Is barley chromosome 4H dicentric?

Is barley chromosome 4H dicentric?: A cytological evidence with common wheat lines carrying deleted 4H chromosomes

Masaya Sakata, Shuhei Nasuda, and Takashi R. Endo

Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Corresponding author: Takashi R. Endo

E-mail: trendo@kais.kyoto-u.ac.jp

We established 60 dissection lines of common wheat carrying segments of a barley chromosome 4H by the gametocidal (Gc) system (Sakata et al. 2010). Although we obtained several additional lines carrying Gc-induced 4H telosomes in the study, we did not use them for PCR analysis because we thought regular 4H telosomes isolated by Islam et al. (1981) would represent those 4H telosomes. However, we noticed that the regular 4HS telosome has a gap near the proximal end (Sakata et al. 2010; Fig. 1B). Among the Gc-induced 4HS telosomes, we found one without the gap (4H-11), and this telosome appeared to be truncated at the centromeric region (Fig. 1E). We examined the centromeric structure of the 4H-11 chromosome by fluorescence in situ hybridization (FISH) using a probe of the barley-specific repetitive sequence (AGGGAG)n reported by Hudakova et al. (2001), together with genomic in situ hybridization (GISH). The FISH analysis showed that the regular 4HS chromosome had a mass of the repeat sequence but that the 4H-11 chromosome had no (AGGGAG)n repeats (Fig. 1F). Sakata et al. (2010) found one EST marker (k04424) that is around the centromeric region; it is absent in the regular 4HL addition line but present in a Gc-induced 4H deletion chromosome 4H-31 that has a small piece of centromeric C-band heterochromatin of the short arm (Fig. 1D). Using this EST marker, we conducted PCR analysis with the line carrying the 4H-11 chromosome and found that the k04424 marker was present in the 4H addition, the regular 4HS addition and 4H-11 lines, but not in the 4HL addition line (Fig. 2). The 4H-11 chromosome was stable in mitosis and meiosis, so that we could establish a stable disomic 4H-11 addition line of a common wheat. This fact indicated that the 4H-11 chromosome retained kinetochore function. Fig. 3 illustrates the above-mentioned results. The breakpoints of the regular 4HS and 4HL telosomes are in the centromeric region adjacent to the to the (AGGGAG)n repeats in the long arm (see Fig. 1C). The breakpoints of the 4H-11 and 4H-31 chromosomes are in the most proximal 4HS C-band heterochromatin, proximal and distal to the k04424 marker, respectively. Fig. 1B shows that the proximal gap in the regular 4HS telosome is the place where the (AGGGAG)n repeats are located. The stretched centromeric region, appears empty in Fig. 1B, is visible in the regular 4HS telosome. The distal terminal of the regular 4HS telosome looks heterochromatic, although there is no such heterochromatic region observed in the normal chromosome 4H (Fig. 1A). This might be due to the folding up of the stretched chromatin after centromere misdivision.

The above-mentioned results suggest that there are two functional centromeres in chromosome 4H. One centromere is distal to the (AGGGAG)n repeats in the short arm, the other is either distal or proximal to the (AGGGAG)n repeats in the long arm. The above conclusion is logical unless we assume the occurrence of an interstitial deletion in the centromeric region or of a neocentromere. Nasuda et al. (2005) reported that the (AGGGAG)n repeats flank the wide centromeric region of barley chromosomes, including chromosome 4H (Fig. 1A). Nasuda et al. (2005) also reported two telosomes of chromosome 7H lacking the (AGGGAG)n repeats, suggesting that the centromeric repetitive sequence is not necessary relevant to the function of kietochore. The 4H-11 chromosome reported here is a second case of the irrelevance of the centromeric repetitive sequence and kietochore function in barley.

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