籼粳杂种不育的遗传分析和候选基因鉴定

doi:10.3864/j.issn.0578-1752.2024.08.001

摘要/Abstract

摘要：

【目的】籼粳亚种间杂交F₁的育性问题严重阻碍了亚种间杂种优势的利用，探究籼粳杂种不育的遗传机理，挖掘新的籼粳杂种不育调控基因，为促进籼粳杂种结实率遗传改良提供理论依据。【方法】粳稻品种Sasanishiki和籼稻品种Habataki杂交后，采用单粒传法自交10代，获得包含95个株系的稳定遗传重组自交系（RIL），基于Illumina平台，对双亲和RIL进行高通量测序，在全基因组水平分析RIL中Habataki血缘的分布与比例，进而鉴定偏分离区域作为潜在的籼粳杂种不育位点。同时，剖析“全球3000份水稻核心种质资源重测序计划”中典型籼粳稻基因组变异数据，对群体水平进一步验证并趋近目标育性基因区域。最终通过序列比对锁定籼粳杂种不育候选基因。应用CRISPR基因编辑技术进行定点基因敲除，对目标基因进行功能验证。【结果】Habataki和Sasanishiki的杂交F₁在穗数、每穗粒数和千粒重上体现出明显的杂种优势，但其结实率显著降低，I₂-KI镜检发现F₁花粉育性显著降低。RIL的全基因组高通量测序在第1、3、5、6、7和12染色体上检测到明显的偏分离，即该区域的基因型趋向于籼稻Habataki。通过序列比对，进一步确定已知育性基因Sc、S5和HSA1为第3、6和12染色体上偏分离区域的目标基因。应用CRISPR基因编辑技术敲除Habataki中Sc-Haba-3的多拷贝，成功改善了其与Sasanishiki杂交F₁的花粉育性和结实率，说明该基因可独立行使功能。同时，在第1染色体的偏分离区域发现Habataki和Sasanishiki之间存在复杂的结构性变异，Sasanishiki基因组中一段24.7 kb包含4个预测基因的片段在Habataki中被一段64.8 kb包含10个预测基因的片段取代，此结构性变异可能参与籼粳杂种育性的调控。【结论】检测到多个籼粳杂种不育相关位点，应用CRISPR基因编辑技术敲除Habataki中Sc的多拷贝成功改善其杂种F₁育性，确定其为水稻亚种间育性改良的目标基因。同时锁定了第1染色体Sd区域的目标基因。

关键词: 水稻, 籼粳杂种不育, 高通量测序, 基因编辑, Sd候选基因

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 F₁fertility 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

许娜, 唐颖, 徐正进, 孙健, 徐铨. 籼粳杂种不育的遗传分析和候选基因鉴定[J]. 中国农业科学, 2024, 57(8): 1417-1429.

XU Na, TANG Ying, XU ZhengJin, SUN Jian, XU Quan. Genetic Analysis and Candidate Gene Identification on Fertility and Inheritance of Hybrid Sterility of XI and GJ Cross[J]. Scientia Agricultura Sinica, 2024, 57(8): 1417-1429.

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