Genetic differentiation for nuclear,mitochondrial and chloroplast genomes in common wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.) and cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The genetic differentiation of nuclear, mitochondrial (mt) and chloroplast (cp) genomes was investigated by Southern and PCR analysis using 75 varieties of cultivated rice ( Oryza sativa L.) and 118 strains of common wild rice (CWR, Oryza rufipogon Griff.) from ten countries of Asia. The distinguishing differences between the Indica and Japonica cultivars were detected both in the nuclear genome and the cytoplasmic genome, confirming that the Indica-Japonica differentiation is of major importance for the three different classes of genome in cultivated rice. This differentiation was also detected in common wild rice with some differences among the genome compartments and the various regions. For nuclear DNA variation, both Indica-like and Japonica-like types were observed in the Chinese CWR, with the latter more-frequent than the former. No Japonica-like type was found in South Asia, and only two strains of the Japonica-like type were detected in Southeast Asia, thus the Indica-like type is the major type among South and Southeast Asian CWR. For mtDNA, only a few strains of the Japonica-like type were detected in CWR. For cpDNA, the Japonica type was predominant among the CWR strains from China, Bangladesh and Burma, while the Indica type was predominant among the CWR strains from Thailand, Malaysia, Cambodia and Sri Lanka, and both types were found in similar frequencies among the Indian CWR. Altogether, however, the degree of Indica-Japonica differentiation in common wild rice was much-less important than that in cultivated rice. Cluster analyses for nuclear and mitochondrial DNA variation revealed that some CWR strains showed large genetic distances from cultivated rice and formed clusters distinct from cultivated rice. Coincidence in the genetic differentiation between the three different classes of genome was much higher in cultivated rice than in CWR. Among the 75 cultivars, about 3/4 entries were "homoeotype" showing congruent results for nuclear, mt and cpDNA regarding the Indica-Japonica differentiation. In CWR, the proportions of homoeotypes were 5.7%, 15% and 48.8% in China, South Asia and Southeast Asia, respectively. Based on the average genetic distance among all the strains of CWR and cultivated rice for nuclear and mitochondrial genomes, the variability of the nuclear genome was found to be higher than that of the mitochondrial genome. The global pattern based on all genomes shows much-more diversification in CWR than that in cultivated rice.     

Fine mapping and identification of a novel locus <Emphasis Type="Italic">qGL12.2</Emphasis> control grain length in wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.)

Key message

A wild rice QTL qGL12.2 for grain length was fine mapped to an 82-kb interval in chromosome 12 containing six candidate genes and none was reported previously.

Abstract

Grain length is an important trait for yield and commercial value in rice. Wild rice seeds have a very slender shape and have many desirable genes that have been lost in cultivated rice during domestication. In this study, we identified a quantitative trait locus, qGL12.2, which controls grain length in wild rice. First, a wild rice chromosome segment substitution line, CSSL41, was selected that has longer glume and grains than does the Oryza sativa indica cultivar, 9311. Next, an F₂ population was constructed from a cross between CSSL41 and 9311. Using the next-generation sequencing combined with bulked-segregant analysis and F₃ recombinants analysis, qGL12.2 was finally fine mapped to an 82-kb interval in chromosome 12. Six candidate genes were found, and no reported grain length genes were found in this interval. Using scanning electron microscopy, we found that CSSL41 cells are significantly longer than those of 9311, but there is no difference in cell widths. These data suggest that qGL12.2 is a novel gene that controls grain cell length in wild rice. Our study provides a new genetic resource for rice breeding and a starting point for functional characterization of the wild rice GL gene.

     

Mapping QTLs influencing rice floral morphology using recombinant inbred lines derived from a cross between <Emphasis Type="Italic">Oryza sativa</Emphasis> L. and <Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff

To understand the genetic basis of floral traits associated with the mating system in rice, we analyzed pistil, stamen and glume traits using a recombinant inbred line population, derived from a cross between an Asian cultivated rice ( Oryza sativa L.), Pei-kuh, and a wild rice ( Oryza rufipogon Griff.), W1944. Quantitative trait loci (QTLs) affecting floral morphology were detected by composite interval mapping using a linkage map constructed using 147 markers, mostly RFLPs. A total of 7, 4, 14 and 6 QTLs were detected for traits related to pistil, stamen, and size and shape of the glume, respectively. Comparison of 31 QTLs affecting these organs revealed ten QTLs affecting the different organs in four adjacent regions on chromosomes 2, 4, 5 and 10, but most QTLs (68%) were located separately on the whole chromosomes. Although four QTLs for stigma breadth, anther length and thickness of lemma and palea explained more than 25% of the total phenotypic variance, most QTLs (87%) had smaller effects. These results suggest that quantitative variation observed for pistil, stamen and glume traits is controlled by several distinct genes with small effects.     

Large-scale DNA polymorphism study of <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Emphasis Type="Italic">O. rufipogon</Emphasis> reveals the origin and divergence of Asian rice

Polymorphism over ∼26 kb of DNA sequence spanning 22 loci and one region distributed on chromosomes 1, 2, 3 and 4 was studied in 30 accessions of cultivated rice, Oryza sativa, and its wild relatives. Phylogenetic analysis using all the DNA sequences suggested that O. sativa ssp. indica and ssp. japonica were independently domesticated from a wild species O. rufipogon. O. sativa ssp. indica contained substantial genetic diversity (π = 0.0024), whereas ssp. japonica exhibited extremely low nucleotide diversity (π = 0.0001) suggesting the origin of the latter from a small number of founders. O. sativa ssp. japonica contained a larger number of derived and fixed non-synonymous substitutions as compared to ssp. indica. Nucleotide diversity and genealogical history substantially varied across the 22 loci. A locus, RLD15 on chromosome 2, showed a distinct genealogy with ssp. japonica sequences distantly separated from those of O. rufipogon and O. sativa ssp. indica. Linkage disequilibrium (LD) was analyzed in two different regions. LD in O. rufipogon decays within 5 kb, whereas it extends to ∼50 kb in O. sativa ssp. indica. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nucleotide polymorphism in the <Emphasis Type="Italic">Adh1</Emphasis> locus region of the wild rice <Emphasis Type="Italic">Oryza rufipogon</Emphasis>

Nucleotide variation in the alcohol dehydrogenase (Adh1) locus region of the wild rice Oryza rufipogon and its related species was analysed to clarify the maintenance mechanism of DNA variation in Oryza species. The estimated nucleotide diversity in the Adh1 locus region of O. rufipogon was 0.002, which was one of the lowest values detected in nuclear loci of plant species investigated so far. Tests of neutrality detected significantly negative deviation from the neutral mutation model for the coding region, especially for replacement sites. When each of the ADH1 domains was considered, significance was detected only for the catalytic domain 1. These results suggest purifying selection in the Adh1 coding region. In the phylogenetic tree of Oryza species based on Adh1 variation, cultivated rice O. sativa subspp. japonica and indica were included in the cluster of O. rufipogon. The genetic distance of the Adh1 region between O. rufipogon and O. sativa was as low as the nucleotide diversity of O. rufipogon. These results imply that O. rufipogon and O. sativa cannot be classified based on the nucleotide variation of Adh1. No replacement divergence between O. rufipogon and the other three A-genome species (O. glumaepatula, O. barthii and O. meridionalis) were detected, indicating that ADH1 is conserved in the A-genome species. On the other hand, between O. rufipogon and the E-genome species O. australiensis, replacement changes were detected only in the catalytic domain 1. The difference in replacement substitutions between the A- and E-genome species may be related to adaptive changes in the ADH1 domains, reflecting environmental differences where the species encounter anaerobic stress.     

Microsatellite analysis of genetic diversity and population genetic structure of a wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.) in China

Genetic diversity and population genetic structure of natural Oryza rufipogon populations in China were studied based on ten microsatellite loci. For a total of 237 individuals of 12 populations collected from four regions, a moderate to high level of genetic diversity was observed at population levels with the number of alleles per locus ( A) ranging from 2 to 18 (average 10.6), and polymorphic loci ( P) from 40.0% to 100% (average 83.3%). The observed heterozygosity ( H(O)) varied from 0.163 to 0.550 with the mean of 0.332, and the expected heterozygosity ( H(E)) from 0.164 to 0.648 with the mean of 0.413. The level of genetic diversity for Guangxi was the highest. These results are in good agreement with previous allozyme and RAPD studies. However, it was unexpected that high genetic differentiation among populations was found ( R(ST) = 0.5199, theta = 0.491), suggesting that about one-half of the genetic variation existed between the populations. Differentiation (pairwise theta) was positively correlated with geographical distance ( r = 0.464), as expected under the isolation by distance model. The habitat destruction and degradation throughout the geographic range of O. rufipogon may be the main factor attributed to high genetic differentiation among populations of O. rufipogon in China.     

Transposition behavior of nonautonomous a <Emphasis Type="Italic">hAT</Emphasis> superfamily transposon <Emphasis Type="Italic">nDart</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Transposable elements (TEs) have a significant impact on the evolution of gene function and genome structures. An endogenous nonautonomous transposable element nDart was discovered in an albino mutant that had an insertion in the Mg-protoporphyrin IX methyltransferase gene in rice. In this study, we elucidated the transposition behavior of nDart, the frequency of nDart transposition and characterized the footprint of nDart. Novel independent nDart insertions in backcrossed progenies were detected by DNA blotting analysis. In addition, germinal excision of nDart occurred at very low frequency compared with that of somatic excision, 0–13.3%, in the nDart1-4(3-2) and nDart1-A loci by a locus-specific PCR strategy. A total of 253 clones from somatic excision at five nDart loci in 10 varieties were determined. nDart rarely caused deletions beyond target site duplication (TSD). The footprint of nDart contained few transversions of nucleotides flanking to both sides of the TSD. The predominant footprint of nDart was an 8-bp addition. Precise excision of nDart was detected at a rate of only 2.2%, which occurred at two loci among the five loci examined. Furthermore, the results in this study revealed that a highly conserved mechanism of transposition is involved between maize Ac/Ds and rice Dart/nDart, which are two-component transposon systems of the hAT superfamily transposons in plant species.     

Mapping of quantitative trait loci controlling low-temperature germinability in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Low-temperature germination is one of the major determinants for stable stand establishment in the direct seeding method in temperate regions, and at high altitudes of tropical regions. Quantitative trait loci (QTLs) controlling low-temperature germinability in rice were identified using 122 backcross inbred lines (BILs) derived from a cross between temperate japonica varieties, Italica Livorno and Hayamasari. The germination rate at 15°C was measured to represent low-temperature germination and used for QTL analysis. The germination rate at 15°C for 7 days of Italica Livorno and Hayamasari was 98.7 and 26.8%, respectively, and that of BILs ranged from 0 to 83.3%. Using restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) markers, we constructed a linkage map which corresponded to about 90% of the rice genome. Three putative QTLs associated with low-temperature germination were detected. The most effective QTL, qLTG-3-1 on chromosome 3, accounted for 35.0% of the total phenotypic variation for low-temperature germinability. Two additional QTLs, qLTG-3-2 on chromosome 3 and qLTG-4 on chromosome 4, were detected and accounted for 17.4 and 5.5% of the total phenotypic variation, respectively. The Italica Livorno alleles in all detected QTLs increased the low-temperature germination rate.Communicated by F. Salamini     

Marker imputation efficiency for genotyping-by-sequencing data in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) and alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>)

Genotyping-by-sequencing (GBS) is a rapid and cost-effective genome-wide genotyping technique applicable whether a reference genome is available or not. Due to the cost-coverage trade-off, however, GBS typically produces large amounts of missing marker genotypes, whose imputation becomes therefore both challenging and critical for later analyses. In this work, the performance of four general imputation methods (K-nearest neighbors, Random Forest, singular value decomposition, and mean value) and two genotype-specific methods (“Beagle” and FILLIN) was measured on GBS data from alfalfa (Medicago sativa L., autotetraploid, heterozygous, without reference genome) and rice (Oryza sativa L., diploid, 100 % homozygous, with reference genome). Alfalfa SNP were aligned on the genome of the closely related species Medicago truncatula L.. Benchmarks consisted in progressive data filtering for marker call rate (up to 70 %) and increasing proportions (up to 20 %) of known genotypes masked for imputation. The relative performance was measured as the total proportion of correctly imputed genotypes, globally and within each genotype class (two homozygotes in rice, two homozygotes and one heterozygote in alfalfa). We found that imputation accuracy was robust to increasing missing rates, and consistently higher in rice than in alfalfa. Accuracy was as high as 90–100 % for the major (most frequent) homozygous genotype, but dropped to 80–90 % (rice) and below 30 % (alfalfa) in the minor homozygous genotype. Beagle was the best performing method, both accuracy- and time-wise, in rice. In alfalfa, KNNI and RFI gave the highest accuracies, but KNNI was much faster.     

Gas exchange,carbon balance and stomatal traits in wild and cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes

Carbon balancing within the plant species is an important feature for climatic adaptability. Photosynthesis and respiration traits are directly linked with carbon balance. These features were studied in 20 wild rice accessions Oryza spp., and cultivars. Wide variation was observed within the wild rice accessions for photosynthetic oxygen evolution or photosynthetic rate (A), dark (R _d), and light induced respiration (LIR) rates, as well as stomatal density and number. The mean rate of A varied from 10.49 μmol O₂ m^?2 s^?1 in cultivated species and 13.09 μmol O₂ m^?2 s^?1 in wild spp., The mean R _d is 2.09 μmol O₂ m^?2 s^?1 and 2.31 μmol O₂ m^?2 s^?1 in cultivated and wild spp., respectively. Light induced Respiration (LIR) was found to be almost twice in wild rice spp., (16.75 μmol O₂ m^?2 s^?1) compared to cultivated Oryza spp., Among the various parameters, this study reveals LIR and A as the key factors for positive carbon balance. Stomatal contribution towards carbon balance appears to be more dependent on abaxial surface where several number of stomata are situated. Correlation analysis indicates that R _d and LIR increase with the increase in A. In this study, O. nivara (CR 100100, CR 100097), O. rufipogon (IR 103404) and O. glumaepatula (IR104387) were identified as potential donors which could be used in rice breeding program. Co-ordination between gas exchange and patchiness in stomatal behaviour appears to be important for carbon balance and environmental adaptation of wild rice accessions, therefore, survival under harsh environment.     

Functional markers for <Emphasis Type="Italic">xa5</Emphasis>-mediated resistance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>, L.)

The recent cloning of several agronomically important genes has facilitated the development of functional markers. These markers reside within the target genes themselves and can be used with great reliability and efficiency to identify favorable alleles in a breeding program. Bacterial blight (BB) is a severe rice disease throughout the world that is controlled primarily through use of resistant cultivars. xa5 is a race-specific, recessive gene mediating resistance to BB. It is widely used in rice breeding programs throughout the tropics. Due to its recessive nature, phenotypic selection for xa5-mediated resistance is both slow and costly. Previously, marker assisted selection (MAS) for this resistance gene was not efficient because it involved markers that were only indirectly linked to xa5 and ran the risk of being separated from the trait by recombination. Recently, the cloning of the gene underlying this trait made it possible to develop functional markers. Here we present a set of CAPS markers for easy, quick and direct identification of cultivars or progeny carrying xa5-mediated resistance and provide evidence that these markers are 100% predictive of the presence of the xa5 allele. These markers are expected to enhance the reliability and cost-effectiveness of MAS for xa5-mediated resistance.     

Knockout of a starch synthase gene <Emphasis Type="Italic">OsSSIIIa</Emphasis>/<Emphasis Type="Italic">Flo5</Emphasis> causes white-core floury endosperm in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1–5°C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.     

QTL mapping and introgression of yield-related traits from <Emphasis Type="Italic">Oryza glumaepatula</Emphasis> to cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) using microsatellite markers

Rice ( Oryza sativa) cultivar development currently faces the task of overcoming yield plateaus, which is difficult due to the narrow genetic base of breeding programs. Oryza glumaepatula is a diploid wild relative of cultivated rice, native to Central and South America, and is therefore a potential source of alleles of agronomic importance to rice breeding programs. We studied 11 agronomic traits in BC(2)F(2) families of the interspecific cross Oryza sativa x O. glumaepatula. Transgressive lines which are almost isogenic to the elite recurrent O. sativa parent were identified for most of these traits. Quantitative trait locus (QTL) analysis was performed by single-point and interval mapping using a molecular map based on 157 microsatellite and STS markers. Marker regions accounting for 14.5 to 72.9% of a phenotypic variation trait were identified in 9 of the 12 rice chromosomes. Positive QTL effects from O. glumaepatula were observed in chromosomal regions associated with tillering and panicle-number traits.     

Assessment of genetic diversity of <Emphasis Type="Italic">Saltol</Emphasis> QTL among the rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes

Eight Saltol quantitative trait locus (QTL) linked simple sequence repeat (SSR) markers of rice (Oryza sativa L.) were used to study the polymorphism of this QTL in 142 diverse rice genotypes that comprised salt tolerant as well as sensitive genotypes. The SSR profiles of the eight markers generated 99 alleles including 20rare alleles and 16 null alleles. RM8094 showed the highest number (13) of alleles followed by RM3412 (12), RM562 (11), RM493 (9) and RM1287 (8) while as, RM10764 and RM10745 showed the lowest number (6) of alleles. Based on the highest number of alleles and PIC value (0.991), we identified RM8094 as suitable marker for discerning salt tolerant genotypes from the sensitive ones. Based upon the haplotype analysis using FL478 as a reference (salt tolerant genotypes containing Saltol QTL), we short listed 68 rice genotypes that may have at least one allele of FL478 haplotype. Further study may confirm that some of these genotypes might have Saltol QTL and can be used as alternative donors in salt tolerant rice breeding programmes.     

Map-based cloning and functional analysis of the chromogen gene <Emphasis Type="Italic">C</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The chromogen gene C is critical for anthocyanin regulation in rice, and apiculus color is an important agronomic trait in selective breeding and variety purification. Mapbased cloning and in-depth functional analysis of the C gene will be useful for understanding the molecular mechanism of anthocyanin biosynthesis and for rice breeding. Japonica landrace Lijiangxintuanheigu (LTH) has red apiculi and purple stigmas. Genetic analysis showed that red apiculus and purple stigma in LTH co-segregated indicating control by a single dominant gene, or by two completely linked genes. Using 1,851 recessive individuals from two F₂ populations, the target gene OsC was delimited to a 70.8 kb interval on chromosome 6 that contains the rice homologue of the maize anthocyanin regulatory gene C1. When the entire OsC gene and its full-length cDNA cloned from LTH were transformed into japonica cultivar Kitaake with colorless apiculi and stigmas all positive transformants had red apiculi but non-colored stigmas, validating that OsC alone was responsible for the apiculus color and represented the functional C gene. OsC was constitutively expressed in all tissues examined, with strongest expression in leaf blades. These results set a foundation to clarify the regulatory mechanisms of OsC in the anthocyanin biosynthetic pathway.     

Mapping of quantitative trait loci associated with ultraviolet-B resistance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The detection of quantitative trait loci (QTLs) associated with UV-B resistance in rice should allow their practical application in breeding for such a complex trait, and may lead to the identification of gene characteristics and functions. Considerable variation in UV-B resistance exists within cultivated rice (Oryza sativa L.), but its detailed genetic control mechanism has not been well elucidated. We detected putative QTLs associated with the resistance to enhanced UV-B radiation in rice, using 98 BC₁F₅ (backcross inbred lines; BILs) derived from a cross between Nipponbare (a resistant japonica rice variety) and Kasalath (a sensitive indica rice variety). We used 245 RFLP markers to construct a framework linkage map. BILs and both parents were grown under visible light with or without supplemental UV-B radiation in a growth chamber. In order to evaluate UV-B resistance, we used the relative fresh weight of aerial parts (RFW) and the relative chlorophyll content of leaf blades (RCC). The BIL population exhibited a wide range of variation in RFW and RCC. Using composite interval mapping with a LOD threshold of 2.9, three putative QTLs associated with both RFW and RCC were detected on chromosomes 1, 3 and 10. Nipponbare alleles at the QTLs on chromosome 1 and 10 increased the RFW and RCC, while the Kasalath allele at the QTL on chromosome 3 increased both traits. Furthermore, the existence of both QTLs on chromosomes 1 and 10 for UV-B resistance was confirmed using chromosome segment substitution lines. Plants with Kasalath alleles at the QTL on chromosome 10 were more sensitive to UV-B radiation than plants with them on chromosome 1. These results also provide the information not only for the improvement of UV-B resistance in rice though marker-associated selection, but also for the identification of UV-B resistance mechanisms by using near-isogenic lines.Communicated by D.J. Mackill     

Mapping of quantitative trait loci for basmati quality traits in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Traditional basmati rice varieties are very low yielding due to their poor harvest index, tendency to lodging and increasing susceptibility to foliar diseases; hence there is a need to develop new varieties combining the grain quality attributes of basmati with high yield potential to fill the demand gap. Genetic control of basmati grain and cooking quality traits is quite complex, but breeding work can be greatly facilitated by use of molecular markers tightly linked to these traits. A set of 209 recombinant inbred lines (RILs) developed from a cross between basmati quality variety Pusa 1121 and a contrasting quality breeding line Pusa 1342, were used to map the quantitative trait loci (QTLs) for seven important quality traits namely grain length (GL), grain breadth (GB), grain length to breadth ratio (LBR), cooked kernel elongation ratio (ELR), amylose content (AC), alkali spreading value (ASV) and aroma. A framework molecular linkage map was constructed using 110 polymorphic simple sequence repeat (SSR) markers distributed over the 12 rice chromosomes. A number of QTLs, including three for GL, two for GB, two for LBR, three for aroma and one each for ELR, AC and ASV were mapped on seven different chromosomes. While location of majority of these QTLs was consistent with the previous reports, one QTL for GL on chromosomes 1, and one QTL each for ELR and aroma on chromosomes 11 and 3, respectively, are being reported here for the first time. Contrary to the earlier reports of monogenic recessive inheritance, the aroma in Pusa 1121 is controlled by at least three genes located on chromosomes 3, 4 and 8, and similar to the reported association of badh2 gene with aroma QTL on chromosome 8, we identified location of badh1 gene in the aroma QTL interval on chromosome 4. A discontinuous 5 + 3 bp deletion in the seventh exon of badh2 gene, though present in all the RILs with high aroma, was not sufficient to impart this trait to the rice grains as many of the RILs possessing this deletion showed only mild or no aroma expression. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.