AUTHOR=Chang Yanan , Tang Huali , Wang Surong , Li Xi , Huang Peipei , Zhang Jiahui , Wang Ke , Yan Yueming , Ye Xingguo 

TITLE=Efficient induction and rapid identification of haploid grains in tetraploid wheat by editing genes TtMTL and pyramiding anthocyanin markers

JOURNAL=Frontiers in Plant Science

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1346364

DOI=10.3389/fpls.2024.1346364

ISSN=1664-462X

ABSTRACT=<p>Doubled haploid (DH) technology provides an effective way to generate homozygous genetic and breeding materials over a short period of time. We produced three types of homozygous <italic>TtMTL</italic> gene-edited mutants (<italic>mtl-a</italic>, <italic>mtl-b</italic>, and <italic>mtl-ab</italic>) by CRISPR/Cas9 in durum wheat. PCR restriction enzymes and sequencing confirmed that the editing efficiency was up to 53.5%. The seed-setting rates of the three types of mutants ranged from 20% to 60%. Abnormal grain phenotypes of kernel, embryo, and both embryo and endosperm abortions were observed in the progenies of the mutants. The average frequency of embryo-less grains was 25.3%. Chromosome counting, guard cell length, and flow cytometry confirmed that the haploid induction rate was in the range of 3%–21% in the cross- and self-pollinated progenies of the <italic>mtl</italic> mutants (<italic>mtl-a</italic> and <italic>mtl-ab</italic>). Furthermore, we co-transformed two vectors, <italic>pCRISPR/Cas9-MTL</italic> and <italic>pBD68-</italic>(<italic>ZmR + ZmC1</italic>), into durum wheat, to pyramide <italic>Ttmtl</italic>-edited mutations and embryo-specifically expressed anthocyanin markers, and developed a homozygous durum haploid inducer with purple embryo (DHIPE). Using DHIPE as the male parent to be crossed with the wild-type Kronos, the grains with white embryos were identified as haploid, while the grains with purple embryos were diploid. These findings will promote the breeding of new tetraploid wheat varieties.</p>