Fine mapping and candidate gene prediction of the photoperiod and thermo-sensitive genic male sterile gene pms1(t) in rice

Abstract

Pei'ai64S, an indica sterile variety with photoperiod and thermo-sensitive genic male sterile (PTGMS) genes, has been widely exploited for commercial seed production for "two-line" hybrid rice in China. One PTGMS gene from Pei'ai64S, pms1(t), was mapped by a strategy of bulked-extreme and recessive-class approach with simple sequence repeat (SSR) and insert and deletion (In-Del) markers. Using linkage analysis for the F(2) mapping population consisting of 320 completely male sterile individuals derived from a cross between Pei'ai64S and 93-11 (indica restorer) lines, the pms1(t) gene was delimited to the region between the RM21242 (0.2 cM) and YF11 (0.2 cM) markers on the short arm of chromosome 7. The interval containing the pms1(t) locus, which was co-segregated with RM6776, is a 101.1 kb region based on the Nipponbare rice genome. Fourteen predicted loci were found in this region by the Institute for Genomic Research (TIGR) Genomic Annotation. Based on the function of the locus LOC_Os07g12130 by bioinformatics analysis, it is predicted to encode a protein containing a Myb-like DNA-binding domain, and may process the transcript with thermosensory response. The reverse transcription-polymerase chain reaction (RT-PCR) results revealed that the mRNA levels of LOC_Os07g12130 were altered in different photoperiod and temperature treatments. Thus, the LOC_Os07g12130 locus is the most likely candidate gene for pms1(t). These results may facilitate not only using the molecular marker assisted selection of PTGMS genes, but also cloning of the pms1(t) gene itself.

Fig. 1

Anthers observed by naked eyes and pollen grains observed under light microscope A1–A4 showed the shapes of anthers from two parents and their F₂ individuals. A1: 93-11 as the fertile control with dispersed normal fertility pollens; A2: F₂ individual with partially dehiscent anther; A3: F₂ individual with no dehiscent anther; A4: Pei′ai64S as the sterile control under the LD/HT condition. B1–B4 and C1–C4 showed anthers and pollen grains stained with I₂-KI solution, corresponding to column specimens of A1–A4. The black-stained pollen grains are considered viable, while shrink and unstained sterile pollen grains are abortive

Fig. 2

BSA analysis for SSR markers PF, PS, BF, BS, and F₁ represent 93-11, Pei′ai64S, fertile bulk, sterile bulk, and F₁ hybrid of a cross of Pei′ai64S/93-11, respectively. The PCR products were separated in non-denatured 0.06 g/ml polyacrylamide gel electrophoresis, and visualized by quick silver-staining. The bands were amplified by two SSR primers (RM21242 and RM432)

Fig. 3

Location of the pms1(t) locus on the molecular linkage map of chromosome 7

Fig. 4

High-resolution genetic and physical map of the pms1(t) locus In the partial genetic map of the molecular markers, the numbers under the long horizontal line indicate the times of recombination events detected between the pms1(t) locus and its corresponding markers. The bold bars represent the BAC clones. The pms1(t) locus was covered by the thick horizontal line between the two vertical dashed lines from RM21242 to YF11, in which the genomic information was based on Nipponbare genome sequence (released 4.0), and some gene annotations of the gene loci represented with black bars were from TIGR database

Fig. 5

Semi-quantitative RT-PCR results of five candidate genes for Pei′ai64S The individuals were planted under LD/HT and SD/LT conditions, respectively. Also, samples were taken at three different stages of the young panicle sizes (2, 10, and 20 cm). LOC_Os07g12130, 12140, 12160, 12170, and 12240 were used as the candidate genes, and ubiquitin as a control in our experiment