Fine Mapping Of Cisc(t), A Gene For Cold-induced Seedling Chlorosis, And Identification Of Its Candidate In Rice

The seedlings of indica rice cultivar Dular may show chlorosis (albino or xantha) under low temperature condition. Our previous genetic studies indicated that this low temperature-induced seedling chlorosis is controlled by a recessive gene, located between SSR markers RM257 and RM242 on the long arm of chromosome 9. We named the gene as cisc(t) temporarily. In this study, based on the previous results, cisc(t) was fine mapped further and its candidate gene was identified by molecular biology technology and bioinformatics methods. Meanwhile, the allelism between Dular and another indica rice cultivar DZ60 on the character of low temperature-induced seedling chlorosis was tested. The main results are as follows:(1) A large F2 population was derived from a cross between Dular and a japonica cultivar Lemont (normal under low temperature). Based on the previous results, polymorphic SSR and InDel markers between the two parents were developed. The genotypes of 2446 F2 chlorotic seedlings were analyzed using these polymorphic markers. As a result, cisc(t) was fine mapped in a 12-kb interval between markers ID18124 and ID18136 on BAC clone OSJNBa0026C08.(2) The fine mapping result indicated that there is only one annotated gene in this interval, which encodes a pentatricopeptide repeat (PPR) protein. Based on the information from three websites, i.e. Rice Genome Annotation, RISe, and KOME, we presumed that there is only one ORF encoding a pentatricopeptide repeat (PPR) protein in the region between markers ID18124 and ID18136. Sequencing analysis indicated that 8 bases were deleted at the 60th base in the Dular allele, resulting in a frame-shift mutation and the loss of function of the gene. This is consistent with the chlorosis mutant phenotype of Dular. In addition, previous studies have shown that many mutants of seedling chlorosis are related to PPR proteins. Hence, we take the PPR gene as the candidate of cisc(t).(3) We found that the seedlings of indica cultivar DZ60 also showed chlorosis under low temperature condition. So, we crossed DZ60 with Dular. We found that the F1 seedlings also exhibited the chlorosis phenotype at 19℃, suggesting that the gene for cold-induced seedling chlorosis in DZ60 was allelic to the cisc(t) gene of Dular. Sequencing analysis indicated that the DZ60 allele was exactly the same as the Dular allele, possessing the same mutation. This suggests that the Dular allele and the DZ60 allele might have the same origin.