Boron (B) toxicity is a significant constraint to cereal production in regions worldwide, including parts of southern Australia. In recent years, much progress has been made by research groups investigating the molecular and physiological mechanisms involved in B toxicity tolerance in both barley (Hordeum vulgare L.) and wheat (Triticum sp. L.). In barley, genes have been identified controlling B tolerance at two of the four known B toxicity tolerance loci, both of which encode B transporters. Progress has also been made towards the identification of genes involved in B toxicity tolerance in wheat. Here we describe the current status of this work, in the context of B toxicity tolerance research in Australia and internationally. We also summarize prospects for breeding new cereal varieties with B toxicity tolerance in the future.
We report here the construction of a new set of chromosome segment substitution lines (CSSLs) in rice, in which the genome of the elite japonica cultivar C418 has been introgressed into the background of the elite indica cultivar 9311. This set was developed by marker aided selection, based on 136 SSR and in/del markers. The introgressed chromosomal segments presented in the 108 CSSLs covered 98.3% of the cultivar C418 genome. The CSSL set was used to genetically analyze variation for 1000-grain weight (TGW) and related traits in two contrasting environments, and led to the identification of 41 quantitative trait loci (QTLs), five of which were expressed in both environments. More detailed mapping of qTGW7 showed that it is co-segregated with RM22034 on the short arm of chromosome 7. The CSSLs varied phenotypically with respect to a number of agronomic traits besides TGW. CSSL populations would be effective in identifying QTLs for these various traits, and could provide germplasm relevant for crop improvement.
Lignin, one of the major components of plant cell walls, has negative impacts on forage digestibility and bioethanol production through fermentation. Understanding the regulatory mechanism of lignin biosynthesis is essential to developing plants with altered lignin contents. The sorghum (Sorghum bicolor [L.] Moench) brown-midrib-6 mutant (bmr-6) has reduced levels of several key enzymes in the lignin biosynthesis pathway. Among the genes encoding these enzymes, the gene for cinnamyl alcohol dehydrogenase (CAD; EC 220.127.116.11) contains a nonsense mutation (responsible for bmr-6) that results in reduced CAD gene transcript levels. We found that the CAD coding region was 4225 bp and consisted of four exons separated by three introns. Bioinformatic analysis of the 5′-flanking region identified several putative binding sites for transcription factors. Moreover, a cross-species comparison of CAD genomic DNA sequences revealed evolutionary and structural variation among plant taxa. The expression level of the CAD gene was much lower in bmr-6 than in normal isoline (N-6) in root, stem, leaf, and midrib. Expression levels of other lignin biosynthesis genes were comparable in bmr-6 and N-6. These results suggest that the depression of CAD enzymatic activity in bmr-6 could affect enzymatic activity and post-translational regulation of other enzymes related to lignin biosynthesis.
The total carotenoid contents in eight sweetpotato (Ipomoea batatas L.) cultivars or breeding lines with yellow flesh were evaluated by absorption spectrophotometry and compared to those of four cultivars with orange flesh. The content ranged from 1.3 mg/100 g dry weight to 3.9 mg/100 g dry weight in yellow-fleshed cultivars and from 13.5 mg/100 g dry weight to 39.9 mg/100 g dry weight in orange-fleshed cultivars. Seventeen carotenoids were detected in yellow- and orange-fleshed sweetpotato by HPLC analysis. The amount of carotenoids analyzed by HPLC correlated highly with the carotenoid content analyzed spectrophotometrically. The main carotenoids were β-carotene 5,8;5′,8′-diepoxide (ca. 32%–51%) and β-cryptoxanthin 5,8-epoxide (ca. 11%–30%) in yellow-fleshed cultivars/lines, while β-carotene (ca. 80%–92%) was dominant in orange-fleshed cultivars. These results suggest that the content of each carotenoid differs according to flesh color, yellow or orange, although the carotenoid component in the yellow and orange flesh was almost identical. The antioxidative activities of carotenoids isolated from yellow-fleshed sweetpotato were analyzed by the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical-scavenging activity. The activity of ipomoeaxanthin A was comparable to β-carotene, and that of β-cryptoxanthin 5,8-epoxide was the lowest. The carotenoids in yellow-fleshed sweetpotato might contribute to the prevention of some diseases due to their antioxidative effect. β-carotene epoxides and β-crypthoxanthin epoxides, which are abundant in the yellow-fleshed cultivars, are noteworthy components for the breeding selection of sweetpotato with deep yellow flesh.
By data mining of EST databases and screening of a genomic library, the cold stimulated gene Lcs19 was isolated from Italian ryegrass (Lolium multiflorum Lam.). The amino acid sequence of the putative LCS19 was 74% identical to wheat (Triticum aestivum) WCS19. Transcripts of Lcs19 were detected after 8 h of cold treatment, and continued to increase up to 48 h after exposure. Lcs19 expression was induced only by cold temperature, and not by high salinity or exogenous abscisic acid. No Lcs19 expression was observed in seedlings that underwent the cold treatment in the dark. Comparison of the promoter of Lcs19 with that of homologous genes of wheat and barley (Hordeum vulgare) showed that regions that include the DRE/CRT cis-acting element sequence were conserved. These results suggest that Lcs19 expression is regulated by signal pathway mediated by the DRE/CRT element and other signal pathways reflecting photosynthetic conditions such as a redox signal.
The green rice leafhopper (GRH), Nephotettix cincticeps Uhler, is a serious insect pest of cultivated rice (Oryza sativa L.) in temperate East Asia. An African rice cultivar, Oryza glaberrima Steud. (IRGC104038), was shown to be highly GRH-resistant at the booting stage. To reveal the genetic basis of the GRH resistance in O. glaberrima, a BC1F1 population derived from a cross between a susceptible japonica variety, Taichung 65 (T65), and O. glaberrima was analyzed by quantitative trait locus (QTL) analysis. A single major QTL for GRH resistance, designated qGRH9, was detected on rice chromosome 9, and three minor QTLs were detected on rice chromosomes 3, 7, and 10. A series of O. glaberrima introgression lines (GILs) containing IRGC104038 chromosome segments in the T65 genetic background were evaluated to confirm the genetic effects of the QTLs, and three GILs carrying qGRH9 showed resistance to GRH. Substitution mapping using the GILs revealed that qGRH9 was located between simple sequence repeat (SSR) markers RM215 and RM2482 in a 1.39-Mbp region on the distal region of the long arm of chromosome 9, and tightly linked to RM7306. These SSR markers maybe useful for marker-assisted selection of qGRH9 for improvement of GRH resistance in rice.
The HYBRID STERILITY 1 gene allele from Oryza glaberrima (S1-g) frequently causes sterility in F1 hybrids with O. sativa allele S1-a. We tested the hypothesis that O. sativa lines homozygous for the O. glaberrima sterile gene alleles would be more compatible with O. glaberrima than similar lines with O. sativa alleles. The S1-g allele was introgressed from O. glaberrima into three O. sativa backgrounds using marker aided selection. These S1-g lines and one check line (Dianjingyou 1) were used as males in interspecific hybridization with numerous cultivars of O. glaberrima and as the recurrent parents in backcrosses. Both the pollen grain fertility and egg fertility of progenies of S1-g lines were higher than those without S1-g in F1 and BC1F1 progenies. Spikelet fertility of BC2F1 progenies with S1-g gave similar results. The results indicated that O. sativa lines carrying S1-g allele from O. glaberrima can be used as bridge parents to significantly improve the fertility of hybrids between O. glaberrima and O. sativa.
Festulolium is an intergeneric hybrid between Festuca and Lolium spp. that has agronomic benefits derived from both grass species. The genomic constitution of this intergeneric hybrid has been examined previously using genomic in situ hybridization (GISH). However, the genomic constitution of the first festulolium cultivar developed in Japan, ‘Tohoku 1’, which was released in 2009, has not yet been determined by GISH. We therefore developed a method for determining the genomic constitution of festulolium hybrids using multi-colored GISH and image analysis. GISH revealed numerous recombinant chromosomes with a relatively small proportion of the Festuca genome in Tohoku 1. Also, discriminating the origin of the species based on staining of the centromeres and nucleolar organizing regions in festulolium by GISH and FISH with an rDNA probe is difficult. The ratio of the Festuca-specific genome (f ratio) was tested for use as an evaluation index for estimating the genomic constitution of festulolium. Although the observed number of Festuca chromatins in Tohoku 1 was reduced during mitosis, the f ratio was stable throughout mitosis, indicating that the f ratio is a useful index for characterizing the festulolium genome.
Low-temperature is one of the most common environmental stresses that affect plant growth and development and places a major limit on plant productivity. Tolerance to low-temperature is an important objective of rice breeding programs in temperate areas and at high altitudes in tropical and sub-tropical areas. Vigorous rice growth during the seedling stage at low-temperature is an important characteristic for stable seedling establishment (SES) in direct seeding methods, in which rice is sown directly into a flooded field. The aim of this study was to identify QTLs controlling SES using 3 mapping populations of backcrossed inbred lines (BILs) with Arroz Da Terra and Italica Livorno as the donor parents. SES was evaluated by a field evaluation system in this study, which is used in current rice breeding programs because of the complexity of environmental conditions in SES in rice cultivation in a paddy field. A total of 9 chromosomal regions for SES were identified, which explained 10.9% to 25.6% of total phenotypic variation. For all of QTLs except for qSES5-2, alleles from the donor parents increased phenotypic values. These QTLs should be useful for the improvement of SES in rice breeding programs in low-temperature regions.
We previously reported spreading of RNA silencing that occurred from 5′ to 3′ direction along the transgene without any effect on transgene methylation by grafting transgenic Nicotiana benthamiana carrying either entire or 5′ region of coat protein gene (CP) of Sweet potato feathery mottle virus (SPFMV). Here, spreading of RNA silencing and transgene methylation was investigated in hybrids between these transgenic lines. When silenced line carrying 5′ region of CP was crossed with a non-silenced line harboring the entire CP, progeny showed very little accumulation of transgene mRNA and siRNAs were detected not only from 5′ region but also from middle and 3′ region of CP. This suggested that RNA silencing spread from 5′ to 3′ region over the entire CP transcript in hybrids. In addition, increased level of cytosine methylation in both symmetrical (CG and CNG) and asymmetrical (CHH) contexts (in the order of frequency, CG > CNG > CHH) were observed in entire transgene in hybrids. Together with the results, it is postulated that transitive silencing can induce spreading of transgene methylation depending on the system implemented reflecting the extent of interaction between silencing signal and transgene DNA in nuclei.
Phosphorus (P) deficiency is a major yield-limiting constraint in wheat production. This situation is further aggravated by the depleting supply of P resources. The ability of wheat to adapt to low P soil is inferior to some wild grass species. In order to locate high P efficiency genes on specific chromosomes of wheat relatives, 107 wheat alien chromosome addition lines were used to investigate P efficiency at the seedling stage. The results showed that introgression of alien chromosomes affected the P uptake and utilization efficiency, which resulted in altered P efficiency. Nine addition lines conferred high P efficiency, seven addition lines conferred high P uptake efficiency, and 39 addition lines conferred high P utilization efficiency. The high P addition lines of selected did not depend on genome type or homologous group. The addition lines with high P efficiency could be used for breeding high P efficiency wheat cultivars through wild hybridization.
The number and distribution of branches in soybean plants influence seed yield through effects on the efficiency of light utilization as well as on tolerance to lodging. We have developed recombinant inbred lines (RILs) from a cross between two experimental determinant lines, which differ in branching number. The 172 RILs were divided into four maturity groups according to their alleles for two maturity loci, E1 and E3, and were planted separately to avoid confounding effects of competition. The late-maturity RIL groups with the E1 genotype were grown in two different locations, whereas the early-maturity RIL groups with the e1 genotype were planted at one location. Analysis of all lines resulted in the identification of five quantitative trait loci (QTLs) for branching number, designated qBr1 to qBr5. Among these QTLs, qBr1 and qBr2 were mapped to the proximal regions of the E1 and E3 loci, respectively. The other three QTLs were mapped to regions distant from any known maturity loci and were detected only in the presence of the E1 genotype, indicating that they interact with qBr1. Our results suggest that branching number might be controlled genetically by the identified QTLs, even though the maturity loci substantially affect branching phenotype.
To reveal the sequence diversity and population structure of Asian rice (Oryza sativa L.) cultivars, we surveyed genome-wide single-nucleotide polymorphisms (SNPs) in 140 diverse accessions. We identified 4357 SNPs distributed on the 12 chromosomes by sequencing PCR amplicons from the exons and introns of anonymous rice genes. We detected 4.87 SNPs per 1 kb genome-wide. By classifying the 140 accessions on the basis of these SNPs, we identified seven cultivar groups that reflected the geographical distribution of the accessions. Three cultivar groups were defined from tropical japonica that corresponded to previous categories, and three indica cultivar groups were also defined within indica. The linkage disequilibrium (LD) distance between SNPs was approximately 250 kb, except for a longer LD detected in the Indica I cultivar group (corresponding to the previously identified aus group). The allele frequency of the SNPs varied among cultivar groups, reflecting the level of genetic diversity in each group. These SNPs for the diverse accessions enhance our understanding of natural variation in rice.
We investigated the effects of ion beams, in comparison to gamma rays, on mutation induction and nuclear DNA content as an index of radiation damage in Chrysanthemum morifolium. Leaf segments were irradiated with 220 MeV carbon ions (mean linear energy transfer = 107 keV/μm), 320 MeV carbon ions (76 keV/μm), 100 MeV helium ions (9 keV/μm), and gamma rays. The nuclear DNA content was measured and frequency of flower color mutations was investigated in regenerated plants. The number of plants with reduced nuclear DNA content increased with increasing irradiation doses of 320 MeV carbon ions, 100 MeV helium ions, and gamma rays. In contrast, they did not increase with 220 MeV carbon ions, even when the dose was increased. Irradiation treatment with 220 and 320 MeV carbon ions and gamma rays had a similar effect on mutation induction, while the effect of 100 MeV helium ions was not as great. Thus, the effects of irradiation treatments on mutation induction and nuclear DNA content differed according to the type of ion beams. 220 MeV carbon ion beam seemed to be the most appropriate among the three types of ion beams because it gave a high mutation frequency with low damage to chromosomes.
In wheat (Triticum aestivum L.), Fusarium head blight (FHB) resistance is an important breeding target to reduce yield losses and mycotoxin contamination. We hypothesized that cleistogamous (CL, closed flowering) cultivars have a lower risk of FHB infection than chasmogamous (CH, opened flowering) cultivars because FHB infection usually occurs in the inside of florets or extracted anthers and FHB resistance in CL cultivars decreases mycotoxin accumulation. To test these hypotheses, we produced 126 recombinant inbred lines (RILs) derived from a cross between common wheat varieties ‘U24’ (CL variety) and ‘Saikai 165’ (CH variety). Although ‘U24’ and ‘Saikai 165’ are both resistant to FHB infection, ‘U24’ is susceptible to FHB spread, grain deterioration, and mycotoxin accumulation compared with ‘Saikai 165’. Among the RILs, there were significant differences in all of the tested traits. Although the CL RILs showed less initial FHB infection than CH RILs, there were no significant differences in grain deterioration and mycotoxin accumulation between the 2 groups. An FHB-resistant quantitative trait locus (QTL) located on chromosome 3BS increased resistance to FHB spread, grain deterioration, and mycotoxin accumulation; however, not all CL RILs with this QTL accumulated less mycotoxin than ‘Saikai 165’.
The genetic relationship among cassumunar gingers (Zingiber cassumunar) in Thailand was assessed by amplified fragment length polymorphism (AFLP). Twelve AFLP primer combinations generated a total of 309 fragments, of which, 242 bands were polymorphic with an average of 20.2 bands per primer pair. Genetic similarities were obtained using Jaccard similarity coefficients, and a phylogenetic tree was constructed using the UPGMA clustering method. Pairwise similarity estimated between cassumunar gingers ranged from 0.7644 to 1.00 with an average of 0.879. Cluster analysis divided the samples into five groups with a high co-phenetic correlation value (r = 0.99). Genetic variability within and among collection regions was estimated by analysis of molecular variance (AMOVA). High molecular variance (84%) was found within samples from the same region. The results implied dispersal of plant materials between collection regions. The genetic similarity assessed by AFLP showed that there are duplicate accessions in the germplasm collection. This genetic information is very useful for germplasm maintenance and a crop improvement program.
The generation of useful mutant alleles of specific genes would accelerate conventional breeding programs in various commercially important crops. Common soybean oil is easily oxidized because it is rich in polyunsaturated fatty acids (PUFAs). Microsomal omega-6 fatty acid desaturase (FAD2), which introduces a second unsaturated bond into oleic acid, is a primary target for elevating oleic acid levels and reducing PUFA levels. The paleopolyploid soybean genome contains five FAD2 gene homologues, at least three of which (GmFAD2-1a, 2-1b, and 2-2a) are functional. In spite of their importance, very little genetic variation has been identified in these genes except in GmFAD2-1a, because fatty acid content is easily affected by environmental conditions such as temperature. Here we isolated novel mutant alleles of GmFAD2-1b from ethyl methanesulfonate-treated soybean mutant populations through Targeting Induced Local Lesions In Genomes (TILLING), a reverse genetic method. Evaluation of enzyme activity in a yeast heterologous expression system suggested that two mutant lines, ‘B12’ and ‘E11’, contain near-null and null alleles, respectively, of GmFAD2-1b. Furthermore, by combining GmFAD2-1a and GmFAD2-1b mutant alleles, we successfully generated soybean lines with >80% oleic acid content. TILLING could provide a practical method for expanding the genetic diversity of polyploid crops.
Wild emmer (Triticum dicoccoides) is a progenitor of tetraploid wheat and currently grows in environments subject to abiotic stresses, including high salinity. Fifty-four genotypes originating from nine geographical populations in Israel, and five standard wheats (three durum and two bread wheats) were assessed for salinity tolerance using supported hydroponics. In this study, we summarize two key components that contribute to salinity tolerance: shoot growth in saline conditions relative to control conditions (relative dry weight); and Na+ accumulation in leaves of salinised plants. An additional third component (shoot growth under control conditions) has an indirect role but is important for salinity tolerance in an agricultural context. Variability in these three components was high. Some genotypes showed high overall relative dry weight, having the ability to maintain growth in moderately saline solution, a low-to-moderate Na+ accumulation, and high biomass production under control conditions. Genotypes from other populations had very high relative dry weight but grew very slowly, so were of limited agricultural relevance. Six selected genotypes possessing useful qualities for at least one of the tested components of salinity tolerance were re-analyzed, and a genotype from Gitit in the eastern Samaria steppes was identified as the most promising salt-tolerant line for further investigation.
Lotus japonicus is a model legume used by researchers worldwide to study nodulation and other aspects of legume biology. We investigated 15 morphological traits in a large collection of wild L. japonicus accessions collected in Japan, and also analyzed the genetic diversity among them, in order to develop a core collection based on genome-wide SSR polymorphisms. We found a wide diversity of morphological traits among the accessions. A total of 321 putative alleles were detected using 25 SSR primer pairs. The average observed and expected heterozygosity scores for individual loci were 0.014 and 0.772, respectively, indicating a high degree of homozygosity. The accessions were divided into 9 clusters based on GST’ and STRUCTURE analyses. In addition, we selected 20 accessions to serve as the representative core collection based on SSR analysis. The range of morphological traits in the core collection was representative of that found in the entire collection. This core collection will be useful for genome-wide studies and data obtained for this model species should lead to numerous practical applications for crop legumes. All accessions from the entire collection are available from Legume Base (http://www.legumebase.brc.miyazaki-u.ac.jp/), a section of the National BioResource Project in Japan.
Using 77 single-nucleotide polymorphic (SNP) markers and dot-blot analysis, we examined 218 rice cultivars, respectively occupying 99% and 92% of the planted areas of non-glutinous and glutinous rice for three consecutive years from 2003 to 2005 in Japan. Among them, 205 cultivars were identified at one time by the genotypes of 18 markers, but 13 cultivars belonged to six groups in which cultivars were indistinguishable from each other. The 205 cultivars were individually distinguished from the others using combinations of up to six markers. This result was considered to be useful for the identification of Japanese commercial rice cultivars, monitoring the contamination of rice with other cultivars, and rice breeding using these cultivars.
A plant of the wheat cultivar Moulin grown in a greenhouse was found to be completely sterile. The study of the F1 and F2 offspring of the plant pollinated by Moulin showed that in field conditions the sterility was partial (fertility reduced by 91.6% with only a few completely sterile plants), recessive and monogenic. The F1 crosses of the mutant Moulin with Ms1b/ms1b Sirocco and Probus monosomic 4B showed that the gene for sterility is not located in chromosome 4B and hence cannot be allelic to the recessive allele ms1b which confers complete male sterility in wheat. The expression of sterility was demonstrated in wheat backgrounds with high levels of fertility. This is the first time partial-monogenic sterility is reported in wheat. This kind of sterility is of no interest for producing wheat F1 hybrids. However, it might be of interest for recurrent selection, or dynamic management of genetic resources where limited intermating is required, as well as in the study of genes involved in microsporogenesis.