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.

Bph3, a major brown planthopper (BPH) resistance locus derived from the rice cultivar Rathu Heenati (RH), has been used as a stable donor of traits that improve highly susceptible aromatic rice varieties in Thailand. Map-based cloning was initiated using a set of isogenic lines (ILs) harboring the major Bph3 locus on chromosome 6. IL genomes were scanned with a 57 K Affymetrix Rice GeneChip to identify the gene responsible for Bph3. Single-feature polymorphism (SFP) mapping was used to localize 84 candidate genes. An expression analysis of 15 selected candidate genes in the aromatic rice cultivar KDML105 (KD) and the ILs under normal conditions revealed two differentially expressed sequences. Following hopper feeding, only one candidate gene, Os04g27430, was differentially expressed. Os04g27430 encodes a putative sesquiterpene synthase (STPS) gene that was induced by BPH feeding in ILs. An antixenosis test in three selected ILs revealed a major role for STPS in insect preference during the first 120 hours of the rice-insect interaction. Functional SNPs in exon 5 that resulted in the deletion of seven amino acids in the susceptible rice line were identified. Moreover, three additional SNPs associated with three transcription binding sites were also identified, which might explain the differential response of Os04g27430 during the anti-feeding test. Os04g27430 is the second known rice STPS induced by BPH. The gene may involve an antixenosis BPH resistance mechanism. The combination of the STPS and the Bph3 locus was more effective than Bph3 alone in the tested ILs.

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.