KEW, UK

Royal Botanic Gardens, Kew¹

SAINT PAUL, MINNESOTA, USA

University of Minnesota² and USDA-ARS³

Adding B-chromosomes of Zea mays L. to the genome of Avena sativa L.

––Kynast RG¹, Galatowitsch MW², Huettl PA², Phillips RL² and Rines HW³

B-chromosomes (Bs) are supernumerary dispensable chromosomes described in hundreds of animal and plant species, including maize (Zea mays L.). However, Bs have not been reported to exist in hexaploid oat (Avena sativa L.).

In order to transfer maize Bs sexually from maize to oat genomes, we chose the maize cultivar ‘Black Mexican Sweet’ (a well known sweet corn line hosting Bs in different numbers) as the B donor (male parent) and the oat cultivars ‘Starter’, ‘Sun II’ and ‘Paul’ as potential B recipients (female parent) for inter-species cross-hybridizations. Since all of these direct crossings of ‘Black Mexican Sweet’ to each of the oat cultivars failed to produce vigorous F₁-offspring, we used in a further experimental series as the male parent a backcross line of the maize inbred 'B73' harboring Bs from ‘Black Mexican Sweet’. The ‘B73^B’ derivative is the 5th backcross generation of the F₁ (‘B73’ ´ ‘Black Mexican Sweet’) hybrid to ‘B73’. BC₅-seeds with hexasomic B addition (BC₅-B73^B, 2n = 2x+6_B = 26) were generously provided by JA Birchler, University of Missouri Columbia. This genotype based on ‘B73’ germplasm seemed more promising because, recently, Kynast et al. (2004, PNAS 101: 9921-9926) had reported the successful crossing of ’B73’ without Bs to different oat genotypes.

All parental plants were cultivated in growth chambers delivering favorable environmental conditions for germination and plant growth and to synchronize the peak of pistil receptiveness in oat plants with the peak of pollen grain release in maize plants. For inter-species crossing, the stigmas of emasculated oat florets were hand-pollinated with freshly shed maize pollen by using a fine camelhair brush. The panicles with pollinated florets were isolated in glassine bags, then 24-48 hours after pollination sprayed with a mixture of 50 ppm 2,4-D and 50 ppm GA₃ and again bagged during further cultivation in the growth chambers.

From 2341 ovaries, 115 immature F₁ (oat ´ maize) embryos (Table 1) were in vitro rescued 14-15 days after pollination. The F₁-embryos were cultivated on modified one-half strength MS-medium. A total of 31 F₁-embryos germinated and developed into vigorous plantlets large enough for molecular and cytogenetic analyses. Plantlets were further grown in growth chambers with optimal growing conditions to produce F₂-seeds for testing (1) fertility of (aneu)haploid oat plants with added maize Bs and (2) transmission of maize Bs to the offspring in an oat background.

Two F₁-plantlets (5811-1 and 5845-1) were found to have retained maize chromosomes in shoot tissues based on results from a PCR assay for ‘Grande1’, a dispersed LTR-type retrotransposon which is abundant on all A-chromosomes (As) and Bs of maize but absent from all chromosomes of the oat genotypes used in our crossing program. PCR assays involving two B-specific markers (primer pair p-2ndb1 + p-2ndb4 and primer pair p-brpt2 + p-taralb1; generously provided by JA Birchler, University of Missouri Columbia) and a selected set of A-specific markers for maize [chromosome arm-specific SSR markers selected from the ‘Maize Genetics and Genomics Database’ (http://www.maizegdb.org/)] showed that in both genotypes the Grande1-positive PCR products represented the presence of maize Bs and not maize As (Figure 1).

Cytological analyses on very young, juvenile plantlets revealed that in the F₁-plant 5811-1 all ten maize As had been eliminated and a complete set of 21 oat chromosomes plus three maize Bs (2n = 3x+3_B = 24) were retained in its primary root meristem. In the primary root meristem of the F₁-plant 5845-1 all ten maize As had been eliminated with a complete set of 21 oat chromosomes and a single maize B retained (2n = 3x+1_B = 22).

Both F₁-plants were kept under short-day conditions to allow plants to tiller extensively for continuing tiller cloning. Tiller cloning provides an extended source of leaves for the extraction of genomic DNA and RNA, and for further seed production. Both genotypes are descended from a (‘Starter’ ´ ‘B73^B’) cross. Hence they represent B additions in a ‘Starter’ background. The phenotypes of both mature F₁-plants did not differ from those of haploids of ‘Starter’ without Bs at any time point during their growth period.

After shifting individual tiller clones into long-day growing conditions, self-pollination produced F₂-seed in both genotypes (Table 1). This fertility could be attributed to the frequent formation of numerically unreduced female and male gametes. High fertility had already been observed in oat haploids of ‘Starter’, ‘Sun II’ and ‘Paul’ without and with individual maize As of ‘B73’ (Rines et al. 1997, In: Jain, Sopory, Veilleux (eds) Kluwer Acad Publishers, Dordrecht, The Netherlands, In vitro haploid production in higher plants 4, 205-221; Kynast et al. 2004, PNAS 101: 9921-9926).

Cytological and molecular analyses of 20 F₂-offspring plants showed that the F₁-plant 5811-1 carrying 3 Bs produced three F₂-plants with 1 B, six F₂-plants with 2 Bs, one F₂-plant with 3 Bs, one F₂-plant with 4 Bs, and nine F₂-plants with highly chimeric root meristems showing cells with 1-5 Bs in different frequencies. All chromosome counts were based on ten cells of root meristems of each F₂-offspring. The presence of Bs in root meristem cells was visualized by GISH at high (85%) stringency using Alexa Fluor 488-labeled genomic DNA of maize as the probe without oat competitor DNA (Figure 2).

For the F₁-plant 5845-1 carrying one B none of the 10 F₂-offspring tested by cytological and molecular means had Bs, indicating a transmission failure. However, since more F₂-seeds were produced, we will analyze a larger offspring population of F₁-plant 5845-1 to evaluate the transmission data.

Taking all the data together, our results showed that Bs from maize ‘Black Mexican Sweet’ can be sexually transferred to the genome of ‘Starter’ oat, and that haploid oat plants hosting one or three maize Bs are fertile and can set seed after self-pollination. In addition, our results of 30 tested F₂-offspring from two maize B-positive F₁-plants showed that maize Bs can be transmitted to offspring even when in the presence of only oat chromosomes.

Table 1: Plant material for crossing three different oat cultivars (2n = 6x = 42) by the maize ‘B73^B’ (2n = 2x+6_B = 26) and results of maize B-positive offspring production

Figure 1: PCR products of B-chromosome-specific markers after electrophoresis in 1.5% agarose; both markers demonstrate the presence of B-chromatin in the two F₁-plants 5811-1 and 5845-1 and the absence of B-chromatin in two examples of B-negative F₁-plants (5845-2 and 5846-1)

Figure 2: Root prometaphase cell from the F₂-plant K1188; the tetrasomic addition of maize B-chromosomes to ‘Starter’ oat is demonstrated by green fluorescence after GISH with Fluor 488-labeled genomic DNA of maize well contrasted against the red-brown counterstained oat chromosomes