本项目以和田紫花苜蓿(Medicago sativa L. cv. Hetian) 为材料，通过对它的Histone H3和CENH3基因家族进行系统分析，了解其所包含的不同Histone H3和CENH3基因的遗传多样性以及它们的表达差异。根据以上结果，用Crispr/Cas9介导的基因编辑技术敲除紫花苜蓿的CENH3基因，挑选适当的Histone H3 基因改造CENH3基因，改造的CENH3基因通过农杆菌介导代替其原始的CENH3基因，此转基因紫花苜蓿可作为四倍体紫花苜蓿单倍体诱导系。此单倍体诱导系与紫花苜蓿不同种质资源杂交能诱导产生二倍体，纯合的二倍体通过染色体加倍为纯合的四倍体，即可作为紫花苜蓿纯合自交系用于杂交优势育种。通过本项目的实施可在较短的育种周期中高效获得紫花苜蓿单倍体和纯合四倍体，也为苜蓿属其它物种获得纯合自交系开拓了思路；这为加快我国优质苜蓿的培育奠定了基础，具有重要的科学意义和研究价值。

In this project, we propose to identify the Histone H3 and CENH3 gene sequences in alfalfa(Medicago sativa L. cv. Hetian) and characterize their differential expression in different tissue and under stress treatments. We will identify the highly expressed Histone H3 and CENH3 genes, which will be used to develop a CENH3-H3 chimerical construct. We will also design multiple guide RNAs targeting on the conserved sequence of all CENH3 genes and knockout all endogenous CENH3 by using the CRISPR/Cas9-mediated gene editing approach. The native promoter-CENH3-H3 chimerical gene and the CRISPR/Cas9 construct will be integrated as a single T-DNA for alfalfa transformation. The transgenic lines will be further characterized to identify homozygous haploid inducer lines. The haploid inducer line will be further crossed with different alfalfa germplasm resources to induce uniparental chromosome elimination. The haploid plants will be chromosome-doubled either naturally or through chemical treatments to generate homozygous tetraploid alfalfa lines. The derived homozygous tetraploid alfalfa lines will be valuable sources for hybrid breeding in the future. The method can greatly shorten the breeding cycle of alfalfa, and lay the foundation for speeding up the breeding process of high quality alfalfa in China.