Finding new loci and genes related to rice chlorophyll content, and providing new theoretical basis for the research on the genetic mechanism of rice chlorophyll content.

A recombinant inbred line (RIL) population containing 186 lines was constructed by crossing the japonica rice TD70 and the indica rice Kasalath with obvious difference in the chlorophyll content of the flag leaf. The two parents and RIL population were re-sequenced to construct a high-density genetic linkage map with 12 328 recombination Bin markers. The RILs and two parents were planted in fields at the Jiangsu Academy of Agricultural Sciences, in Nanjing in 2011 and 2020. The contents of chlorophyll of flag leaves were directly measured using the chlorophyll meter SPAD-502 on the 3^rd day after heading. QTLs that control the chlorophyll content of the flag leaf at the heading stage of rice were detected by IciMappingv3.4 software with inclusive compound interval mapping method. The photosynthesis parameters of 20 SPAD extreme strains in the RIL population were measured with a portable photosynthesis system.

19 QTLs controlling chlorophyll content of flag leaves were detected on 9 chromosomes except Chr.8, Chr.9 and Chr.10 in two years. The phenotype variation explained (PVE) of single QTL ranged from 3.09% to 13.13%, LOD value ranged from 2.74 to 14.08. After comparing the physical positions, 10 QTLs were found to locate in the same interval or adjacent to previously QTLs. qCHL2-1 and qCHL5-1 were detected every year showing their genetic stability. qCHL2-1 was mapped between the 7.63-7.71 Mb on chromosome 2, and the two-year LOD values are 14.08 and 7.93 with the PVE 13.13% and 7.94%, respectively. qCHL5-1 was mapped between the 23.44-23.49 Mb on chromosome 5, and the two-year LOD values are 4.31 and 3.76, respectively. After the annotation and sequences analysis of genes located in the region of qCHL2-1 and qCHL5-1, two genes, Os02g0236000 and Os05g0476700, were found to be associated with chlorophyll content of flag leaves in the rice. There are differences in sequences of the two genes between TD70 and Kasalath. Os02g0236000 is the AAT1 gene encoding the Aspartate Aminotransferase, which is an important enzyme in nitrogen metabolism and related to protein and amino acid content of rice. Os05g0476700 encodes protein relating to spotted leaf, which might associate with leaf color. Based on the mutation of AAT1 at CDS+273 bp, the haplotypes of ATT1 were classified in RIL population. Among the 20 extreme SPAD RIL lines, there were significant differences between different haplotype of ATT1 in SPAD value, chlorophyll content, water use efficiency, transpiration rate, stomatal conductance and net photosynthetic rate of flag leaf.

19 QTLs associated with chlorophyll content in flag leaf at heading stage of rice were detected and two stable QTL loci, qCHL2-1 and qCHL5-1 were identified. Two candidate genes were obtained after annotation and sequence comparison. One of them, ATT1, was considered as the most possible candidate gene after effort analysis of different haplotypes in photosynthetic efficiency. The QTLs and gene we obtained could be used for subsequent functional studies of flag leaf chlorophyll regulation and molecular marker breeding.