Optimizing commercial Arabica coffee quality by integrating flavor precursors with anaerobic germination strategy.

This study attempted to improve commercial Arabica coffee quality by integrating flavor precursors with anaerobic germination. Using raw coffee beans as materials, anaerobic germination was conducted with 5 g/100 g of flavor precursors (sucrose, glucose, fructose). The chemical composition and sensory quality of roasted coffee beans were analyzed. Results showed that adding flavor precursors facilitated the harmonization of water-soluble chemical components and altered aroma characteristics. Specifically, the inclusion of flavor precursors significantly increased the levels of 5-Hydroxymethylfurfural and volatile aldehydes. Principal component analysis (PCA) on chemical composition dataset revealed 48.7% variability. Sensory analysis, employing the Specialty Coffee Association (SCA) cupping protocol, demonstrated that combining flavor precursors with anaerobic germination transformed coffee flavor properties, enhanced quality, and substantially increased sensory scores (p < 0.05). Sucrose supplementation produced the highest sensory score and intensified fruity flavor attributes. Therefore, adding different flavor precursors forms distinct flavor characteristics, conducive to further improving the quality of germinated coffee.

Identification of Key Genes and Pathways for Anaerobic Germination Tolerance in Rice Using Weighted Gene Co-Expression Network Analysis (WGCNA) in Association with Quantitative Trait Locus (QTL) Mapping.

BACKGROUND: Rice is one of the most important food crops in the world, and with the development of direct seeding methods for rice, exposure to anaerobic stress has become a major factor limiting its growth. RESULTS: In this experiment, we tested the tolerance to anaerobic germination of rice varieties NIP and HD84, and they were used as parents to construct a DH (doubled-haploid) population. The transcriptomes of NIP (highly tolerant) and HD86 (intolerant), and their progeny HR (highly tolerant) and NHR (intolerant) were sequenced from normal and anaerobic environments. The differentially-expressed genes (DEGs) were subjected to GO (Gene ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), and WGCNA analyses. QTL mapping of the DH population identified tolerance to anaerobic germination-related chromosomal segments. The transcriptome results from 24 samples were combined with the anaerobic stress QTL results for 159 DH population lines to construct a metabolic network to identify key pathways and a gene interaction network to study the key genes. Essential genes were initially subjected to rigorous functional validation, followed by a comprehensive analysis aimed at elucidating their potential utility in domestication and breeding practices, particularly focusing on the exploitation of dominant haplotypes. CONCLUSION: The results show that pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are the starting signals of energy metabolism for coleoptile length growth, the auxin transporter EXPA is the determining signal for coleoptile length growth. The pivotal genes Os05g0498700 and Os01g0866100 exert a negative regulatory influence on coleoptile length, ultimately enhancing tolerance to anaerobic germination in rice. Analyses of breeding potential underscore the additional value of Os05g0498700-hyp2 and Os01g0866100-hyp2, highlighting their potential utility in further improving rice through breeding programs. The results of our study will provide a theoretical basis for breeding anaerobic-tolerant rice varieties.

GWAS Combined with WGCNA of Transcriptome and Metabolome to Excavate Key Candidate Genes for Rice Anaerobic Germination.

Direct seeding of rice is a lightweight and simple cultivation method, which can effectively promote rice production. Anaerobic germination tolerance is one of the main traits of rice adaptability to direct seeding. The mining of related genetic loci, analysis of anaerobic traits and screening of tolerance genes provided valuable genetic resources for improving the anaerobic germination ability of direct seeding rice. This study conducted a dynamic genome-wide association study (GWAS) based on coleoptile-related traits of 591 rice natural populations, and a total of 317 SNP sites were detected. Integrated dynamic widely targeted metabolomics analysis, we found that xanthine, L-alanine and GABA may be key biomarkers that are sensitive and respond strongly to hypoxic stress perception. By WGCNA analysis of targeted metabolomics and transcriptomics, a total of 3 modules were obtained that were significantly correlated with the above three marker metabolites, namely dark green, dark gray and light green modules, respectively, and several key structural genes of OsAlaAT1, OsGAD4, OsAAH and Os09g0424600 that may affect hypoxic germination were screened from the 3 modules. Among them, OsAlaAT1 (Os10g0390500), located in Chr10-12877840, which is within the GWAS location range of CVAN3d, is considered to be a more reliable candidate gene. Overall, in addition to providing new insight into the metabolic regulation of L-alanine, GABA and xanthine during hypoxic germination of rice. This study also provided a reference for the basic theoretical research and breeding application research on the related traits of anaerobic germination in direct-seeding rice.

Genomic landscape of the OsTPP7 gene in its haplotype diversity and association with anaerobic germination tolerance in rice.

Early season flooding is a major constraint in direct-seeded rice, as rice genotypes vary in their coleoptile length during anoxia. Trehalose-6-phosphate phosphatase 7 (OsTPP7, Os09g0369400) has been identified as the genetic determinant for anaerobic germination (AG) and coleoptile elongation during flooding. We evaluated the coleoptile length of a diverse rice panel under normal and flooded conditions and investigated the Korean rice collection of 475 accessions to understand its genetic variation, population genetics, evolutionary relationships, and haplotypes in the OsTPP7 gene. Most accessions displayed enhanced flooded coleoptile lengths, with the temperate japonica ecotype exhibiting the highest average values for normal and flooded conditions. Positive Tajima's D values in indica, admixture, and tropical japonica ecotypes suggested balancing selection or population expansion. Haplotype analysis revealed 18 haplotypes, with three in cultivated accessions, 13 in the wild type, and two in both. Hap_1 was found mostly in japonica, while Hap-2 and Hap_3 were more prevalent in indica accessions. Further phenotypic performance of major haplotypes showed significant differences in flooded coleoptile length, flooding tolerance index, and shoot length between Hap_1 and Hap_2/3. These findings could be valuable for future selective rice breeding and the development of efficient haplotype-based breeding strategies for improving flood tolerance.

Genome-wide association study reveals novel genetic loci involved in anaerobic germination tolerance in Indica rice.

Increasing numbers of rice farmers are adopting methods of direct seeding in flooded paddy fields to save costs associated with labor and transplanting. Successful seedling establishment under anoxic conditions requires rapid coleoptile growth to access oxygen near the water surface. It is important to identify relevant genetic loci for coleoptile growth in rice. In this study, the coleoptile length (CL), coleoptile surface area (CSA), coleoptile volume (CV), and coleoptile diameter (CD) of a germplasm collection consisting of 200 cultivars growing in a low-oxygen environment for 6 days varied extensively. A genome-wide association study (GWAS) was performed using 161,657 high-quality single nucleotide polymorphisms (SNPs), which were obtained via genotyping by sequencing (GBS). A total of 96 target trait-associated loci were detected, of which 14 were detected repeatedly in both the wet and dry seasons. For these 14 loci, 384 genes were located within a 200-kb genomic region (± 100 kb from the peak SNP). In addition, 12,084 differentially expressed genes (DEGs) were identified using transcriptome expression profiling. Based on the GWAS and expression profiling, we further narrowed the candidate genes down to 111. Among the 111 candidate DEGs, Os02g0285300, Os02g0639300, Os04g0671300, Os06g0702600, Os06g0707300, and Os12g0145700 were the most promising candidates associated with anaerobic germination. In addition, we performed a detailed analysis of OsTPP7 sequences from 29 samples in our panel containing 200 diverse germplasms. A total of 11 mutation sites were identified, and four haplotypes were obtained. We found that 7 varieties with the OsTPP7-1 haplotype had higher phenotypic values. This work broadens our understanding of the genetic control of germination tolerance of anaerobic conditions. This study also provides a material basis for breeding superior direct-seeded rice varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01345-1.

Unraveling the genetic potential of native rice (Oryza sativa L.) landraces for tolerance to early-stage submergence.

Direct-seeded rice (DSR) is a promising alternative to the traditional puddled rice system. It has become more popular among rice growers as a result of socioeconomic shifts and global climate change. Although DSR offers advantages, rice plants experience greater anaerobic stress at sowing from unpredicted rainfall. Rice is unique among cereals in its ability to germinate under anaerobiosis. The coleoptile of rice rapidly elongates above the water surface to obtain more oxygen and enhance vigorous seedling growth. A panel of 115 landraces and four check varieties were subjected to anaerobic stress with a water level of 10 cm for up to 15 days. The present study observed significant variation in anaerobic germination percentage (AGP) (10%-100%) and anaerobic vigor index (AVI) (150-4,433). Landraces Karuthakar, Poovan samba, Mattaikar, Edakkal, Manvilayan, and Varappu kudainchan were identified as genotypes tolerant to early water submergence. The shoot and root length of susceptible landraces were significantly lower than the tolerant landraces under hypoxia condition, implying that landraces with longer shoots and roots had a higher survival rate. The response index substantiated this. The results clearly show that tolerant and moderately tolerant landraces possessed higher mean values for root and shoot lengths than susceptible landraces. The landraces grouped under the long-bold category had superior AGP and AVI scores to other grain type groups. This raises the possibility that differences in kernel breadth, which is linked to grain type, could affect anaerobic germination potential. Molecular confirmation using gene-specific markers, viz., DFR, TTP_G4, RM478, RM208, and RM24161, for which the polymorphic information content (PIC) value ranged from 0.36 (RM478) to 0.68 (RM206) suggests that this diverse panel of landraces must be assessed further using advanced molecular tools to precisely clarify the genetic mechanism behind this phenomenon. The tolerant landraces thus identified may become donors in breeding programs. The introduction of these traits would contribute to the development of rice varieties tolerant to anaerobic stress, resulting in sustainable yields. This solution could promote the DSR system across the world.

The Impact of Carbohydrate Management on Coleoptile Elongation in Anaerobically Germinating Seeds of Rice (Oryza sativa L.) under Light and Dark Cycles.

The ability of rice to elongate coleoptiles under oxygen deprivation is a determinant of anaerobic germination tolerance, critical for successful direct seeding. Most studies on anaerobic coleoptile elongation have been performed under constant darkness or in flooded soils because a drilling method was the primary approach for direct seeding of rice. However, aerial seeding is becoming popular, in which seeds which land on flooded soils are exposed to light during the daytime. Here, we investigated physiological mechanisms underlying anaerobic elongation of coleoptiles under light and dark cycles. This study identified two novel varieties, LG and L202, enabling the development of long coleoptiles under oxygen limitation, comparable to well-characterized varieties with strong anaerobic germination tolerance. Germination experiments using these two tolerant and two intolerant varieties, including Takanari and IR64, revealed that light and dark cycles increased coleoptile length in LG, Takanari, and IR64 relative to constant darkness. Interestingly, even in intolerant lines, dramatic starch breakdown and soluble carbohydrate accumulation occurred under oxygen limitation. However, intolerant lines were more susceptible to a representative soluble sugar, glucose, than tolerant lines under oxygen deprivation, suggesting that coleoptile growth can be inhibited in intolerant lines due to hypersensitivity to soluble sugars accumulated in anaerobically germinating seeds.

Comparative transcriptomic analysis of germinating rice seedlings to individual and combined anaerobic and cold stress.

BACKGROUND: Rice is one of the most important cereals consumed worldwide. Two major abiotic factors affecting rice plants in different growth stages are flooding stress and cold stress. These abiotic stresses can take place independently or simultaneously and significantly affect rice plants during germination and seedling growth. Fortunately, a wide array of phenotypic responses conferring flooding stress and chilling stress tolerance exist within the rice germplasm, indicating the presence of different molecular mechanisms underlying tolerance to these stresses. Understanding these differences may assist in developing improved rice cultivars having higher tolerance to both stresses. In this study, we conducted a comparative global gene expression analysis of two rice genotypes with contrasting phenotypes under cold stress, anaerobic stress, and combined cold and anaerobic stress during germination. RESULTS: The differential gene expression analysis revealed that 5571 differentially expressed genes (DEGs), 7206 DEGs, and 13279 DEGs were identified under anaerobic stress, cold stress, and combined stress, respectively. Genes involved in the carbohydrate metabolic process, glucosyltransferase activity, regulation of nitrogen compound metabolic process, protein metabolic process, lipid metabolic process, cellular nitrogen compound biosynthetic process, lipid biosynthetic process, and a microtubule-based process were enriched across all stresses. Notably, the common Gene Ontology (GO) analysis identified three hub genes, namely Os08g0176800 (similar to mRNA-associated protein mrnp 41), Os11g0454200 (dehydrin), and OS10g0505900 (expressed protein). CONCLUSION: A large number of differentially expressed genes were identified under anaerobic, cold conditions during germination and the combination of the two stress conditions in rice. These results will assist in the identification of promising candidate genes for possible manipulation toward rice crops that are more tolerant under flooding and cold during germination, both independently and concurrently.

Anaerobic germination of green coffee beans: A novel strategy to improve the quality of commercial Arabica coffee.

This study aimed to improve the brewing quality of commercial Arabica coffee through anaerobic germination. Changes in important compounds and cupping scores of germination roasting coffee with different germination degrees were investigated by 1H NMR, HS-SPME-GC-MS and sensory analysis. Statistical analysis of multivariate analysis results indicated that 6 water-soluble chemical components and 8 volatile chemical components have the potential to be markers of germinated roasting coffee. In addition, germination significantly reduced caffeine content and acrylamide formation in roasted coffee. Sensory analysis according to the Specialty Coffee Association (SCA) cupping protocol demonstrated that anaerobic germination modified flavor attributes, improved the quality, and increased sensory scores. Furthermore, anaerobic sprouting increased fruity descriptors, but over-sprouting did not improve overall attributes while producing both fermentative and vegetable descriptors. Therefore, suitable anaerobic germination of green coffee beans can be used as a new strategy to improve the flavor of commercial Arabica coffee.

High-density genetic mapping identified QTLs for anaerobic germination tolerance in rice.

The tolerance of rice anaerobic germination (AG) is the main limiting factor for direct seeding application, yet the genetics mechanism is still in its infancy. In the study, recombinant inbred lines population of TD70 Japonica cultivar and Kasalath Indica cultivar, was employed to construct a high-density genetic map by whole genome re-sequencing. As a result, a genetic map containing 12,328 bin-markers was constructed and a total of 50 QTLs were then detected for CL(coleoptile length), CD (coleoptile diameter), CSA (coleoptile surface area) and CV (coleoptile volume) related traits in the two stages of anaerobic treatment using complete interval mapping method (inclusive composite interval mapping, ICIM). Among the four traits associated with coleoptile, coleoptile volume had the largest number of QTLs (17), followed by coleoptile diameter (16), and coleoptile length had 5 QTLs. These QTLs could explain phenotypic contribution rates ranging from 0.34% to 11.17% and LOD values ranging from 2.52 to 11.57. Combined with transcriptome analysis, 31 candidate genes were identified. Furthermore, 12 stable QTLs were used to detect the aggregation effect analysis. Besides, It was found that individuals with more aggregation synergistic alleles had higher phenotypic values in different environments. Totally, high-density genetic map, QTL mapping and aggregation effect analysis of different loci related to the anaerobic germination of rice seeds were conducted to lay a foundation for the fine mapping of related genes in subsequent assisted breeding.

Recent Advances of Genetic Resources, Genes and Genetic Approaches for Flooding Tolerance in Rice.

Flooding is one of the most hazardous natural disasters and a major stress constraint to rice production throughout the world, which results in huge economic losses. The frequency and duration of flooding is predicted to increase in near future as a result of global climate change. Breeding of flooding tolerance in rice is a challenging task because of the complexity of the component traits, screening technique, environmental factors and genetic interactions. A great progress has been made during last two decades to find out the flooding tolerance mechanism in rice. An important breakthrough in submergence research was achieved by the identification of major quantitative trait locus (QTL) SUB1 in rice chromosomes that acts as the primary contributor for tolerance. This enabled the use of marker-assisted backcrossing (MABC) to transfer SUB1 QTL into popular varieties which showed yield advantages in flood prone areas. However, SUB1 varieties are not always tolerant to stagnant flooding and flooding during germination stage. So, gene pyramiding approach can be used by combining several important traits to develop new breeding rice lines that confer tolerances to different types of flooding. This review highlights the important germplasm/genetic resources of rice to different types of flooding stress. A brief discussion on the genes and genetic mechanism in rice exhibited to different types of flooding tolerance was discussed for the development of flood tolerant rice variety. Further research on developing multiple stresses tolerant rice can be achieved by combining SUB1 with other tolerance traits/genes for wider adaptation in the rain-fed rice ecosystems.

Enriched-GWAS and Transcriptome Analysis to Refine and Characterize a Major QTL for Anaerobic Germination Tolerance in Rice.

Tolerance of anaerobic germination (AG) is a key trait in the development of direct seeded rice. Through rapid and sustained coleoptile elongation, AG tolerance enables robust seedling establishment under flooded conditions. Previous attempts to fine map and characterize AG2 (qAG7.1), a major centromere-spanning AG tolerance QTL, derived from the indica variety Ma-Zhan Red, have failed. Here, a novel approach of "enriched haplotype" genome-wide association study based on the Ma-Zhan Red haplotype in the AG2 region was successfully used to narrow down AG2 from more than 7 Mb to less than 0.7 Mb. The AG2 peak region contained 27 genes, including the Rc gene, responsible for red pericarp development in pigmented rice. Through comparative variant and transcriptome analysis between AG tolerant donors and susceptible accessions several candidate genes potentially controlling AG2 were identified, among them several regulatory genes. Genome-wide comparative transcriptome analysis suggested differential regulation of sugar metabolism, particularly trehalose metabolism, as well as differential regulation of cell wall modification and chloroplast development to be implicated in AG tolerance mechanisms.

Genetic Mapping by Sequencing More Precisely Detects Loci Responsible for Anaerobic Germination Tolerance in Rice.

Direct seeded rice (DSR) is a mainstay for planting rice in the Americas, and it is rapidly becoming more popular in Asia. It is essential to develop rice varieties that are suitable for this type of production system. ASD1, a landrace from India, possesses several traits desirable for direct-seeded fields, including tolerance to anaerobic germination (AG). To map the genetic basis of its tolerance, we examined a population of 200 F2:3 families derived from a cross between IR64 and ASD1 using the restriction site-associated DNA sequencing (RAD-seq) technology. This genotyping platform enabled the identification of 1921 single nucleotide polymorphism (SNP) markers to construct a high-resolution genetic linkage map with an average interval of 0.9 cM. Two significant quantitative trait loci (QTLs) were detected on chromosomes 7 and 9, qAG7 and qAG9, with LOD scores of 7.1 and 15.0 and R2 values of 15.1 and 29.4, respectively. Here, we obtained more precise locations of the QTLs than traditional simple sequence repeat and low-density SNP genotyping methods and may help further dissect the genetic factors of these QTLs.

Identification of QTLs associated with the anaerobic germination potential using a set of Oryza nivara introgression lines.

BACKGROUND: Rice (Oryza sativa L.) is an important crop and a staple food for half of the population around the world. The recent water and labor shortages are encouraging farmers to shift from traditional transplanting to direct-seeding. However, poor germination and slow elongation of the coleoptile constrains large-scale application of direct-seeding. OBJECTIVE: This study was aimed to investigate the genetic basis of the anaerobic germination (AG) potential using a set of Oryza nivara (O. nivara) introgression lines (ILs). METHODS: In this study, a total of 131 ILs were developed by introducing O. nivara chromosome segments into the elite indica rice variety 93-11 through advanced backcrossing and repeated selfing. A high-density genetic map has been previously constructed with 1,070 bin-markers. The seeds of ILs were germinated and used to measure coleoptile length under normal and anaerobic conditions. QTLs associated with AG potential were determined in rice. RESULTS: Based on the high-density genetic map of the IL population, two QTLs, qAGP1 and qAGP3 associated with AG tolerance were characterized and located on chromosomes 1 and 3, respectively. Each QTL explained 15% of the phenotypic variance. Specifically, the O. nivara-derived chromosome segments of the two QTLs were positively tolerance to anaerobic condition by increasing coleoptile length. In a further analysis of public transcriptome data, a total of 26 and 36 genes within qAGP1 and qAGP3 were transcriptionally induced by anaerobic stress, respectively. CONCLUSIONS: Utilization of O. nivara-derived alleles at qAGP1 and qAGP3 can potentially enhance tolerance to anaerobic stress at the germination stage in rice, thereby accelerating breeding of rice varieties to be more adaptative for direct-seeding.

Dynamic genome-wide association analysis and identification of candidate genes involved in anaerobic germination tolerance in rice.

BACKGROUND: In Asian rice production, an increasing number of countries now choose the direct seeding mode because of rising costs, labour shortages and water shortages. The ability of rice seeds to undergo anaerobic germination (AG) plays an important role in the success of direct seeding. RESULTS: In this study, we used 2,123,725 single nucleotide polymorphism (SNP) markers based on resequencing to conduct a dynamic genome-wide association study (GWAS) of coleoptile length (CL) and coleoptile diameter (CD) in 209 natural rice populations. A total of 26 SNP loci were detected in these two phenotypes, of which 5 overlapped with previously reported loci (S1_ 39674301, S6_ 20797781, S7_ 18722403, S8_ 9946213, S11_ 19165397), and two sites were detected repeatedly at different time points (S3_ 24689629 and S5_ 27918754). We suggest that these 7 loci (-log10 (P) value > 7.3271) are the key sites that affect AG tolerance. To screen the candidate genes more effectively, we sequenced the transcriptome of the flooding-tolerant variety R151 in six key stages, including anaerobic (AN) and the oxygen conversion point (AN-A), and obtained high-quality differential expression profiles. Four reliable candidate genes were identified: Os01g0911700 (OsVP1), Os05g0560900 (OsGA2ox8), Os05g0562200 (OsDi19-1) and Os06g0548200. Then qRT-PCR and LC-MS/ MS targeting metabolite detection technology were used to further verify that the up-regulated expression of these four candidate genes was closely related to AG. CONCLUSION: The four novel candidate genes were associated with gibberellin (GA) and abscisic acid (ABA) regulation and cell wall metabolism under oxygen-deficiency conditions and promoted coleoptile elongation while avoiding adverse effects, allowing the coleoptile to obtain oxygen, escape the low-oxygen environment and germinate rapidly. The results of this study improve our understanding of the genetic basis of AG in rice seeds, which is conducive to the selection of flooding-tolerant varieties suitable for direct seeding.

The Molecular Regulatory Pathways and Metabolic Adaptation in the Seed Germination and Early Seedling Growth of Rice in Response to Low O2 Stress.

As sessile organisms, flooding/submergence is one of the major abiotic stresses for higher plants, with deleterious effects on their growth and survival. Therefore, flooding/submergence is a large challenge for agriculture in lowland areas worldwide. Long-term flooding/submergence can cause severe hypoxia stress to crop plants and can result in substantial yield loss. Rice has evolved distinct adaptive strategies in response to low oxygen (O2) stress caused by flooding/submergence circumstances. Recently, direct seeding practice has been increasing in popularity due to its advantages of reducing cultivation cost and labor. However, establishment and growth of the seedlings from seed germination under the submergence condition are large obstacles for rice in direct seeding practice. The physiological and molecular regulatory mechanisms underlying tolerant and sensitive phenotypes in rice have been extensively investigated. Here, this review focuses on the progress of recent advances in the studies of the molecular mechanisms and metabolic adaptions underlying anaerobic germination (AG) and coleoptile elongation. Further, we highlight the prospect of introducing quantitative trait loci (QTL) for AG into rice mega varieties to ensure the compatibility of flooding/submergence tolerance traits and yield stability, thereby advancing the direct seeding practice and facilitating future breeding improvement.

Genome-wide association studies using 50 K rice genic SNP chip unveil genetic architecture for anaerobic germination of deep-water rice population of Assam, India.

North Eastern part of India such as Assam is inundated by flood every year where the farmers are forced to grow the traditional tall deep-water rice. Genetic improvement of this type of rice is slow because of insufficient knowledge about their genetic architecture and population structure. In the present investigation, the genetic diversity architecture of 94 deep-water rice genotypes of Assam and association mapping strategy was, for the first time, applied to determine the significant SNPs and genes for deep-water rice. These genotypes are known for their unique elongation ability under deep-water condition. The anaerobic germination (AG) related trait-associated genes identified here can provide affluent resources for rice breeding especially in flood-prone areas. We investigated the genome-wide association studies (GWAS) using 50 K rice genic SNP chip across 94 deep-water rice genotypes collected from different flood-prone districts/villages of Assam. Population structure and diversity analysis revealed that these genotypes were stratified into four sub-populations. Using GWAS approach, 20 significant genes were identified and found to be associated with AG-related traits. Of them, two most relevant genes (OsXDH1and SSXT) have been identified which explain phenotypic variability (R2 > 20%) in the population. These genes were located in Chr 3 (LOC_Os03g31550) which encodes for enzyme xanthine dehydrogenase 1(OsXDH1) and in Chr 12 (LOC_Os12g31350) which encodes for SSXT family protein. Both of these genes were found to be associated with anaerobic response index (increase in the coleoptile length under water in anaerobic condition with respect to control), respectively. Interestingly, OsXDH1is involved in purine catabolism pathway and acts as a scavenger of reactive oxygen species in plants, whereas SSXT is GRF1-interacting factor 3. These two candidate genes associated with AG of deep-water rice have been found to be reported for the first time. Thus, this study provides a greater resource for breeders not only for improvement of deep-water rice, but also for AG tolerant variety useful for direct-seeded rice in flood-affected areas.

Trait-based mapping to identify the genetic factors underlying anaerobic germination of rice: Phenotyping, GXE, and QTL mapping.

BACKGROUND: Anaerobic germination is one of the most important traits for rice under direct-seeded conditions. The trait reduces risk of crop failure due to waterlogged conditions after seeding and allows water to be used as a means of weed control. The identification of QTLs and causal genes for anaerobic germination will facilitate breeding for improved direct-seeded rice varieties. In this study, we explored a BC1F2:3 population developed from a cross between BJ1, an indica landrace, and NSIC Rc222, a high-yielding recurrent parent. The population was phenotyped under different screening methods (anaerobic screenhouse, anaerobic tray, and aerobic screenhouse) to establish the relationship among the methods and to identify the most suitable screening method, followed by bulk segregant analysis (BSA) to identify large-effect QTLs. RESULTS: The study showed high heritability for survival (SUR) under all three phenotyping conditions. Although high correlation was observed within screening environments between survival at 14 and 21 days after seeding, the correlation across environments was low. Germination under aerobic and anaerobic conditions showed very low correlation, indicating the independence of their genetic control. The results were further confirmed through AMMI analysis. Four significant markers with an effect on anaerobic germination were identified through BSA. CIM analysis revealed qAG1-2, qAG6-2, qAG7-4, and qAG10-1 having significant effects on the trait. qAG6-2 and qAG10-1 were consistent across screening conditions and seedling age while qAG1-2 and qAG7-4 were specific to screening methods. All QTLs showed an effect when survival across all screening methods was analyzed. Together, the QTLs explained 39 to 55% of the phenotypic variation for survival under anaerobic conditions. No QTL effects were observed under aerobic conditions. CONCLUSIONS: The study helped us understand the effect of phenotyping method on anaerobic germination, which will lead to better phenotyping for this trait in future studies. The QTLs identified through this study will allow the improvement of breeding lines for the trait through marker-assisted selection or through forward breeding approaches such as genomic selection. The high frequency of the BJ1 allele of these QTLs will enhance the robustness of germination under anaerobic conditions in inbred and hybrid rice varieties.

QTL mapping for tolerance to anaerobic germination in rice from IR64 and the aus landrace Kharsu 80A.

Direct seeding of rice often results in poor crop establishment due to unlevelled fields, unpredicted heavy rains after sowing, and weed and pest invasion. Thus, it is important to develop varieties able to tolerate flooding during germination, also known as anaerobic germination (AG), to address these constraints. A study was conducted to identify QTLs associated with AG tolerance from an IR64/Kharsu 80A F2:3 mapping population using 190 lines phenotyped for seedling survival under the stress. Genotyping was performed using a genomewide 384-plex Indica/Indica SNP set. Four QTLs derived from Kharsu 80A providing increased tolerance to anaerobic germination were identified: three on chromosome 7 (qAG7.1, qAG7.2 and qAG7.3) and one on chromosome 3 (qAG3), with LOD values ranging from 5.7 to 7.7, and phenotypic variance explained (R2) from 8.1% to 12.6%. The QTLs identified in this study can be further investigated to better understand the genetic bases of AG tolerance in rice, and used for marker-assisted selection to develop more robust direct-seeded rice varieties.

Deciphering Genetics Underlying Stable Anaerobic Germination in Rice: Phenotyping, QTL Identification, and Interaction Analysis.

Anaerobic germination (AG) is an important trait for direct-seeded rice (DSR) to be successful. Rice usually has low germination under anaerobic conditions, which leads to a poor crop stand in DSR when rain occurs after seeding. The ability of rice to germinate under water reduces the risk of poor crop stand. Further, this allows the use of water as a method of weed control. The identification of the genetic factors leading to high anaerobic germination is required to develop improved DSR varieties. In the present study, two BC1F2:3 mapping families involving a common parent with anaerobic germination potential, Kalarata, an indica landrace, and two recurrent parents, NSIC Rc222 and NSIC Rc238, were used. Phenotyping was done under two environmental conditions and genotyping was carried out through the KASP SNP genotyping platform. A total of 185 and 189 individuals genotyped with 170 and 179 polymorphic SNPs were used for QTL analysis for the two populations, Kalarata/NSIC Rc238 and Kalarata/NSIC Rc222, respectively. A total of five QTLs on chromosomes 3, 5, 6, 7, and 8 for survival (SUR) and four QTLs on chromosomes 1, 3 (two locations), and 7 for the trait seedling height (SH) across the populations and over the screening conditions were identified. Except for the QTLs on chromosomes 5 and 8, the parent with AG potential, Kalarata, contributed all the other QTLs. Among the five QTLs for SUR, the second-largest QTL (qSUR6-1) was novel for AG potential in rice, showing a stable expression in terms of genetic background and screening conditions explaining 11.96% to 16.01% of the phenotypic variation. The QTL for SH (qSH1-1) was also novel. Considering different genetic backgrounds and different screening conditions, the QTLs identified for the trait SUR explained phenotypic variation in the range of 57.60% to 73.09% while that for the trait SH ranged from 13.53% to 34.30%.