Backki Kim

An interspecific (Capsicum annuum ×C. chinese) F2 linkage map in pepper using RFLP and AFLP markers

Abstract We have constructed a molecular linkage map of pepper (Capsicum spp.) in an interspecific F2 population of 107 plants with 150 RFLP and 430 AFLP markers. The resulting linkage map consists of 11 large (206–60.3 cM) and 5 small (32.6–10.3 cM) linkage groups covering 1,320 cM with an average map distance between framework markers of 7.5 cM. Most (80%) of the RFLP markers were pepper-derived clones, and these markers were evenly distributed across the genome. By using 30 primer combinations, we were able to generate 444 AFLP markers in the F2 population. The majority of the AFLP markers clustered in each linkage group, although PstI/MseI markers were more evenly distributed than EcoRI/MseI markers within the linkage groups. Genes for the biosynthesis of carotenoids and capsaicinoids were mapped on our linkage map. This map will provide the basis of studying secondary metabolites in pepper.

Complete chloroplast and ribosomal sequences for 30 accessions elucidate evolution of Oryza AA genome species.

Cytoplasmic chloroplast (cp) genomes and nuclear ribosomal DNA (nR) are the primary sequences used to understand plant diversity and evolution. We introduce a high-throughput method to simultaneously obtain complete cp and nR sequences using Illumina platform whole-genome sequence. We applied the method to 30 rice specimens belonging to nine Oryza species. Concurrent phylogenomic analysis using cp and nR of several of specimens of the same Oryza AA genome species provides insight into the evolution and domestication of cultivated rice, clarifying three ambiguous but important issues in the evolution of wild Oryza species. First, cp-based trees clearly classify each lineage but can be biased by inter-subspecies cross-hybridization events during speciation. Second, O. glumaepatula, a South American wild rice, includes two cytoplasm types, one of which is derived from a recent interspecies hybridization with O. longistminata. Third, the Australian O. rufipogan-type rice is a perennial form of O. meridionalis.

Analysis of segregation distortion and its relationship to hybrid barriers in rice

BackgroundSegregation distortion (SD) is a frequently observed occurrence in mapping populations generated from crosses involving divergent genotypes. In the present study, ten genetic linkage maps constructed from reciprocal F2 and BC1F1 mapping populations derived from the parents Dasanbyeo (indica) and Ilpumbyeo (japonica) were used to identify the distribution, effect, and magnitude of the genetic factors underlying the mechanisms of SD between the two subspecies.ResultsSD loci detected in the present study were affected by male function, female function, and zygotic selection. The most pronounced SD loci were mapped to chromosome 3 (transmitted through male gametes), chromosome 5 (transmitted through male gametes), and chromosome 6 (transmitted through female gametes). The level of SD in BC1F1 populations which defined by chi-square value independence multiple tests was relatively low in comparison to F2 populations. Dasanbyeo alleles were transmitted at a higher frequency in both F2 and BC1F1 populations, suggesting that indic a alleles are strongly favored in inter-subspecific crosses in rice. SD loci in the present study corresponded to previously reported loci for reproductive barriers. In addition, new SD loci were detected on chromosomes 2 and 12.ConclusionThe identification of the distribution of SD and the effect of genetic factors causing SD in genetic mapping populations provides an opportunity to survey the whole genome for new SD loci and their relationships to reproductive barriers. This provides a basis for future research on the elucidation of the genetic mechanisms underlying SD in rice, and will be useful in molecular breeding programs.

QTLs for hybrid fertility and their association with female and male sterility in rice

Hybrid sterility is one of the major barriers to the application of wide crosses in plant breeding and is commonly encountered in crosses between indica and japonica rice varieties. Ten mapping populations comprised of two reciprocal F2 and eight BC1F1 populations generated from the cross between Ilpumbyeo (japonica) and Dasanbyeo (indica) were used to identify QTLs and to interpret the gametophytic factors involved in hybrid fertility or sterility between two subspecies. Frame maps were constructed using a total of 107 and 144 STS markers covering 12 rice chromosomes in two reciprocal F2 and eight BC1F1 populations, respectively. A total of 15 main-effect QTLs and 17 significant digenic-epistatic interactions controlling spikelet fertility (SF) were resolved in the entire genome map of F2 and BC1F1 populations. Among detected QTLs responsible for hybrid fertility, four QTLs, qSF5.1 and qSF5.2 on chromosome 5, qSF6.2 on chromosome 6, and qSF12.2 on chromosome 12, were identified as major QTLs since they were located at corresponding positions in at least three mapping populations. Loci qSF5.1, qSF6.1 and qSF6.2 were responsible for both female and male sterility, whereas qSF3.1, qSF7 and qSF12.2 affected the spikelet fertility only through embryosac factors, and qSF9.1 did through pollen factors. Five new QTLs identified in this study will be helpful for understanding the hybrid sterility and for breeding programs via inter-subspecific hybridization.

Influence of Multi-Gene Allele Combinations on Grain Size of Rice and Development of a Regression Equation Model to Predict Grain Parameters

BackgroundGrain size is one of the key factors determining yield and quality in rice. A large number of genes are involved in the regulation of grain size parameters such as grain length and grain width. Different alleles of these genes have different impacts on the grain size traits under their control. However, the combined influence of multiple alleles of different genes on grain size remains to be investigated. Six key genes known to influence grain size were investigated in this study: GS3, GS5, GS6, GW2, qSW5/GW5, and GW8/OsSPL16. Allele and grain measurement data were used to develop a regression equation model that can be used for molecular breeding of rice with desired grain characteristics.ResultsA total of 215 diverse rice germplasms, which originated from or were developed in 28 rice-consuming countries, were used in this study. Genotyping analysis demonstrated that a relatively small number of allele combinations were preserved in the diverse population and that these allele combinations were significantly associated with differences in grain size. Furthermore, in several cases, variation at a single gene was sufficient to influence grain size, even when the alleles of other genes remained constant. The data were used to develop a regression equation model for prediction of rice grain size, and this was tested using data from a further 34 germplasms. The model was significantly correlated with three of the four grain size-related traits examined in this study.ConclusionRice grain size is strongly influenced by specific combinations of alleles from six different genes. A regression equation model developed from allele and grain measurement data can be used in rice breeding programs for the development of new rice varieties with desired grain size and shape.

Sugary Endosperm is Modulated by Starch Branching Enzyme IIa in Rice ( Oryza sativa L.)

BackgroundStarch biosynthesis is one of the most important pathways that determine both grain quality and yield in rice (Oryza sativa L.). Sugary endosperm, sugary-1 (sug-1), is a mutant trait for starch biosynthesis. Rice plants carrying sug-1 produce grains that accumulate water-soluble carbohydrates instead of starch, even after maturity. Although this trait enhances the diversity of grain quality, sugary endosperm rice has hardly been commercialized due to the severely wrinkled grains and subsequent problems in milling. This study was conducted to identify the genes responsible for the sug-h phenotype through a map-based cloning technology.ResultsWe induced a mild sugary mutant, sugary-h (sug-h) through the chemical mutagenesis on the Korean japonica cultivar Hwacheong. Grains of the sug-h mutant were translucent and amber-colored, and the endosperm appeared less wrinkled than sug-1, whereas the soluble sugar content was fairly high. These characteristics confer greater marketability to the sug-h mutant. Genetic analyses indicated that the sug-h mutant phenotype was controlled by a complementary interaction of two recessive genes, Isoamylase1 (OsISA1), which was reported previously, and Starch branching enzyme IIa (OsBEIIa), which was newly identified in this study. Complementation tests indicated that OsBEIIa regulated the properties of sugary endosperm.ConclusionsComplementary interactions between the starch biosynthesis genes OsISA1 and OsBEIIa determine the mild sugary endosperm mutant, sugary-h, in rice. Our finding may facilitate the breeding of sugaryendosperm rice for commercial benefit.

Genome-wide analyses of late pollen-preferred genes conserved in various rice cultivars and functional identification of a gene involved in the key processes of late pollen development

BackgroundUnderstanding late pollen development, including the maturation and pollination process, is a key component in maintaining crop yields. Transcriptome data obtained through microarray or RNA-seq technologies can provide useful insight into those developmental processes. Six series of microarray data from a public transcriptome database, the Gene Expression Omnibus of the National Center for Biotechnology Information, are related to anther and pollen development.ResultsWe performed a systematic and functional study across the rice genome of genes that are preferentially expressed in the late stages of pollen development, including maturation and germination. By comparing the transcriptomes of sporophytes and male gametes over time, we identified 627 late pollen-preferred genes that are conserved among japonica and indica rice cultivars. Functional classification analysis with a MapMan tool kit revealed a significant association between cell wall organization/metabolism and mature pollen grains. Comparative analysis of rice and Arabidopsis demonstrated that genes involved in cell wall modifications and the metabolism of major carbohydrates are unique to rice. We used the GUS reporter system to monitor the expression of eight of those genes. In addition, we evaluated the significance of our candidate genes, using T-DNA insertional mutant population and the CRISPR/Cas9 system. Mutants from T-DNA insertion and CRISPR/Cas9 systems of a rice gene encoding glycerophosphoryl diester phosphodiesterase are defective in their male gamete transfer.ConclusionThrough the global analyses of the late pollen-preferred genes from rice, we found several biological features of these genes. First, biological process related to cell wall organization and modification is over-represented in these genes to support rapid tube growth. Second, comparative analysis of late pollen preferred genes between rice and Arabidopsis provide a significant insight on the evolutional disparateness in cell wall biogenesis and storage reserves of pollen. In addition, these candidates might be useful targets for future examinations of late pollen development, and will be a valuable resource for accelerating the understanding of molecular mechanisms for pollen maturation and germination processes in rice.

Identification and quantification of flavonoids in yellow grain mutant of rice (Oryza sativa L.)

Flavonoids are naturally occurring phenolic compounds with potential health-promoting activities. Although anthocyanins and phenolic acids in coloured rice have been investigated, few studies have focused on flavonoids. Herein, we analysed flavonoids in a yellow grain rice mutant using UHPLC-DAD-ESI-Q-TOF-MS, and identified 19 flavonoids by comparing retention times and accurate mass measurements. Among them, six flavonoids, isoorientin, isoorientin 2″-O-glucoside, vitexin 2″-O-glucoside, isovitexin, isoscoparin 2″-O-glucoside and isoscoparin, were isolated and fully identified from the yellow grain rice mutant, and the levels were significantly higher than wild-type, with isoorientin particularly abundant in mutant embryo. Significant differences in total phenolic compounds and antioxidant activity were observed in mutant rice by DPPH, FRAP and TEAC assays. The results suggest that the representative six flavonoids may play an important role in colouration and antioxidant activity of embryo and endosperm tissue. The findings provide insight into flavonoid biosynthesis and the possibility of improving functionality in rice.

Identification of quantitative trait loci for panicle length and yield related traits under different water and P application conditions in tropical region in rice (Oryza sativa L.)

Climate change is projected to have a serious impact on the yield potential of rice in tropical as well as in temperate countries. It is therefore essential to develop rice varieties which are climate change ready and with stable yield when grown under low inputs of irrigation water and fertilizer. In this study, the effects of the shift from temperate to tropical environment as well as the different levels of water regime-phosphorus application were evaluated using a set of temperate recombinant inbred lines (RILs) derived from a cross between Dasanbyeo (Tongil-type indica) and TR22183 (temperate japonica). Here, we have identified genetic mechanisms for yield stability mainly by observing the panicle length in the RILs and the parental lines. TR22183 grown in the Philippines showed no reduction in panicle length whereas the Dasanbyeo exhibited a considerable reduction in panicle length when grown in the Philippines compared to those grown in Korea. In the RILs, a total of 18 QTLs for panicle length were identified across 12 chromosomes except in chromosomes 6 and 7. There were six interesting panicle length QTLs, qPL1.4, qPL2.1, qPL2.2, qPL4.1, qPL9.2, and qPL11.2 on chromosomes 1, 2, 4, 9, and 11 respectively. They were clustered together with other yield-related QTLs such as spikelet number and grain number in two different years. Except for qPL2.1, all the beneficial alleles originated from TR22183. The panicle length QTLs were identified across different water-P treatments. Interestingly, qPL1.4, qPL2.1, qPL4.1, and qPL11.2 were constantly detected in the low-input tropical condition. No QTL for panicle length was identified in the parallel experiment conducted under temperate conditions in Korea suggesting that the QTLs identified in tropical conditions could be useful in breeding programs to develop rice varieties that have stable yield potential under a warming temperate climate.

Genome-wide transcriptome comparison of flag leaves among japonica and indica varieties

Flag leaves in crops are one of the key organs determining grain yield, which significantly affects total yield. However, our understanding of the molecular and genetic regulation of flag leaves is very limited. To provide a genome-wide view of gene expression in flag leaves associated with grain yield, we compared the flag leaves of rice varieties with different yield potentials, such as Hwacheong (moderate yield), Milyang23 (high yield), Dasan (high yield), and IR64 (high yield), using an Agilent 8x60K microarray. As a result, we identified 245 genes that were up-regulated in high yield potential varieties compared to Hwacheong, along with 293 genes that were up-regulated in Hwacheong. GO enrichment analysis of the selected candidate genes revealed that the thiamin biosynthetic process and the sucrose metabolic process were the most enriched terms in flag leaves from the high yield potential varieties, while phosphate transport and the chitin catabolic process terms were the most significant in flag leaves of Hwacheong. In addition, MapMan analysis suggested that the biotic stress response and auxin signaling are important in Hwacheong, while the heat stress response, calcium and G-protein signaling are necessary in other high yield potential varieties. The functions of 11 of our candidate genes have been previously characterized in genetic and molecular biological studies and most of them are related to tolerance against environmental challenges or yield, thereby indicating the potential significance of our candidate genes in further applications.