Quantitative Trait Loci and Maternal Effects Affecting the Strong Grain Dormancy of Wild Barley (Hordeum vulgare ssp. spontaneum).

Wild barley (Hordeum vulgare ssp. spontaneum) has strong grain dormancy, a trait that may enhance its survival in non-cultivated environments; by contrast, cultivated barley (Hordeum vulgare ssp. vulgare) has weaker dormancy, allowing uniform germination in cultivation. Malting barley cultivars have been bred for especially weak dormancy to optimize their use in malt production. Here, we analyzed the genetic mechanism of this difference in seed dormancy, using recombinant inbred lines (RILs) derived from a cross between the wild barley accession 'H602' and the malting barley cultivar 'Kanto Nakate Gold (KNG)'. Grains of H602 and KNG harvested at physiological maturity and dried at 30°C for 7 days had germination of approximately 0 and 100%, respectively. Analysis of quantitative trait loci (QTL) affecting grain dormancy identified the well-known major dormancy QTL SD1 and SD2 (located near the centromeric region and at the distal end of the long arm of chromosome 5H, respectively), and QTL at the end of the long arm of chromosome 4H and in the middle of the long arm of chromosome 5H. We designated these four QTL Qsd1-OK, Qsd2-OK, Qsdw-4H, and Qsdw-5H, and they explained approximately 6, 38, 3, and 13% of the total phenotypic variation, respectively. RILs carrying H602 alleles showed increased dormancy levels for all QTL. The QTL acted additively and did not show epistasis or QTL-environment interactions. Comparison of QTL locations indicated that all QTL except Qsdw-5H are likely the same as the QTL previously detected in the doubled haploid population from a cross between the malting cultivar 'Haruna Nijo' and 'H602.' We further examined Qsd2-OK and Qsdw-5H by analyzing the segregation of phenotypes and genotypes of F2 progenies derived from crosses between RILs carrying specific segments of chromosome 5H from H602 in the KNG background. This analysis confirmed that the two genomic regions corresponding to these QTL are involved in the regulation of grain dormancy. Germination tests of F1 grains derived from reciprocal crosses between H602 and KNG revealed that the H602 strong dormancy phenotype shows maternal inheritance with incomplete dominance. These results provide new insight into the mechanisms regulating grain dormancy in barley.

Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat.

Evolution of the Grain Dispersal System in Barley.

About 12,000 years ago in the Near East, humans began the transition from hunter-gathering to agriculture-based societies. Barley was a founder crop in this process, and the most important steps in its domestication were mutations in two adjacent, dominant, and complementary genes, through which grains were retained on the inflorescence at maturity, enabling effective harvesting. Independent recessive mutations in each of these genes caused cell wall thickening in a highly specific grain "disarticulation zone," converting the brittle floral axis (the rachis) of the wild-type into a tough, non-brittle form that promoted grain retention. By tracing the evolutionary history of allelic variation in both genes, we conclude that spatially and temporally independent selections of germplasm with a non-brittle rachis were made during the domestication of barley by farmers in the southern and northern regions of the Levant, actions that made a major contribution to the emergence of early agrarian societies.

High-resolution genetic mapping and physical map construction for the fertility restorer Rfm1 locus in barley.

KEY MESSAGE: High-resolution genetic linkage mapping and BAC physical mapping narrowed the fertility restorer locus Rfm1 in barley to a sub-centimorgan genetic interval and a 208-kb physical interval. Rfm1 restores the fertility of msm1 and msm2 male-sterile cytoplasms in barley. The fertility restoration gene is located on the short arm of chromosome 6H (6HS), and we pursued a positional cloning of this gene. Starting from a previous result that has delimited Rfm1 within a 10.8 cM region on 6HS, we developed novel CAPS and SSR markers tightly linked to the gene in barley using the sequence information from the syntenic region of rice and barley genome assemblies. Next, we performed fine mapping of the Rfm1 locus. To isolate recombinants, we surveyed 3,638 F2 plants derived from a cross between the CMS strain and the Rf strain with adjacent markers (NAS2090 and NAS1080). This analysis identified 175 recombinant plants from the F2 population to build a high-resolution map with nine markers tightly linked to the Rfm1 locus. Rfm1 was located within the 0.14 cM region delimited by two markers (NAS9113 and NAS9200). Using these flanking markers as well as marker cosegregating with Rfm1 (NAS9133), we screened the BAC libraries of the cultivar Morex, an rfm1 carrier. We isolated 11 BAC clones and constructed a BAC physical map using their fingerprints. Finally, we delimited the Rfm1 locus encompassing the rfm1 allele on a 208-kb contig composed of three minimally overlapping BAC clones. This precise localization of the Rfm1 locus in the barley genome is expected to greatly accelerate the future map-based cloning of the Rfm1 gene by sequence analysis and its genetic transformation for the complementation of cytoplasmic male-sterile plants.

An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice.

Land plants have developed a cuticle preventing uncontrolled water loss. Here we report that an ATP-binding cassette (ABC) subfamily G (ABCG) full transporter is required for leaf water conservation in both wild barley and rice. A spontaneous mutation, eibi1.b, in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. Map-based cloning revealed that Eibi1 encodes an HvABCG31 full transporter. The gene was highly expressed in the elongation zone of a growing leaf (the site of cutin synthesis), and its gene product also was localized in developing, but not in mature tissue. A de novo wild barley mutant named "eibi1.c," along with two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves. HvABCG31 is hypothesized to function as a transporter involved in cutin formation. Homologs of HvABCG31 were found in green algae, moss, and lycopods, indicating that this full transporter is highly conserved in the evolution of land plants.

Cleistogamous flowering in barley arises from the suppression of microRNA-guided HvAP2 mRNA cleavage.

The cleistogamous flower sheds its pollen before opening, forcing plants with this habit to be almost entirely autogamous. Cleistogamy also provides a means of escape from cereal head blight infection and minimizes pollen-mediated gene flow. The lodicule in cleistogamous barley is atrophied. We have isolated cleistogamy 1 (Cly1) by positional cloning and show that it encodes a transcription factor containing two AP2 domains and a putative microRNA miR172 targeting site, which is an ortholog of Arabidopsis thaliana AP2. The expression of Cly1 was concentrated within the lodicule primordia. We established a perfect association between a synonymous nucleotide substitution at the miR172 targeting site and cleistogamy. Cleavage of mRNA directed by miR172 was detectable only in a noncleistogamous background. We conclude that the miR172-derived down-regulation of Cly1 promotes the development of the lodicules, thereby ensuring noncleistogamy, although the single nucleotide change at the miR172 targeting site results in the failure of the lodicules to develop properly, producing the cleistogamous phenotype.