Fine-mapping And Candidate Gene Prediction For Fertility Restoration Gene Rf10 And Rf11 Of Maize CMS-S

Maize is an important food crop with a wide range of usage,including feed,industrial processing and energy raw materials.The development of maize male sterile lines is of great significance to maize hybrid seed production.Of the three types of cytoplasmic male sterility in maize,CMS-S has the largest members.With genome-wide association study on fertility restoration of maize CMS-S,fertility restoration was found to be regulated by multiple loci with main and minor effects in our lab previously.In this study,two partial fertility restorer lines of maize CMS-S（the fertility restoration genes were named Rf10 and Rf11,respectively）and CMS lines were used to construct backcross（BC）populations.Combined with BSR-Seq analysis,the two restorer genes were fine mapped with the BC populations,and possible candidate genes were predicted.Based on the comparative analysis of the differentially expressed genes（DEG）of populations including Rf10,Rf11 and Rf3,the genetic mechanism of their fertility restoration was discussed.The main results are as follows:1.Field fertility investigation was conducted on the backcross populations including Rf10 or Rf11.Sown in spring,anthers in the BC₁population of Rf10 were completely exposed in the fertile individuals with different degree of dehiscence,or not exposed at all in the sterile individuals;anthers in the BC₄population of Rf11 were also completely exposed（fertile individuals）or not exposed at all（sterile individuals）,and partical pollen shedding were observed in the exposed anthers.As sown in autumn,anthers in the Rf10 and Rf11 populations were not dehisced at all,indicating that high temperature at the booting stage influenced anther dehiscence.It is also found that,pollen fertility in the fertile plants of the BC₁population of Rf10 varied in a larger range,suggesting that the population includes other fertility restoration loci with small effectes besides Rf10.The chi-square test analysis revealed that fertility segregation in the populations fit to 1:1,indicating that fertility in the populations of Rf10 or Rf11 was controlled by a dominant restorer gene.2.Preliminarily mapping of Rf10 with the BSR-Seq method located the main restorer gene in the region same as the previously identified Rf10,and the fertility restoration performance was also similar.Thus they are the same gene.Seven pairs of SNP markers and three pairs of Indel markers were used to screen recombinants in the BC₁and BC₂populations,and Rf10 was then fine-mapped between the two markers S865 and G39600 on chromosome 3 with a physical distance of 71.59kb.This region contains 5 predicted genes,of which Zm00001d039597 encodes a FLZ zinc finger domain and is rich in glycine（GRP）at the N-terminus.Zm00001d039597 is speculated as a candidate gene of Rf10.3.BSR-Seq comparative analysis with two BC populations of Rf10 and Rf3revealed a total of 353 DEG.Of them,a total of 265 DEG were expressed in fertile plants in both populations.Functional annotation analysis found that these genes are mainly related to cell wall expansion,carbohydrate metabolism,programmed death,transcription factors or signal transduction.35 and 7 specifically DEG were up-regulated in the fertile plants of Rf3 and Rf10,respectively.The genes specifically up-regulated in the fertile plants of Rf10 encode P450 proteins,expansins,SLAC,UBL1B and a legume lectin.UBP1B is a member of plant SUMO（small ubiquitin-related modifier）proteases,and is related to stress regulation.Mutations of some members of SUMO were found to affect the function of pollen development.Thus,it can be deduced that Rf10 mediated fertility restoration of CMS-S would be related to the SUMO system and stress would be also involved into this process.4.Preliminarily mapping for Rf11 with BSR-Seq method was conducted and verified with SNP markers.Rf11 was mapped on 5.04 bin.Three pairs of SNP markers and one pair of Indel markers were then used to fine-map Rf11 with BC₃and BC₄populations,and it was narrowed between the markers of S1478 and G1614 with a physical distance of 800 kb.The Rf11 fine-mapped region contains 10 predicted genes.Of them Zm00001d016149 encodes a heavy metal transport/detoxification superfamily protein,which is specifically expressed in anthers and pollen.RNA-Seq analysis revealed that expression of this gene in the fertile pools was significantly higher than that in the sterile pools.Subcellular location prediction found that it is located in the nucleus,cytoplasm and mitochondria.Therefore,it is preliminarily speculated to be a candidate gene of Rf11.5.Totally 59 DEG were identified to be up-regulated in the fertile pools of the Rf11 population by the BSR-Seq analysis using leaves,and 25 DEG were down-regulated in the fertile pools of the Rf11 population.Most of the up-regulated DEG encode carbohydrate synthesis-related proteins,phosphorylation-related enzymes,transcription factors,respiratory chain ATP metabolism-related enzymes,and PPR proteins.These genes may play important roles in the development of anthers or pollens.These results are of great significance for further revealing the fertility restoration mechanism of CMS-S,and provide new reference means and theoretical basis for its practical application in hybrid breeding.