The world-renowned Thai Hom Mali Rice has been the most important aromatic rice originating in Thailand. The aromatic variety was collected from Chachoengsao, a central province, and after pure-line selection, it was officially named as Khao Dawk Mali 105, (KDML105). Because of its superb fragrance and cooking quality, KDML105 has been a model variety for studying genes controlling grain quality and aroma. The aromatic gene was cloned in KDML105, as an amino aldehyde dehydrogenase (AMADH) or better known as BADH2 located on chromosome 8. Later on, all other aromatic rice genes were discovered as allelic to the AMADH. As a selection of local landrace variety found in rainfed areas, the Thai Jasmine rice showed adaptive advantages over improved irrigated rice in less fertile lowland rainfed conditions. Because KDML105 was susceptible to most diseases and insect pests, marker-assisted backcross selection (MABC) was used for the genetic improvement since 2000. After nearly 17 years of MABC for integrating new traits into KDML105, a new generation of KDML105, designated HM84, was developed which maintains the cooking quality and fragrance, and has gained advantages during flash flooding, disease, and insect outbreak.

Background: Pyramiding multiple genes into a desirable genetic background can take years to accomplish. In this paper, a pseudo-backcrossing scheme was designed to shorten the backcrossing cycle needed. PinK3, an aromatic and potentially high-yielding rice variety—although one that is intolerant to flash flooding (Sub) and susceptible to bacterial leaf blight (BB), leaf-neck blast (BL) and the brown planthopper (BPH)—was used as a genetic basis for significant improvements through gene pyramiding.Results: Four resistance donors with five target genes (Sub1A-C, xa5, Xa21, TPSandSSIIa) and three QTLs (qBph3, qBL1 and qBL11) were backcrossed individually using markers into the pseudo-recurrent parent ‘PinK3’ via one cycle of backcrossing followed by two cycles of pseudo-backcrossing and three selfings with rigorous foreground marker-assisted selection. In total, 29 pseudo-backcross inbred lines (BILs) were developed. Genome composition was surveyed using 61 simple sequence repeats (SSRs), 35 of which were located on six carrier chromosomes, with the remainder located on six non-carrier chromosomes. The recurrent genome content (%RGC) and donor genome content (%DGC), which were based on the physical positions of BC₁F₂, ranged from 69.99 to 88.98% and 11.02 to 30.01%, respectively. For the pseudo-BC₃F₃BILs, the %RGC and %DGC ranged from 74.50 to 81.30% and 18.70 to 25.50%, respectively. These results indicated that without direct background selection, no further increases in %RGC were obtained during pseudo-backcrossing, whereas rigorous foreground marker-assisted selection tended to reduce linkage drag during pseudo-backcrossing. The evaluation of new traits in selected pseudo-BC₃F₃BILs indicated significant improvements in resistance to BB, BL, BPH and Sub compared with PinK3, as well as significant improvements in grain yield (21-68%) over the donors, although yield was 7-26% lower than in ‘PinK3’. All pyramided lines were aromatic and exhibited improved starch profiles, rendering them suitable for industrial food applications.Conclusions: Results show that our new pyramiding platform, which is based on marker-assisted pseudo-backcrossing, can fix five target genes and three QTLs into a high-yielding pseudo-recurrent background within seven breeding cycles in four years. This multiple pseudo-backcrossing platform decreases the time required to generate new rice varieties exhibiting complex, durable resistance to biotic and abiotic stresses in backgrounds with desirable qualities.

The development of rice varieties with broad-spectrum resistance to insect pests is the most promising approach for controlling a fast evolving insect pest such as the brown planthopper (BPH). To cope with rapid evolution, discovering new sources of broad-spectrum resistance genes is the ultimate goal. We used a forward genetics approach to identify BPH resistance genes in rice (Oryza sativa L.) using double digest restriction site-associated DNA sequencing (ddRADseq) for quantitative trait loci (QTL)-seq of the backcross inbred lines (BILs) derived from a cross between the BPH-susceptible cultivar KDML105 and BPH-resistant cultivar Rathu Heenati (RH). Two major genomic regions, located between 5.78-7.78 Mb (QBPH4.1) and 15.22-17.22 Mb (QBPH4.2) on rice chromosome 4, showed association with BPH resistance in both pooled BILs and individual highly resistant and susceptible BILs. The two most significant candidate resistance genes located within the QBPH4.1 and QBPH4.2 windows were lectin receptor kinase 3 (OsLecRK3) and sesquiterpene synthase 2 (OsSTPS2), respectively. Functional markers identified in these two genes were used for reverse screening 9323 lines of the fast neutron (FN)-mutagenized population developed from the BPH-susceptible, purple-pigmented, indica cultivar Jao Hom Nin (JHN). Nineteen FN-mutagenized lines (0.24%) carried mutations in the OsLecRK3 and/or OsSTPS2 gene. Among these mutant lines, only one highly resistant line (JHN4) and three moderately resistant lines (JHN09962, JHN12005, and JHN19525) were identified using three active, local BPH populations. The 19 mutant lines together with three randomly selected mutant lines, which did not harbor mutations in the two target genes, were screened further for mutations in six known BPH resistance genes including BPH9, BPH14, BPH18, BPH26, BPH29, and BPH32. Multiple single nucleotide polymorphisms (SNPs) and insertion-deletion (Indel) mutations were identified, which formed gene-specific haplotype patterns (HPs) essential for broad-spectrum resistance to BPH in both BILs and JHN mutant populations. On the one hand, HPs of OsLekRK2-3, OsSTPS2, and BPH32 determined broad-spectrum resistance to BPH among RH-derived BILs. On the other hand, in the JHN mutant population, BPH9 together with seven significant genes on chromosome 4 played a crucial role in BPH resistance.

Background: Rice contains the lowest grain Fe content among cereals. One biological limiting factor is the tolerance of rice to Fe toxicity. Reverse and forward genetic screenings were used to identify tolerance to Fe toxicity in 4,500 M₄ lines irradiated by fast neutrons (FN). Findings: Fe-tolerant mutants were successfully isolated. In the forward screen, we selected five highly tolerant and four highly intolerant mutants based on the response of seedlings to 300 ppm Fe. Reverse screening based on the polymorphic coding sequence of seven Fe homeostatic genes detected by denaturing high performance liquid chromatography (dHPLC) revealed MuFRO1, a mutant for OsFRO1 (LOC_Os04g36720). The MuFRO1 mutant tolerated Fe toxicity in the vegetative stage and had 21-30% more grain Fe content than its wild type. All five highly Fe-tolerant mutants have the same haplotype as the MuFRO1, confirming the important role of OsFRO1 in Fe homeostasis in rice. Conclusions: FN radiation generated extreme Fe-tolerant mutants capable of tolerating different levels of Fe toxicity in the lowland rice environment. Mutants from both reverse and forward screens suggested a role for OsFRO1 in seedling tolerance to Fe toxicity. The MuFRO1 mutant could facilitate rice production in the high-Fe soil found in Southeast Asia.

Rice is a staple food for more than half of the world’s population. Modern rice varieties have been developed for high yield and quality; however, there has been a substantial loss of diversity. A greater number of genetically dynamic landraces could offer valuable and useful genetic resources for rice improvement. In this study, the genetic diversity and population structure of 365 accessions of lowland and upland landraces from four populations from different geographical regions of Thailand were investigated using 75 SNP markers. Clustering analyses using maximum likelihood, Principal Coordinate Analysis (PCoA), and Discriminant Analysis of Principal Components (DAPC) clustered these landraces into two main groups, corresponding to indica and japonica groups. The indica group was further clustered into two subgroups according to the DAPC and STRUCTURE analyses (K = 3). The analysis of molecular variance (AMOVA) analysis results revealed that 91% of the variation was distributed among individuals, suggesting a high degree of genetic differentiation among rice accessions within the populations. Pairwise F_ST showed the greatest genetic differentiation between the northeastern and southern populations and the smallest genetic differentiation between the northern and northeastern populations. Isolation-by-distance analysis based on a Mantel test indicated a significant relationship between the genetic distance and geographic distance among the Thai rice landraces. The results from this study provide insight into the genetic diversity of Thai rice germplasm, which will enhance the germplasm characterization, conservation, and utilization in rice genetics and breeding.