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, 104 (4), 1424-9

Six-rowed Barley Originated From a Mutation in a Homeodomain-Leucine Zipper I-class Homeobox Gene

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Six-rowed Barley Originated From a Mutation in a Homeodomain-Leucine Zipper I-class Homeobox Gene

Takao Komatsuda et al. Proc Natl Acad Sci U S A.

Abstract

Increased seed production has been a common goal during the domestication of cereal crops, and early cultivators of barley (Hordeum vulgare ssp. vulgare) selected a phenotype with a six-rowed spike that stably produced three times the usual grain number. This improved yield established barley as a founder crop for the Near Eastern Neolithic civilization. The barley spike has one central and two lateral spikelets at each rachis node. The wild-type progenitor (H. vulgare ssp. spontaneum) has a two-rowed phenotype, with additional, strictly rudimentary, lateral rows; this natural adaptation is advantageous for seed dispersal after shattering. Until recently, the origin of the six-rowed phenotype remained unknown. In the present study, we isolated vrs1 (six-rowed spike 1), the gene responsible for the six-rowed spike in barley, by means of positional cloning. The wild-type Vrs1 allele (for two-rowed barley) encodes a transcription factor that includes a homeodomain with a closely linked leucine zipper motif. Expression of Vrs1 was strictly localized in the lateral-spikelet primordia of immature spikes, suggesting that the VRS1 protein suppresses development of the lateral rows. Loss of function of Vrs1 resulted in complete conversion of the rudimentary lateral spikelets in two-rowed barley into fully developed fertile spikelets in the six-rowed phenotype. Phylogenetic analysis demonstrated that the six-rowed phenotype originated repeatedly, at different times and in different regions, through independent mutations of Vrs1.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Map-based cloning of barley six-rowed spike gene vrs1. (A) Two-rowed spike. (B) Six-rowed spike. (C–G) One central and two lateral spikelets at a rachis node. (C) Ethiopian landrace var. deficiens; rudimentary lateral spikelets (Vrs1.t). (D) Two-rowed cultivar var. distichon; sterile lateral spikelets (Vrs1.b). (E) Wild barley var. spontaneum; sterile lateral spikelets (Vrs1.b). (F) Wild barley var. proskowetzii; short-awned or tip-pointed lateral spikelets (Vrs1.p). (G) Six-rowed cultivar convar. vulgare; fully fertile and awned lateral spikelets (vrs1.a). (H and I) Staminate floret of lateral spikelet (H) and hermaphroditic floret (I) in central spikelet in Vrs1.b two-rowed cultivar (D). (Scale bars: 2 mm.) (J) High-resolution linkage map and physical map. Six BAC clones (red) were fully sequenced. Open circles indicate markers uniquely assigned to chromosome 2H, of which genetically mapped markers are connected with the high-resolution map by dotted lines. Filled circles indicate repeated markers used for BAC connection. M669N11 and M185K11 are shown head-to-tail, separated by a vertical broken line.
Fig. 2.
Fig. 2.
Expression pattern of Vrs1 in two-rowed barley. (A) Expression of Vrs1 in immature inflorescences of different developmental stages (1–50 mm). Single-stranded cDNA synthesized from total RNA by using reverse transcriptase was subjected to RT-PCR analysis using primers specific to the 3′-UTR sequence. The barley actin gene was used as a control. (B–F) RNA in situ hybridization analysis of Vrs1. (B and C) Longitudinal serial sections along the row of lateral (B) and central (C) spikelet primordia at glume primordium stage. Red arrowheads in (B) indicate Vrs1 expression. (D–F) Transverse sections at double-ridge stage (D), triple-mound stage (E), and glume primordium stage (F). Red arrowheads indicate lateral spikelet primordia, and black arrowheads indicate central spikelet primordia. Broken lines in F correspond to longitudinal sectioning shown in B and C. (Scale bars: B and C, 500 μm; D–F, 100 μm.)
Fig. 3.
Fig. 3.
Analysis of mutants allelic to vrs1. (A) Lesions at Vrs1 detected in 48 mutants. Arrows pointing down indicate amino acid substitutions, arrows pointing up with a solid line indicate new stop codons, and the three arrows pointing up with a broken line indicate single-nucleotide substitutions in the introns with a changed splicing. The arrowheads and horizontal broken lines indicate deletions, in which five mutants have a partial deletion and seven mutants have a complete deletion of Vrs1. (B) RT-PCR analysis of nine mutant lines (including a New Golden mutant, NG M13) that did not show any lesions on the Vrs1. Four two-rowed cultivars and a deletion mutant (hex-v.3) were included as positive and negative controls, respectively. NG, New Golden. Total RNA was extracted from immature inflorescences 2–3 mm long.
Fig. 4.
Fig. 4.
Molecular events occurred in six-rowed barley. (A) Haplotype analysis of the Vrs1 region in two- and six-rowed cultivars. (B) Phylogenetic tree of Vrs1 alleles illustrating the three independent origins of six-rowed barley. Three wild barley lines (“OUH” identifiers) are outgroups. (C) RT-PCR analysis of Vrs1 expression in immature inflorescences of six-rowed and two-rowed barley.

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