Effects of winter flooding on weedy rice (Oryza sativa L.)

Weedy rice (Oryza sativa L.), characterised by competitiveness, seed longevity, and dormancy is a troublesome weed to rice fields. Furthermore, its close botanical affinity to cultivated rice makes its control particularly difficult. However, winter flooding of rice fields can be an efficient technique to control weedy rice infestation by promoting weed seed decay, animal predation, or germination.     

Diversity of weedy red rice (Oryza sativa L.) in Arkansas,U.S.A. in relation to weed management

Weedy rice, specifically red rice (Oryza sativa L.), is a major weed in rice which causes up to 80% yield loss and reduction of grain quality. Red rice accessions from Arkansas, U.S.A., were characterized to classify red rice accessions into certain phenotypic groups relevant to weedy rice management. The red rice accessions were 70% strawhull, 22% blackhull, 7% brownhull and <1% goldhull. Generally, blackhull red rice was the tallest (139 cm) and strawhull the shortest (133 cm) among all accessions. Blackhull red rice had more tillers (102/plant), smaller flag leaves (13 cm wide, 34 cm long), and flowered later (1225 heat units) than strawhull red rice which had 85 tillers/plant, 15 cm-wide and 34 cm-long flag leaves, and flowered after accumulating 1195 heat units. Morphological differences between accessions within each hull type were highly significant, showing great diversity within a hull color group as indicated by large ranges in traits. For example, blackhulls were 75–190 cm tall with 18–69 cm long flag leaves, 21–188 tillers and produced 40–949 g seed. Strawhulls were 46–189 cm tall with 18–66 cm flag leaf length, 16–172 tillers and produced 100–608 g seed. Some traits, such as seed production, differed widely between accessions within each hull color group such that the average seed production/accession for blackhull did not differ from that of strawhull weedy rice (196 vs. 192 g/plant). The onset of flowering among all accessions ranged from 56 to 126 d after planting. Red rice accessions formed six phenotypic clusters generally segregated by plant size or flowering time. Each morphotype would have different competitive abilities; thus, weedy rice management could be geared toward plant types. Highly competitive plant types would require intensive control measures to minimize yield losses and reduce the soil seed bank. Flowering dates impact stewardship strategies for herbicide-resistant or any genetically modified rice.     

Differential tolerance of weedy red rice (Oryza sativa L.) from Arkansas, USA to glyphosate

Red rice (Oryza sativa L.), a weedy relative of cultivated rice (O. sativa), is a troublesome weed in rice in the southern United States. It is also very problematic in other world regions where rice is grown. There are many types of red rice. Some have white pericarp. In this paper, the term ‘red rice’ will be used to refer to the weedy types that occur in this region of the southern USA. Because of the great diversity in morphology and phenology of red rice populations in the USA, we hypothesized that these have differential tolerance to glyphosate. Red rice is subjected to selection pressure by glyphosate, which is the principal herbicide used in glyphosate-resistant soybean planted after rice. Two hundred-fifteen red rice accessions were collected in Arkansas, USA between 2002 and 2003 and evaluated for glyphosate tolerance between 2003 and 2006. The commercial dose of glyphosate controlled red rice accessions 81-100% regardless of application timing, by 30 d after treatment. The number of survivors that produced seeds and the seeds produced per survivor differed between accessions. Application of the full dose at the tillering stage was more effective than at the 3- to 4-leaf stages. At half the field dose, 40 to 54% of survivors produced seed, with germination capacity of 30-100%. The lowest dose of glyphosate, 225 g a.e. ha⁻¹, resulted in the widest range of control ratings from 30 to 100%. The red rice accessions differentiated into six tolerance categories. A tolerance map was generated and will be used in educating growers about the proper use of glyphosate for red rice management.     

Metabolite profiling of colored rice (Oryza sativa L.) grains

HPLC analysis of dehulled red, black and non-colored indica and japonica rice subspecies revealed significant differences in the contents of the anthocyanins cyanidin-3-glucoside and peonidin-3-glucoside. The rice materials were subjected to a comparative capillary gas chromatography-based metabolite profiling approach. The employed extraction and fractionation protocol enables the analysis of a broad spectrum of lipophilic and hydrophilic low molecular weight constituents from different chemical classes. The method covers not only primary metabolites (e.g. sugars, fatty acids) but also nutritionally relevant constituents (e.g. α-tocopherol, γ-aminobutyric acid). Statistical assessment of the data via principal component analysis and agglomerative hierarchical clustering revealed a distinct grouping of the different colored rice subspecies on the basis of their metabolite profiles. Compared to non-colored and red rice, black rice exhibited, in particular, higher levels of fatty acid methyl esters, free fatty acids, organic acids and amino acids.     

Polymorphisms in the ALS gene of weedy rice (Oryza sativa L.) accessions with differential tolerance to imazethapyr

Herbicide-resistant Clearfield™ rice technology allows the use of ALS inhibitors to control weedy rice. Weedy rice plants have differential tolerance to imazethapyr, perhaps due to ALS polymorphisms. We aimed to assess ALS polymorphisms in weedy rice accessions from Arkansas, USA, with differential tolerance to imazethapyr in seedling growth bioassays. Six base changes were identified in the ALS of 14 weedy rice accessions. Three of these nucleotide changes resulted in amino acid substitutions — Pro⁹³Thr, Glu⁶³⁰Asp, and Val⁶⁶⁹Met — in four accessions: Ark-4, Ark-9, Poi-1 and Poi-6. The GR₅₀ values and inhibition of root and shoot growth (%) of these accessions differed. The Glu⁶³⁰Asp substitution occurs in the herbicide binding domain B and Val⁶⁶⁹Met occurs at the C-terminal tail where the co-factor binds. Variability in weedy rice ALS exists, but polymorphism patterns did not relate to tolerance levels. The observed mutations presented the possibility that sustained selection pressure will eventually lead to selection of a herbicide-tolerant individual that will be the progenitor of a resistant population. Concomitantly, pollen-mediated gene flow from Clearfield™ rice to weedy rice will lead to the evolution of ALS-resistant weedy rice populations.     

An SNP downstream of the OsBEIIb gene is significantly associated with amylose content and viscosity properties in rice (Oryza sativa L.)

Allele mining in starch synthesis-related genes (SSRGs) has facilitated the discovery of desired natural sequence variations for eating quality in rice. This study investigated the sequence variations from 10 SSRGs, and further evaluated their relationship with the amylose content (AC) and rapid viscosity analysis profiles in a global collection of rice accessions by association mapping (AM). In total, 83 sequence variations were found in 10 sequenced amplicons, including 73 single nucleotide polymorphisms (SNPs), eight insertion-deletions (InDels) and two polymorphic simple sequence repeats (SSRs). Four subpopulations were identified by population structure analysis based on 170 genome-wide SSR genotypes. AM revealed 11 significant associations between three phenotypic indices and three sequence variations. One SNP with a g/c transversion at the 63rd nucleotide downstream of the OsBEIIb gene termination codon on rice chromosome 2 was significantly associated with multiple trait indices in both the general linear and mixed linear models (GLM and MLM), including the final viscosity (p < 0.001, R² = 23.87%) in both 2009 and 2010, and AC (p < 0.01, R² = 11.25%) and trough viscosity (p < 0.01, R² = 20.43) in 2010. This study provides a new perspective of allele mining for breeding strategies based on marker-assisted selection.     

Distribution of phytic acid and mineral elements in three indica rice (Oryza sativa L.) cultivars

Three indica rice cultivars with different grain shapes were selected to determine the milling characteristics and distribution of phytic acid (PA) and six mineral elements including magnesium (Mg), calcium (Ca), manganese (Mn), iron (Fe), zinc (Zn), and selenium (Se). Milling characteristics were quite different among the rice cultivars with different physical dimensions. Similar milling times did not necessarily result in the same degree of milling (DOM) for different rice cultivars. The concentrations of phytic acid and minerals decreased with an increased DOM. These results also showed that phytic acid and minerals except for Zn and Se were not evenly distributed and highly concentrated in the outer layer (0% < DOM < 15%) of the rice kernel. In contrast, Zn and Se seem to be relatively evenly distributed in the grain. Optimum DOM of about 14% was detected for Zhenong 7 (long and slender grain); 10% was found for Zhenong 60 (medium-grain) and 9% for Zhenong 34 (short and round grain). The information generated in this experiment could be useful to optimize milling procedures for maximum removal of phytic acid, minimum mineral losses and weight loss in indica rice cultivars with different grain shapes.     

Agronomic,molecular and antioxidative characterization of red- and purple-pericarp rice (Oryza sativa L.) mutants in Taiwan

Pigmentation of rice grain is controlled by Ra, Rc and Rd genes, and the expressions of these genes differ among red-, purple- and white-pericarp varieties. The present study examined the grain yield and the expression of Ra, Rc and Rd genes in non-waxy white-pericarp rice SA418 and waxy white-pericarp rice SA419 and their respective red-pericarp and purple-pericarp mutants with waxy or non-waxy endosperm. Significant variations in 100-grains weight and grain yield were observed among the tested mutants. The Ra was expressed in purple-pericarp mutants, while the Rc was expressed in red-pericarp mutants. The total phenolics, total flavonoids, total anthocyanins and total proanthocyanidins contents and antioxidant activities in the bran part also differed among the mutants. Non-waxy red-pericarp mutant M-69 had heavier 100-grains weight (2.86 g), contained more total phenolics (49.37 mg g⁻¹ bran dry weight) and produced higher grain yield (6.93 t ha⁻¹) than white-pericarp rices SA418 (2.43 g, 2.89 mg g⁻¹ bran dry weight and 2.80 t ha⁻¹, respectively) and SA419 (2.62 g, 2.20 mg g⁻¹ bran dry weight and 6.73 t ha⁻¹, respectively). Thus, the polished rice grain of M-69 can be used for staple food consumption, and its bran parts are useful for producing health-promoting by-product.     

Genomic regions associated with the degree of red coloration in pericarp of rice (Oryza sativa L.)

The degree of red coloration (DRC) in pericarp of rice depends on the content of flavonoid compounds which have beneficial health effects for humans. In this study, 182 backcross-recombinant inbred lines (BILs) derived from Koshihikari (white pericarp)/Kasalath (red pericarp)//Koshihikari were used to detect the genomic regions associated with DRC through the QTL mapping approach. As a result, a total of four genomic regions were found to associate with DRC on chromosomes 1, 7, 9 and 11, respectively. Interestingly, the two genomic regions having the largest effects corresponded to previously characterized Rc and Rd genes on chromosome 7 and 1, respectively. In addition, two novel genomic regions having minor effects on DRC and located on chromosomes 9 and 11, respectively, are reported here for the first time. These results and the identification of tightly linked molecular markers that flank the genomic regions provide an opportunity for marker-aided improvement of red coloration in pericarp of rice.     

Root growth dynamics and stomatal behaviour of rice (Oryza sativa L.) grown under aerobic and flooded conditions

Aerobic rice culture is a new technology designed to reduce water use, but the vulnerability of rice to aerobic condition has limited its development. The objective of this study was to characterize the root growth and stomatal behaviour of four rice cultivars grown in flooded and aerobic culture for 2 years. In aerobic culture, where the soil water potential at 20-cm depth averaged between −15 and −30 kPa, total root biomass was significantly lower than in flooded culture for the whole growth period, owing to a reduction in root biomass in the surface layer. Dry-matter partitioning to roots decreased, but the ratio of deep root biomass to total root biomass tended to be higher in aerobic culture than in flooded culture. The low root-to-shoot ratio and poor root growth in the surface layer in aerobic culture are attributable to the considerable reduction in adventitious root number. As a result, the varietal difference in total root biomass was due largely to individual root growth in aerobic culture. Stomatal closure was distinct at the vegetative stage in aerobic culture, even when the soil water potential was near field capacity, partly because of the poor rooting vigour. When the soil water potential at 20-cm depth was below −50 kPa, the stomatal behaviour reflected the root growth in the subsurface layer. These results suggest the role of vigorous root growth in soil water uptake and hence, in maintaining transpiration in aerobic rice culture.     

Comparative metabolic profiling of pigmented rice (Oryza sativa L.) cultivars reveals primary metabolites are correlated with secondary metabolites

Gas-chromatography coupled with time-of-flight mass spectrometry (GC-TOFMS) was used to analyze the relationships between primary metabolites and phenolic acids in rice (Oryza sativa L.), including six black cultivars and one white cultivar. A total of 52 metabolites were identified, including 45 primary metabolites and seven phenolic acids from rice seeds. The metabolite profiles were subjected to data mining processes, including principal component analysis (PCA), Pearson's correlation analysis, and hierarchical clustering analysis (HCA). PCA could fully distinguish between these cultivars. HCA of these metabolites resulted in clusters derived from common or closely related biochemical pathways. There was a positive relationship between all phenolic and shikimic acids. Projection to latent structure using partial least squares (PLS) was applied to predict the total phenolic content based on primary metabolite profiles from rice grain. The predictive model showed good fit and predictability. The GC-TOFMS-based metabolic profiling approach could be used as an alternative method to predict food quality and identify metabolic links in complex biological systems.     

Effect of salinity on yield and 2-acetyl-1-pyrroline content in the grains of three fragrant rice cultivars (Oryza sativa L.) in Camargue (France)

Aromatic quality of rice grains is known to vary greatly with environmental factors and cultivation methods. Among the environmental factors, soil salinity is thought to have a positive impact on the content of 2-acetyl-1-pyrroline (2AP) in grains, the key volatile compound of rice aroma. This study compared 2AP content in grains of three improved fragrant rice (Oryza sativa L.) varieties grown in two fields, differing mainly in their soil salinity level. The impact of salinity on yield and main yield components was also investigated to understand the relationship between aromatic quality and yield build-up. Soil salinity was monitored by measuring the electrical conductivity (EC) of soil solution samples extracted every week. 2AP content in grains was determined by a newly developed stable isotope dilution method involving solid-phase microextraction (SPME) and GC MS/MS analysis. The results showed an increase of 2AP content in grains with salinity for the three varieties. The relationship between 2AP and mean EC of the crop fitted a single model for the three varieties (R² = 0.728). In contrast, the impact of salinity on yield and yield components differed greatly between the three varieties. One variety appeared to be very sensitive to salt stress, with significant yield loss up to 40%, while the two other varieties proved to be resistant to the salinity levels experienced by the plants, with no significant yield loss or even higher yields in saline conditions. Nevertheless, the three varieties presented a significant negative correlation between 1000 grain weight (TGW) and the mean EC of the crop, and between TGW and 2AP content. It was concluded that the increase of 2AP content with salinity resulted partially from a 2AP concentration mechanism in smaller size grains. The direct effect of salinity on 2AP synthesis through stimulation of the proline metabolism is further discussed.     

Identification of novel glutelin subunits and a comparison of glutelin composition between japonica and indica rice (Oryza sativa L.)

Glutelin, a major protein in rice grains, is encoded by a multigene family. However, its protein composition is not well characterised. Here, we identified and characterised two novel glutelin subunits, GluBX and GluC. The individual glutelin subunits of japonica cv. Nipponbare and indica cv. 93-11 rice were analysed using 2-dimensional gel electrophoresis, LC–MS/MS, and Western blotting. Comparison of the glutelin profiles between three japonica and three indica cultivars indicated two distinct subunits (GluA-1 and GluA-3 isomers) and a distinction in the subunit composition (notably GluA-3 and Lys-rich GluB-1 components) of these two subspecies. Sequence alignment revealed different nutritional (Lys residues) and functional (Cys residues) characteristics between the type-A and type-B glutelin subfamilies. We also analysed amino acid and total protein contents of the grains in thirty-five cultivars, and we demonstrated that the Lys-rich glutelin composition of indica cultivars is superior to that of japonica cultivars. The Lys-rich and Cys-poor GluBX subunit is a native protein and is a high nutritional protein in grains. Our combined approaches for the identification of glutelin subunits have revealed the nutritional characteristics of individual subunits in rice, and this knowledge will provide new insights for improving grain quality during rice breeding.     

Regional scale distribution of imidazolinone herbicide-resistant alleles in red rice (Oryza sativa L.) determined through SNP markers

Red rice is the main weed in rice paddy fields. Imidazolinone herbicides in resistant rice cultivars currently provide a unique opportunity to control red rice in large-scale rice fields. However, the continuous use of this technology has resulted in imidazolinone-resistant red rice biotypes. This study aimed to identify the mechanism of herbicide resistance and the frequency and spatial distribution of the known imidazolinone herbicide-resistant alleles in red rice. The nucleotide sequence of the ALS gene indicated that the G₆₅₄E, S₆₅₃D and A₁₂₂T mutations are present in the imidazolinone herbicide-resistant rice cultivars IRGA 422 CL, SATOR CL and PUITÁ INTA CL, respectively. This information and the nucleotide sequence surrounding these mutations were used for the development of single nucleotide polymorphism (SNP) molecular markers to identify the possible mutations that confer herbicide resistance in red rice. This analysis was carried out in a total of 481 plants from 38 populations collected as individuals that escaped control with the herbicides imazethapyr and imazapic in rice paddy fields in Southern Brazil. The G₆₅₄E mutation was the most frequent, being found in 100% and 90.9% of the populations in the 2006/2007 and 20007/2008 seasons, respectively. In addition, the S₆₅₃D and A₁₂₂T mutations were also present either alone or as double or triple mutations in some plants. Target site insensitivity is the predominant mechanism of resistance in red rice resistant to imidazolinone herbicides in Southern Brazil. The high frequency of the S₆₅₃D mutation, the same mutation responsible for the resistance in the rice cultivar largely used in Southern Brazil, indicates that gene flow is occurring from the rice cultivar to red rice. Management practices related to increasing crop sanitation and decreasing of herbicide selection pressure through crop rotation should be enforced to prevent the evolution of herbicide resistance in red rice.     

Identification and quantification of phenolic acids and anthocyanins as antioxidants in bran,embryo and endosperm of white,red and black rice kernels (Oryza sativa L.)

Whole rice grain comprising endosperm, embryo (or germ), and bran has potential beneficial health effects in addition to provision of nutrients. The distribution of phenolic acids and anthocyanins in endosperm, embryo, and bran of white, red, and black rice grains was investigated in this study. The total phenolic content (TPC) was highest in the bran, averaging 7.35 mg GAE/g and contributing 60%, 86% and 84% of phenolics in white, red and black rice. The average TPC of the embryo and endosperm were 2.79 and 0.11 mg GAE/g accounting for 17% and 23%, 4% and 10% and 7% and 9% in white, red and black rice, respectively. The antioxidant capacity determined using DPPH and ORAC displayed a trend similar to TPC. Free/conjugated phenolic acids in white, red and black rice bran accounted for 41%, 65% and 85% of total acids. Bound phenolic acids in rice bran accounted for 90% of total acids in whole grain. Cis-p-coumaric was detected in bound form in bran while cis-sinapic acid was detected in the free/conjugated form in embryo and bran. Cyanidin-3-O-glucoside and peonidin-3-O-glucoside were identified mainly in black rice bran as the total anthocyanins. Cyanindin-3-O-rutinoside was also detected in black rice bran.     

Antioxidant,anti-amylase and anti-glycation potential of brans of some Sri Lankan traditional and improved rice (Oryza sativa L.) varieties

Brans of 23 traditional and 12 improved (both red and white) rice varieties in Sri Lanka were screened for anti-amylase and anti-glycation activities in vitro. Varieties which showed the highest inhibitory activities at screening were further investigated for anti-glucosidase and glycation reversing as anti-diabetic properties. The same varieties were studied for selected antioxidant properties. Significantly high anti-amylase and anti-glycation activities were observed for bran extracts of red varieties compared to white varieties at screening. Traditional red rice varieties, Masuran, Sudu Heeneti, Dik Wee and Goda Heeneti, exhibited significant and dose dependent anti-amylase, anti-glycation and glycation reversing activities. These varieties also showed marked antioxidant properties. It is concluded that brans of Sri Lankan traditional red rice varieties Masuran, Sudu Heeneti, Dik Wee and Goda Heeneti may be potential food supplements for diabetes.     

Identification of deletion mutants with improved performance under water-limited environments in rice (Oryza sativa L.)

Rice (Oryza sativa L.) is a semi-aquatic member of the grass family that is poorly adapted to dry environments and has greater sensitivity to water-deficits than other important cereals in this family. To increase productivity in aerobic or water-limited environments rice must overcome its adaptations to flooded environments. Deletion mutants offer an alternative genetic resource for improving drought tolerance. Almost 3500 IR64 deletion mutants were screened under vegetative and reproductive stage drought stress in the field and evaluated for leaf drying and/or grain yield. Seven novel conditional mutants of rice which showed gain of function through continued growth as drought stress developed compared to the wild type were identified. Mutant recovery rate was 0.1%. Further evaluation of putative drought mutants revealed that their average shoot biomass at maturity and grain yield per plant under stress exceeded those of the wild type by two-fold. Studies under controlled conditions confirmed mutants to have continued growth of both roots and shoots as drought developed compared to the wild type, and a tendency for greater water extraction. We propose that deletions in these mutants have affected a regulator of the highly conservative growth response common to irrigated lowland rice cultivars. Our results suggest that screening deletion mutants for performance under managed drought stress in the field could be a highly effective way to identify valuable genetic resources for improved drought response and aerobic adaptation in rice.     

Use of propanil and quinclorac tank mixtures for broadleaf weed control on rice (Oryza sativa) levees

Broadleaf weed control on rice levees is an emerging problem faced by growers and consultants in Arkansas, USA. Field experiments were conducted at Lonoke and Stuttgart, Arkansas, in 2007 and 2008 to evaluate the effectiveness of various postemergence herbicides applied alone or in tank mixture with propanil or quinclorac for large-sized broadleaf weed control on rice levees. Rice injury was minimal (≤5%) from all herbicides at 2 weeks after treatment (WAT), and no injury was observed at 4 WAT. Sida spinosa (prickly sida) and Amaranthus palmeri (Palmer amaranth) were the most difficult-to-control weeds on levees. Of the herbicides applied alone, 2,4-D generally supplied the highest and most consistent weed control across the six species evaluated. Quinclorac was generally a better tank-mix partner than propanil for control of the weed spectrum evaluated. Propanil at 4.48 kg/ha lowered the activity of several systemic herbicides on S. spinosa, Polygonum pensylvanicum (Pennsylvania smartweed), and Ipomoea wrightii (palmleaf morningglory). Quinclorac plus 2,4-D was the most consistent tank mixture, providing more than 80% control of all weeds at 2 and 4 WAT, except A. palmeri.     

Temperature during grain ripening affects the ratio of type-II/type-I protein body and starch pasting properties of rice (Oryza sativa L.)

The starch and protein properties of rice grain are important factors for sake brewing and these properties are reported to be influenced by temperatures during grain ripening. Amylose content, nitrogen content, protein composition, pasting properties measured by a Rapid Visco Analyser (RVA), and their relationship to temperatures during ripening were investigated in a rice cultivar, Yamadanishiki, which was grown under various conditions in the same experimental field. The average temperature after heading was significantly correlated with amylose content and RVA properties, but not with nitrogen content. Under high temperatures during ripening, a decrease in prolamin, which accumulated in type I protein body (PB-I), and an increase in glutelin, which deposits in type II PB (PB-II), were recognized. The ratio of PB-II/PB-I and RVA pasting temperatures were distinctly increased as the temperature became higher. High temperatures during grain ripening would lead to difficulties in digesting steamed rice grains by Aspergillus oryzae, together with ease in digesting rice protein. The average temperature of 11–20 days after heading showed a higher correlation coefficient than that of 1–10 or 21–30 days, implying that temperatures during the middle stage of grain development would be important in determining the rice component that relates to brewing properties.     

Potential of Waxy gene microsatellite and single-nucleotide polymorphisms to develop japonica varieties with desired amylose levels in rice (Oryza sativa L.)

The Waxy (Wx) gene is responsible for the synthesis of amylose, a key determinant of the cooking and processing qualities of rice. Polymorphisms of CT-microsatellite and G–T single-nucleotide polymorphism (SNP) in the Wx gene and their relationship to amylose content (Ac) were explored using 178 non-waxy rice genotypes. Nine Wx microsatellite alleles, namely (CT)10 and 11, and (CT)14–20 were identified and 11 haplotypes were recognised by different combinations of CT-microsatellite and G–T SNP. Amylose content analysed in a random set of 39 genotypes was correlated with different microsatellite alleles/haplotypes. The highest Ac levels (>30%) correlated with (CT)10 and 16, high (26–30%) with (CT)11, 15 and 20, and intermediate (21–25%) with (CT)14, in all cases with G at the G–T SNP. The CT-classes (CT)17 and 18 (mean Ac value of 21%), could be subdivided into low amylose haplotypes (16–20%) for 17T and 18T and intermediate amylose haplotypes (21–25%) for 17G and 18G. The use of haplotypes proved to discriminate between intermediate and low amylose accessions within the same microsatellite class. Analyses of a segregating population of a cross between low and high Ac parents showed that CT-microsatellite may help to classify breeding lines and identify pollen contamination. We suggest that CT-microsatellite together with G–T SNP may be used as molecular marker by breeders to develop varieties with desired amylose levels.