Michael J. Thomson

Caught Red-Handed: Rc Encodes a Basic Helix-Loop-Helix Protein Conditioning Red Pericarp in Rice

Rc is a domestication-related gene required for red pericarp in rice (Oryza sativa). The red grain color is ubiquitous among the wild ancestors of O. sativa, in which it is closely associated with seed shattering and dormancy. Rc encodes a basic helix-loop-helix (bHLH) protein that was fine-mapped to an 18.5-kb region on rice chromosome 7 using a cross between Oryza rufipogon (red pericarp) and O. sativa cv Jefferson (white pericarp). Sequencing of the alleles from both mapping parents as well as from two independent genetic stocks of Rc revealed that the dominant red allele differed from the recessive white allele by a 14-bp deletion within exon 6 that knocked out the bHLH domain of the protein. A premature stop codon was identified in the second mutant stock that had a light red pericarp. RT-PCR experiments confirmed that the Rc gene was expressed in both red- and white-grained rice but that a shortened transcript was present in white varieties. Phylogenetic analysis, supported by comparative mapping in rice and maize (Zea mays), showed that Rc, a positive regulator of proanthocyanidin, is orthologous with INTENSIFIER1, a negative regulator of anthocyanin production in maize, and is not in the same clade as rice bHLH anthocyanin regulators.

Genetic and genomic approaches to develop rice germplasm for problem soils

Soils that contain toxic amounts of minerals or are deficient in essential plant nutrients are widespread globally and seriously constrain rice production. New methods are necessary to incorporate the complex adaptive traits associated with tolerance of these abiotic stresses, while simultaneously retaining the high yield potential of rice varieties when conditions are favorable. Significant progress in the genetic characterization of stress response pathways and recent advances in genomics have provided powerful tools for in-depth dissection of tolerance mechanisms. Additionally, tolerance of most of these abiotic stresses in rice is controlled by a few QTLs with large effects despite the intricacy of the numerous traits involved. Genetic dissection of these QTLs and their incorporation into high-yielding varieties will significantly enhance and stabilize rice productivity in these problem soils. Current efforts at IRRI and in rice breeding programs worldwide are seeking to explore diverse germplasm collections and genetically dissect the causal mechanisms of tolerance to facilitate their use in breeding. This review focuses on salinity and P and Zn deficiency as the major problems encountered in rice soils, and examines current understanding of the mechanisms involved and efforts toward germplasm improvement.

Characterizing the Saltol Quantitative Trait Locus for Salinity Tolerance in Rice

This study characterized Pokkali-derived quantitative trait loci (QTLs) for seedling stage salinity tolerance in preparation for use in marker-assisted breeding. An analysis of 100 SSR markers on 140 IR29/Pokkali recombinant inbred lines (RILs) confirmed the location of the Saltol QTL on chromosome 1 and identified additional QTLs associated with tolerance. Analysis of a series of backcross lines and near-isogenic lines (NILs) developed to better characterize the effect of the Saltol locus revealed that Saltol mainly acted to control shoot Na+/K+ homeostasis. Multiple QTLs were required to acquire a high level of tolerance. Unexpectedly, multiple Pokkali alleles at Saltol were detected within the RIL population and between backcross lines, and representative lines were compared with seven Pokkali accessions to better characterize this allelic variation. Thus, while the Saltol locus presents a complex scenario, it provides an opportunity for marker-assisted backcrossing to improve salt tolerance of popular varieties followed by targeting multiple loci through QTL pyramiding for areas with higher salt stress.

Global Dissemination of a Single Mutation Conferring White Pericarp in Rice

Here we report that the change from the red seeds of wild rice to the white seeds of cultivated rice (Oryza sativa) resulted from the strong selective sweep of a single mutation, a frame-shift deletion within the Rc gene that is found in 97.9% of white rice varieties today. A second mutation, also within Rc, is present in less than 3% of white accessions surveyed. Haplotype analysis revealed that the predominant mutation originated in the japonica subspecies and crossed both geographic and sterility barriers to move into the indica subspecies. A little less than one Mb of japonica DNA hitchhiked with the rc allele into most indica varieties, suggesting that other linked domestication alleles may have been transferred from japonica to indica along with white pericarp color. Our finding provides evidence of active cultural exchange among ancient farmers over the course of rice domestication coupled with very strong, positive selection for a single white allele in both subspecies of O. sativa.

Genetic diversity analysis of traditional and improved Indonesian rice (Oryza sativa L.) germplasm using microsatellite markers

The archipelago of Indonesia has a long history of rice production across a broad range of rice-growing environments resulting in a diverse array of local Indonesian rice varieties. Although some have been incorporated into modern breeding programs, the vast majority of these landraces remain untapped. To better understand this rich source of genetic diversity we have characterized 330 rice accessions, including 246 Indonesian landraces and 63 Indonesian improved cultivars, using 30 fluorescently-labeled microsatellite markers. The landraces were selected across 21 provinces and include representatives of the classical subpopulations of cere, bulu, and gundil rices. A total of 394 alleles were detected at the 30 simple sequence repeat loci, with an average number of 13 alleles per locus across all accessions, and an average polymorphism information content value of 0.66. Genetic diversity analysis characterized the Indonesian landraces as 68% indica and 32% tropical japonica, with an indica gene diversity of 0.53 and a tropical japonica gene diversity of 0.56, and a Fst of 0.38 between the two groups. All of the improved varieties sampled were indica, and had an average gene diversity of 0.46. A set of high quality Indonesian varieties, including Rojolele, formed a separate cluster within the tropical japonicas. This germplasm presents a valuable source of diversity for future breeding and association mapping efforts.

Through the genetic bottleneck: O. rufipogon as a source of trait-enhancing alleles for O. sativa

This paper summarizes results from a decade of collaborative research using advanced backcross (AB) populations to a) identify quantitative trait loci (QTL) associated with improved performance in rice and to b) clone genes underlying key QTLs of interest. We demonstrate that AB-QTL analysis is capable of (1) successfully uncovering positive alleles in wild germplasm that were not obvious based on the phenotype of the parent (2) offering an estimation of the breeding value of exotic germplasm, (3) generating near isogenic lines that can be used as the basis for gene isolation and also as parents for further crossing in a variety development program and (4) providing gene-based markers for targeted introgression of alleles using marker-assisted-selection (MAS). Knowledge gained from studies examining the population structure and evolutionary history of rice is helping to illuminate a long-term strategy for exploiting and simultaneously preserving the well-partitioned gene pools in rice.

Multi-parent advanced generation inter-cross (MAGIC) populations in rice: progress and potential for genetics research and breeding

BackgroundThis article describes the development of Multi-parent Advanced Generation Inter-Cross populations (MAGIC) in rice and discusses potential applications for mapping quantitative trait loci (QTLs) and for rice varietal development. We have developed 4 multi-parent populations: indica MAGIC (8 indica parents); MAGIC plus (8 indica parents with two additional rounds of 8-way F1 inter-crossing); japonica MAGIC (8 japonica parents); and Global MAGIC (16 parents – 8 indica and 8 japonica). The parents used in creating these populations are improved varieties with desirable traits for biotic and abiotic stress tolerance, yield, and grain quality. The purpose is to fine map QTLs for multiple traits and to directly and indirectly use the highly recombined lines in breeding programs. These MAGIC populations provide a useful germplasm resource with diverse allelic combinations to be exploited by the rice community.ResultsThe indica MAGIC population is the most advanced of the MAGIC populations developed thus far and comprises 1328 lines produced by single seed descent (SSD). At the S4 stage of SSD a subset (200 lines) of this population was genotyped using a genotyping-by-sequencing (GBS) approach and was phenotyped for multiple traits, including: blast and bacterial blight resistance, salinity and submergence tolerance, and grain quality. Genome-wide association mapping identified several known major genes and QTLs including Sub1 associated with submergence tolerance and Xa4 and xa5 associated with resistance to bacterial blight. Moreover, the genome-wide association study (GWAS) results also identified potentially novel loci associated with essential traits for rice improvement.ConclusionThe MAGIC populations serve a dual purpose: permanent mapping populations for precise QTL mapping and for direct and indirect use in variety development. Unlike a set of naturally diverse germplasm, this population is tailor-made for breeders with a combination of useful traits derived from multiple elite breeding lines. The MAGIC populations also present opportunities for studying the interactions of genome introgressions and chromosomal recombination.

High-Throughput SNP Genotyping to Accelerate Crop Improvement

Recent advances in next-generation sequencing (NGS) and single nucleotide polymorphism (SNP) genotyping promise to greatly accelerate crop improvement if properly deployed. High-throughput SNP genotyping offers a number of advantages over previous marker systems, including an abundance of markers, rapid processing of large populations, a variety of genotyping systems to meet different needs, and straightforward allele calling and database storage due to the bi-allelic nature of SNP markers. NGS technologies have enabled rapid whole genome sequencing, providing extensive SNP discovery pools to select informative markers for different sets of germplasm. Highly multiplexed fixed array platforms have enabled powerful approaches such as genome-wide association studies. On the other hand, routine deployment of trait-specific SNP markers requires flexible, low-cost systems for genotyping smaller numbers of SNPs across large breeding populations, using platforms such as Fluidigms Dynamic Arrays™, Douglas Scientifics Array Tape™, and LGCs automated systems for running KASP™ markers. At the same time, genotyping by sequencing (GBS) is rapidly becoming popular for low-cost high-density genome-wide scans through multiplexed sequencing. This review will discuss the range of options available to modern breeders for integrating SNP markers into their programs, whether by outsourcing to service providers or setting up in-house genotyping facilities, and will provide an example of SNP deployment for rice research and breeding as demonstrated by the Genotyping Services Lab at the International Rice Research Institute.

Response to zinc deficiency of two rice lines with contrasting tolerance is determined by root growth maintenance and organic acid exudation rates, and not by zinc-transporter activity

*Zinc (Zn)-deficient soils constrain rice (Oryza sativa) production and cause Zn malnutrition. The identification of Zn-deficiency-tolerant rice lines indicates that breeding might overcome these constraints. Here, we seek to identify processes underlying Zn-deficiency tolerance in rice at the physiological and transcriptional levels. *A Zn-deficiency-tolerant line RIL46 acquires Zn more efficiently and produces more biomass than its nontolerant maternal line (IR74) at low [Zn](ext) under field conditions. We tested if this was the result of increased expression of Zn(2+) transporters; increased root exudation of deoxymugineic acid (DMA) or low-molecular-weight organic acids (LMWOAs); and/or increased root production. Experiments were performed in field and controlled environment conditions. *There was little genotypic variation in transcript abundance of Zn-responsive root Zn(2+)-transporters between the RIL46 and IR74. However, root exudation of DMA and LMWOA was greater in RIL46, coinciding with increased root expression of putative ligand-efflux genes. Adventitious root production was maintained in RIL46 at low [Zn](ext), correlating with altered expression of root-specific auxin-responsive genes. *Zinc-deficiency tolerance in RIL46 is most likely the result of maintenance of root growth, increased efflux of Zn ligands, and increased uptake of Zn-ligand complexes at low [Zn](ext); these traits are potential breeding targets.

Substitution Mapping of dth1.1, a Flowering-Time Quantitative Trait Locus (QTL) Associated With Transgressive Variation in Rice, Reveals Multiple Sub-QTL

A quantitative trait locus (QTL), dth1.1, was associated with transgressive variation for days to heading in an advanced backcross population derived from the Oryza sativa variety Jefferson and an accession of the wild rice relative Oryza rufipogon. A series of near-isogenic lines (NILs) containing different O. rufipogon introgressions across the target region were constructed to dissect dth1.1 using substitution mapping. In contrast to the late-flowering O. rufipogon parent, O. rufipogon alleles in the substitution lines caused early flowering under both short- and long-day lengths and provided evidence for at least two distinct sub-QTL: dth1.1a and dth1.1b. Potential candidate genes underlying these sub-QTL include genes with sequence similarity to Arabidopsis GI, FT, SOC1, and EMF1, and Pharbitis nil PNZIP. Evidence from families with nontarget O. rufipogon introgressions in combination with dth1.1 alleles also detected an early flowering QTL on chromosome 4 and a late-flowering QTL on chromosome 6 and provided evidence for additional sub-QTL in the dth1.1 region. The availability of a series of near-isogenic lines with alleles introgressed from a wild relative of rice provides an opportunity to better understand the molecular basis of transgressive variation in a quantitative trait.