New semi-dwarfing alleles with increased coleoptile length by gene editing of gibberellin 3-oxidase 1 using CRISPR-Cas9 in barley (Hordeum vulgare L.).

The green revolution was based on genetic modification of the gibberellin (GA) hormone system with "dwarfing" gene mutations that reduces GA signals, conferring shorter stature, thus enabling plant adaptation to modern farming conditions. Strong GA-related mutants with shorter stature often have reduced coleoptile length, discounting yield gain due to their unsatisfactory seedling emergence under drought conditions. Here we present gibberellin (GA) 3-oxidase1 (GA3ox1) as an alternative semi-dwarfing gene in barley that combines an optimal reduction in plant height without restricting coleoptile and seedling growth. Using large-scale field trials with an extensive collection of barley accessions, we showed that a natural GA3ox1 haplotype moderately reduced plant height by 5-10 cm. We used CRISPR/Cas9 technology, generated several novel GA3ox1 mutants and validated the function of GA3ox1. We showed that altered GA3ox1 activities changed the level of active GA isoforms and consequently increased coleoptile length by an average of 8.2 mm, which could provide essential adaptation to maintain yield under climate change. We revealed that CRISPR/Cas9-induced GA3ox1 mutations increased seed dormancy to an ideal level that could benefit the malting industry. We conclude that selecting HvGA3ox1 alleles offers a new opportunity for developing barley varieties with optimal stature, longer coleoptile and additional agronomic traits.

Effects of dwarfing allele sd1-d originating from 'Dee-geo-woo-gen' and its tall alleles SD1-in and SD1-ja on morphological characteristics concerning dry-matter production and photosynthesis on the genetic background of indica-rice IR36.

sd1-d has been utilized to develop short-culmed indica varieties adaptable to higher fertilizer-applications. Its tall alleles SD1-in and SD1-ja are harbored in indica and japonica subspecies, respectively. SD1-in possesses a higher effect on elongating culm than SD1-ja. The sd1-d of indica IR36 was substituted with SD1-in or SD1-ja through recurrent backcrossing with IR36, and two tall isogenic lines ("5867-36" and "Koshi-36") were developed. IR36, 5867-36 and Koshi-36 were grown in a paddy field, and the effects of sd1-d, SD1-in and SD1-ja on morphological characteristics concerning dry-matter production and photosynthesis were compared mutually. sd1-d diminished dry weight of total brown rice/m2 and total dry matter weights, but enhanced harvest indexes, compared with SD1-in. In IR36, shorter lengths of the first (flag) to third leaves, and more panicle-bearing stems, caused by sd1-d, compared with SD1-in-carrying 5867-36, and erect first leaves, not caused by sd1-d, could construct the canopy structure appropriate for obtaining a high rate of photosynthesis at an optimum LAI. Koshi-36 could be used for a mid-mother line to develop indica varieties adaptable to middle and low fertilizer-applications, due to higher effect of SD1-ja on yielding ability, compared with that of sd1-d, no breaking-type lodging, and resistances to diseases and pests.

Effects of tall alleles SD1-in and SD1-ja to the dwarfing allele sd1-d originating from 'Dee-geo-woo-gen' on yield and related traits on the genetic background of indica IR36 in rice.

sd1-d originating from 'Dee-geo-woo-gen' has been utilized to develop short-culmed indica varieties adaptable to higher fertilizer-application. Its tall alleles SD1-in and SD1-ja are harbored in indica and japonica subspecies, respectively. The sd1-d of indica IR36 was substituted with SD1-in or SD1-ja by recurrent backcrossing with IR36, and two tall isogenic lines ("5867-36" and "Koshi-36") were developed. IR36, 5867-36 and Koshi-36 were grown in a paddy field in three years, and yield and related traits were measured, the effects of SD1-in and SD1-ja on yielding ability and related characteristics were examined on the genetic background of IR 36. SD1-in decreased panicle number per m2 but increased spikelet number per panicle, ripened-grain percentage and 1000-grain weight, compared with sd1-d, resulting in the increase of yield. The increase of 1000-grain weight by SD1-in, caused by the increases of length, width and thickness of grain, was due to the increases of the length and width of lemma. SD1-ja did not significantly affect yield, mainly because the decrease of panicle number per m2 was compensated by the enlarged 1000-grain weight owing to the increase of lemma length. Serious lodging was observed in long-culmed 5867-36, suggesting that sd1-d is indispensable for indica breeding programs.

Resolution of quantitative resistance to clubroot into QTL-specific metabolic modules.

Plant disease resistance is often under quantitative genetic control. Thus, in a given interaction, plant cellular responses to infection are influenced by resistance or susceptibility alleles at different loci. In this study, a genetic linkage analysis was used to address the complexity of the metabolic responses of Brassica napus roots to infection by Plasmodiophora brassicae. Metabolome profiling and pathogen quantification in a segregating progeny allowed a comparative mapping of quantitative trait loci (QTLs) involved in resistance and in metabolic adjustments. Distinct metabolic modules were associated with each resistance QTL, suggesting the involvement of different underlying cellular mechanisms. This approach highlighted the possible role of gluconasturtiin and two unknown metabolites in the resistance conferred by two QTLs on chromosomes C03 and C09, respectively. Only two susceptibility biomarkers (glycine and glutathione) were simultaneously linked to the three main resistance QTLs, suggesting the central role of these compounds in the interaction. By contrast, several genotype-specific metabolic responses to infection were genetically unconnected to resistance or susceptibility. Likewise, variations of root sugar profiles, which might have influenced pathogen nutrition, were not found to be related to resistance QTLs. This work illustrates how genetic metabolomics can help to understand plant stress responses and their possible links with disease.

Genetic mapping and a new PCR-based marker linked to a dwarfing gene in oat (Avena sativa L.).

Short straw is a desired trait in cultivated hexaploid oat (Avena sativa L.) for some production environments. Marker-assisted selection, a key tool for achieving this objective, is limited by a lack of mapping data and available markers. Here, bulked-segregant analysis was used to identify PCR-based markers associated with a dwarfing gene. Genetic analysis identified a monogenic dominant inheritance of one dwarfing gene from WAOAT2132, temporarily designated DwWA. A simple sequence repeat (SSR) marker (AME117) that was already available and a new codominant PCR-based marker (bi17) developed by homologous cloning in the present study were both associated with the dwarfing gene. The two markers were located 21 and 1.2 cM from DwWA, respectively. The bi17 marker was mapped to neighboring SNP markers on chromosome 18D of the oat consensus map. Since Dw6 was previously mapped on chromosome 18, and since our new marker bi17 is also diagnostic for NILs generated for Dw6, there is strong evidence that the dwarfing gene identified in WAOAT2132 is Dw6. The newly developed markers could find applications in the identification of this gene in oat germplasm and in the fine mapping or positional cloning of the gene.

Identification of tillering node proteins differentially accumulated in barley recombinant inbred lines with different juvenile growth habits.

Barley (Hordeum vulgare L.) is an important cereal crop grown for both the feed and malting industries. The allelic dwarfing gene sdw1/denso has been used throughout the world to develop commercial barley varieties. Proteomic analysis offers a new approach to identify a broad spectrum of genes that are expressed in the living system. Two-dimensional electrophoresis and mass spectrometry were applied to investigate changes in protein abundance associated with different juvenile growth habit as effect of the denso locus in barley homozygous lines derived from a Maresi × Pomo cross combination. A total of 31 protein spots were revealed that demonstrate quantitative differences in protein abundance between the analyzed plants with different juvenile growth habit, and these protein spots were selected to be identified by mass spectrometry. Identification was successful for 27 spots, and functional annotations of proteins revealed that most of them are involved in metabolism and disease/defense-related processes. Functions of the identified proteins and their probable influence on the growth habit in barley are discussed.

Position Validation of the Dwarfing Gene Dw6 in Oat (Avena sativa L.) and Its Correlated Effects on Agronomic Traits.

An F6 : 8 recombinant inbred line (RIL) population derived from the cross between WAOAT2132 (Dw6) and Caracas along with the two parents were used to evaluate the genetic effects of Dw6 dwarfing gene on plant height and other agronomic traits in oat (Avena sativa L.) across three environments, and develop closely linked markers for marker-assisted selection (MAS) for Dw6. The two parents differed in all investigated agronomic traits except for the number of whorls. The RIL lines showed a bimodal distribution for plant height in all three tested environments, supporting the height of this population was controlled by a single gene. Dw6 significantly reduced plant height (37.66∼44.29%) and panicle length (13.99∼22.10%) but without compromising the coleoptile length which was often positively associated with the reduced stature caused by dwarfing genes. Dw6 has also strong negative effects on hundred kernel weight (14.00∼29.55%), and kernel length (4.21∼9.47%), whereas the effects of Dw6 on the kernel width were not uniform across three environments. By contrast, lines with Dw6 produced more productive tillers (10.11∼10.53%) than lines without Dw6. All these together suggested the potential yield penalty associated with Dw6 might be partially due to the decrease of kernel weight which is attributed largely to the reduction of kernel length. Eighty-one simple sequence repeat (SSR) primer pairs from chromosome 6D were tested, five of them were polymorphic in two parents and in two contrasting bulks, confirming the 6D location of Dw6. By using the five polymorphic markers, Dw6 was mapped to an interval of 1.0 cM flanked by markers SSR83 and SSR120. Caution should be applied in using this information since maker order conflicts were observed. The close linkages of these two markers to Dw6 were further validated in a range of oat lines. The newly developed markers will provide a solid basis for future efforts both in the identification of Dw6 in oat germplasm and in the determination of the nature of the gene through positional cloning.

Structural Characterization of ABCB1, the Gene Underlying the d2 Dwarf Phenotype in Pearl Millet, Cenchrus Americanus (L.) Morrone.

Pearl millet is an important food crop in arid and semi-arid regions of South Asia and sub-Saharan Africa and is grown in Australia and the United States as a summer fodder crop. The d2 dwarf germplasm has been widely used in the last half-century to develop high-performing pearl millet hybrids. We previously mapped the d2 phenotype to a 1.6 cM region in linkage group (LG) 4 and identified the ABCB1 gene as a candidate underlying the trait. Here, we report the sequence, structure and expression of ABCB1 in tall (D2D2) and d2 dwarf (d2d2) germplasm. The ABCB1 allele in d2 dwarfs differs from that in tall inbreds by the presence of two different high copy transposable elements, one in the coding region and the second located 664 bp upstream of the ATG start codon. These transposons were present in all d2 dwarfs tested that were reported to be of independent origin and absent in the analyzed wild-type tall germplasm. We also compared the expression profile of this gene in different organs of multiple tall and d2 dwarf inbreds, including the near-isogenic inbreds at the d2 locus, Tift 23B (D2D2) and Tift 23DB (d2d2). Heterologous transformation of the tall (Ca_ABCB1) and the d2 dwarf (Ca_abcb1) pearl millet alleles in the Arabidopsis double mutant abcb1abcb19 showed that the pearl millet D2 but not the d2 allele complements the Arabidopsis abcb1 mutation. Our studies also show the importance of the COOH-terminal 22 amino acids of the ABCB1 protein in either protein function or stability.

Pleiotropic effects of the sdw1 locus in barley populations representing different rounds of recombination

Background In the present study populations, representing different rounds of recombination were used for the analysis of phenotypic effects associated with the sdw1/denso locus. Other studies have mostly focused only on one type of population. Many different QTLs mapped at the same position as the sdw1/denso locus may indicate a pleiotropy of this gene or a tight linkage between genes conditioning quantitative traits. To date, results of studies have not unequivocally proven either of these two phenomena. Results Both breeding and molecular mapping experiments were undertaken to examine 200 single seed descent (SSD) and 60 doubled haploid (DH) lines obtained from the Maresi (with a semi-dwarfing gene) and Pomo cross combination. They were evaluated for the type of juvenile growth habit and certain agronomic traits were measured after harvesting. The estimates of mean values, standard errors and significance of effects were analyzed. In terms of the analyzed characteristics, the greatest variability was obtained for genotypes with the prostrate growth habit. Microsatellite markers (SSR) were also used to identify co-segregation with the sdw1/denso locus and Bmag0013, Bmag0877, Bmag0306b markers were linked the closest. A partial linkage map of chromosome 3H with the sdw1/denso semi-dwarfing gene was constructed and QTLs were identified. Conclusions Our experiments confirmed the impact of the semi-dwarfing gene on plant height, heading and flowering date both in SSD and DH populations, which may indicate pleiotropy. Moreover, a partial linkage between sdw1/denso locus and grain weight per spike and 1000-grain weight was found in the SSD population.

Identification of alternate dwarfing gene sources to widely used Dee-Gee-Woo-Gen allele of sd1 gene by molecular and biochemical assays in rice ( Oryza sativa L. )

After the success of IR8 and TN1, breeders depended heavily on these two rice cultivars for source of short stature led to the narrow genetic base to majority of present day rice varieties, as far as sd1 (semi-dwarf1) gene is concerned. In addition, analysis of genetic lineage of the majority of the cultivated rice varieties in tropical Asia reveals that sd1 from DGWG (Dee-Gee-Woo-Gen) is the major source of dwarfing gene. Such high amount of genetic homogeneity renders rice plants vulnerable to epidemic of diseases and insect pests. In the current study, we made an attempt to identify the alternate sources of DGWG allele of sd1 gene by characterizing 29 induced and 3 spontaneous dwarf accessions employing marker for DGWG allele of sd1 gene and exogenous application of gibberellic acid (GA3). When occurrence of DGWG allele of sd1 gene and GA3 response were analyzed together, existence of two kinds of dwarfs was noticed viz., dwarf accessions with DGWG allele and dwarf accessions without DGWG allele of sd1 allele exhibiting varying responses to GA3. As many as 22 of 32 dwarf accessions showed absence of DGWG allele of sd1 gene with varying response to GA3 could be used as excellent alternate sources for DGWG allele of sd1 gene. These dwarf accessions could be used for broadening the genetic base for the plant height and thereby minimize the risk of genetic vulnerability. Our strategy of combining molecular and biochemical assays can be efficiently used for identifying alternate dwarfing gene sources to the Green Revolution gene sd1.