Identification Of M5P And Y_Ⅱ-1 New Type Male-sterile Cytoplasms And Screening Of RAPD Markers For CMS-associated Fragments In Maize(Zea Mays L.)

We designed three sets of PCR specific primers for T-, C-, and S-type cytoplasms according to the published mtDNA(mitochondrial DNA) sequences in the GenBank(M12582, S81074, AF008647). We used a PCR assay for rapid discrimination of M5P and YⅡ-1 new type male-sterile cytoplasm. We will afford a reliable proof for taking advantage of M5P and YⅡ-1 new type male-sterile cytoplasm. Meanwhile, YⅡ-1B77 male sterile line, B77 maintainer line and F1 were used as samples in this experiment. We have screened their mtDNA sequence differential fragments by RAPD(Randomly Amplified Polymorphic DNA) marker and demonstrated mtDNA structure changed after adding fertility restorer genes to male-sterile line. This changes are possible associated with male-sterility. The results show as follows:1. Identification of new type male-sterile cytoplasmsThree pairs of PCR primers for chimeric regions of mtDNA sequence regions of maize CMS-T(CMSTF+CMSTR), CMS-C(CMSCF+CMSCR), and CMS-S(CMSSF+ CMSSR) were designed. Amplified results by PCR from mtDNA with two sets of homonucleus-alloplasmic male sterile lines with B77 and U8112 nuclear background with six different types of cytoplasm(N, T, C, S, YⅡ-1, M5P ) indicated: no primer pairs amplified fragments from the mitochondria DNA of two sets of inbred line B77 and U8112 with N-type cytoplasm. The primer set CMSTF and CMSTR yielded only a 440bp DNA fragment in B77 and U8112 with T-type cytoplasm, while the primer sets CMSSF and CMSSR, CMSCF and CMSCR yielded unique 799bp and 398bp DNA fragments with S-type cytoplasm and C-, YⅡ-1-, M5P-type respectively. However, there are the same amplified results by PCR from total leaf DNA and total seed DNA as template of the inbred line B77 and U8112 nuclear background with six different types of cytoplasm(N, T, C, S, YⅡ-1, M5P ). This result showed that M5P and YⅡ-1 new type male-sterile cytoplasm may belong to C group male-sterile cytoplasm.2. Investigated of screening of RAPD markers1.1 Optimize the reaction conditions of RAPDWe perform a series of experiments by changing the concentrations of Mg2+, dNTPs, Taq enzyme, template DNA and primers. The results show the optimum conditions in 25μl total volume by RAPD amplified from mtDNA as template in maize: Mg2+ concentration 2.0mmol/l ; dNTPs concentration 100μmol/l; Taq enzyme concentration 1.5U; template DNA concentration 20～100ng; primer concentration 0.2μmol/l.1.2 The result of screening of RAPD primersUnder the optimum conditions, YⅡ-1-B77 male sterile line, B77 maintainer line and F1 hybrid(YⅡ-1-B77 male sterile line×W341 restorer line) mtDNA are screened by 30 RAPD primers. As a result, we found two differential fragments from mtDNA amplified by primer S8: S8-2200 and S8-2500; Three differential fragments from mtDNA amplified by primer S116: S116-2200, S116-2500 and S-3500; A differential fragments from mtDNA amplified by primer S183: S183-3500. Of differential fragments, S8-2500 and S116-2500 fragments were amplified by mtDNA as template extracted from YⅡ-1-B77male sterile line and F1 hybrid. However, two fragments could not been amplified by mtDNA extracted in B77 maintainer line. S8-2200, S116-2200, S116-3500 and S183-3500, four specific fragments were only amplified by extraction of mtDNA as template from YⅡ-1-B77 male sterile line. But four specific fragments could not been found in the profile of amplified products by RAPD from mtDNA in B77 maintainer line and F1 hybrid. This study indicated two specific fragments S8-2500 and S116-2500 were only found probably in the profile of products amplified by RAPD from mtDNA in YⅡ-1-B77 male sterile line and F1 hybrid. Maybe four chimeric fragments S8-2200, S116-2200, S116-3500 and S183-3500 were only found in mtDNA amplified profile extracted from YⅡ-1-B77 male sterile line.