Sorghum bicolor - an important species for comparative grass genomics and a source of beneficial genes for agriculture

A high-resolution genetic, physical, and cytological map of the sorghum genome is being assembled using AFLP DNA marker technology, six-dimensional pooling of BAC libraries, cDNA mapping technology, and cytogenetic analysis. Recent advances in sorghum comparative genomics and gene-transfer technology are accelerating the discovery and utilization of valuable sorghum genes and alleles.     

Phytochrome B and shade signals regulate phytochrome A expression

The control of phytochrome A expression at the protein and mRNA levels was investigated in wild-type and phyB-1 mutant sorghum ( Sorghum bicolor [L.] Moench). PHYA mRNA abundance follows a diurnal rhythm in both genotypes, with maximal accumulation near the latter part of the light period. PHYA mRNA is more abundant in the phyB-1 mutant. The level of PHYA message correlates with both R : FR and photon flux density in wild-type, but only with photon flux density in the phyB-1 mutant. The differences in mRNA abundance are reflected in the level of phyA protein, which is elevated in the phyB-1 mutant and accumulates under low photon flux density. During de-etiolation, PHYA message accumulation is initially repressed solely by a very low fluence response (VLFR) presumably mediated by phyA. The phyB-mediated low fluence response maintains the repression of accumulation past the time controlled by the VLFR. With repetitive photoperiods, the transition from the etiolated growth form to autotrophic competency is accompanied by a transition from light-induced reduction of PHYA mRNA abundance to enhanced accumulation during the light period. The loss of phyB function allows partial de-repression of PHYA message accumulation under repetitive photoperiods, resulting in plants deficient in phyB but enriched in phyA. The modification of PHYA mRNA and protein levels in the phyB-1 mutant documented in this study may help clarify the molecular basis of the phyB-1 phenotype. The tailoring of phyA abundance in wild-type to the time of day and shade signals suggests a plastic role for this pigment in controlling development in light-grown plants.     

Integrated karyotyping of sorghum by in situ hybridization of landed BACs.

The reliability of genome analysis and proficiency of genetic manipulation are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, and orientation with respect to telomeres. We have endeavored to establish means to enable these steps in sorghum (Sorghum bicolor (L.) Moench), the genome of which contains ca. 780 Mbp spread across n = 10 chromosomes. Our approach relies on fluorescence in situ hybridization (FISH) and integrated structural genomic resources, including large-insert genomic clones in bacterial artificial chromosome (BAC) libraries. To develop robust FISH probes, we selected sorghum BACs by association with molecular markers that map near the ends of linkage groups, in regions inferred to be high in recombination. Overall, we selected 22 BACs that encompass the 10 linkage groups. As a prelude to development of a multiprobe FISH cocktail, we evaluated BAC-derived probes individually and in small groups. Biotin- and digoxygenin-labeled probes were made directly from the BAC clones and hybridized in situ to chromosomes without using suppressive unlabelled C0t-1 DNA. Based on FISH-signal strength and the relative degree of background signal, we judged 19 BAC-derived probes to be satisfactory. Based on their relative position, and collective association with all 10 linkage groups, we chose 17 of the 19 BACs to develop a 17-locus probe cocktail for dual-color detection. FISH of the cocktail allowed simultaneous identification of all 10 chromosomes. The results indicate that linkage and physical maps of sorghum allow facile selection of BAC clones according to position and FISH-signal quality. This capability will enable development of a high-quality molecular cytogenetic map and an integrated genomics system for sorghum, without need of chromosome flow sorting or microdissection. Moreover, transgeneric FISH experiments suggest that the sorghum system might be applicable to other Gramineae.     

Molecular mapping of the rf1 gene for pollen fertility restoration in sorghum (Sorghum bicolor L.)

We report the molecular mapping of a gene for pollen fertility in A1 (milo) type cytoplasm of sorghum using AFLP and SSR marker analysis. DNA from an F₂ population comprised of 84 individuals was screened with AFLP genetic markers to detect polymorphic DNAs linked to fertility restoration. Fifteen AFLP markers were linked to fertility restoration from the initial screening with 49 unique AFLP primer combinations (+3/+3 selective bases). As many of these AFLP markers had been previously mapped to a high-density genetic map of sorghum, the target gene (rf1) could be mapped to linkage group H. Confirmation of the map location of rf1 was obtained by demonstrating that additional linkage group-H markers (SSR, STS, AFLP) were linked to fertility restoration. The closest marker, AFLP Xtxa2582, mapped within 2.4 cM of the target loci while two SSRs, Xtxp18 and Xtxp250, flanked the rf1 locus at 12 cM and 10.8 cM, respectively. The availability of molecular markers will facilitate the selection of pollen fertility restoration in sorghum inbred-line development and provide the foundation for map-based gene isolation. Received: 22 August 2000 / Accepted: 18 October 2000     

Genetic Regulation of Development in Sorghum bicolor (X. Greatly Attenuated Photoperiod Sensitivity in a Phytochrome-Deficient Sorghum Possessing a Biological Clock but Lacking a Red Light-High Irradiance Response)

The role of a light-stable, 123-kD phytochrome in the biological clock, in photoperiodic flowering and shoot growth in extended photoperiods, and in the red light-high irradiance response was studied in Sorghum bicolor using a phytochrome-deficient mutant, 58M (ma3R ma3R), and a near-isogenic wild-type cultivar, 100M (Ma3 Ma3). Since chlorophyll a/b-binding protein mRNA and ribulose bisphosphate carboxylase small subunit mRNA cycled in a circadian fashion in both 58M and 100M grown in constant light, the 123-kD phytochrome absent from 58M does not appear necessary for expression or entrainment of a functional biological clock. Although 58M previously appeared photoperiod insensitive in 12-h photoperiods, extending the photoperiod up to 24 h delayed floral initiation for up to 2 weeks but did not much affect shoot elongation. Thus, although 58M flowers early in intermediate photoperiods, a residual photoperiod sensitivity remains that presumably is not due to the missing 123-kD phytochrome. Since rapid shoot elongation persists in 58M under extended photoperiods despite delayed floral initiation, long photoperiods uncouple those processes. The observed absence of a red light-high irradiance response in 58M, in contrast to the presence of the response in 100M, strengthens the suggestion that the 123-kD phytochrome missing from 58M is a phyB.     

High Throughput BAC DNA Isolation for Physical Map Construction of Sorghum (Sorghum bicolor)

With the aim of constructing a physical map of sorghum, we developed a rapid, high throughput approach for isolating BAC DNA suitable for restriction endonuclease digestion fingerprinting, PCR- based STS-content mapping, and BAC-end sequencing. The system utilizes a programmable 96 channel liquid handling system and associated accessories that permit bacterial cultivation and DNA isolation in 96-well plate format. This protocol details culture conditions that optimize bacterial growth in deep-well plates and criteria for BAC DNA isolation to obtain high yields of quality BAC DNA. The system is robust, accurate, and relatively cost-effective. The BAC DNA isolation system has been tested during efforts to construct a physical map of sorghum.     

Lipoxygenase gene expression is modulated in plants by water deficit, wounding, and methyl jasmonate

Summary Two classes of lipoxygenase (LOX) cDNAs, designated loxA and loxB, were isolated from soybean. A third lipoxygenase cDNA, loxP1, was isolated from pea. The deduced amino acid sequences of loxA and loxB show 61–74% identity with those of soybean seed LOXs. loxA and loxB mRNAs are abundant in roots and non-growing regions of seedling hypocotyls. Lower levels of these mRNAs are found in hypocotyl growing regions. Exposure of soybean seedlings to water deficit causes a rapid increase in loxA and loxB mRNAs in the elongating hypocotyl region. Similarly, loxP1 mRNA levels increase rapidly when pea plants are wilted. loxA and loxB mRNA levels also increase in wounded soybean leaves, and these mRNAs accumulate in soybean suspension cultures treated with 20 M methyl jasmonate. These results demonstrate that LOX gene expression is modulated in response to water deficit and wounding and suggest a role for lipoxygenase in plant responses to these stresses.