A PCR-marker for the CMS1 inducing cytoplasm in chives derived from recombination events affecting the mitochondrial gene atp9

Abstract

The complete coding and 3;-flanking region of the mitochondrial gene atp9 of chives ( Allium schoenoprasum L.) was determined in order to develop primers that allow the identification of atp9-related sequences in subsequent PCR-amplifications. One of these sequences is of a chimerical nature, consisting of atp9-homologous regions on its end, interrupted by an insertion that is composed of one atp6-homologous part and one part of unknown origin. This PCR-fragment is 762 bp in size and exclusively amplified in the sterility inducing cytoplasm of CMS(1). Thus it can be used as a PCR-marker in order to distinguish this cytoplasm type from the remaining cytoplasm types of chives. The chimerical marker sequence forms a putative open reading frame ( orfA501), from which CMS(1) might originate.

The fertility restorer genes X and T alter the transcripts of a novel mitochondrial gene implicated in CMS1 in chives ( Allium schoenoprasum L.)

“…The previously known sequence of the CMS 1 configuration ended in a region homologous to atp 9 (see Introduction; Engelke and Tatlioglu 2002). However, extension of the 3¢-region by 3¢RACE showed that the atp 9 homologous portion of the sequence actually ends upstream of the transcript termini.…”

“…In the first case the orf would comprise 780 bp, in the latter case 744 bp. The next inframe ATG codon results in the previously described orf 501 (Engelke and Tatlioglu 2002). The predicted proteins encoded by these orf s consist of 260 (29 kDa), 248 (27.5 kDa), and 167 amino acids (19 kDa), respectively, leaving aside possible alterations due to RNA editing.…”

“…This marker amplifies the expected (wild-type) 152-bp fragment of atp 9 from all five cytoplasm types, but an additional 762-bp fragment is obtained from cytoplasm type (S 1 ). Sequencing of this marker fragment confirmed its chimeric nature; it consists of atp 9-homologous regions at each end, separated by a segment composed of an atp 6-homologous sequence and a sequence of unknown origin (Engelke and Tatlioglu 2002).…”

“…Cloning of PCR products was performed using the TOPO TA cloning kit (Invitrogen) as previously described (Engelke and Tatlioglu 2002). Plasmids were recovered using the QiaPrep-Mini kit (Qiagen) and sequencing was carried out by MWG Biotech.…”

“…PCR amplification of such sequences using atp 9-derived primers was attempted in the various cytoplasm types, and a PCR marker that allows the differentiation of the CMS 1 -inducing cytoplasm was developed (Engelke and Tatlioglu 2002). This marker amplifies the expected (wild-type) 152-bp fragment of atp 9 from all five cytoplasm types, but an additional 762-bp fragment is obtained from cytoplasm type (S 1 ).…”