Gene isolation and characterization of a dominant dwarf mutant, D-h, in rice

Rihua Piao,Sang-Ho Chu,Yoye Yu,Donggwan Kim,Sunghan Kim,Hee-Jong Koh 한국육종학회 2013 한국육종학회 심포지엄 Vol.2013 No.07

Plant height is an important agronomic trait that affects grain yield. Previously, we reported a novel semi-dominant dwarf mutant, D-h, derived from chemical mutagenesis using N-methyl-N-nitrosourea(MNU) on a japonica rice cultivar, Hwacheongbyeo. In this study, we cloned the gene responsible for the dwarf mutant using the map-based approach. Fine mapping revealed that the mutant gene was located on the short arm of chromosome 1 in a 48 kb region. Sequencing of the candidate genes and rapid amplification of cDNA ends-polymerase chain reaction(RACE-PCR) analyses identified the gene, d-h, which encodes a protein of unknown function, but whose sequence is conserved in other cereal crops. Real-time (RT)-PCR analysis and promoter activity assay showed that the d-h gene was primarily expressed in the nodes and the panicle. In the D-h mutant plant, the gene was found to carry a 63-bp deletion in the ORF region, which was confirmed to be directly responsible for the mutant’s gain of a functional phenotype by subsequent transgenic experiments. Since the mutant plants exhibit a defect in the GA response, but not in the GA synthetic pathway, it appears that the d-h gene may be involved in a GA signaling pathway.

Fine mapping and characterization of small round grain erect panicle mutant in rice

Yogendra Bordiya,Rihua Piao,Backki Kim,Hong-Yeol Kim,Hee-Jong Koh 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

Grain size is one of the most important trait determining yield in cereal crops, apart from number of grains per panicle, number of panicles per plant and 1000 seed weight. Other than grain characteristics, plant architecture is another very important factor influencing yield by affecting the amount plant surface area directly exposed to the sun light. Erect panicle is important morphological characteristic which helps in enhancing the yield by allowing sun light to fall directly on leaves unlike curved panicle which blocks sunlight and consequently reduce photosynthesis. A small round grain and erect panicle mutant was obtained by treating Hwacheong rice (japonica) with MNU (N-methyl-N-nitrosourea) chemical mutagen. Through bulked segregant analysis (BSA) using STS (Sequence-Tagged Sites) and SS-STS (Sub-species Specific Sequence-Tagged Site) markers we located the mutated gene on the long arm of chromosome 7 and narrowed down candidate region to 168.75kbp through fine mapping. Mutant manifested characteristics like reduced grain size and plant height, dense and erect panicle and relatively erect plant compared to the wild type. When we crossed the mutant with its parent (Hwacheong), F1 panicle and grain characteristics showed intermediate phenotype, therefore, we concluded that wild type allele of this gene shows incomplete dominance. Scanning electron microscopy(SEM) result shows that increase in width of mutant grain, which changes its shape, is due to increase in width of glume cells. Phenotypic examination shows that dense and erect panicle phenotype is result of reduction in length of rachis, primary and secondary branch.

Phenotypic characterization and genetic mapping of an open-hull sterile mutant in rice.

Gileung Lee,Yoye Yu,Rihua Piao,Aeri Han,Sunghan Kim,Hee-Jong Koh 한국육종학회 2013 한국육종학회 심포지엄 Vol.2013 No.07

Rice hulls remain closed throughout the ripening period to maintain internal humidity of the grains. An Open-hull sterile mutant was induced by N-methyl-N-nitrosourea(MNU) treatment on Sinsunchalbyeo rice, a japonica type. This mutant showed open hulls even in the ripening stages and fully mature grains. In addition, several altered characteristics were observed, including of narrowed palea, decreased grain size, partial pollen sterility and erect panicle. Microscopic analysis showed that the palea was positioned slightly inside the lemma, and the size of palea decreased in the mutant. Genetic analysis of F2 and F3 segregation populations derived from the cross between the Open-hull sterile mutant (Oryza sativa ssp. japonica) and Milyang23 (O. sativa ssp. indica) indicated that the Open-hull trait was controlled by a single recessive allele. The fine-mapping with STS (sequence tagged site) markers revealed that the mutant gene was located on the short arm of chromosome 3. We were able to narrow it down until 30.6Kb where three candidate genes were found.

Fine mapping and candidate gene analysis of a new mutant gene for panicle apical abortion in rice

Md. Babul Akter,Rihua Piao,Backki Kim,Yunjoo Lee,Eunbyeol Koh,Hee-Jong Koh 한국육종학회 2013 한국육종학회 심포지엄 Vol.2013 No.07

The architecture of rice panicle is primarily determined by the arrangement of branches and spikelets, and it directly affects grain yield. We identified a mutant for panicle apical abortion from a japonica cultivar Hwacheongbyeo treated with N-methyl-N-nitrosourea. Under normal growth conditions, the mutant had multiple abnormal phenotypes, such as a slight reduction in plant height, narrow and dark green leaf blades, and small erect panicles with clear panicle apical abortion compared to the wild-type plants. Genetic analysis revealed that the panicle apical abortion was controlled by a single recessive gene, which is tentatively designated as paa. The paa gene was fine mapped at an interval of 71 kb flanked by STS markers aptn3 and S6685-1 at the long arm of chromosome 4. Sequence analysis of the candidate genes within the delimited region showed a single base-pair change corresponding to an amino acid substitution from glycine to glutamic acid. We expect that the paa gene will be a clue to uncover the molecular mechanism of panicle apical abortion and to maintain the panicle identity for grain yield in rice breeding programs.

Development of diagnostic DNA markers for eating quality of indica rice

Yunjoo Lee,Puji Lestari,Rihua Piao,Eunbyeol Koh,Sunmi Jang,Hee-Jong Koh 한국육종학회 2014 한국육종학회 심포지엄 Vol.2014 No.07

Rice eating quality is considered to be one of the top priorities in determining the agronomical value of rice. Thus the rapid evaluation of eating quality at early breeding generations in breeding programs for better eating quality is of great importance. However, it has been limited due to the complex nature of eating quality and the absence of standard evaluation method. In our previous study, we developed a evaluation method with a set of DNA markers that allows to predict the eating quality for japonica rices. Here we successfully developed another marker set for the eating quality of indica rices. We used multiple regression analysis to test 54 markers, which were preselected for their possible association with eating quality, using 24 indica varieties with different palatability scores. Of these markers, eighteen markers were found to be significantly associated with palatability according to sensory evaluation. Accordingly, a marker set in the model regression equation with a high R2 (0.997) was formulated to estimate indica rice palatability. Validation suggests that markers and the statistical parameters formulated by the equation could be a potential tool to predict the palatability of cooked Indonesian indica rice and could be reliable in developing country-dependent model equations for eating quality. This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ008125), Rural Development Administration, Republic of Korea.

Genotypic variation of embryo dent of rice grains

Yunjoo Lee,Gileung Lee,Rihua Piao,Sunmi Jang,Hee-Jong Koh 한국육종학회 2013 한국육종학회 심포지엄 Vol.2013 No.07

As the market demand on functionality rice has been increasing, embryo rice in which embryo residue remains even after milling has come to comsumers’ attention because rice embryo contains several functionality components. Consequently, development of rice varieties for higher rate of embryo adhesion to grains after milling has become one of the breeding objectives for quality improvement. In this study, we observed embryo dent of 49 commercial varieties and analyzed the relationship between embryo dent and grain size and shape. Embryo dent of rice grains varied 0.27 (Keunnun)~0.59 (Daerip 1) mm. Varieties Jinbu, Jinbo, Heugseol, Obong, Unkwang, and Cheongnam showed relatively deeper embryo dent, suggesting that they will be applicable in breeding for embryo rice. Embryo dent was correlated positively with grain width (r=0.53**) and grain size(r=0.34*), and negatively with grain width/length ratio (r= -0.38**). Strategies for breeding embryo rice were discussed in relation to embryo dent, grain size and shape.

Characterization and genetic analysis of Hwacheong sugary-2 mutant in rice

Yun Joo Lee,Min-Seon Choi,Rihua Piao,Eunbyeol Koh,Hee-Jong Koh 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

Two sugary mutants, Hwacheong sugary-1 (Hsu1) and Hwacheong sugary-2 (Hsu2) were obtained by chemical mutagenesis from japonica cultivar, Hwacheongbyeo. Sugary mutants exhibited wrinkled and translucent grain with high soluble sugar content. In addition, amber-colored endosperm of sugary mutants was loosely packed due to abnormal starch granules compared to densely packed wild-type. Especially, the grain of Hsu2 mutant was less wrinkled than that of Hsu1, thus Hsu2 can be polished easily. Previous studies reveal that su1 mutant was resulted from mutation in gene for a debranching enzyme, isoamylase but the sequence of the mutated gene has not been identified. To identify the sequence of sugary genes, the map-based cloning strategy was applied. The genetic study revealed that the phenotype of Hsu2 mutant was controlled by two recessive genes. Interestingly, one of the genes was located on chromosome 8 at the position of isoamylase which was known as su-1. This indicates that mutation in isoamylase gene causes sugary endosperm characteristics. However we found different mutation points between the Hsu1 and Hsu2. The point mutation in Hsu1 was occurred at 10th exon whereas the other mutation related with Hsu2 was occurred at 15th exon. As mentioned above, the Hsu2 mutant has less wrinkled shape and less soluble sugar content than the Hsu1 mutant. Thus, we hypothesize that the other gene controlling Hsu2 mutant phenotype may have a role in weakening the effect of the su-1. Further study on the other gene associated with the Hsu2 phenotype is in progress.

RESEARCH ARTICLES : Phenotypic Characterization and Genetic Mapping of An Open-hull Sterile Mutant in Rice

( Yoye Yu ),( Rihua Piao ),( Wenzhu Jiang ),( Sunghan Kim ),( Hee Jong Koh ) 한국육종학회 2013 Plant Breeding and Biotechnology Vol.1 No.1

Rice hulls remain closed throughout the ripening period to maintain internal humidity of the grains. An Open-hull sterile mutant was induced by N-methyl-N-nitrosourea (MNU) treatment on Sinsunchalbyeo rice, a japonica type. This mutant showed Open hulls even in the ripening stage and fully mature grains. In addition, several altered characteristics were observed, including narrowed palea, decreased grain size, partial pollen sterility and erect panicle. Microscopic analysis showed that the palea was positioned slightly inside the lemma, and the size of palea decreased in mutant. Genetic analysis of F2 and F3 populations derived from the cross between the Open-hull sterile mutant (Oryza sativa ssp. japonica) and Milyang23 (O. sativa ssp. indica) indicated that the open-hull trait was controlled by a single recessive allele. Fine-mapping with STS (sequence tagged site) markers revealed that the mutant gene was located on the short arm of chromosome 3. The location of the gene was further narrowed down to 30.6 kb where three candidate genes were found.

Identification and Characterization of <i>LARGE EMBRYO</i> , a New Gene Controlling Embryo Size in Rice ( <i>Oryza sativa</i> L.)

Lee, Gileung,Piao, Rihua,Lee, Yunjoo,Kim, Backki,Seo, Jeonghwan,Lee, Dongryung,Jang, Su,Jin, Zhuo,Lee, Choonseok,Chin, Joong Hyoun,Koh, Hee-Jong Springer US 2019 Rice Vol.12 No.1

<P><B>Background</B></P><P>Although embryo accounts for only 2–3% of the total weight of a rice grain, it is a good source of various nutrients for human health. Because enlarged embryo size causes increase of the amount of nutrients and bioactive compounds stored within rice grain, giant embryo mutants of rice (<I>Oryza sativa</I> L.) are excellent genetic resources for improving the nutritional value of rice grains.</P><P><B>Results</B></P><P>Three giant embryo mutants, including <I>large embryo</I> (<I>le</I>), <I>giant embryo</I> (<I>ge</I>) and <I>super</I>-<I>giant embryo</I> (<I>ge</I><SUP><I>s</I></SUP>), with variable embryo size were used in this study. We investigated whether genes controlling embryo size in these mutants (<I>le</I>, <I>ge</I> and <I>ge</I><SUP><I>s</I></SUP>) were allelic to each other. Although <I>ge</I> and <I>ge</I><SUP><I>s</I></SUP> was allelic to <I>GIANT EMBRY</I> (<I>GE</I>), <I>le</I> was not allelic to <I>ge</I> and <I>ge</I><SUP><I>s</I></SUP> in allelism test. The <I>GE</I> gene carried a unique nucleotide substitution in each of the two mutants (<I>ge</I> and <I>ge</I><SUP><I>s</I></SUP>), resulting in non-synonymous mutations in exon 2 of <I>GE</I> in both mutants. However, the <I>GE</I> gene of the <I>le</I> mutant did not carry any mutation, suggesting that the enlarged embryo phenotype of <I>le</I> was governed by another gene. Using map-based cloning, we mapped the <I>LE</I> gene to the short arm of chromosome 3. The <I>le</I> mutant showed mild enlargement in embryo size, which resulted from an increase in the size of scutellar parenchyma cells. The <I>LE</I> encodes a C3HC4-type RING finger protein and was expressed to relatively high levels in seeds at a late developmental stage. Knockdown of <I>LE</I> expression using RNA interference increased the embryo size of rice grains, confirming the role of <I>LE</I> in determining the embryo size.</P><P><B>Conclusion</B></P><P>Overall, we identified a new gene controlling embryo size in rice. Phenotypic and molecular characterization results suggest that the <I>le</I> mutant will serve as a valuable resource for developing new rice cultivars with large embryos and nutrient-dense grains.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1186/s12284-019-0277-y) contains supplementary material, which is available to authorized users.</P>

Fine mapping and candidate gene analysis of a new mutant gene for panicle apical abortion in rice

Akter, Md. Babul,Piao, Rihua,Kim, Backki,Lee, Yunjoo,Koh, Eunbyeol,Koh, Hee-Jong Springer-Verlag 2014 Euphytica Vol.197 No.3