Oat-maize chromosome addition lines: a new system for mapping the maize genome

Abstract

Novel plants with individual maize chromosomes added to a complete oat genome have been recovered via embryo rescue from oat (Avena sativa L., 2n = 6x = 42) x maize (Zea mays L., 2n = 20) crosses. An oat-maize disomic addition line possessing 21 pairs of oat chromosomes and one maize chromosome 9 pair was used to construct a cosmid library. A multiprobe (mixture of labeled fragments used as a probe) of highly repetitive maize-specific sequences was used to selectively isolate cosmid clones containing maize genomic DNA. Hybridization of individual maize cosmid clones or their subcloned fragments to maize and oat genomic DNA revealed that most high, middle, or low copy number DNA sequences are maize-specific. Such DNA markers allow the identification of maize genomic DNA in an oat genomic background. Chimeric cosmid clones were not found; apparently, significant exchanges of genetic material had not occurred between the maize-addition chromosome and the oat genome in these novel plants or in the cloning process. About 95% of clones selected at random from a maize genomic cosmid library could be detected by the multiprobe. The ability to selectively detect maize sequences in an oat background enables us to consider oat as a host for the cloning of specific maize chromosomes or maize chromosome segments. Introgressing maize chromosome segments into the oat genome via irradiation should allow the construction of a library of overlapping fragments for each maize chromosome to be used for developing a physical map of the maize genome.

Figure 1

Blot hybridization of a labeled multiprobe, composed of 22 maize-specific repetitive DNA sequences, to genomic DNA digests of 2 maize and 2 oat varieties. Strong signal is seen over lanes with maize DNA but not those with oat DNA. (a) EtdBr-stained 0.8% agarose gel after separation of DNA samples cut with EcoRI. (b) Autoradiogram after hybridization to the P-32 labeled multiprobe.

Figure 2

Blot panel of 29 maize-specific cosmid clones isolated from a cosmid library of an oat–maize chromosome 9 addition line. (a) Cosmids (–29) are cut by EcoRI restriction enzyme and size-fractionated in an 0.85% agarose gel stained with EtdBr (M = molecular weight marker, 1-kb ladder). (b) Labeled, total genomic maize DNA as a probe shows strong, medium, or weak hybridization to almost 90% of all EcoRI subfragments present in the cosmid clones. (c) Labeled multiprobe of highly repeated maize DNA sequences shows hybridization to from one to five EcoRI fragments in each lane.

Figure 3

Hybridization of unique and low-copy-number DNA sequences isolated from maize-specific cosmids shown in Fig. 2 to a blot panel of oat–maize chromosome addition lines carrying maize chromosomes 9, 7, 4, 3, and 2, respectively; m1 and m2 are maize stocks A188 and Seneca 60; o1 and o2 are oat stocks Sun II and Starter-1. (a) The 2.5-kb EcoRI fragment from cosmid 15 shows one band on the chromosome 9 addition line. An additional polymorphic band is present in the parental stocks of maize. (b) The 1.8-kb EcoRI fragment from cosmid 28 detects two bands. One band, polymorphic between m1 and m2, is present in the chromosome 9 addition line. An additional nonpolymorphic band is present on chromosome 9 and in both parental stocks. (c) The 2.1-kb EcoRI fragment from cosmid 10 shows one band on chromosome 9. (d) The 1.4-kb EcoRI fragment from cosmid 20 detects about 20 bands in parental maize stocks and several bands among the chromosome addition lines. The band pattern is chromosome-specific. No cross-hybridization occurred to oat DNA in a–d. (e) The 2.9-kb EcoRI fragment from cosmid 6 detects several polymorphic bands in parental maize stocks. One nonpolymorphic band is seen in maize, oat, and chromosomes 9, 7, 4, and 2 addition lines. Additional bands are seen in other chromosome addition lines.

Figure 4

Hybridization of medium and highly repetitive cloned maize DNA sequences to a blot panel of oat–maize chromosome addition lines carrying maize chromosomes 9, 7, 4, 3, and 2, respectively; m1 and m2 are maize stocks A188 and Seneca 60; o1 and o2 are oat stocks Sun II and Starter-1. (a) EtdBr-stained 0.8% agarose gel; lane M is a 1-kb molecular weight marker ladder. (b) The 0.7-kb EcoRI fragment of cosmid 10 shows multiple bands on different maize chromosomes as well as one common band for all chromosomes. (c) Cosmid 1, with a 40-kb insertion, shows strong hybridization signal over maize (lines m1 and m2) and over chromosome addition lines 9, 7, 4, 3, and 2; no hybridization is seen over oat DNA (lines o1 and o2). (d) With the 185-bp knob repeat, most of the hybridization signal is located on chromosome 9. No hybridization to oat DNA is detected.