Identification and Genetic Analysis of the Yellow Leaf Mutant yl(t) in Rice (Oryza sativa)
KANG Wei-Wei1,2, LI Zhe-Li1,3, YI Zi-Li2, SUN Zhi-Zhong1, SHENG Xia-Bing1,2, HUANG An-Ping4, DUAN Mei-Juan3,*, TAN Yan-Ning1,*
1 State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha 410125, China; 2 College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China; 3 College of Agronomy Science, Hunan Agricultural University, Changsha 410128, China; 4 Hunan Agricultural Biotechnology Research Institute, Changsha 410125, China
Abstract：Leaf color mutants are ideal genetic resources to reveal the mechanisms underlying chlorophyll biosynthesis. Here, a yellow leaf mutant yl(t), identified from indica rice (Oryza sativa) T98B was analyzed on phenotype, physiology characteristics and mutation mechanism. The results showed that, the seedlings of yl(t) presented yellow leaves with a significant decrease of 53.02%, 55.35% and 46.88% in the contents of total chlorophyll, chlorophyll a and chlorophyll b, respectively, compared with T98B. When it grew to mature, the chlorophyll content increased. Further genetic analysis showed yl(t) was controlled by a nuclear recessive gene. By employing a F2 population from yl(t) crossed with the O. sativa subsp. japonica cv. Nipponbare (NPB) which presents normally green leaf, yl(t) was fine mapped in a 42 kb region on chromosome 6. Fragment sequencing confirmed that the heme oxygenase 1 gene OsHO1 (LOC_Os06g40080.1) would be the candidate of yl(t) that happened to mutate at the splicing site of exon2/intron2 (AG/GT) with a deletion of 'T'. Moreover, this point was proved to be associated with variation in leaf color by a CAPS marker's verification. Later, by means of 3' rapid amplification of cDNA ends (RACE), the full-length CDS of OsHO1 in yl(t) was cloned. Compared with T98B, the transcription of OsHO1 in yl(t) terminates at the 20~22 bp (TAA) on the second intron, thereby causing a reduction of 179 bp in the CDS sequence. Inferred by DNAMAN and CD-search, 59 amino acid residues at the C-terminus of OsHO1 in yl(t) would be missed to impair the ability of heme binding. In addition, qRT-PCR analysigbbbs found several key genes involving in chlorophyll biosynthesis expressed differently in yl(t). The OsHemA (hemin A gene) that encodes glutamyl-tRNA reductase was up-regulated, while OsCAO1 (chlorophyllide a oxygenase 1 gene) and OsNOL (non-yellow coloring1-like gene) that encodes chlorophyll b reductase expressed lower. This study reveals yl(t) is an allele of OsHO1 to mutate at splicing site, and provides a new germplasm for further understanding of the biological function of OsHO1.
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