34 29.00Dudheswar 107 140.5 10.5 24.5 2.70 173 0.35 25.58Masino 108 137 9.2 23.0 1.68 90 0.34 14.06Shantibhog 111.5 144.0 11.0 21.5 2.16 120.5 0.34 21.11Mean 109.9 140.8 10.1 24.6 2.07 133.7 0.34 20.08SD 2.94 2.97 1.91 2.64 0.40 51.27 0.02 6.74SE 0.60 0.61 0.39 0.54 0.08 10.47 0.00 1.37Minimum 106.00 136.00 6.60 19.00 1.01 50.00 0.30 7.08Maximum 115.00 146.00 14.00 30.00 2.70 210.00 0.38 29.45P-value 0.048 0.018 0.038 0.001 0.000 0.000 0.001 0.000

DF, days to 50% flowering; DM, days to maturity; PP, panicles/plant; PL, panicle length; PW, panicle weight (g); GP, grains/panicle; HI, harvestindex; GY, grain yield/plant (g); SE, standard error.

Comparative analysis of agro-morphology, grain quality and aroma traits 3

as per the PCR assay. In addition to 8-bp deletion in exon 7,the �Gobindabhog� line had a T/A SNP and addition of AT in

intron 8. Similar T/A SNP was also observed in �Tulsibhog�.Among the three non-scented lines, �Dudheswar� and �Shan-tibhog� revealed a deletion of T in intron 6 while �Masino� hada G/T SNP in the same intron (Fig. 3).

Discussion

Improvement of grain quality is one of the most importantobjectives in rice breeding. Moreover, aromatic rices are

usually poor yielder. Aromatic rice varieties in general are tallstatured, have fewer number of panicles, lower grain yields andsusceptible to lodging. Morphological traits are useful forpreliminary evaluation and could be used as general approach

for assessing genetic diversity among morphologically distin-guishable aromatic rice cultivars (Hien et al. 2007). Glaszmann

Table 3: Mean values of seven grain quality traits for 12 rice varieties

Variety GL GB GLB GW ASV AC

Basmati 385 7.15 1.90 3.8 19.2 3.2 19.2Gobindabhog 4.35 1.90 2.3 15.7 2.8 18.3Kalikhasa 6.05 1.90 3.2 17.6 3.1 19.1Khaskani 4.60 2.05 2.2 18.1 3.3 19.7Madhuri 6.15 1.85 3.3 18.2 3.2 19.9Randhunipagal 4.85 2.00 2.4 15.7 2.5 15.8Taroari Basmati 6.80 1.75 3.9 18.7 3.2 19.8Tulaipanji 6.00 1.90 3.2 17.4 3.3 19.5Tulsibhog 5.50 2.05 2.7 17.3 3.2 16.9Dudheswar 6.10 1.85 3.3 17.7 3.2 17.0Masino 5.75 1.90 3.0 18.0 2.5 17.7Shantibhog 5.10 1.90 2.6 17.8 3.2 17.7Mean 5.70 1.91 3.05 17.62 3.14 18.43SD 0.85 0.12 0.56 1.07 0.30 1.36SE 0.17 0.02 0.11 0.22 0.06 0.28Minimum 4.20 1.70 2.10 15.30 2.35 15.46Maximum 7.30 2.10 4.12 19.50 3.35 20.20P-value 0.000 0.371 0.000 0.000 0.000 0.000

GL, grain length (mm); GB, grain breadth (mm); GLB, grain length/breadth; GW, 1000-grain weight (g); ASV, alkali spreading value; AC,amylose content; PP, panicles/plant; SE, standard error.

Fig. 1: Grouping of the twelve rice varieties on the basis ofstandardized squared Euclidean distance applied towards hierarchicalanalysis

Table 4: Pearson�s correlation between agro-morphological and grain quality traits

Traits

DF DM PP PL PW GP GL GB GLB GW HI ASV AC

DF –DM 0.779** –PP )0.081 )0.081 –PL )0.402 )0.289 )0.444* –PW )0.582** )0.157 0.177 0.102 –GP )0.348 0.071 0.099 0.182 0.539** –GL )0.161 )0.387 )0.387 0.321 )0.070 )0.580** –GB )0.392 )0.183 0.163 0.427* 0.177 0.674** )0.605* –GLB )0.032 )0.273 )0.374 0.172 )0.090 )0.650** 0.979** )0.750** –GW )0.287 )0.566** )0.211 0.130 0.186 )0.474* 0.752** )0.384 0.728** –HI )0.488* )0.457* 0.381 0.128 0.269 )0.339 0.222 )0.011 0.193 0.386 –ASV 0.020 )0.009 0.260 0.192 0.308 )0.109 0.302 )0.071 0.276 0.496* 0.393 –AC 0.300 )0.165 0.114 )0.225 )0.196 )0.548** 0.386 )0.403 0.430* 0.600** 0.178 0.519* –GY )0.379 )0.028 0.480* 0.036 0.578** 0.897** )0.575** 0.626** )0.639** )0.416 )0.136 0.059 )0.470*

AC, amylose content; ASV, alkali spreading value; DF, days to 50% flowering; DM, days to maturity; GB, grain breadth; GL, grain length;GLB, grain length-to-breadth ratio; GP, grains/panicle; GW, 1000-grain weight; GY, grain yield/plant; HI, harvest index; PL, panicle length; PP,panicles/plant; PW, panicle weight.*P = 0.05; **P = 0.01.

4 S . Roy , A . Baner j e e , B . K . Senapat i e t a l .

(1987), based on isozyme diversity, first reported that aromatic

rice varieties fall into separate group in comparison with indicaand japonica rice. The non-functional BADH2 interferes inpollen tube development and may be regarded as one of thereasons for low grain yield in aromatic rice varieties (Bradbury

et al. 2008). The clustering of the lines enables the selection ofparents based on wider intercluster distance (Mishra et al.2003, Chaturvedi and Mourya 2005). In addition, for improv-

ing the yield potential of aromatic rice varieties, the correla-tions between the grain yield and its components need to bewell understood.

The knowledge of the correlation among agro-morpholog-ical and grain quality traits is vital for choosing most efficientselection criteria. The correlation analysis indicated that

selection for longer grains would result in a negative responseto GB and would increase GLB. Koutroubas et al. (2004) alsoreported similar correlations. The relationship between GPand grain size/shape indicated that genes controlling grain size

also influence GP as suggested earlier by Chandraratna (1964).The negative correlations between GL and GB may resultfrom the linkage of length and width genes or pleiotropy

(McKenzie and Rutger 1983). Similar association between GL,GB, GLB and GW was recorded earlier in 28 aromatic/qualityrice cultivars by Roy et al. (2009). In this present study, AC

was positively correlated with GLB. Earlier, Koutroubas et al.(2004) also reported a similar correlation between AC andGLB, but a negative association between AC and GW. Wefound that AC had a positive correlation with GW. ASV, an

indicator of gelatinization temperature, showed significantlypositive association with GW and AC. The complex relation-ships between these traits indicated a breeder could expect

some concurrent increase in AC and ASV or gelatinizationtemperature when he selects long grains and slender shape.

All the aromatic rice included in this study had characteristic

8-bp deletion in BADH2. With an exception to the 8-bpdeletion reported as the genetic cause for aroma, Sakthivelet al. (2009b) did not found this deletion in some indigenous

aromatic rice genotypes of India. Similar exceptions were alsoreported in some fragrant varieties (Kuo et al. 2005, Navarroet al. 2007, Fitzgerald et al. 2008, Shi et al. 2008). Thesestudies indicated the existence of allelic/genetic diversity for

fragrance in aromatic rice gene pool. In our study, we foundthat four (�Dudheswar�, �Masino�, �Tulaipanji� and �Tulsibhog�)among the twelve rice cultivars had mild aroma level, and

according to PCR analysis along with sequence analysis, only

Fig. 3: Multiple alignment of the BADH2 gene sequences showing8-bp deletion and three SNPs in exon 7 of the aromatic rice varieties.Only variable positions are shown

Fig. 2: Agarose gel showing polymerase chain reaction amplificationof BADH2 using four gene-specific primers. Lane 1–3, aromatic ricevarieties viz. �Taroari Basmati�, �Gobindabhog� and �Tualipanji�; Lane4, a negative control (water); Lane 5–7, non-aromatic rice varieties viz.�Dudheswar�, �Masino� and �Shantibhog� M, molecular weight marker

Comparative analysis of agro-morphology, grain quality and aroma traits 5

two cultivars, that is �Tulaipanji� and �Tulsibhog�, were aromatic(Table 1). As BADH2 seems to explain the accumulation of2-AP in most (but not all) aromatic rice varieties, theinvolvement of other gene(s) in fragrance development and

their interaction with the environment could not be taken intoconsideration. Further characterization of other gene(s) infragrance development will lead to a complete understanding

of the variation in aroma level.The results of our study suggest that selection for grain

quality using conventional breeding methods could be opti-

mized by understanding the correlations among the yield andgrain quality components. The best strategy for aromatic/quality rice breeding would be to improve number of grainsper panicle. The primers reported by Bradbury et al. (2005b)

could be used effectively for aroma genotyping of bothBasmati and traditional aromatic/quality rice varieties. TheBADH2 mutation in exon 7 is the major cause of aroma in rice,

and this region is highly conserved among the rice genotypes,although the involvement of other genes also to be consideredfor the variation in the level of aroma in rice varieties.

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