Development of KASP marker for cytoplasmic male sterility in Nicotiana tabacum and utilization in trait introgression

Cytoplasmic male sterility (CMS) is an important trait for large-scale hybrid seed production which avoids manual emasculation and undesired horizontal spread of pollen.Rearrangements in mitochondrial genome in terms of deletions and insertions are frequent causes leading to CMS. Mitochondrial ATP synthase is a multisubunit molecular machine which is involved in synthesis of ATP. In this study, three mutations in ATPase subunit 6 were identified and their cosegregation with male sterility was established using tobacco male sterile hybrids and Nicotiana suvaolensis. A breeder friendly Kompetitive allele specific polymerase chain reaction (KASP) SNP marker was developed for high throughput and quick genotyping. Introgression of this trait into selected germplasm lines (n = 9) was achieved based on foreground for CMS and background selection for recurrent parent using KASP marker and 50K custom tobacco SNP array, respectively. Analysis of genotyping data from SNP array revealed the presence of 88–99% of recurrent parent genome in BC3F1 plants. The selected BC3F1 plants exhibit CMS and are indistinguishable from the fertile recurrent parent (germplasm) in terms of plant morphology.

Nucleotide sequence polymorphism in the RFL-PPR genes of potato

Cytoplasmic male sterility (CMS) is widely used for hybrid seed production in cultivated Solanaceae species. However, there is very limited information about CMS-Rf genetic systems in potato (Solanum tuberosum). Studying the CMS-Rf systems in potato is both of theoretical and practical significance due to the emergence of a new revolutionary strategy of reinventing potato as adiploid inbred line-based crop to develop F1 hybrid seed potato breeding (Lindhout et al. 2011; Jansky et al. 2016). To search for potato Rf gene candidates, the comparative genetic approach was applied. Based on similarity to petunia Rf-PPR592 gene, 38 fragments were identified in five loci of the whole-genome nucleotide sequence of the accession DM 1-3 516 R44 S. tuberosum Phureja group (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The putative encoded mitochondrial proteins have 589–597 amino acid residues, similarto RF-PPR proteins of petunia and chili pepper and contain 14 or 15 PPR motifs. Primers have been developed flanking the most variable 782–865 bp regions of the selected loci, and polymorphism of the cloned fragments has been investigated in a subset of nine potato genotypes. The amplified fragments included seven or eight PPR motifs and lacked introns. The SNP frequencies ranged from 7.0 to 19.8% depending on the locus, while the ratio of nonsynonymous to synonymous substitutions varied between 0.9 and 2.1.Positions 1, 3 and 6 were the most variable in the studied PPR motifs. Our results demonstrated that the analysed sequences belong to the RFL-PPR gene subfamily and may be considered as Rf gene candidates in potato.

RNA editing analysis of ATP synthase genes in the cotton cytoplasmic male sterile line H276A

BACKGROUND: Pollen development is an energy-consuming process that particularly occurs during meiosis. Low levels of adenosine triphosphate (ATP) may cause cell death, resulting in CMS (cytoplasmic male sterility). DNA sequence differences in ATP synthase genes have been revealed between the N- and S-cytoplasms in the cotton CMS system. However, very few data are available at the RNA level. In this study, we compared five ATP synthase genes in the H276A, H276B and fertile F1 (H276A/H268) lines using RNA editing, RNA blotting and quantitative real time-PCR (qRT-PCR) to explore their contribution to CMS. A molecular marker for identifying male sterile cytoplasm (MSC) was also developed. RESULTS: RNA blotting revealed the absence of any novel orf for the ATP synthase gene sequence in the three lines. Forty-one RNA editing sites were identified in the coding sequences. RNA editing showed that proteins had 32.43% higher hydrophobicity and that 39.02% of RNA editing sites had proline converted to leucine. Two new stop codons were detected in atp6 and atp9 by RNA editing. Real-time qRT-PCR data showed that the atp1, atp6, atp8, and atp9 genes had substantially lower expression levels in H276A compared with those in H276B. By contrast, the expression levels of all five genes were increased in F1 (H276A/H268). Moreover, a molecular marker based on a 6-bp deletion upstream of atp8 in H276A was developed to identify male sterile cytoplasm (MSC) in cotton. CONCLUSIONS: Our data substantially contributes to the understanding of the function of ATP synthase genes in cotton CMS. Therefore, we suggest that ATP synthase genes might be an indirect cause of cotton CMS. Further research is needed to investigate the relationship among ATP synthase genes in cotton CMS.

cDNA-AFLP Reveals Variation of Sequence Region trnF-ndhJ in Cytoplasmic Male Sterile Chinese Cabbage-pak-choi / 生物化学与生物物理进展

By means of cDNA amplified fragment length polymorphism(cDNA-AFLP) technique, a fragment P1708 was amplified from Polima cytoplasmic male sterile Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinenesis Makino, syn, B. rapa L. ssp. chinenesis) 'Bpol97-05A'. RT-PCR showed that this fragment was specifically expressed in male sterile material. Sequencing and BLAST search in GenBank database indicated that P1708 had 100% homolog with chloroplast ndhJ-trnF gene region except a 54 bp insertion. Gene specific primer pairs were synthesized according to ndhJ-trnF gene region and two fragments about 1 900 bp were amplified respectively using genomic DNA templates of Polima cabbage and male fertile oilseed rape. The sequencing results showed that the gene region ndhJ-trnF of Polima cabbage contained two 54 bp repeats and some variation sites. The repeat part shared the same sequence as trnF gene except three bases at 5′ ends. For the insertion of 108 bp sequence, a new open reading frame was created.

Mitochondrial ATP synthase genes may be implicated in cytoplasmic male sterility in Sorghum bicolor.

Incompatible nuclear-cytoplasmic interactions are responsible for the phenomenon of cytoplasmic male sterility in plants. We have analysed male sterile (2077A, 296A), maintainer fertile (2077B, 296B) and fertility restored (2077R, 296R) lines of sorghum for the restriction fragment locations of various mitochondrial genes and their transcripts. We report here a polymorphism in genes related to the ATP synthase complex between two different cytoplasms from the A and Β set of lines of 2077 and 296. There is also a difference in the transcript size of the atpA gene between the A and Β cytoplasms. We propose that incompatibility in nuclear cytoplasmic interactions may be explained in terms of incompatible subunits being synthesized by the mitochondria and nucleus for a multisubunit complex of the mitochondrial membrane such as ATPase.

Mitochondrial genome organization and cytoplasmic male sterility in plants.

Plant mitochondrial genomes are much larger and more complex than those of other eukaryotic organisms. They contain a very active recombination system and have a multipartite genome organization with a master circle resolving into two or more subgenomic circles by recombination through repeated sequences. Their protein coding capacity is very low and is comparable to that of animal and fungal systems. Several subunits of mitochondrial functional complexes, a complete set of tRNAs and 26S, 18S and 5S rRNAs are coded by the plant mitochondrial genome. The protein coding genes contain group II introns. The organelle genome contains stretches of DNA sequences homologous to chloroplast DNA. It also contains actively transcribed DNA sequences having open reading frames. Plasmid like DNA molecules are found in mitochondria of some plants Cytoplasmic male sterility in plants, characterized by failure to produce functional pollen grains, is a maternally inherited trait. This phenomenon has been found in many species of plants and is conveniently used for hybrid plant production. The genetic determinants for cytoplasmic male sterility reside in the mitochondrial genome. Some species of plants exhibit more than one type of cytoplasmic male sterility. Several nuclear genes are known to control expression of cytoplasmic male sterility. Different cytoplasmic male sterility types are distinguished by their specific nuclear genes (rfs) which restore pollen fertility. Cytoplasmic male sterility types are also characterized by mitochondrial DNA restriction fragment length polymorphism patterns, variations in mitochondrial RNAs, differences in protein synthetic profiles, differences in sensitivity to fungal toxins and insecticides, presence of plasmid DNAs or RNAs and also presence of certain unique sequences in the genome. Recently nuclear male sterility systems based on (i) over expression of agrobacterial rol C gene and (ii) anther specific expression of an RNase gene have been developed in tobacco and Brassica by genetic engineering methods.