Determination of cytoplasmic male sterile factors in onion plants (Allium cepa L.) using PCR-RFLP and SNP markers.
We have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker that can distinguish male-fertile (N) and male-sterile (S) cytoplasm in onions. The PCR-RFLP marker was located in a chloroplast psbA gene amplicon. Digesting the amplicons from different cytoplasm-containing varieties with the restriction enzyme MspI revealed that N-cytoplasm plants have a functional MspI site (CCGG), whereas the S-cytoplasm plants has a substitution in that site (CTGG), and thus no MspI target. The results obtained using this PCR-RFLP marker to distinguish between cytoplasmic male sterile factors in 35 onion varieties corresponded with those using a CMS-specific sequence-characterized amplified region (SCAR) marker. Moreover, the PCR-RFLP marker can identify N- ot S-cytoplasms in DNA sample mixtures in which they are in up to a 10-fold minority, indicating that use of the marker has high diagnostic precision. We also demonstrated the usefulness of the SNP detected in the psbA gene for high-throughput discrimination of CMS factors using Real-time PCR and a TaqMan probe assay.
Cho
Kwang-Soo
KS
National Institute of Highland Agriculture, Rural Development Administration, Pyeongchang 232-955, Korea. kscho@rda.go.kr
Yang
Tae-Jin
TJ
Hong
Su-Young
SY
Kwon
Young-Seok
YS
Woo
Jong-Gyu
JG
Park
Hyo-Guen
HG
D000483:Alleles; D003593:Cytoplasm / Q000235:genetics*; D017343:Genes, Plant / Q000235:genetics*; D005819:Genetic Markers; D019697:Onions / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide
D005819:Genetic Markers