Identification of cytoplasm types in rapeseed (Brassica napus L.) accessions by a multiplex PCR assay

Abstract

Cytoplasmic male sterility (CMS) has widely been used as an efficient pollination control system in rapeseed hybrid production. Identification of cytoplasm type of rapeseed accessions is becoming the most important basic work for hybrid-rapeseed breeding. In this study, we report a simple multiplex PCR method to distinguish the existing common cytoplasm resources, Pol, Nap, Cam, Ogu and Ogu-NWSUAF cytoplasm, in rapeseed. Cytoplasm type of 35 F(1) hybrids and 140 rapeseed open pollinated varieties or breeding lines in our rapeseed breeding programme were tested by this method. The results indicated that 10 of 35 F(1) hybrids are the Nap, and 25 the Pol cytoplasm type, which is consistent with the information provided by the breeders. Out of 140 accessions tested, 100 (71.4%), 21 (15%) and 19 (13.6%) accessions possess Nap, Cam and Pol cytoplasm, respectively. All 19 accessions with Pol cytoplasm are from China. Pedigree analysis indicated that these accessions with Pol cytoplasm were either restorers for Pol CMS, including Shaan 2C, Huiyehui, 220, etc. or derived from hybrids with Pol CMS as female parent. Our molecular results are consistent with those of the classical testcross, suggesting the reliability of this method. The multiplex PCR assay method can be applied to CMS "three-line" breeding, selection and validation of hybrid rapeseed.

Cytological observation of anther structure and genetic investigation of a new type of cytoplasmic male sterile 0A193‐CMS in Brassica rapa L.

“…, Zhao et al. ) in the present study. As expected, a specific amplification product of about 465 bp was yielded in RF04(R), which was specific for orf138 ( ogu CMS type), 1102 bp in Westar, which was specific for orf222 ( nap CMS type), and 747 bp in Zhong 9A, which was specific for orf224 ( pol CMS type).…”

“…The result of multiplex PCR (Zhao et al. ) and molecular cloning and sequencing of the specific PCR bands revealed that the cytoplasm of this male sterile accession was identical to that of nap CMS. Together, this male sterile accession belonged to the nap CMS type, and its fertility was controlled by a pair of genic recessive genes.…”

“…The associated genes for CMS were reported in Brassica include orf138 for ogu CMS (Duroc et al 2005), orf222/nad5c/orf139 for nap CMS (L'Homme and Brown 1993) and orf224/atp6 for pol CMS (L'Homme et al 1997). Three pairs of primers that were specifically designed for orf138, orf222 and orf224 were used to amplify male sterile accessions 2A345 and 2A397 together with RF04(R) (ogu CMS type), Westar (nap CMS type) and Zhong 9A (pol CMS type) through the multiple PCR (Wei et al 2005, Zhao et al 2010) in the present study. As expected, a specific amplification product of about 465 bp was yielded in RF04(R), which was specific for orf138 (ogu CMS type), 1102 bp in Westar, which was specific for orf222 (nap CMS type), and 747 bp in Zhong 9A, which was specific for orf224 (pol CMS type).…”

Sequence analysis of the mtDNA region correlated with Shaan 2A cytoplasmic male sterility in rapeseed (Brassica napus L.)

“…Several CMS systems as Ogu-INRA, MSL and Pol are already used in hybrid breeding of Brassica napus and commercial cultivars based on Ogu-INRA, MSL and Safecross MS are registered and cultivated in the Czech Republic. In China, 70% rapeseed cultivars are based on CMS, mainly the Pol or Shaan 2A systems that both arose spontaneously within the species (Zhao et al 2010). The orf224-1 gene has been isolated as a Shaan 2A CMS-associated gene (Wang et al 2002).…”

“…Genomic DNA was isolated according to CTAB procedure described by Doyle and Doyle (1987). Total gDNA has been used for PCR because it has already been confirmed that gDNA is mixed with mtDNA and PCR amplification of mtDNA orf genes is possible (Zhao et al 2010). The concentration and quality of DNA was determined with a BioMate 5 spectrophotometer.…”