Genetic Analysis Of Male Sterility Line K932S In Maize

Maize?Zea mays L.?is one of the crops which uses male sterile?MS?in production earliest.Application of the male sterility not only save costs of seed production,but also is an effective way to improve seed quality.The breeders obtained a lot of maize MS materials by various ways.However,the application of these materials is limited,because the fertility stability,the cytoplasmic malady and the abortion mechanism are still not clearly understood.Therefore,it is of a great theoretical and practical significance to create,identify and research more new male sterility in maize.K932S is a new male sterile material obtained by spontaneous mutation.In this study,we take the sibling population K932MS?composed of 50%sterile line K932S and 50%fertile line K932F?,K932S crossed with normal inbred lines as F₁ and reciprocal F₁?F₂?populations,BC1 and BC2populations,the restorer line K932R as research materials.By using genetic identification,cytology observation,physiological and biochemical determination and RNA-seq analysis to clarify the hereditary mode,abortive period and features,physiological and biochemical properties,and the relationship of differentially expressed genes?DEGs?of key metabolism pathways with abortion of K932S,then provide theoretical basis for further research and application.The main results are as follows:?1?There was no phenotypic difference between K932S and K932F at vegetative growth stage,but K932S had degeneration anther without pollen grains in pollinating period.The segregation ratio of fertile plants to sterile plants was 1:1 in the sibling progenies,while it was 3:1 in self-crossing progenies of K932F at different locations,years and seasons.Sterility of K932S could be restored among reciprocal progenies when seven normal inbred lines were used as females crossed with K932S�A9-2 fertility plant respectively;30 testers crossed with K932S showed that,one inbred line could restore the fertility of K932S completely,21 testers maintained the male sterility,while the other eight testers presented an unstable effect on the fertility;as changes in the ncelar background of backcross offspring,the fertility performance ofK932S will be changed also,means that the fertility was affected by nuclear-cytoplasmic interactions.Hence,K932S is nuclear-cytoplasmic male sterility mutant with pollen-free and inheritance stablely,and the sterile nuclear gene is controlled by recessive single gene.?2?Based on the special restoration group identification and PCR identification with specific primers,it was found that C type restore line Zifeng1 could restore K932S fertility,and K932MS had the same PCR band with CMS-C_Mo17.Cytological observations of anther and pollen development showed,K932S began to abortion at dyad with tapetum expanded radically and vacuolated,disintegration delay,filled the entire chamber gradually;the dyads and tetrads were abnormal shapes,the volume of early mononuclear microspores was further reduced,then it became collapsed and disappeared gradually.Indicate that K932S belongs to C type cytoplasmic male sterility with abortion from dyad stage,the abortive characteristic is different from the typic C-type sterile materials.?3?Determinations of physiological and biochemical properties showed that,compared with fertile anther,the contents of nutriments including soluble sugar is less in sterile anther significantly at four developing stages,CAT and SOD activity are significantly less,however,the POD activity is significantly higher at the sporogenous cell period and tetrad stage.The nutrients in K932S was insufficient seriously,affected the normal development of anthers and microspores;simultaneously,the activity changing of key enzymes including CAT,SOD and POD of antioxidant system,is not beneficaial to maintain the dynamic equilibrium of the content of active oxygen effectively.That may be the typical physiological and biochemical properties of K932S abortion.?4?RNA-seq results showed that a total of 2962 DEGs were screened.K932S compared to K932R,there were 68,101,85 and 1145 up-regulated,and 83,179,79 and1222 down-regulated respectively at the pollen mother cells,dyad,tetrad and early mononuclear microspore stage.At the four stages,the significant GO terms enriched by the DEGs were 0,7,4 and 158 respectively,mainly including pollen development,DNA replication,hydrolase activity and amino compounds biosynthesis.It is suggested that dyad and early mononuclear microspore stage is the critical period of abortion.?5?KEGG pathway analytical results,there were 48,104,56 and 1467 DEGs enriched at 28,46,25 and 118 pathways at the four stages respectively.These pathways mainly incluted starch and sucrose metabolism,prolin metabolism at the pollen mother cell period,DNA replication and cutin,suberin and waxy biosynthesis at the dyad period,peroxisomes and flavonoid biosynthesis at the tetrad period,and the ribosom and amino acid biosynthesis at the early mononuclear microspore prophase.It is speculated that starch and sucrose metabolism,cutin,suberin and waxy biosynthesis,and amino acid biosynthesis pathways are related to abortion.?6?Selected five key DEGs in amino acid biosynthesis pathway,the relative expression of qRT-PCR analysis were consistent with the results of RNA-seq.The contents of prolin,tyrosine and alanine of anther in K932S were significantly less than that in K932R at the four stages,verified that the amino acid biosynthesis is closely relative to abortion of K932S.?7?The relations of DEGs in key metabolism pathways with K932S abortion progress,the up-regulated genes related to tapetum,caursed the tapetum PCD abnormal,disintegration delay.The down-regulated genes related to cutin,suberin and waxy,resulted in insufficient synthesis of these substances,causing the anther degeneretion;the down-regulated genes related to starch and sucrose metabolism and amino acids biosynthesis,caused insufficient of energy and nutrients including amino acid,then the microspores became deformed and disappeared.These results lay a solid foundation for further revealing the abortive mechanism of K932S.