Genome sequencing identified novel mechanisms underlying virescent mutation in upland cotton Gossypiuma hirsutum

Virescent mutation broadly exists in plants and is an ideal experimental material to investigate regulatory mechanisms underlying chlorophyll synthesis, photosynthesis and plant growth. Up to date, the molecular mechanisms in two virescent mutations have been clarified in cottons (Gossypiuma hirsutum). A virescent mutation has been found in the cotton strain Sumian 22, and the underlying molecular mechanisms have been studied.

The virescent mutant and wild type (WT) of Sumian 22 were cross-bred, and the F₁ population were self-pollinated to calculate the segregation ratio. Green and yellow leaves from F₂ populations were subjected to genome sequencing and bulked-segregant analysis was performed to screen mutations. Real-time quantitative PCR (RT-qPCR) were performed to identify genes in relations to chlorophyll synthesis. Intermediate products for chlorophyll synthesis were determined to validate the RT-qPCR results.

The segregation ratio of green and virescent plants in F2 population complied with 3:1. Compared with WT, a 0.34 Mb highly mutated interval was identified on the chromosome D10 in mutant, which contained 31 genes. Among them, only ABCI1 displayed significantly lower levels in mutant than in WT. Meanwhile, the contents of Mg-protoporphyrin IX, protochlorophyllide, chlorophyll a and b were all significantly lower in mutant than in WT, which were consistent with the inhibited levels of ABCI1. In addition, a mutation from A to T at the -317 bp position from the start codon of ABCI1 was observed in the genome sequence of mutant.

Inhibited transcription of ABCI1 might be the mechanism causing virescent mutation in Sumian 22 cotton, which reduced the transportation of protoporphyrin IX to plastid, and then inhibited Mg-protoporphyrin IX, Protochlorophyllide and finally chlorophyll synthesis. These results provided novel insights into the molecular mechanisms underlying virescent mutation in cotton.