Identification of genes and SNP markers asociated with aroma trait in Thai fragrant local rice

Abstract

Thailand is located within a center of diversity and origin of cultivated rice (Oryza sativa L.), resulting in a diverse array of rice varieties, particularly landrace rice, which remains diverse within and between populations. Local rice is considered a significant genetic resource, possessing unique genetic characteristics that enable adaptation to local environments, resistance to diseases and pests, and some populations are known for their aromatic qualities. This genetic diversity and special quality traits can serve as valuable resources for future studies, development, and conservation of rice varieties before they disappear due to societal changes and the introduction of new breeding programs. The aroma is one of the qualities that make highly valued fragrant rice. It is an important economic trait that affects consumption and helps determine rice prices in both domestic and international markets. Fragrant rice is known for its excellent eating quality, both softness and aroma, making it increasingly popular and desirable. The aroma of rice is derived from the combination of various volatile compounds, with the main aroma compound found in fragrant rice being 2-acetyl-1-pyrroline (2AP), which is controlled by the recessive badh2 gene. Mutations on the Badh2 gene lead to the accumulation of 2AP. The fragrance characteristics are influenced not only by genetic factors but also by environmental conditions, particularly the management of nutrients. Additionally, molecular markers are now used in the plant breeding and selection of aromatic rice varieties, as they allow for the identification of desired traits at the genotype, allowing for quick and accurate selection of desired characteristics. Thus, this thesis examined the effect of nutrient management on aroma traits in Thai fragrant rice. In addition, molecular studies were conducted to identify SNPs on genes associated with aroma traits in Thai fragrant rice for developing SNP markers which will be used in selecting aromatic rice varieties in the future. The evaluation of the quality and diversity of the Badh2 gene in aromatic rice landraces can be utilized as a genetic basis for selection and improvement of aromatic rice varieties which may also be developed to become a pure-line rice variety. The 22 rice seed samples from farmers' storage facilities include 9 from the north, 8 from the northeast, and 5 from the south of Thailand plus two advanced breeding lines and three check varieties were evaluated for seed morphology and grain quality, and their Badh2 gene covering intron 4 to intron 8 was re-sequenced. Almost all of the landraces were classified as large grain types with medium to high gelatinization temperature. The variation in the Badh2 gene by haplotype analysis correlated with grain aroma by sensory evaluation. The badh2-E7 was found in haplotype 1 that had a strong aroma in KH, NDLP, and PLD just as in KDML105 and moderately aromatic BNM4, SKH, and BNM-CMU, along with PTT1. While 3 haplotypes had different positions of SNP on the Badh2 gene with a varied results of the sensory test. The present results suggested that rice varieties in the same group as the check varieties KDML105 and PTT1 could be potentially introduced as genetic resources for the fragrant rice breeding program or could be developed into highly palatable cultivars with geographical indications (GI) to increase the income for the highland farmer. However, besides genetic factors related to gene expression and various enzymes in the 2AP synthesis pathways, environmental factors such as temperature, fertilizer management, and storage also influence the quantity of 2AP. Fertilizer management is another key external factor affecting aroma quantity. Previous studies have identified that nitrogen and silicon fertilizers not only increase yield but also affect the aroma characteristics in some aromatic rice varieties. This study examines the effect of nitrogen fertilizer on proline concentration and grain quality in rice landrace, as well as the expression of the gene associated with 2AP biosynthesis pathways using the quantitative RT-PCR technique. BNM4, KDML105, PTT1, and SPR1 were grown under waterlogged conditions with nitrogen fertilizer applications. N fertilizer as urea (46%N) was applied at 30, 60, 120 kg ha-1 by splitting it into three applications at 15, 30, and 45 days after transplanting. N fertilizer increased yield in all four varieties and affected amylose, but did not influence aroma traits when compared within the varieties. Additionally, there was no effect of nitrogen on the expression of the Badh2 and P5CS genes; only differences were observed among the varieties. Furthermore, the study examined the effect of silicon on yield, seed quality, and gene expression in BNM4, KDML105, PTT1, and SPR1, which were grown in pots with 13 kg of soil under waterlogged conditions. Silicon (Si) fertilizer (Na2SiO3·5H2O) was applied at 0, 75, 150, and 225 mg kg-1 soil (designated Si0, Si75, Si150, and Si225, respectively) with two splits equally at the maximum tillering and booting stage. When compared to Si0 (no silicon fertilizer), fragrant rice varieties were found to differ markedly in the effect of Si on yield and their quality. BNM4 and KDML105 increased yield with increasing Si to Si225, while the yield of PTT1 and SPR1 reached the maximum at Si150. KDML105 represents the type with a high 2AP concentration when Si supply is low, with Si application increasing 2AP only slightly. BNM4 with increased Si fertilizer showed only slight increases in 2AP concentration and decreased when Si fertilizer was applied to Si225. While PTT1 responded to a decrease in 2AP concentration when Si fertilizer was increased. Si affected the gene expression levels of the genes associated with the 2AP biosynthesis pathway differently in each rice variety. When compared to Si0, the expression level of Badh2, DAO, ProDH, and P5CS increased, except for the expression of the ProDH gene in KDML105, which showed a decrease. Additionally, a decrease in the expression level of the DAO gene was observed when silicon fertilizer was applied at the highest level (225 mg/kg of soil) in PTT1. Conversely, the expression level of the OsLsi6 gene, which is related to silicon transport in plants, increased with the application of silicon fertilizer in all four rice varieties. The accumulation of 2AP in fragrant rice under Si fertilizer application primarily occurred through the upregulation of Badh2, DAO, ProDH, and P5CS gene expression. These results suggest that nitrogen fertilization is suitable for increasing yield, while silicon fertilizer is effective for enhancing fragrance, with only a slight impact on yield and differing in each species. Molecular studies have identified several genes in the 2AP biosynthesis pathways. Gene expression and nucleotide sequences have been studied using Next Generation Sequencing (NGS) technologies such as Whole Genome Sequencing (WGS). Therefore, studying nucleotide sequences to identify differences in base sequences at single positions (SNP) and developing molecular markers specific to genes controlling aroma characteristics in aromatic rice, including genes involved in 2AP synthesis, is a valuable option for selecting and improving aromatic rice varieties. By studying the genes involved in the 2AP synthesis process, apart from the Badh2 gene, SNP molecular markers (Kompetitive Allele-Specific PCR; KASP) can be designed and developed through nucleotide sequence analysis using Whole-genome sequencing techniques in BNM4 and KHCMU (classified as Indica type and Tropical Japonica type, respectively) compared with the Nipponbare reference genome (Japonica type). A total of 503 SNPs loci were identified in 16 genes associated with 2AP biosynthesis pathways, including the polyamine pathway and glycolysis pathway. Forty-five KASP markers from the coding sequence on nine chromosomes, including P5CS2, P5CR, TPI, OAT, SSATB, SSATA, P5CS, SPDS6, DAO, BADH2, PAO6.9, PAO6.9.8, and ProDH, were screened in 13 rice varieties, which were assessed for phenotype and badh2-E7 genotype. The badh2-E7 deletion was found in seven rice varieties exhibiting moderately aromatic and strongly aromatic traits. However, most KASP markers could not differentiate between fragrant rice varieties. Only two KASP markers were able to distinguish between landrace and elite rice varieties. Thus, to obtain information and molecular markers that can differentiate aromatic rice in genes associated with 2AP biosynthesis pathways, other techniques such as Transcriptome or RNA sequencing, should be used study intermediates in the 2AP synthesis process. The results of this study will provide valuable insights and serve as a guide for appropriate fertilizer management to enhance yield and grain quality, particularly the fragrance characteristics of Thai fragrant rice landraces. This aims to increase income for highland farmers and meet consumers' demands for fragrant rice. Additionally, the analyzed nucleotide sequence could serve as a preliminary database for further research and development of molecular markers. Furthermore, the study of intermediates and enzyme metabolites in the 2AP synthesis has been included.