Fine Mapping Of Rf3 Gene Based On Target BAC Contig Sequences In Maize

Maize is one of the most important food and fodder crops in the world,and it is also one of the important model plants for plant genetics research.Genomics of maize is the forefront field of molecular biology.In early 2008,a first draft of the sequence of the maize inbred line B73 genome was released.That is useful for maize function genomics and it is the foundation for researching maize genomic structure and gene cloning.The development of BAC library especially the BAC library construction of inbred line B73 and BAC sequencing provided an effective tool for physical mapping, developing of molecular markers for gene mapping,gene map-based cloning.Plant cytoplasmic male sterility(CMS) is the basis of utilizing heterosis.CMS/Rf system has been used extensively in breeding for the production of hybrid crops and maize is one of the foremost crops.S-CMS is the biggest one of the cytoplasmic male sterility,but it is unstable.So it is important to clone its restore gene Rf3.Several attempts have been made to fine map Rf3 gene,however it is not yet near enough to map-based clone the gene.In this study,based on the research of S-CMS before,we screened SSR and STS markers polymorphic between near isogenic lines(NILs) S-Mo17^Rf3Rf3 and S-Mo17^rf3rf3.A BC₁F₁ population was constructed.The sequences of BACs in Contig108 in 2.09bin were analyzed of repetitive sequences,SSR motifs,predicting of potential genes and annotation. Some primary results are as follows:1.The BC₁F₁ population(S-Mo17^rf3rf3×S-Mo17^Rf3Rf3×N-Mo17^rf3rf3 were constructed using the NIL S-Mo17^Rf3Rf3 and S-Mo17^rf3rf3.According to their ability in shedding pollen and ability of pollen grains,each plant was scored as male semi-fertile or sterile.The segregation ratio fits 1:1,it confirmed that fertility restoration was conditioned by one dominant restorer gene in this research.2.Using markers UMC2184 and SCARE12M7 around Rf3 locus in 2.09bin for preliminary mapping Rf3 gene and to insure Rf3 gene was in Contig108.3.Using Repeat Masker software analyze sequences in Contig108 found that 62.82%of sequences contained repetitive elements,92.62%of repetitive elements were retroelements and most of retroelements were LTR.Gypsy/DIRS1 and Ty1/Copia occupied 46.26%and 39.22%,respectively.This was agreed with other's research and it indicated that the structure of whole genome could be understood through analyzing BAC sequences.4.Using SSRScan and SSRIT software analysis BAC sequences found that there were 221 SSR sequences and there was one SSR every 60.0kb.5.Utilized Clemson University's platform synthesized 63 pairs of primers,and 22 pairs showed polymorphism between S-Mo17^Rf3Rf3and S-Mo17^rf3rf3,the efficiency was 35%.9 of 22 SSR markers were used to map Rf3 gene,and the closest were N7 and N8, they located in different side of Rf3 gene,both of the distances was 1.7cM.6.According to the preliminary results of Rf3 mapping,153 pairs of primers were synthesized and 5 of them showed polymorphism between S-Mo17^Rf3Rf3and S-Mo17^rf3rf3. 4 STS markers used to construct genetic linkage map.Markers N10,N11 cosegregated with N8,the distances around Rf3 gene was 1.7 cM.Markers N9 cosegregated with N12, they were 0.7 cM away from Rf3 locus.7.High-quality BAC sequences were predicted of potential genes using FGENESH program.The predicted genes then annotated with Blastp and InterProScan software.A total of 406 predicted genes were annotated.There were 16 PPR genes in 8 BACs.But the PPR genes related to rice Rf-1 gene in 7of the 8 BACs.In addition,the two BACs that might be contained Rf3 gene did not involve any PPR gene.Compared mitochondrial genome with nuclear genome BAC sequences of maize S-CMS found that the two BAC sequences were high colinearity with mitochondrial genome.That confirmed the mapping result and it was essential to construct the BAC library of inbred line Mo 17.