Screening And Analysing The Candidate Gene Of Gal-S Conferring Unilateral Cross Incompatibility In Maize

Maize(Zea mays L.) is a hermaphrodite and cross-pollinated crop, usually selfing and crossing both could set seeds successfully. Many popcorn strains carry Ga gene conferring unilateral cross incompatibility, controlled by the gametophyte. This cause the phenomenon that popcorn strains cannot be fertilized by pollen of dent and flint maize although the reciprocal crosses are successful. Material studied in this experiment is a Chinese popcorn strain SDGa25 carring the strongest allele of Gal (Gal-S). Currently, the molecular cloning of Gal-S has not been reported both at home and abroad. The study has important theoretical and practical significance. The research got the following results:1. The waxy strain JKN2000F and dent strain 178 are the Ga1-M has been proved. The cross-allelic experimental analysis shows that segregating populations BC1F1 solid ratio of 1:0.The datas indicates Ga1-M from different strains are not allelic.2. Through new single phenotypic groups, and molecular marker-assisted selecting recombinants, the Gal-S gene was mapped between markers A5 and A7, the physical distance is about 237 Kb reference B73 RefGen_v2 sequence, narrowing original positioning range about 453Kb.3. SDG25 BAC library contains 307,200 monoclonal, coveraging the corn genome 10 times. The super pool, sub-pool and monoclonal molecular were screened through marker-assisted. After verification, we obtained two monoclonal L9 and 10, who owns the largest inserts and covers fine positioning range. The sequencing was completed.4. According to the BAC sequencing and the B73 sequence provided by the database MaizeGDB online, three functional genes was predicted. They are GRMZM2G419836, GRMZM2G027021, GRMZM2G039983.The predicted genes GRMZM2G419836 gene has 2 exons,1 transcript. It contains a thioredoxin (thioredoxin, Trx) domain, regulating the enzyme activity of protein.The predicted GRMZM2G027021 owns 13 exons,5 transcripts. It’s GTP-binding protein hflX, may affecting the formation and growth of pollen tubes. The expression level is highest in leaf-20 days, relatively high in the post-emergence stems and the four-leaf stage seedlings, while virtually no in the endosperm expression level.The predicted genes GRMZM2G039983 has 6 exons,3 transcripts. As the homology to WDL1 of Arabidopsis, it can be the targeting protein for Xklp2, involving microtubules formation. The RNA-Seq data showed that the expression level is highest in the emergence, relatively high in the four-leaf stage seedlings, while virtually no in the endosperm expression level. Sequence analysis showed that the gene containing one or two Q (glutamine) difference between Gal-S, Gal-M materials and common corn, which may cause disruption in pollen-pistil interactions. Based on three transcripts of GRMZM2G039983, different primers were designed. Using the amplification of RNA and cDNA or RT-PCR, three transcripts of the predicted genes are expressed in different tissues and have different expression levels.Digested and obtained the BAC containing two predicted genes into pCAMBIA1300 Agrobacterium vector, then transformed into corn, the work is ongoing.