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Identification of Maize Rf4-Restorer Lines and Development of a CAPS Marker for Rf4

Rf4 is one of the dominant restorer genes for maize C-type cytoplasmic male sterility (CMS-C), which has significant value in hybrid maize seed production. However, the highly complex fertility restoration mechanism of CMS-C makes it difficult to screen Rf4-restorer lines, and insufficient Rf4-restorer lines limit its use in current agricultural production. To search for Rf4-restorer lines, in this study, the genotypes of eighteen inbred maize lines at the Rf4 locus were analyzed based on the male fertility investigation of hybrid F1, the genetic analysis of F2 populations, molecular marker mapping, allelic tests, and Rf4 genomic sequence analysis. Our results indicated that of the eighteen maize inbred lines, ten were able to completely rescue CMS-C line CHuangzaosi (CHZS) male sterility. A genetic analysis showed that DNA598, PHT77, 78551S, and LH212Ht only contained one dominant restorer gene each, and the molecular-marker mapping indicated that their restorer genes were located at the short arm of chromosome 8. The allelic testing of the fertility of the restorer (Rf) demonstrated that the restorer gene of twelve inbred lines, including DAN598, PHT77, 78551S, and LH212Ht, was allelic to one restorer gene of A619. Furthermore, the genomic sequence alignment of Rf4 revealed that there were two different amino acids in the coding sequence between the A619 (Rf4Rf4) restorer lines and four CMS-C lines (rf4rf4). For the crucial S1596 site variation (TTT/TAC), DAN598, PHT77, 78551S, and LH212Ht shared the same bases (TTT) with A619 and encoded phenylalanine, while the four CMS-C sterile lines had the TAC and encoded tyrosine. Our results revealed that these tester lines, DNA598, PHT77, 78551S, and LH212Ht, were the Rf4-restorer lines. Additionally, derived from the sequence variants of Rf4, 39 possible Rf4-restorer lines from 129 inbred maize lines were detected. Furthermore, we developed a Cleaved Amplified Polymorphism Sequences (CAPS) marker based on the S1596 variations. The PCR amplification product of S1596 (TAC) was digested by the TatI endonuclease into two bands with sizes of ~260 bp and ~100 bp. In comparison, when S1596 was TTT, the PCR product could not be digested. In conclusion, in this study, we identified various Rf4-restorer lines for maize CMS-C and developed a molecular marker for Rf4. The reported results will contribute to the popularization and application of Rf4 in hybrid maize-seed production.