Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (8): 1417-1429.doi: 10.3864/j.issn.0578-1752.2024.08.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Genetic Analysis and Candidate Gene Identification on Fertility and Inheritance of Hybrid Sterility of XI and GJ Cross

XU Na(), TANG Ying, XU ZhengJin, SUN Jian(), XU Quan()   

  1. Rice Research Institute, Shenyang Agricultural University, Shenyang 110866
  • Received:2023-10-23 Accepted:2023-11-29 Online:2024-04-16 Published:2024-04-24
  • Contact: SUN Jian, XU Quan

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Abstract:

【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 identified 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.

Key words: rice, hybrid sterility, high throughput sequencing, gene editing, Sd candidate genes

Fig. 1

The heterosis of the cross between Sasa and Haba A: Plant architecture; B: Panicle architecture; C: Plant height; D: Panicle number; E: Grain number per panicle; F: 1000 grain weight; G: Setting rate. Different letters indicate significant differences (P<0.05). The same as below"

Fig. 2

The fertile pollen of Sasa, Haba and F1 plants A: Pollen phenotypes of Sasa; B: Pollen phenotypes of Haba; C: Pollen phenotypes of F1 (Sasa/Haba); D: The fertile pollen rate of Sasa, F1, and Haba"

Fig. 3

The genetic analysis of the hybrid sterility between Sasa and Haba A: The genetic map of RILs; B: The Haba pedigree introgression ratio of RILs; C: The loci corresponding to the XI/GJ hybrid sterile"

Fig. 4

The sequence differences of XI/GJ hybrid sterile related locus between Sasa and Haba A: The sequence differences of DPL1 and DPL2 between Sasa and Haba; B: The sequence differences of SaF and SaM between Sasa and Haba; C: The structure variation of Sc between Sasa and Haba; D: The sequence differences of S5 between Sasa and Haba; E: The structure variation of RHS12 between Sasa and Haba; F: The sequence differences of HSA1a and HSA1b between Sasa and Haba"

Fig. 5

The sequence difference and expression pattern of Sc locus between Sasa and Haba A: The multiple copies of Sc in Haba; B: The expression pattern of Sc in Sasa and Haba"

Fig. 6

The sequence and phenotypes of Sc-haba-3 CRISPR gene edited plants in Haba A: Editing site specific to Sc-Haba-3, and the sequence of CRISPR edited plants; B; Pollen phenotypes of F1(Sasa/Haba), F1(Sasa/CR-1), and F1(Sasa/CR-2); C: The relative expression level of Sc-Sasa in Sasa, F1(Sasa/Haba), F1(Sasa/CR-1), and F1(Sasa/CR-2); D: The fertile pollen rate of F1(Sasa/Haba), F1(Sasa/CR-1), and F1(Sasa/CR-2); E: The setting rate of F1(Sasa/Haba), F1(Sasa/CR-1), and F1(Sasa/CR-2)"

Fig. 7

The candidate gene of Sasa and Haba hybrid sterility locus Sd A: Chr1's 100 kb window and 10 kb sliding population differentiation Fst region; The pink region represents the upstream and downstream 1 Mb genomic region radiated by the Fst differentiation signal peak at a 3.8 Mb physical location B: The Haba pedigree introgression in Chr.1 of RILs; C: The structure variation of candidate region of Sd between Sasa and Haba"

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