Genetic Analysis and Candidate Gene Identification on Fertility and Inheritance of Hybrid Sterility of XI and GJ Cross
[Objective]The F1 hybrid sterility between Ⅺ/indica and GJ/japonica severely hinders the utilization of hybrid advantage between subspecies.Exploring the genetic mechanism and identifying new regulatory genes for XI/GJ hybrid sterility will provide theoretical basis for promoting genetic improvement of XI/GJ hybrid seed setting rate.[Method]A series of stable genetic recombination inbred lines(RILs)containing 95 plant lines were derived from the cross between Ⅺ variety Habataki and GJ variety Sasanishiki after 10 generations inbred using single seed descent method.High throughput sequencing was performed on both parents and RILs on the Illumina platform,and the distribution of Habataki pedigree in RILs was analyzed at the whole genome level.The segregation distortion regions were identified,and hybrid sterile related gene loci were screened within the segregation distortion regions,then identifiied candidate genes through sequence alignment comparison.The targeted gene was knockout to verify the function using CRISPR gene editing technology.[Result]The hybrid F1 plants derived from the cross between Habataki and Sasanishiki showed significant heterosis in panicles,grains per panicle,and thousand grain weight,but its seed setting rate significantly decreased.I2-KI microscopy revealed a significant decrease in F1 pollen fertility.High throughput sequencing of the entire genome of RILs revealed significant segregation distortion on Chr.1,Chr.3,Chr.5,Chr.6,Chr.7,and Chr.12,indicating that the genotype in this region tends towards the Habataki.Sequence alignment comparison revealed that Sc,S5,and HSA1 are target genes for the segregation distortion on Chr.3,Chr.6,and Chr.12.The CRISPR gene editing mutants with a knock-out Sc-Haba-3 allele in Habataki successfully improved the pollen fertility and seed setting rate of F1 hybrid with Sasanishiki.A complex structural variation was found between Sasanishiki and Habataki in the segregation distortion of Chr.1.A 24.7 kb segment containing 4 predicted genes in the Sasanishiki genome was replaced by a 64.8 kb segment containing 10 predicted genes in Habataki,the structural variation may involve in controlling the hybrid sterility of XI and GJ cross.[Conclusion]This study detected multiple XI/GJ hybrid infertility related loci,and successfully improved F1 fertility by using CRISPR gene editing to knock out multiple copies of Sc in Habataki,locking in the target gene in the Sd region of Chr.1.
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