1. Physical Mapping of the ASGR-Carrier Chromosome Reveals Collinearity to Chromosome 2 of Sorghum and Foxtail Millet Outside the ASGR Sirjan K. Sapkota, Peggy Ozias-Akins, Joann A. Conner Department of Horticulture, UGA
Introduction
Gametophytic apomixis is a process of asexual reproduction through seeds in which embryos are produced from egg cells of ovules without meiosis and fertilization of gametes. Endosperm development in apomictic seed is either autonomous or by fertilization of the polar nuclei. Since progeny from apomictic plants are genetically identical to the maternal plant, apomixis holds as a promising mechanism to fix hybrid vigor in crop species. Gametophytic apomixis can be further subdivided into diplospory and apospory1.
Apospory in Pennisetum squamulatum and Cenchrus ciliaris is controlled by a genetic locus transmitted as a large (>50 Mb), hemizygous, heterochromatic chromosomal block called apospory-specific genomic region (ASGR)2-4. In P. squamulatum, the ASGR is located near the telomere on the short arm of the ASGR-carrier chromosome (ASGR-cc)3.
Sequence analysis of partially sequenced ASGR-linked BAC clones showed the presence of multiple regions of microsynteny but did not exhibit any large-scale collinearity with the rice or sorghum genome sequences5.
Fluorescence in situ hybridization (FISH) identified a backcross 8 (BC8) line 58 plant, originating from a cross between P. squamulatum and P. glaucum, to contain only the ASGR-cc from P. squamulatum6.
Ovule transcriptome analysis comparing sequences from apomictic BC8 line 58 and P. squamulatum identified 49 ASGR-cc specific transcripts. Only one of the 49 ovule transcripts mapping to the ASGR-cc mapped to the ASGR using SCAR markers. SCAR polymorphism was not detected for the remaining transcripts7.
A comparative genomic hybridization (CGH) experiment using apomictic and sexual BC8 line 58 DNA identified enrichment in sorghum chromosome 2 for the apomictic sample (Unpublished; Figure 1). In addition, BlastN analysis of the ASGR-cc transcripts to the sorghum and foxtail millet genomic sequences exhibited highest similarity to chromosome 2 in sorghum and foxtail millet in a collinear fashion.
e. p036L06
d. p258L05
c. p236E19
b. p142D19
h. p285J18
f. p220A02
g. Inverted DAPI of ‘f’
i. p181G17
Figure 2. Physical location of the ASGR-cc linked BACs on the ASGR-cc. a, Inset shows a separate image of the ASGR-cc for p057M05. Figure b-i, shows only the ASGR-cc. White and yellow arrows represent the ASGR and centromere on the ASGR-cc, respectively. Red arrows show the signals (green) from the ASGR-cc linked BAC clones (captioned below image).
a. p057M05
Centromere h f i e c a b
Sorghum 2 (~80 Mb)
Chromosome 2 peak
ASGR
ASGR-cc (~200 Mb)
Foxtail millet 2 (~50 Mb) h f i e d c a b
Figure 1. Graph showing enrichment of apomictic but not sexual signal on sorghum chromosome 2 during CGH analysis.
Figure 3. Collinear alignment between ASGR-cc and chromosome 2 of sorghum and foxtail millet. Letters indicate relative position of putative orthologous transcribed genes (linked to the BACs in figure 2) in sorghum and foxtail millet chromosome 2.
References
Ozias-Akins, P Critical Reviews in Plant Sciences. 25:
Ozias-Akins et al PNAS. 95 (9):
Akiyama et al Plant Physiology. 134:
Akiyama et al Theor. Appl. Genet. 111(6):
Conner et al Plant Physiology. 147(3):
Singh et al Crop Science. 50(3):
Zeng et al BMC Genomics. 12:206.
Fukui, K pp Plant Chromosome Laboratory methods. CRC Press, Boca Raton, FL.
Kirov et al Molecular Cytogenetics. 7(1): 1-20.
Zhong et al Plant Molecular Biology Reporter. 14(3):
Results and conclusion
40 and 44 out of 49 ASGR-cc transcripts showed highest BlastN similarity
(>e-20) to chromosome 2 of sorghum and foxtail genome sequences, respectively.
Seven low copy, one medium copy (p142D19) and one high copy (p181G17) ASGR-cc linked BACs were mapped to the ASGR-cc through dual-color FISH.
Two BACs (p285J18 and p220A02) were mapped to the short-arm (ASGR arm), five BACs (p036L06, p258L05, p236E19, p057M05 and p142D19) were mapped on the long arm (non-ASGR arm), and one BAC (p181G17) gave multiple signals around the centromere of the ASGR-cc (Figure 2).
The mapping of the ASGR-cc linked BACs showed macro-collinearity with sorghum and foxtail millet chromosome 2, outside the ASGR (Figure 3).
In conclusion, the ASGR-carrier chromosome is evolutionarily similar to chromosome 2 of sorghum and foxtail millet outside of the ASGR.
Materials and Methods
Plant material: root tips were collected from apomictic BC8 line 58.
BlastN search was done using ovule transcripts7 against sorghum and foxtail millet reference genomic sequences.
SCAR markers7 from the ASGR-cc were used to identify ASGR-cc linked BAC clones.
Chromosome spreads were prepared by enzyme maceration/air-drying8 or “SteamDrop”9 method.
Physical mapping was done by dual-color FISH with ASGR and ASGR-cc linked BAC clones according to Zhong et al (1996)10 with modifications.
Probes were detected using Texas Red and FITC-conjugated antibodies.