Rice mutants, selected under severe drought stress, show reduced stomatal density and improved water use efficiency under restricted water conditions.

Introduction: Rice is among the least water-use-efficient crops, and rice plants utilise most of their water uptake for transpirational cooling via stomata. To improve water-use efficiency (WUE) in rice, reducing stomatal density and size could help optimise transpiration and photosynthesis. Methodology: In this study, we compared two series of purple rice stomata mutants: the Stomatal Model Mutant (SMM) identified by microscopic observation of flag-leaf stomata, and the Drought-selected Model Mutant (DMM) generated through screening under severe water stress. After undergoing two rounds of severe water stress between -60 to -80 Ym, right before the R1-2 reproductive stage, three DMMs were selected based on their rapid recovery rate and % filled-grain percentage. Result: The three DMMs displayed 618-697 stomatal units per mm2, similar to the SMMs low-density stomata mutant (JHN 8756 (LD)). Furthermore, the four SMMs, three DMMs and the Jao Hom Nin wild type (JHN WT) were treated with two restricted water condition schemes from seedlings to harvest. The total amount of irrigation and precipitation during the experiment was 78.1 L/plant (69.1 mm/plant) for the less restricted water condition (LR) and 47.5 L/plant (42 mm/plant) for the more restricted water condition (MR). Water condition treatments had no effects on stomatal density and stomatal index. In contrast, genotypes and restricted water condition schemes affected plant height, tillers/plant, % filled grains and shoot dry weight (SDW). The three DMMs and the JHN 8756 (LD), the SMM's low-density stomata mutant, displayed greater resilience towards more restricted water conditions than the SMMs and the JHN wild type. Particularly, DMMs were tolerant to more restricted water condition treatments, showing no SDW penalties. Together, the DMMs and the JHN 8756 (LD) displayed higher WUE under these conditions of more restricted water conditions. Conclusion: A rigorous screening process to distinguish tolerant mutants with a rapid drought recovery rate from severe water stress could pave the way to isolate more mutants with better stomatal functionality and resilience in preparation for imminent climate changes.

Metabolomics approach to identify key volatile aromas in Thai colored rice cultivars.

In addition to white jasmine rice, Thailand has many native-colored rice varieties with numerous health benefits and the potential to become a global economic crop. However, the chemical characteristics of aromatic substances in native-colored rice are still mostly unknown. This study aimed to identify the key volatile aroma compounds and the biosynthetic pathways possibly involved in their formation in Thai native-colored rice varieties, and thus leading to the search for potential genetic markers for breeding colored rice with better aromatic properties. Twenty-three rice varieties in four categories: aromatic white, aromatic black, non-aromatic black, and non-aromatic red, were investigated (n=10 per variety). Seed husks were removed before the analysis of rice volatile aromas by static headspace gas chromatography-mass spectrometry. Untargeted metabolomics approach was used to discover the key volatile compounds in colored rice. Forty-eight compounds were detected. Thirty-eight of the 48 compounds significantly differed among groups at p<0.05, 28 of which at p<0.0001, with the non-aromatic black and red rice containing much lower content of most volatile constituents than the aromatic black and white rice. Focusing on the aromatic black rice, the samples appeared to contain high level of both compound groups of aldehydes (3-methylbutanal, 2-methylbutanal, 2-methylpropanal, pentanal, hexanal) and alcohols (butane-2,3-diol, pentan-1-ol, hexan-1-ol). Biosynthetically, these distinctive black-rice volatile compounds were proposed to be formed from the metabolic degradation of branched-chain amino acids (L-leucine, L-isoleucine and L-valine) and polyunsaturated fatty acids (linoleic acid and α-linolenic acid), involving the branched-chain aminotransferases and keto-acid decarboxylases and the 9-lipoxygonases and 13-lipoxygeases, respectively. The proposed degradative pathways of amino acids and fatty acids were well agreed with the profiles key volatile compounds detected in the Thai native-colored rice varieties.

Induced Genetic Variations in Stomatal Density and Size of Rice Strongly Affects Water Use Efficiency and Responses to Drought Stresses.

Rice (Oryza sativa L.) is an important food crop relied upon by billions of people worldwide. However, with increasing pressure from climate change and rapid population growth, cultivation is very water-intensive. Therefore, it is critical to produce rice that is high-yielding and genetically more water-use efficient. Here, using the stabilized fast-neutron mutagenized population of Jao Hom Nin (JHN) - a popular purple rice cultivar - we microscopically examined hundreds of flag leaves to identify four stomatal model mutants with either high density (HD) or low density (LD) stomata, and small-sized (SS) or large-sized (LS) stomata. With similar genetic background and uniformity, the stomatal model mutants were used to understand the role of stomatal variants on physiological responses to abiotic stress. Our results show that SS and HD respond better to increasing CO2 concentration and HD has higher stomatal conductance (gs) compared to the other stomatal model mutants, although the effects on gas exchange or overall plant performance were small under greenhouse conditions. In addition, the results of our drought experiments suggest that LD and SS can better adapt to restricted water conditions, and LD showed higher water use efficiency (WUE) and biomass/plant than other stomatal model mutants under long-term restricted water treatment. Finally, our study suggests that reducing stomata density and size may play a promising role for further work on developing a climate-ready rice variety to adapt to drought and heat stress. We propose that low stomata density and small size have high potential as genetic donors for improving WUE in climate-ready rice.

Identification of spontaneous mutation for broad-spectrum brown planthopper resistance in a large, long-term fast neutron mutagenized rice population.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Thai Hom Mali Rice: Origin and Breeding for Subsistence Rainfed Lowland Rice System.

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.

Forward screening for seedling tolerance to Fe toxicity reveals a polymorphic mutation in ferric chelate reductase in rice.

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.

Pseudo-backcrossing design for rapidly pyramiding multiple traits into a preferential rice variety.

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, TPS and SSIIa) 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.