Sang-Nag Ahn

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.

Development of a research platform for dissecting phenotype-genotype associations in rice (Oryza spp.).

We present an overview of a research platform that provides essential germplasm, genotypic and phenotypic data and analytical tools for dissecting phenotype–genotype associations in rice. These resources include a diversity panel of 400 Oryza sativa and 100 Oryza rufipogon accessions that have been purified by single seed descent, a custom-designed Affymetrix array consisting of 44,100 SNPs, an Illumina GoldenGate assay consisting of 1,536 SNPs, and a suite of low-resolution 384-SNP assays for the Illumina BeadXpress Reader that are designed for applications in breeding, genetics and germplasm management. Our long-term goal is to empower basic research discoveries in rice by linking sequence diversity with physiological, morphological, and agronomic variation. This research platform will also help increase breeding efficiency by providing a database of diversity information that will enable researchers to identify useful DNA polymorphisms in genes and germplasm of interest and convert that information into cost-effective tools for applied plant improvement.

Phytochelatin–metal(loid) transport into vacuoles shows different substrate preferences in barley and Arabidopsis

Cadmium (Cd) and arsenic (As) are toxic to all living organisms, including plants and humans. In plants, Cd and As are detoxified by phytochelatins (PCs) and metal(loid)-chelating peptides and by sequestering PC-metal(loid) complexes in vacuoles. Consistent differences have been observed between As and Cd detoxification. Whereas chelation of Cd by PCs is largely sufficient to detoxify Cd, As-PC complexes must be sequestered into vacuoles to be fully detoxified. It is not clear whether this difference in detoxification pathways is ubiquitous among plants or varies across species. Here, we have conducted a PC transport study using vacuoles isolated from Arabidopsis and barley. Arabidopsis vacuoles accumulated low levels of PC2 -Cd, and vesicles from yeast cells expressing either AtABCC1 or AtABCC2 exhibited negligible PC2 -Cd transport activity compared with PC2 -As. In contrast, barley vacuoles readily accumulated comparable levels of PC2 -Cd and PC2 -As. PC transport in barley vacuoles was inhibited by vanadate, but not by ammonium, suggesting the involvement of ABC-type transporters. Interestingly, barley vacuoles exhibited enhanced PC2 transport activity when essential metal ions, such as Zn(II), Cu(II) and Mn(II), were added to the transport assay, suggesting that PCs might contribute to the homeostasis of essential metals and detoxification of non-essential toxic metal(loid)s.

Mapping QTLs for salt tolerance in an introgression line population between Japonica cultivars in rice

QTL analysis of physiological traits related to salt tolerance was carried out using 117 BC3F5 lines derived from a cross between “Ilpumbyeo” as a recurrent parent and “Moroberekan” as a donor parent. The 117 introgression lines with the parents were evaluated for five traits; dry weight, fresh weight, leaf area, seedling height, and survival rate under control and salinity conditions (55 mM) at the seedling stage. To identify QTLs related to salt tolerance, 125 SSR markers showing polymorphisms between the parents were genotyped for the 117 BC3F5 lines. A total of eight QTLs were detected on chromosomes 1, 6, and 7. These include two QTLs on chromosomes 6 and 7 for reduction rate of dry weight (R2 = 10.2∼13.6%), three QTLs on chromosomes 1, 6, and 7 for reduction rate of fresh weight (R2 = 10.9∼13.9 %), two QTLs on chromosomes 1 and 7 for reduction rate of leaf area (R2 = 12.1%), and one QTL on chromosome 7 for reduction rate of seedling height (R2 = 10.5%). The Moroberekan alleles contributed the positive effect at these eight QTL loci. Although the parents, Ilpumbyeo and Moroberekan, were not salt tolerant as the salt tolerant check variety, Pokkali, some lines displayed a similar level of tolerance as Pokkali. The effect of the QTL on chromosome 7 was further confirmed by evaluating four lines containing the target QTL for fresh and dry weight, turgid weight, and relative water content (RWC). Significant differences between each line and Ilpumbyeo were detected for dry and fresh weight, and RWC values, and these results seem to indicate the beneficial effect of the Moroberekam alleles for salt tolerance. A set of introgression lines are being developed containing only few chromosomal segments from Moroberekan in the Ilpumbyeo background. These would be useful in developing salt tolerant lines in the breeding program.

Relationship between genetic divergence and hybrid performance in japonica rice grown in a cold water-irrigated field

The present study was carried out to evaluate genetic divergence among eleven japonica rice cultivars and to assess the relationship between genetic distance and hybrid performance in partial non-reciprocal crosses among them. The 44 F1 hybrids along with the eleven parents were evaluated for five cold tolerance-related traits; discoloration at seedling stage, days to heading, culm length, fertility, and spikelets per panicle in a cold water screening nursery (17 °C). The eleven parents were examined for DNA polymorphism using amplified fragment length polymorphisms(AFLPs). A total of 855 polymorphic variants were generated and based on the polymorphism data, genetic distances (GDs) ranged from 0.023 to0.524. Very little heterosis was observed in hybrids for most of the traits,whereas heterosis was high for fertility. The correlation values of GDs with F1 performance were mostly non-significant except for discoloration and fertility. The correlations of GDs with mid-parent and better-parent heterosis were not significant and proved to be of no predictive value. Our results indicate that GDs based on AFLP markers are not useful for predicting heterosis for cold tolerance in japonica hybrids.

Mapping and characterization of quantitative trait loci for mesocotyl elongation in rice (Oryza sativa L.)

Mesocotyl elongation is an important trait for seedling emergence in direct-seeding cultivation in rice. In this study, a backcross inbred line (BIL) population from a cross between Kasalath and Nipponbare was employed to map quantitative trait loci (QTLs) for mesocotyl elongation. A total of 5 QTLs for mesocotyl length were identified on chromosomes 1, 3, 7, 9, and 12 in 2 independent experiments. At all QTL, the Kasalath alleles contributed to an increase in mesocotyl length. Two QTLs (qMel-1 and qMel-3) on chromosomes 1 and 3 were consistently detected in both experiments. To fine map the QTLs, a cross was made between 2 chromosome segment substitution lines (CSSL-6 and CSSL-15), each harboring the Kasalath allele across the qMel-1 and qMel-3 regions, and an F2:3 population was developed. A two-way ANOVA indicated that no epistatic interaction was detected between the 2 QTLs in the F2 population (P = 0.31). Moreover, analysis of two F3 near-isogenic lines (NILs) derived from the same cross, indicated that the 2 QTLs act additively in distinct or complementary pathways in controlling mesocotyl elongation. Substitution mapping indicated that the qMel-1 QTL was located between the 2 SSR markers RM5448 and RM5310, which are 3,799-kb apart, and that the qMel-3 QTL was located between the 2 SSR markers RM3513 and RM1238, which are 6,964-kb apart. To our knowledge, this is the first report to fine-map QTLs for mesocotyl elongation and to analyze their interaction.

Fine mapping of grain weight QTL, tgw11 using near isogenic lines from a cross between Oryza sativa and O. grandiglumis

In our previous study, we reported the grain weight (GW) QTL, tgw11 in isogenic lines derived from a cross between Oryza sativa ssp. Japonica cv. Hwaseong and O. grandiglumis. The O. grandiglumis allele at tgw11 decreased GW in the Hwaseong background. To fine-map tgw11, one F5 plant homozygous for the O. grandiglumis DNA in the target region on chromosome 11 was selected from F4 line, CR1242 segregating for tgw11 and crossed with Hwaseong to produce secondary F2 and F3 populations. QTL analysis using 760 F2 plants confirmed the existence of tgw11 with an R2 value of 15.0%. This QTL explained 32.2% of the phenotypic variance for GW in 91 F3 lines. Substitution mapping with 65 F3 lines with informative recombination breakpoints in the target region was carried out to narrow down the position of the tgw11. The result indicated that tgw11 was located in the 900-kb interval between two SSR markers, RM224 and RM27358. QTLs for grain width and grain thickness were also located in the same interval suggesting that a single gene is involved in controlling these three traits. Analysis of F3 lines indicated that the variation in TGW is associated with variation in grain shape, specifically grain thickness and grain width. Genetic analysis indicated that the O. grandiglumis allele for small seed was dominant over the Hwaseong allele. SSR markers tightly linked to the GW QTL would be useful in marker-assisted selection for variation in GW in breeding program.

Comprehensive identification of LMW-GS genes and their protein products in a common wheat variety

Although it is well known that low-molecular-weight glutenin subunits (LMW-GS) from wheat affect bread and noodle processing quality, the function of specific LMW-GS proteins remains unclear. It is important to find the genes that correspond to individual LMW-GS proteins in order to understand the functions of specific proteins. The objective of this study was to link LMW-GS genes and haplotypes characterized using well known Glu-A3, Glu-B3, and Glu-D3 gene-specific primers to their protein products in a single wheat variety. A total of 36 LMW-GS genes and pseudogenes were amplified from the Korean cultivar Keumkang. These include 11 Glu-3 gene haplotypes, two from the Glu-A3 locus, two from the Glu-B3 locus, and seven from the Glu-D3 locus. To establish relationships between gene haplotypes and their protein products, a glutenin protein fraction was separated by two-dimensional gel electrophoresis (2-DGE) and 17 protein spots were analyzed by N-terminal amino acid sequencing and tandem mass spectrometry (MS/MS). LMW-GS proteins were identified that corresponded to all Glu-3 gene haplotypes except the pseudogenes. This is the first report of the comprehensive characterization of LMW-GS genes and their corresponding proteins in a single wheat cultivar. Our approach will be useful to understand the contributions of individual LMW-GS to the end-use quality of flour.

QTL dissection of agronomic and domestication traits using introgression lines carrying wild rice (Oryza rufipogon Griff.) segments in cultivated rice (O. sativa L.) background

The nearly isogenic nature of the introgression lines (ILs) provides a relative advantage over other segregating populations in the rapid implementation of pyramiding approach through crosses and marker analysis. A set of 126 ILs carrying various introgressed segments from a presumed wild progenitor, Oryza rufipogon Griff. Acc. W1944 in the background of an elite Korea japonica cultivar (O. sativa L.), Hwayeongbyeo, was constructed using the marker assisted selection (MAS) technique combined with repeated backcrosses. The 126 ILs have different W1944 segments on each chromosome, with 100% coverage of wild segments on chromosome 1, while for chromosome 10, the coverage was only 33.3%. The mean number of homozygous and heterozygous donor segments were 3 (ranging from 0 to 7) and 4.7 (ranging from 1 to 14.5), respectively, and 31.6% of introgressed segments had sizes of less than 10.5 cM. A total of 41 quantitative trait loci (QTLs) and two loci associated with pericarp coloration were identified. The number of QTLs per trait ranged from 2 to 6. Phenotypic variance associated with each QTL varied from 9.1 to 52.2%, with an average of 17.1%. For 11 (26.8%) of the QTLs detected in this study, the O. rufipogon-derived alleles contributed the desired agronomic effect despite the overall inferior characteristics of the wild phenotype. Favorable alleles from the O. rufipogon accession were identified for panicle number, panicle length, days to heading, secondary branches, spikelets per panicle, and 1,000-grain weight.

Nuclear and chloroplast diversity and phenotypic distribution of rice (Oryza sativa L.) germplasm from the democratic people’s republic of Korea (DPRK; North Korea)

BackgroundRice accounts for 43% of staple food production in the Democratic People’s Republic of Korea (DPRK). The most widely planted rice varieties were developed from a limited number of ancestral lines that were repeatedly used as parents in breeding programs. However, detailed pedigrees are not publicly available and little is known about the genetic, phenotypic, and geographical variation of DPRK varieties.ResultsWe evaluated 80 O. sativa accessions from the DPRK, consisting of 67 improved varieties and 13 landraces. Based on nuclear SSR analysis, we divide the varieties into two genetic groups: Group 1 corresponds to the temperate japonica subpopulation and represents 78.75% of the accessions, while Group 2 shares recent ancestry with indica varieties. Interestingly, members of Group 1 are less diverse than Group 2 at the nuclear level, but are more diverse at the chloroplast level. All Group 2 varieties share a single Japonica maternal-haplotype, while Group 1 varieties trace maternal ancestry to both Japonica and Indica. Phenotypically, members of Group 1 have shorter grains than Group 2, and varieties from breeding programs have thicker and wider grains than landraces. Improved varieties in Group 1 also show similar and/or better levels of cold tolerance for most traits, except for spikelet number per panicle. Finally, geographic analysis demonstrates that the majority of genetic variation is located within regions that have the most intensive rice cultivation, including the Western territories near the capital city Pyungyang. This is consistent with the conscious and highly centralized role of human selection in determining local dispersion patterns of rice in the DPRK.ConclusionsDiversity studies of DPRK rice germplasm revealed two genetic groups. The most widely planted group has a narrow genetic base and would benefit from the introduction of new genetic variation from cold tolerant landraces, wild accessions, and/or cultivated gene pools to enhance yield potential and performance.