Cloning And Functional Analysis Of Barley Semi-dwarf Gene Sdw1/Denso

Semi-dwarf genes have also been extensively explored in barley breeding programs and more than 30 types of them have been described. The sdwl/denso gene has been one of the most successful semi-dwarf genes used in Europe, American and Australia, while most of short straw Chinese barley cultivars carry the uzul gene. The main phenotypic effect of the semi-dwarf gene sdwl/denso is a 10-20 cm reduction of plant height, depending on environmental conditions. Besides, there are some deleterious effects associated with sdwl/denso gene, such as late heading and maturity, decreased thousand-grain weight. As for the relationship between grain yield and sdwl/denso, both positive and negative were reported depending on different genetic backgrounds. However, with its suitable semi-dwarf phenotype and potentially increased yield, sdwl/denso has been introduced to numerous cultivars. There are at least four independent alleles based on the allelism test done up to date. Interestingly, the denso mutant has been used in malting barley breeding, while the sdwl has been limited to use in feed barley breeding. Therefore, it is necessary to isolate sdwl/denso gene and elucidate its semi-dwarf mechanism, which could help for understanding its effect on agronomic traits and provide a tool to select this gene in barley breeding programs.1. Cloning the candidate gene for semi-dwarf gene sdwl/denso in barleyComparative genomic analysis revealed that the sdwl/denso gene was located in the syntenic region of the rice semi-dwarf gene sdl on chromosome 1. The sdl gene encodes a GA 20-oxidase. Part of barley sequence from the gene ortholog of rice sdl was isolated using polymerase chain reaction （PCR） and the gene was named as HvGA20ox2. The complete genome sequence of HvGA20ox2 （Mloc₅6462） was identified after BLAST against Gramene （http://www.gramene.org/） based on the sequence of HvGA20ox2 from PCR method. The barley and rice genes showed a similar gene structure consisting of three exons and two introns. The predicted protein of barley HvGA20x2 gene was more similar to wheat GA20ox2b and Aegilops GA20ox2 （94.1% and 95.7% identity, respectively）. One SNP and one InDel marker were developed in barley varieties Baudin and AC Metcalfe, respectively and Baudin is kwnon to contain the denso semi-dwarf gene. Both markers were co-segregated and mapped to chromosome 3HL in a doubled haploid （DH） population of Baudin× AC Metcalfe with 178 DH lines. Furthermore, plant height was associated with the markers in Baudin x AC Metcalfe DH population. These results suggested that sdwl/denso gene in barley is the most likely ortholog of the sdl in rice. Isolation of HvGA20ox2 will facilitate understanding of the molecular mechanism controlling semi-dwarf phenotype and provide a diagnostic marker for selection of semi-dwarf gene in barley.2. Expression level of HvGA20ox2 is associated with many agronomic and quality traits in barley.Semi-quantitative RT-PCR revealed that HvGA20ox2 was expressed at different levels in various organs of barley. Transcriptional levels were reduced in leaf blade, sheath, stem, rachis and unopened flower in the barley variety Baudin with the denso allele. Subsequently, the relative expression levels of HvGA20ox2 were determined by quantitative real-time PCR in the above-mentioned DH population and mapped as a quantitative trait. A single expression quantitative trait locus （eQTL） was identified and mapped to its structural gene region on chromosome 3H. The eQTL was co-located with QTLs for height, yield, development score, hectolitre weight and grain plumpness, indicating the many QTLs for agronomic traits identified in this study are due to pleiotropic effects of HvGA20ox2. QTL mapping and associated analysis showed that low expression level of HvGA20ox2 reduced plant height and improved grain yield in the DH population. Although sdwl/denso gene is related to low grain weight and high screening, the head length was similar between the semi-dwarf and tall genotypes. As a result, the semi-dwarf gene increased grain yield through increasing the number of effective tillers.3. Identification and functional analysis of different sdwl/denso allelesPCR method showed that HvGA20ox2 is lost in both sdwl the mutants Jotun and Riso no.9265. Sequence analysis revealed the denso allele is due to a 7 bp （GACTCCC） deletion in exon 1. The InDel-denso molecular marker was developed based on the 7 bp deletion and was used to distinguished denso and sdwl alleles. The marker was further tested in 535 barley varieties, and found that denso allele was used in the barley breeding of Gansu and Yunnan Province.In addition, a gene expression marker （GEM） was also developed to screen denso and sdwl alleles. After detected flanking gene of HvGA20ox2, different sources of sdwl alleles from Jotun and Riso no.9265, respectively, displayed distinct chromosome deletion in the HvGA20ox2 region. Riso no.9265 has a completely deleted Morex_contig₄0861 including both HvGA20ox2 and Mloc₅6463 genes, while Jotun had at least 5 genes deletion containing Morex_contig₄0861. Therefore, flanking genes of Morex_contig₄0861 can be used to identify Jotun and Riso no.9265 alleles. All of allelic analysis showed independent HvGA20ox2 mutations in sdwl/denso mutants, which confirmed that sdwl/denso was HvGA20oxzBioactive gibberellins are not only essential regulators for barley growth and development, but are also essential for malting and brewing. The 7 bp deletion in denso allele has an internal ATG site and possibly produces a truncated protein with conserved domains correlated with 2-oxoglutarate and GA substrate combination. As a result, the denso allele may still has partial function of GA 20-oxidase. On the other hand, the deletion of the HvGA20ox2 gene in sdwl allele indicates totally loss function of GA 20-oxidase. Therefore, the denso mutant is still widely acceptable as malting barley The sdwl mutant is doomed to be used in feed barley breeding due to its serious shortage of gibberellin.4. Functional analysis of HvGA20ox2Expression level of GA dioxygenase genes in stems of Bomi and Riso no.9265 were determined at the initial jointing stage. The relative mRNA expression of HvGA20ox1 and HvGA3ox2 were dramatically increased in the mutant Riso no.9265 compared with those of Bomi. On the other hand, the mRNA expression of HvGA2ox3 was decreased in the Riso no.9265. These results suggested a type of feedback regulation from the low bioactive GA because of the null mutation of HvGA20ox2. The GA-over-production phenotype was found in HvGA20ox2 transgenic Arabidopsis. The phenotype was characterized by elongated hypocotyl and stems, early flowering, and higher growth rate. In addition, barley GA20ox2 could recover AtGA20oxj lose-of-function mutant. HvGA20ox2 transgenic Arabidopsis had higher GA contents, especially for GA1+3 compared with the control. The increased GAs is associated with the changed phenotype. The transgenic lines over-expressing barley GA 20-oxidases also showed a decrease in AtGA20ox1, AtGA20ox2, AtGA3ox1 and an increase in AtGA2ox2 expression, while ga5-3 was just opposite in the expression of these genes, which could be attributed to feedback from an increased bioactive GAs in HyGA20ox2 over-expressing transgenic Arabidopsis or decreased bioactive GAs in ga5-3, respectively. These results suggest that HvGA20ox2 is the orthologous of AtGA20ox1 and acts as the oxidase at carbon-20 of GA biosynthetic pathway. Due to the deficiency of HvGA20ox2 and later heading in Riso no.9265, we deduced that the sdw1/denso allele itself affects heading date owing to its reduced endogenous GA concentration.