Microsatellite genetic linkage maps of myrobalan plum and an almond-peach hybrid--location of root-knot nematode resistance genes.

Inheritance and linkage studies were carried out with microsatellite [or simple sequence repeat (SSR)] markers in a F(1) progeny including 101 individuals of a cross between Myrobalan plum ( Prunus cerasifera Ehrh) clone P.2175 and the almond (Prunus dulcis Mill.)-peach ( Prunus persica L. Batsch) hybrid clone GN22 ["Garfi" (G) almond x "Nemared" (N) peach]. This three-way interspecific Prunus progeny was produced in order to associate high root-knot nematode (RKN) resistances from Myrobalan and peach with other favorable traits for Prunus rootstocks from plum, peach and almond. The RKN resistance genes, Ma from the Myrobalan plum clone P.2175 and R(MiaNem) from the 'N' peach, are each heterozygous in the parents P.2175 and GN22, respectively. Two hundred and seventy seven Prunus SSRs were tested for their polymorphism. One genetic map was constructed for each parent according to the "double pseudo-testcross" analysis model. The Ma gene and 93 markers [two sequence characterized amplified regions (SCARs), 91 SSRs] were placed on the P.2175 Myrobalan map covering 524.8 cM. The R(MiaNem) gene, the Gr gene controlling the color of peach leaves, and 166 markers (one SCAR, 165 SSRs) were mapped to seven linkage groups instead of the expected eight in Prunus. Markers belonging to groups 6 and 8 in previous maps formed a single group in the GN22 map. A reciprocal translocation, already reported in a G x N F(2), was detected near the Gr gene. By separating markers from linkage groups 6 and 8 from the GN22 map, it was possible to compare the eight homologous linkage groups between the two maps using the 68 SSR markers heterozygous in both parents (anchor loci). All but one of these 68 anchor markers are in the same order in the Myrobalan plum map and in the almond-peach map, as expected from the high level of synteny within Prunus. The Ma and R(MiaNem)genes confirmed their previous location in the Myrobalan linkage group 7 and in the GN22 linkage group 2, respectively. Using a GN22 F(2) progeny of 78 individuals, a microsatellite map of linkage group 2 was also constructed and provided additional evidence for the telomeric position of R(MiaNem) in group 2 of the Prunus genome.

Nuclear genes from Tx CMS maintainer lines are unable to maintain atp6 RNA editing in any anther cell-type in the sorghum bicolor A3 cytoplasm.

RNA editing and cytoplasmic male sterility are two important phenomena associated with higher plant mitochondria. We recently have shown a potential function of RNA editing in CMS development. The frequency of atp6 RNA editing was specifically reduced in anthers of male-sterile Sorghum bicolor, which increased in frequency in partially restored progeny. Here we present data that show that the loss of RNA editing capability also occurs in a second nuclear background that allows the expression of male sterility. Loss of RNA editing thus appears to be associated with unique combinations of male-sterile cytoplasm and non-restoring nuclear backgrounds. In addition, the reduction of RNA editing affects both gametophytic and sporophytic anther cell-types but not other floral tissues. An analysis of F(2) plants exhibiting different levels of fertility indicates a co-segregation of fertility restoration and atp6 RNA editing. The atp6 transcript abundance is similar in seedlings and anthers of male-sterile, partially restored, and male-fertile lines and thus is not associated with loss of atp6 RNA editing in anthers. A model for RNA editing and male sterility based on the data available is presented. Functional correlations with other CMS systems are also discussed.

A unique two-gene gametophytic male sterility system in sorghum involving a possible role of RNA editing in fertility restoration.

The sorghum line IS1112C carries a male sterility-inducing cytoplasm when introduced into nuclear backgrounds that do not include fertility restoration genes. An mtDNA chimeric configuration resulting from recombination/duplication with atp9 resulted in the formation of orf107, a chimeric open reading frame. Transcription of orf107 is driven by three promoters, and abundant whole-length transcripts are detected in male-sterile lines. Fertility restoration is exacted through a unique two-gene gametophytic system requiring complementary action of genes designated Rf3 and Rf4. In male-sterile lines carrying Rf3, or lines restored to fertility, an enhanced nucleolytic transcript processing activity is targeted within orf107, cleaving 75% of whole-length transcripts. Rf3 thus confers or regulates the nucleolytic processing activity. A correlation between the frequency of RNA editing at two sites in orf107 and transcript processing suggests that processing may be dependent on templates edited at these sites. In addition, editing of atp6 transcripts is specifically reduced in anthers/pollen of male-sterile lines. Partially restored F1s and segregating F2s exhibit atp6 editing frequencies consistent with the possibility that Rf4 may confer the restitution of normal editing frequency. Thus RNA editing may be involved in features of fertility restoration in this unusual system.

Interaction of mitochondrial RNA editing and nucleolytic processing in the restoration of male fertility in sorghum.

Nucleolytic processing of transcripts within mitochondrial orf107, associated with male sterility in sorghum, is regulated by the fertility restoration gene Rf3, conferring 75% cleavage of whole-length transcripts. Two transcript editing sites are 81% and 61% edited in rf3rf3 lines, while these sites are 41% and 10% edited in the remaining whole-length transcripts in an Rf3Rf3 line. RNA editing and processing efficiency in F1 progeny were similar to the Rf3Rf3 parent, and analyses of backcross progeny indicated that all rf3rf3 lines were characterized by high editing efficiency. We postulate that highly edited transcripts within the population are quickly processed in lines carrying Rf3, generating a residual population of poorly edited transcripts. Thus, action of Rf3 may have no direct affect on RNA editing, and may be dependent on a substrate of highly edited transcripts. These data indicate a potentially novel role of RNA editing in gene expression through an influence on the efficiency of transcript processing.

Cell type-specific loss of atp6 RNA editing in cytoplasmic male sterile Sorghum bicolor.

RNA editing and cytoplasmic male sterility are two important phenomena in higher plant mitochondria. To determine whether correlations might exist between the two, RNA editing in different tissues of Sorghum bicolor was compared employing reverse transcription-PCR and subsequent sequence analysis. In etiolated shoots, RNA editing of transcripts of plant mitochondrial atp6, atp9, nad3, nad4, and rps12 genes was identical among fertile or cytoplasmic male sterile plants. We then established a protocol for mitochondrial RNA isolation from plant anthers and pollen to include in these studies. Whereas RNA editing of atp9, nad3, nad4, and rps12 transcripts in anthers was similar to etiolated shoots, mitochondrial atp6 RNA editing was strongly reduced in anthers of the A3Tx398 male sterile line of S. bicolor. atp6 transcripts of wheat and selected plastid transcripts in S. bicolor showed normal RNA editing, indicating that loss of atp6 RNA editing is specific for cytoplasmic male sterility S. bicolor mitochondria. Restoration of fertility in F1 and F2 lines correlated with an increase in RNA editing of atp6 transcripts. Our data suggest that loss of atp6 RNA editing contributes to or causes cytoplasmic male sterility in S. bicolor. Further analysis of the mechanism of cell type-specific loss of atp6 RNA editing activity may advance our understanding of the mechanism of RNA editing.

Mitochondrial RNA editing is sequence specific and independent of transcript abundance in Sorghum bicolor.

The DNA sequence which encodes the amino-terminal extension to the conserved core of the atp6-1 gene found in line IS1112C is absent from Tx398. Sequences further upstream are present in Tx398, but at a different genomic location. The atp6-2 genes are present in similar copy numbers in IS1112C and A3Tx398, a near-isogenic line carrying the IS1112C cytoplasm in a Tx398 background. However, transcript abundance of atp6-2 in these lines is about ten-times higher than that of atp6-1. RNA editing of atp6-1 transcripts is identical to that of atp6-2 and therefore sequence specific. A single non-silent editing site in the unique atp6-1 pre-piece sequence may indicate mitochondrial-guided RNA editing. While in Petunia the abundance and RNA editing of a transcript are correlated, we show here that RNA editing is independent of transcript abundance and is sequence specific in Sorghum.