ORIGINAL ARTICLE

Fine mapping of grain length QTLs on chromosomes1 and 7 in Basmati rice (Oryza sativa L.)

Rakesh Singh & Ashok Kumar Singh &

Tilak Raj Sharma & Aqbal Singh & Nagendra K. Singh

Received: 30 April 2011 /Accepted: 20 August 2011 /Published online: 1 October 2011# Society for Plant Biochemistry and Biotechnology 2011

Abstract Grain dimensions (length, breadth and length/breadth ratio) are important quality attributes of Basmatirice for its high consumer acceptance. Earlier we identifiedtwo significant quantitative trait loci (QTL) intervals onchromosomes 1 and 7 for grain dimensions in Basmati riceusing a population of recombinant inbred lines (RILs) fromcross between Basmati variety Pusa 1121 and a short grainnon-aromatic variety Pusa 1342. For fine mapping of theseQTLs, 184 F6 RILs were grown and phenotyped in thenormal rice growing season at two different locations.Forty-nine new SSR markers targeting these QTL intervalswere tested and nine were found polymorphic between theparents. Using revised genetic maps adding new markers,the grain length QTL qGRL1.1 on chromosome 1 wasnarrowed down to 108 kbp from the earlier reported6,133 kbp. There were total 13 predicted gene models inthis interval which includes the probable candidate gene forthe exceptionally high grain length of Basmati variety Pusa1121. Similarly, two tandem QTL intervals qGRL7.1 andqGRL7.2 on chromosome 7 were merged into a single one

narrowed down to 2,390 kbp from the earlier reportedlength of 5,269 kbp. This region of chromosome 7 also hasco-localized QTLs for grain breadth and length to breadthratio. SSR markers tightly linked to the QTL at a mapdistance of ≤0.2 cM were developed for the qGRL1.1 andqGRL7.1 loci that could be used for marker-assistedbreeding. Validation of earlier published markers for thegrain length gene GS3 on chromosome 3 showed nodifference between Pusa 1121 and Pusa 1342, highlightingthe significance of qGRL1.1 and qGRL7.1 for the extragrain length of Basmati variety Pusa 1121.

Keywords Basmati . Grain length breadth . Rice . QTL

AbbreviationsQTL Qauntitative trait lociSSR Simple sequence repeatsRIL recombinant inbred linesGRL, GL, GS Grain lengthGBR, GB Grain breadthLBR Grain length to breadth ratio

Introduction

Grain size and shape determine the appearance of rice andaffect milling, cooking and eating qualities, and aretherefore important quality traits in rice breeding programs(Luo and Lin 1990). Since rice grain length and breadth arequantitatively inherited, it is difficult for the breeders toefficiently improve grain appearance using conventionalselection methods (Mckenzie and Rutger 1983; Tan et al.

R. Singh : T. R. Sharma :A. Singh :N. K. Singh (*)NRC on Plant Biotechnology,Indian Agricultural Research Institute,New Delhi 110 012, Indiae-mail: nksingh@nrcpb.org

A. K. SinghDivision of Genetics, Indian Agricultural Research Institute,New Delhi 110 012, India

Present Address:R. SinghNRC on DNA Fingerprinting, NBPGR,New Delhi 110 012, India

J. Plant Biochem. Biotechnol. (July–Dec 2012) 21(2):157–166DOI 10.1007/s13562-011-0080-3

2000). It would be particularly helpful for enhancingbreeding efficiency to use molecular markers closely linkedto the genes or major quantitative trait loci (QTLs) for graindimensions for selecting target genotypes directly in theearly generations of breeding cycle. Unlike other cereals,much of the rice produced in the world is cooked andconsumed as whole kernel therefore grain shape and sizeare important quality parameters for consumer acceptance.A study was conducted by Suwannaporn and Linnemann(2008) on rice eating quality among consumers in differentrice grain preference countries, which showed that ricetexture, was the best discriminator. It could correctly predictconsumers from non-specific grain preference, short grainpreference and basmati preference at 63, 71 and 81%,respectively. Consumers from non rice-eating countriespreferred harder and less sticky rice than traditional riceconsuming countries. For rice millers, the proportion ofhead and broken rice on milling is an importantattribute which is affected by the grain shape and size(Singh et al. 2002). Long slender grains normally havehigher breakage than short bold grains and consequentlyhave a lower head rice recovery. Recently, genetic andmolecular basis of rice yield has been reviewed thorough-ly and grain length, grain breadth controlling gene wereconsidered important for rice yield also (Xing and Zhang2010).

Lin et al. (1995) identified twelve QTLs for graindimensions, including five for grain length, two for grainbreadth, and five for grain thickness on chromosomes 5, 6and 7. Huang et al. (1997) identified a major QTL affectinggrain length on chromosome 3 and a minor one onchromosome 10 and two QTLs for grain breadth onchromosomes 2 and 3. Redona and Mackill (1998)identified seven QTLs for grain length on chromosomes2, 3, 4 and 7 that together explained 42% of the phenotypicvariation. Xu et al. (2004) identified eight QTLs for grainlength explaining 65.9% of the phenotypic variations, twoof these with major effect were located on chromosomes 3and 5. Wan et al. (2005) reported twenty two QTLs forgrain dimension distributed over eight rice chromosomes.Thus, grain dimension QTLs have been identified onalmost all the 12 rice chromosomes, but accurate chromo-somal locations have been determined only for a smallnumber of major consistent QTLs. These include GW2, gl-3/GS3, qSW5/GW5 and grl7-1/qGL7/qGL7-2. The GW2gene controlling grain width and weight is located onchromosome 2 and encodes a previously unknown RINGtype E3 ubiquitin ligase (Song et al. 2007). She et al.(2010) has identified a mutation (flo2) that resulted inreduced grain size and starch quality. Map based cloningand subsequent over expression of FLO2 gene was shownto enlarge the size of grain significantly. This shows thatFLO2 plays a pivotal role in determining the rice grain size

and starch quality by affecting storage substance accumu-lation in the endosperm. The gl-3 locus controlling grainlength was fine mapped within an 87.5 kbp segment nearthe centromere of rice chromosome 3 (Wan et al. 2006).Subsequently, GS3 gene encoding a trans-membraneprotein was identified in a 7.9 kb DNA fragment close tothe location of gl-3 locus (Fan et al. 2006, 2009). The geneunderlying QTL qSW5/GW5 for grain width on ricechromosome 5 has also been cloned and is associated withhigher spikelet volume and cell number in the outer glumes(Shomura et al. 2008; Weng et al. 2008). A consistent QTLfor grain length has been mapped on rice chromosome 7(grl7-1, qGL7, qGL7-2) using three different mappingpopulations (Amaravathi et al. 2008; Bai et al. 2010; Shaoet al. 2010).

The objective of present study was to narrow down theintervals of two grain length QTLs reported in Basmati riceby Amaravathi et al. (2008) using the RIL populationderived from cross between Pusa 1121 and Pusa 1342 bytargeted marker enrichment of the QTL regions.

Materials and methods

Plant material

A RIL population derived from cross between Pusa 1121,an aromatic Basmati rice variety developed at IndianAgricultural Research Institute (IARI), New Delhi (Singhet al. 2002), and Pusa 1342 a short grain non-aromatic linewas used. In the normal Kharif season total 184 F6 RILswere grown at IARI, New Delhi and Regional station,Karnal.

Phenotyping for grain dimensions

Dehulling of rough paddy was carried out with dehuller(model no. NF 271, 0.5HP motor, 1,400 rpm) and polishedwith Satake polisher (model no. TM05C at 750–1,450 rpm). Ten fully polished grains were photographedwith gel documentation system (Alpha imager) and Imagepro plus software version 4.1 (Win 95/98/NT 4.0, MediaCybernetics) was employed to measure length and breadthof the grains. Before taking the measurements, the softwarewas calibrated and cross checked with Vernier calipers andphoto enlarger readings for the grain dimensions. Advancedfiltering and segmentation techniques in the softwarehelped separate overlapping objects, complete grain bound-aries, and recognize clusters. Once segmented, eachgrouping can be colour-coded for quick visual identificationand class verification. It defines features in the sample, andautomatically takes measurements on selected features suchas best-fit arc, best-fit circle, and best-fit line. Unlike

158 J. Plant Biochem. Biotechnol. (July–Dec 2012) 21(2):157–166

manual or other tedious methods, Image-Pro Plus enabledquick calculation of area, length and breadth.

SSR maker development

We used “SSR finder” software for the primer design(http://hornbill.cspp.latrobe.edu.au). The physical intervals

for the grain length QTLs on chromosomes 1 (RM226–RM104) and 7 (RM11–RM505 and RM505–RM336)reported earlier by Amaravathi et al. (2008), were identifiedby BLAST search in the rice chromosome pseudomoleculeswith the primer sequences of the flanking makers. The partsof pseudomolecule sequences representing QTL intervalswere downloaded from the Vanshanudhan website (www.

8.258.00

7.757.50

7.257.00

6.756.50

6.256.00

5.755.50

5.255.00

50

40

30

20

10

08.00

7.757.50

7.257.00

6.756.50

6.256.00

5.755.50

5.255.00

4.754.50

40

30

20

10

0

2.252.132.001.881.751.631.50

100

80

60

40

20

02.752.502.252.001.751.501.25

100

80

60

40

20

0

5.50

5.25

5.00

4.75

4.50

4.25

4.00

3.75

3.50

3.25

3.00

2.75

2.50

2.25

2.00

50

40

30

20

10

0

(a) (b)

(c) (d)

P1 (7.45) P2P)30.6( 2 (6.15)

P1 (7.6)

P2 (1.93)

P1 (1.8)

P1 (1.88)

P2 (2.15)

(e) (f)

P1 (4.13) P2 (3.13)

P1 (4.08)

P2 (2.88)

Fig. 1 Frequency distributionof lines with different grainlength (a) Delhi (b) Karnal;grain breadth (c) Delhi (d)Karnal and length/breadth ratio(e) Delhi (f) Karnal in 184 RILsderived from Pusa 1121x Pusa1342 cross

J. Plant Biochem. Biotechnol. (July–Dec 2012) 21(2):157–166 159

nrcpb.org) and SSR primers were designed. Further theprimers were checked for their unique hits in the ricegenome using BLAST search available at the Gramenewebsite (www.gramene.org). Primers showing unique hitson the desired chromosome as well as in the whole ricegenome were used for parental polymorphism survey.

DNA extraction and PCR amplification

DNA was extracted from rice leaves using the standardcetyl-trimethyl ammonium bromide method (Murray andThompson 1980). PCR reactions were set up in 10 μlvolume containing 50 ng of rice genomic DNA, 5 pmole(~13 ng) each of forward and reverse primers, 0.1 mMdNTPs, 1x PCR buffer (10 mM Tris, pH 8.0, 50 mM KCland 50 mM ammonium sulphate), 1.8 mM MgCl2, and 0.2unit of Taq DNA polymerase (Fermentas, USA). Thecocktail was subjected to PCR amplification in a thermalcycler (Bio-Rad, USA). The PCR cycling conditionsinvolved initial denaturation at 94°C for 5 min followedby 35 cycles of denaturation at 94°C for 30 sec, primerannealing at 45–65°C for 30 sec, and primer extension at72°C for 2 min. This was followed by a final extension stepat 72°C for 7 min and storage at 4°C until electrophoresis.PCR products were separated in 4% metaphor agarose or10% polyacrylamide gel and photographed using a geldocumentation system. Gradient PCR was done for all the60 SSR primers including the GS3 locus primers tooptimize the annealing temperature (Ta). Genotyping ofparents was done at standardized Ta with all the designedSSR and GS3 makers and 184 RILs were genotyped using16 polymorphic markers (Table 2).

Construction of linkage map and QTL mapping

MAPMAKER software version 3.0b (Lander et al. 1987)was used for the construction of genetic linkage map.Kosambi map function was used for the map construction.QTL analysis was conducted using the QTL cartographerversion 2.5 (Basten et al. 1994; Basten et al. 2004), MultiPoint version 1.1 (Mester et al. 2003) was used for drawing

the chromosome maps. Permutation test was performed toestimate appropriate significant threshold for compositeinterval mapping (Deorge and Churchill 1996). Based on1000 permutations, a LOD threshold of 2.5 was set todeclare a significant QTL.

Results and discussion

Phenotyping for grain length and breadth in the RILpopulation

Phenotyping of the parents and RIL population for grainlength, breadth and length/breadth (L/B) ratio was done onseeds collected from single plants. Grain length, breadthand L/B ratio were categorized according to Cruz andKhush (2000). Grain length of Pusa 1121 was 7.45 mm andthat of Pusa 1342 was 6.03 mm at New Delhi. In Karnalenvironment, the grain length of Pusa 1121 and Pusa 1342was 7.6 and 6.13 mm, respectively. The RIL populationshowed significant variation for grain length, ranging frommedium to very long grains in both the environments atNew Delhi and Karnal. Grain length in the RILs rangedfrom 4.9 mm to 8.3 mm with a population mean of6.62 mm at New Delhi and from 4.6 to 8.1 mm with apopulation mean of 6.80 mm at Karnal (Fig. 1a, b). Most ofthe RILs showed grain length within the parental range buttwenty nine of the RILs showed transgressive segregationtowards shorter grains than Pusa 1342 and fourteen linesshowed grain size longer than Pusa 1121 at New Delhi.Twenty three of the RILs showed transgressive segregationtowards shorter grain length and nine lines had longergrains than Pusa 1121 at Karnal.

Grain breadth for Pusa 1121 was 1.81 mm and that forPusa 1342 was 1.93 mm at New Delhi. In Karnalenvironment, grain breadth for Pusa 1121 and Pusa 1342was 1.88 and 2.15, respectively. In the New Delhienvironment, the mean grain breadth for the RILs was1.82 mm with a range of 1.5 to 2.3 mm, approximately51.08% of the RILs showed grain breadth above or belowthe parental values (Fig. 1c). Total 33.69% of the RILs had

Table 1 Number of SSR markers analysed for rice chromosomes 1 and 7, for fine mapping of grain dimension QTLs qGRL-1.1, qGRL-7.1 andqGRL-7.2 and validation of grain length gene GS3 on chromosome 3

Chromosome QTL name Maker Interval/BAC clone Physical length(kbp)

No. of markerstested

No. of polymorphicmarkers

1 qGRL-1.1 RM226–RM104 6133 11 4

7 qGRL-7.1 RM11–RM505 5269 38 5qGRL-7.2 RM505–RM336

3 qgl3/GS3 OSJNBa0090P23, OSJNBa0002D18, OSJNBa0030J19 423 11 3

Total 60 12

160 J. Plant Biochem. Biotechnol. (July–Dec 2012) 21(2):157–166

grains thinner than Pusa 1121 and 17.39% of the RILs hadgrains thicker than Pusa 1342, indicating high transgressivesegregation. Similarly, at Karnal station 51.6% of the RILshad grain breadth above or below the parental values,35.86% of RILs with thinner grains than Pusa 1121 and15.76% RILs with thicker grains than Pusa 1342 parent,indicating a similar pattern to that observed at New Delhi(Fig. 1d).

The mean L/B ratio was 4.13 for Pusa 1121 and 3.13 forPusa 1342 at New Delhi and 4.08 for Pusa 1121 and 2.88for Pusa 1342 at Karnal. In the RIL population it rangedfrom 2.39 to 5.13 with a population mean of 3.66 (Fig. 1e)at New Delhi and 2.02 to 5.62 with population mean of3.59 at Karnal (Fig. 1f). The frequency distribution of L/Bratio displayed a continuous variation similar to that ofgrain length with 23.91% and 19.56% of the RILs showingtransgressive segregation at Delhi and Karnal stations,respectively.

Linkage map construction and mapping of grain lengthQTLs on chromosomes 1 and 7

Forty-nine newly designed SSR markers (CHR series) wereused for fine mapping of the three grain dimension QTLs(qGRL1.1, qGRL7.1 and qGRL7.2). Polymorphism surveyrevealed that nine of these (18.4%) were polymorphicbetween the two parents (Table 1). All the 184 RILs weregenotyped using the nine polymorphic SSR markers; theirPCR product size ranged between 153 to 399 bp (Table 2).For the grain length QTL qGRL1.1, four polymorphicmarkers were easily scored in the RILs (Fig. 2). Similarly,five polymorphic markers in the qGRL7.1 and qGRL7.2intervals on chromosome 7 were also easily scored in the RILpopulation. The RILs had achieved more than 95% homozy-gosity at the SSR loci analysed; indicating that almost wholegenome of the RILs has reached appreciable level ofhomozygosity desired for a reproducible phenotyping.

Deviation of observed frequencies of alleles at individualSSR loci from the expected 1:1 Mendelian ratio in a RILpopulation is segregation distortion that adversely affectsthe QTL mapping (Lyttle 1991; Xu et al. 2004). Hence,segregation ratio was calculated for each of the poly-morphic SSR makers in the 184 RILs. Out of the nine

Tab

le2

Primer

sequ

encesof

thepo

lymorph

icSSRandSTSmarkers,theirrepeat

motifsandop

timum

amplificationtemperature

(Ta)

used

inthefine

mapping

ofgraindimension

QTLsqG

RL-

1.1,

qLBR-7.1,qL

BR-7.2

andthreemarkers

fortheGS3

locus

Primer

Id.

SSRmotif

Chrom

.No.

Forwardprim

erReverse

prim

erTa(0C)

Phy

sicalpo

sitio

n(bp)

Produ

ctsize

(bp)

CHR01

_6(G

CG)n

1GCACAACATCTA

CTCATTTCC

CCATCTCTCTCTCTCTCCTCT

46.7

38,449

,028

399

CHR01

_8(CCT)n

1GGGTCTCTCTTCTTGCTACTC

TTCTTTGTGTTGGGTTTA

CAG

60.0

38,474

,035

153

CHR01

_2(AT)n

1TA

ACAGCCGACCAATA

ACC

GCCTCTA

CCTGAGACTGAC

60.8

38,486

,041

228

CHR01

_1(AT)n

1TGTCGGAAATA

GAAAGAGAGA

AGGGATA

GTA

AGGTTGAGGTG

60.8

38,785

,913

355

CHR00

7_19

(CT)n

7CTGGTCTCTA

TTTCATTCCTG

TATGCTA

ACTA

CCTGCCAACT

50.5

21,630

,770

314

CHR07

_24

(GCA)n

7TCTGGTTGATTA

TTTGTCGTT

TAGTGTGTGTGTGTGTGGTTT

53.4

21,632

,624

379

CHR07

_34

(CGC)n

7CGCTCTCCAGTCTCCAGT

GGGTA

GAAGTCGTCGCAG

60.7

22,127

,494

348

CHR07

_28

(AG)n

7CGATGGGATA

CGAACACT

TGAACACGGAGATGAACAC

50.5

22,425

,743

378

CHR07

_1(AT)n

7AGATA

CTCATA

GCACGACGAA

GCTCTCCTCATA

CAGCCTA

C60

.022

,693

,688

242

GS6

STS

3AGCAAAGCTGGAACGAAGAG

TAAATTA

CGCCGTGTCAACG

59.0

16,305

,161

148

GS9

STS

3GCAACCAAGTCCACGCTAAT

TAGCCGAAGATCAGCCTCCT

59.0

16,690

,353

170

GS19

CAPS

3TCTGCTTGCGGTTA

TCTGTA

TTA

GGTCCCTTTTCTCGTCC

60.0

16,728

,195

1224

M P2 P1 RILs

Fig. 2 Genotyping of parents and RILs using SSR primer CHR1_2 in10% PAGE showing 1:1 segregation. M=100 bp DNA ladder; P1=Pusa 1121; P2=Pusa 1342

J. Plant Biochem. Biotechnol. (July–Dec 2012) 21(2):157–166 161

markers, six segregated in the expected 1:1 ratio at a cutoffP value of 0.01. One marker, namely CHR7_1 showing

significant segregation distortion (χ2=14.57), was excludedfrom the map construction.

Fig. 3 Genetic maps of rice chromosomess (a) 1, (b) 7 and (c) 3 constructed using MultiPoint Software. The order of markers in the genetic mapswere forced according to their physical position in the rice chromosome pseudomolecules build 6.1 (IRGSP 2005)

Table 3 Fine mapped grain dimension QTLs using 184 RILs derived from cross between rice varieties Pusa1121 and Pusa1342 using additionalmarkers

Trait QTL Id. Chromosome Marker interval N.L.M. (cM) N.R.M. (cM) LOD R2 Additive Allele source

Grain Length qGRL-1.1 1 CHR1_1-RM431 0.2069 0.1769 2.87 0.108 2.00 Pusa 1121

qGRL-7.1 7 CHR7_34-RM505 0.282 0.094 3.18 0.152 2.49 Pusa 1121

Grain Breadth qGRB-7.1 7 CHR7_34- RM505 0.02 0.06 2.95 0.086 0.487 Pusa 1342

Grain L/B ratio qLBR-7.1 7 CHR7_34- RM505 0.02 0.06 2.95 0.086 −0.487 Pusa 1121

LOD: Log10 (probability of linkage/probability of no linkage);

R2 : Percentage of variation accounted for by the QTL;

N.R.M. and N.L.M = nearest right marker and nearest left marker genetic distance;

Additive = additive effect expressed in terms of estimated change in phenotype expected from introgression of Pusa 1121 allele.

162 J. Plant Biochem. Biotechnol. (July–Dec 2012) 21(2):157–166

Linkage maps for the three chromosomes harboringgrain dimension QTLs were constructed using Kosambifunction. Nine new random SSR markers were used forremapping of the QTL intervals qGRL1.1, qGRL7.1,qGRL7.2 on chromosome 1 and 7. Genetic distance (incM) between markers was estimated by Multipoint soft-ware. During the revised map construction all the twentyeight makers on these two chromosomes reported earlier byAmaravathi et al. (2008) were merged with the eight newmarkers by fixing the order of the makers according to theirphysical location in the pseudomolecules because newmarkers were targeted specifically to the mapped QTLintervals (Table 1). Two makers from the earlier map andtwo new makers added for fine mapping did not showsignificant genetic linkage in the revised map constructedbased on 184 lines and hence these were dropped from therevised map of chromosome 7 (Fig. 3).

The marker interval RM226–RM104 for grain lengthQTL qGRL1.1 on rice chromosome 1 was remapped withfour additional markers resulting in a revised map ofchromosome 1 with total twenty-one markers. Phenotypicdata from both New Delhi and Karnal locations showed aconsistent and stable QTL qGRL1.1 for grain length in anarrowed down marker interval RM431-CHR1_1 (Fig. 3a).The QTL cartographer detected qGRL1.1 with a significantLOD score of 2.87 explaining 10.8% of the total pheno-typic variation for grain length (Table 3). The earlierreported 6.13 Mbp physical interval of the QTL wasnarrowed down to only 108 kbp (Fig. 3a). There were totalthirteen predicted gene models in this interval whichincludes the probable candidate gene for the exceptionallyhigh grain length of the Basmati variety Pusa 1121(Table 4).Pectin acetylesterase and Serine/threonine protein kinasehas been found as expressed gene out of thirteen genesreported in this interval. Pectin esterases plays an importantrole in determining the extent to which pectin is accessibleto degradation by cell wall hydrolyzing enzymes and Pectinacetylesterase was found responsible for cell expansion inbarley (Radchuk et al. 2011). Wen et al. (1999) showed thatthe partial inhibition of pectin methylesterase by antisenseRNA reduced root elongation in transgenic pea hairy roots.Since pectin acetyl eaterase has been found responsible forcell wall expansion therefore, it has some relation with thegrain length but its role in rice grain length needs to bevalidated.

The two tandem QTL intervals, mapped earlier on ricechromosome 7 between markers RM11–RM505 andRM505–RM336 were remapped including two new SSRmakers in and around this interval, making a total of elevenmarkers for this chromosome (Fig. 3b). In both thephenotyping environments a consistent common QTLregion was identified for grain length, breadth and L/Bratio (qGRL7.1, qGBR7.1, qLBR7.1) in a narrowed down T

able

4Listof

genesidentifiedin

theqG

RL1.1locatedon

chromosom

e1(38,78

5,91

3–38

,893

,994

bp)

S.No.

Locus

name

Geneprod

uct

CDScoordinate

Nucleotide

leng

th

1LOC_O

S01

g668

20Inactiv

ereceptor

kinase

At1g2

7190

precursor,pu

tativ

e,expressed

3879

4795–3

8795

634

840

2LOC_O

S01

g668

30Pectin

acetylesterase

domaincontaining

protein,

expressed

3880

2865–3

8799

913

1242

3LOC_O

S01

g668

40Pectin

acetylesterase

domaincontaining

protein

3880

9408–3

8806

512

1203

4LOC_O

S01

g668

50Pectin

acetylesterase

domaincontaining

protein,

expressed

3881

4136–3

881130

712

09

5LOC_O

S01

g668

60Serine/threon

ineproteinkinase,pu

tativ

e,expressed

3881

7924–3

8820

785

1503

6LOC_O

S01

g668

70Transpo

sonprotein,

putativ

e,CACTA

,En/Spm

sub-class

3883

8958–3

8825

440

7740

7LOC_O

S01

g668

90BTBZ1—

Bric-a-Brac,

Tramtrack,

andBroad

Com

plex

BTBdo

main

with

TAZzinc

fing

erandCalmod

ulin-binding

domains,expressed

3884

3395–3

8845

343

1242

8LOC_O

S01

g669

00Exp

ressed

protein

3885

3234–3

8851

953

903

9LOC_O

S01

g669

10Hyp

othetical

protein

3885

9171–3

8858

370

378

10LOC_O

S01

g669

20Ser/Thr

proteinph

osph

atasefamily

protein,

putativ

e,expressed

3886

3024–3

8866

487

2202

11LOC_O

S01

g669

30Transpo

sonprotein,

putativ

e,CACTA

,En/Spm

sub-class,expressed

3887

1705–3

8870

855

756

12LOC_O

S01

g669

40Kinase,

pfkB

family,pu

tativ

e,expressed

3888

6456–3

8884

295

972

13LOC_O

S01

g669

50Hyp

othetical

protein

3888

8202–3

8887

762

441

J. Plant Biochem. Biotechnol. (July–Dec 2012) 21(2):157–166 163

marker interval CHR7_34-RM505 (Table 3). After additionof two new markers the two consecutive intervals reportedearlier (Amaravathi 2005) got merged in to a single intervalof a much reduced physical length of 2399 kbp (CHR7_34-RM505). The grain length QTL qGRL7.1 was identifiedwith a LOD score of 3.18 and explained 15.2% of thephenotypic variation (Tables 3). The QTLs for grainbreadth (qGBR7.1) and length/breadth ratio (qLBR7.1)were also identified in the same marker interval withsignificant LOD scores of 2.95 and 2.95, respectively,explaining 8.6% of the phenotypic variations (Table 3).After first reports by Lin et al. (1995), Redona and Mackill(1998) and Amaravathi (2005), QTLs for grain length andgrain shape has also been fine mapped independently byBai et al. 2010, and Shao et al. 2010, in the same region ofrice chromosome 7. The physical interval considered forfine mapping of grain length, breadth and grain length/breadth ratio QTLs on chromosome 7 between markerRM11 and RM336 mapped by Amaravathi et al. (2008)was 5.20 Mbp, which was narrowed down to 2.39Mbpbetween markers CHR7_34 and RM505 after addition ofnew SSR markers in the present study. Our fine mapped

QTL region lies between 22.127–24.526 Mbp whereas,Shao et al. (2010) has mapped it between 24.657–24.93Mbp hence newly reported QTL is 130.39 kbp apart fromqGL7-2. Bai et al. (2010) has mapped qGL7 on samechromosome between interval 28.224–28.482 Mbp. Thenewly reported QTL needs further addition of new markersso that exact position of QTL responsible for grain lengthcan be accurately asserted. Therefore at this stage it maynot be called as qGL7-3.

Validation of GS3 locus markers for rice grain length

A consistent QTL for grain length has been reported byseveral workers on rice chromosome 3 and has beendescribed as the major gene (GS3) associated with thedomestication of rice (Kovach et al. 2010). Fan et al. (2006)developed several markers for fine mapping of the GS3gene on chromosome 3. Eleven of these markers werescreened in the present segregating population but onlythree were polymorphic between the parental lines (Table 2).Two Indel (GS06 and GS09) and one CAPS (GS19) markershowed clear polymorphism, Since CAPS (GS19) marker

Fig. 4 Validation of thegrain length gene GS3polymorphism in the parentallines of mapping population,Pusa 1121 and Pusa 1342 usingSF28 CAPS marker

164 J. Plant Biochem. Biotechnol. (July–Dec 2012) 21(2):157–166

was not segregating in 1:1 ratio, therefore it was notincluded during final map construction. Besides this, sixmore SSR markers (CHR series) were mapped on thechromosome 3 (Singh et al. 2007). Total eight polymorphicmarkers (GS and CHR series) were combined with sevenRM series markers mapped on the chromosome 3 byAmaravathi et al. (2008). However, no significant QTL forgrain length was obtained in either of the two environ-ments. This showed that the GS3 gene for grain lengthreported by Fan et al. (2006) was not responsible for thegrain length difference in our mapping population. Theshort grain parent Pusa 1342 also has a slender grain shapewhile presence of wild type allele of the GS3 gene actuallyleads to short roundish grains. To further support thisconclusion, the GS3 gene based CAPS marker SF28,developed by Fan et al. (2009) was used on the parentallines along with positive and negative controls. Our resultsclearly indicate that both the parents of our mappingpopulation, Pusa 1121 and Pusa 1342, contain the wildtype GS3 allele (Fig. 4). That is why we could not identifyany QTL for grain length on chromosome 3 in ourpopulation despite having large difference in grain size.Several researchers have mapped a major QTL for grainlength on chromosome 3 (Aluko et al. 2004; Wan et al.2005; Lin et al. 1995; Bai et al. 2010; Shao et al. 2010), butin the present study no QTL was obtained on Chromosome3. The grain length QTLs mapped on chromosomes 1 and 7were both contributed by Pusa 1121 (Table 3). Similarly,QTLs for grain breadth have been mapped on ricechromosome 2, 3, 5, 6, 7, 8 (Tan et al. 2000; Redona andMacKill 1998; Bai et al. 2010; Rabiei et al. 2004), but inour mapping population we found a consistent QTL onlyon chromosome 7.

In the present study grain length QTLs on chromosome1 and 7 were fine mapped to 108 kbp and 2,399 kbp,respectively. List of predicted genes in these fine mappedQTL regions of qGRL1.1, qGRL7.1, qGRB7.1 andqLBR7.1can be easily obtained from the rice genomedatabase for further validation in functional complementa-tion studies. Gene-based markers can be used in a large F2fine mapping population to look for rare recombination inan attempt to define the actual genes for rice grain length,breadth and length/breadth ratio in these QTL regions andtheir further use in the rice breeding programs. Thepreviously identified grain length gene GS3 was invariantin this population. The grain length QTL on chromosome 1is reported for the first time in Pusa 1121 and it mayresponsible for the exceptional grain length of this Basmativariety in addition to the GS3 and qGRL7.1 loci.

Acknowledgements Authors are grateful to the Indian Council ofAgricultural Research for financial support under the Network Projectfor Transgenics in Crops.

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