Gene Pyramiding for Brown Planthopper Resistance-related Traits, Early Maturity and Aroma of Rice Assisted by Molecular and Phenotypic Markers

The brown planthopper (BPH)-resistant rice, early maturity, and aromatic are expected by both farmers and consumers. The traits have been combined through gene pyramiding and some promising rice genotypes obtained. However the genetic control of BPH resistance and maturity is quantitatively inherited, it is therefore both molecular and phenotypic assessments would be tremendously helpful in selecting promising genotypes. The study aimed to obtain genotypes with such valuable traits. Rice lines were analyzed using molecular markers i.e., RM586, RM589, RM8213 (BPH resistant gene markers); RM7610 and RM19414 (early maturity markers), and IFAP (Internal Fragrant Antisense Primer) for detecting aromatic, and INSP (Internal Non-fragrant Sense Primer) for non-aromatic rice. Phenotypic assessment was performed for brown planthopper resistant-related traits, such as chlorophyll content, stomatal conductance, and trichome density. Other evaluations were heading date and aroma (using 1.7% KOH solution). Results showed that molecular markers for evaluating BPH resistance genes (Bph3, Bph4, Qbph4, and Bph17), aroma (fgr gene), and heading date (Hd2 and Hd3 genes) could differentiate genotypes, and they serve as perfect markers, except for heading date markers. Seven genotypes i.e., #2, #3, #4, #5, #6, #10, and #11 were related to all traits expected based on molecular marker analysis. Meanwhile, genotypes #1, #2, #4, #6, and #11 were similar to their parents based on phenotypic analysis. Pyramiding program based on molecular and phenotypic markers enables us to combine three valuable traits into one rice genotype as presented in this study

Sensory Test and Molecular Marker Based-Selection for Aromatic Rice in the F3 Progenies

Aromatic rice is a special type of rice that highly preferred by people in Asia due to the presence of aroma. Aroma in rice is determined by 2-acetyl-1-pyrroline (2AP) compound which is controlled by a recessive fgr gene. A hybridization between cv. Sintanur (aromatic rice) and PTB33 (non-aromatic, resistant to brown planthopper/BPH) has been done in order to develop aromatic rice lines that resistant to BPH. In the F2 progeny, molecular marker-based selection and bioassay for the brown planthopper resistant lines have been carried out; however selection for the aromatic trait has not been performed yet. The objective of this study was to obtain the F3 progeny’s individual with aromatic trait. Sensory test was conducted by KOH 1.7% solution, meanwhile molecular markers applied were ESP (External Antisense Primer), IFAP (Internal Fragrant Antisense Primer), INSP (Internal Non fragrant Sense Primer) and EAP (External Antisense Primer). Eighty-eight plants from two selected (SP#31 and SP#224) F3 lines progenies derived from cv. Sintanur and PTB33 have been evaluated in this study. Detection by molecular markers found seventy-five genotypes (85.23%) were homozygous recessive (aromatic rice) and one was heterozygous (non-aromatic). Eighty-five (96.59%) genotypes were aromatic as detected by sensory test alone. Seventy-two (81.82%) genotypes were categorized as aromatic rice based on sensory test and molecular markers. Due to inconsistency results from each method alone, it is advised both methods to be applied to ensure the reliability and the accuracy since aroma in rice is affected by genetic composition and environment conditions. Selected genotypes will be continued for breeding program in developing aromatic rice with improved agronomic traits.

Identification of fgr gene and preliminary evaluation for agronomic traits of pandan sticky rice variety (Oryza sativa L.)

Rice (Oryza sativa L.) is the most important food crop in Vietnam particularly in the Mekong Delta. Screening of good quality and high yield rice varieties are needed for rice production in Vietnam. The purpose of this study was to use molecular techniques to identify fgr gene and to evaluate preliminarily agronomic traits of pandan sticky rice variety. The results showed that this variety contained fgr gene. The fragrant allele was amplified by ESP - IFAP primers with a product of 255 bp in size. Similarly, evaluation of agronomic traits showed that the pandan sticky rice variety displayed many desirable characteristics such as plant height of 108 cm, panicle length of 25.6 cm, seed/panicle 135, 100-grain weight 2.07 g, pandan smell, level 2 of alkali digestion, gel consistency 93 mm. These results were useful information and could be applied for improving and providing pandan sticky rice variety for rice production.

Rapid and targeted introgression of fgr gene through marker-assisted backcrossing in rice (Oryza sativa L.)

While it is crucial for developing countries like Malaysia to achieve self-sufficiency in rice (Oryza sativa L.), it is equally critical to be able to produce high-quality rice, specifically fragrant rice, which demands are often met through importation. The present study was aimed at developing high-yielding fragrant rice, in a timely and cost-effective manner. A marker-assisted backcross (MABC) approach was optimised to introgress the fragrance gene (fgr) into two high-yielding Malaysian varieties, MR84 and MR219, within two years utilising less than 50 molecular markers. Coupled with phenotypic screening, one single foreground marker (fgr-SNP) and 48 background markers were selected and utilised, revealing recovery of at least 90% of recurrent parent genome (RPG) in merely two backcross generations. Collectively, the yield potential of the developed BC2F2 lines (BLs) was higher (P > 0.05) than the donor parent, MRQ74, and similar (P < 0.05) to both the recurrent parents, MR84 and MR219. In addition, some of the developed BLs showed good grain quality, such as having long grain. We believe that this is the first report comprising the validation and utilisation of the single functional marker system (fgr-SNP) in introgressing the fgr gene into different rice varieties.

Analysis and comparison of fragrant gene sequence in some rice cultivars

It is known that the fragrant trait in rice (Oryza sativa L.) is largely controlled by fgr gene on chromosome 8 and it has been specified that the existence of an 8 bp deletion and three single nucleotide polymorphism (SNP) in exon 7 is effective on this trait. In this study, sequence alignment analysis of fgr exon7 on chromosome 8 for 11 different fragrant and non-fragrant cultivars revealed that 5 aromatic rice cultivars carried 3 SNPs and 8 bp deletion in exon7 which terminates prematurely at a TAA stop codon. However, 5 of the non-aromatics showed a sequence identical to the published Nipponbare, being non-fragrant Japonica variety sequence. An exception among them was Bejar, which had 8 bp deletion and 3SNPs but it was non-aromatic. Sequencing can determine nucleotide alignment of a gene and give beneficial information about gene function. In silico prediction showed proteins sequences alignment of fgr gene for Khazar and Domsiah genotypes were different. Betaine aldehyde dehydrogenase complete enzyme belongs to Khazar non-fragrant genotype that has complete length and 503 amino acids while non-functional BADH2 enzyme for Domsiah fragrant genotype has 251 amino acids that result in accumulate 2-acetyl-1-pyrroline (2AP) and produces aroma in fragrant genotypes.

Genetic Analysis of Aromatic and Quality Rice Germplasm using Microsatellite Markers

Twenty rice genotypes were analysed with three SSR primers viz., RM223, RM515 and RM342 for detection of the fgr gene responsible for aroma. The primers RM223 and RM515 identified eight and seven genotypes, respectively as having the fgr locus. Presence of the specific allele of the RM342 amplicon, also correlated with strong aroma in two and with weak aroma in five genotypes. The banding pattern for the primer RM342 was different in genotypes with no aroma. Among the three primers (RM223, RM515 and RM342), RM223 detected the highest number of fgr loci in the 20 rice genotypes. In total 11 genotypes were identified as having strong aroma genes by the three primers. These accessions also showed high yields and therefore may prove to be a good source for producing aromatic rice. DOI: http://dx.doi.org/10.3329/ptcb.v22i1.11262Plant Tissue Cult. & Biotech. 22(1): 65-71, 2012 (June)