Inter- and intraspecific sequence variations of the chloroplast genome in wild and cultivated Raphanus

To assess the differentiation of the chloroplast genome in wild and cultivated species of Raphanus , nucleotide sequence polymorphisms were investigated for approximately 2 kbp ranging from trnL (UAA) to psbG in R. raphanistrum and R. sativus . Eighteen plants of wild species, 10 Japanese wild radish plants ( R. sativus ), and 31 cultivated plants were used for sequence analysis. Intraspecific variations of the chloroplast genome were present both in wild and cultivated Raphanus . All three genes investigated ( trnL , trnF and ndhJ ) contained nucleotide substitutions within the genus. Whereas, larger numbers of mutations were observed in the intergenic regions. Using the detected variations, the 59 radish plants were classified into 11 haplotypes, seven of which were unique to wild species. Among the haplotypes, one type corresponded completely with the Ogura male sterile cytoplasm. All the cultivated radishes belonged to one of four types, of which three were also observed in Japanese wild radish. The haplotypes were classified into four groups by cluster analysis, and the distribution in the dendrogram confirmed that cultivated radish has multiple origins. On the other hand, the seven haplotypes uniquely observed in R. raphanistrum were considered as useful materials to provide genetic diversity of cytoplasm for breeding of cultivated radishes.     

Assessing and broadening genetic diversity of a rapeseed germplasm collection

Assessing the level of genetic diversity within a germplasm collection contributes to evaluating the potential for its utilization as a gene pool to improve the performance of cultivars. In this study, 45 high-quality simple sequence repeat (SSR) markers were screened and used to estimate the genetic base of a world-wide collection of 248 rapeseed (Brassica napus) inbred lines. For the whole collection, the genetic diversity of A genome was higher than that of C genome. The genetic diversity of C genome for the semi-winter type was the lowest among the different germplasm types. Because B. oleracea is usually used to broaden the genetic diversity of C genome in rapeseed, we evaluated the potential of 25 wild B. oleracea lines. More allelic variations and a higher genetic diversity were observed in B. oleracea than in rapeseed. One B. oleracea line and one oilseed B. rapa line were used to generate a resynthesized Brassica napus line, which was then crossed with six semi-winter rapeseed cultivars to produce 7 F₁ hybrids. Not only the allele introgression but also mutations were observed in the hybrids, resulting in significant improvement of the genetic base.     

Determination of genetic variation and taxonomy in lentil (Lens Miller) species by chloroplast DNA polymorphism

Summary Chloroplast DNA restriction fragment lenght polymorphisms (RFLP) were used to examine the taxonomic relationships of cultivated and wild lentil (Lens Miller) species and identify the extent of genetic variation in this genus. Twelve accessions representing all Lens subspecies were digested with four hexanucleotide-recognizing restriction endonucleases. These digests randomly surveyed 540 base pairs, or 0.4% of the approximately 125 kilobase lentil chloroplast genome. A high degree of gragment length conservation was seen among members of crossability group I, i.e., L. c. ssp. culinaris, L. c. ssp. orientalis and L. c. ssp. odemensis. Accessions of the two subspecies comprising crossability group II, i.e., L. n. ssp. nigricans and L. n. ssp. ervoides, showed the greatest amount of variation when compared to the cultivated lentil, L. c. ssp. culinaris. Limited variation was observed within subspecies except for L. n. ssp. nigricans, where accessions of the normal cytotype were highly polymorphic to those of the differentiated cytotype. Chloroplast DNA RFLPs reaffirm hypotheses that propose L. c. ssp. orientalis as the progenitor to the cultivated lentil. The implications of this study on taxonomy and genetic resources is also discussed.     

Complexity of indica-japonica varietal differentiation in Bangladesh rice landraces revealed by microsatellite markers

To understand the genetic diversity and indica-japonica differentiation in Bangladesh rice varieties, a total of 151 accessions of rice varieties mostly Bangladesh traditional varieties including Aus, Boro, broadcast Aman, transplant Aman and Rayada varietal groups were genotyped using 47 rice nuclear SSRs. As a result, three distinct groups were detected by cluster analysis, corresponding to indica, Aus and japonica rice. Among deepwater rice varieties analyzed some having particular morphological features that mainly corresponded to the japonica varietal group. Some small seeded and aromatic varieties from Bangladesh also corresponded to the japonica varietal group. This research for the first time establishes that the japonica varietal group is a prominent component of traditional varieties in Bangladesh, particularly in deepwater areas.     

De novo assembly of the complete organelle genome sequences of azuki bean (Vigna angularis) using next-generation sequencers

Since chloroplasts and mitochondria are maternally inherited and have unique features in evolution, DNA sequences of those organelle genomes have been broadly used in phylogenetic studies. Thanks to recent progress in next-generation sequencer (NGS) technology, whole-genome sequencing can be easily performed. Here, using NGS data generated by Roche GS Titanium and Illumina Hiseq 2000, we performed a hybrid assembly of organelle genome sequences of Vigna angularis (azuki bean). Both the mitochondrial genome (mtDNA) and the chloroplast genome (cpDNA) of V. angularis have very similar size and gene content to those of V. radiata (mungbean). However, in structure, mtDNA sequences have undergone many recombination events after divergence from the common ancestor of V. angularis and V. radiata, whereas cpDNAs are almost identical between the two. The stability of cpDNAs and the variability of mtDNAs was further confirmed by comparative analysis of Vigna organelles with model plants Lotus japonicus and Arabidopsis thaliana.     

Persistent C genome chromosome regions identified by SSR analysis in backcross progenies between Brassica juncea and B. napus

Given that feral transgenic canola (Brassica napus) from spilled seeds has been found outside of farmer’s fields and that B. juncea is distributed worldwide, it is possible that introgression to B. juncea from B. napus has occurred. To investigate such introgression, we characterized the persistence of B. napus C genome chromosome (C-chromosome) regions in backcross progenies by B. napus C-chromosome specific simple sequence repeat (SSR) markers. We produced backcross progenies from B. juncea and F₁ hybrid of B. juncea × B. napus to evaluate persistence of C-chromosome region, and screened 83 markers from a set of reported C-chromosome specific SSR markers. Eighty-five percent of the SSR markers were deleted in the BC₁ obtained from B. juncea × F₁ hybrid, and this BC₁ exhibited a plant type like that of B. juncea. Most markers were deleted in BC₂ and BC₃ plants, with only two markers persisting in the BC₃. These results indicate a small possibility of persistence of C-chromosome regions in our backcross progenies. Knowledge about the persistence of B. napus C-chromosome regions in backcross progenies may contribute to shed light on gene introgression.     

Diversity and population structure of black soybean landraces originating from Tanba and neighboring regions

Black soybean landraces that had been cultivated in Tanba region and the neighboring regions and conserved black soybean landraces, including those from other regions in Japan, were used in this study. The polymorphisms of 78 SSR markers in nuclear DNA and 6 SSRs in chloroplast DNA were analyzed in the black soybean landrace populations. The result of phylogenic analysis revealed that the black soybeans can be classified into six clades. The landraces originating from Tanba region were classed into first and second clades, and two chloroplast genotypes were found in the population of black soybeans from the Tanba region. Genotype A chloroplast was predominantly identified in major populations of the Tanba, while genotype B was widely distributed in the black soybean population. Population structure analysis in the Japanese black soybean accessions inferred there are six groups. The black soybean landrace from the Tanba region was classified into three groups, mainly corresponding to the distance-based phylogenic results. The two groups were probably derived from different ancestors with Type A and B chloroplast genomes, respectively, whereas the other group showed both types of chloroplast genome. The admixture situations suggested that the landraces in the main group have been widely cultivated in Tanba region, while the landraces that belong to other groups were cultivated in localized area. Several phenotypes were compared among genotype groups, dividing into two sub-groups: founder sub-group and admixed sub-group. Phenotypic differences were observed between founder landraces in group 1 and group 3. On the other hand, landraces in admixture landraces in group 1 and group 2 segregated for several traits, while founder landraces in group 1 were stabled for each trait. These observations suggest that gene flow events have occurred between different founder landraces.     

Phylogenetic analysis of AGAMOUS sequences reveals the origin of the diploid and tetraploid forms of self-pollinating wild buckwheat,Fagopyrum homotropicum Ohnishi

Fagopyrum homotropicum Ohnishi is a self-pollinating wild buckwheat species indigenous to eastern Tibet and the Yunnan and Sichuan Provinces of China. It is useful breeding material for shifting cultivated buckwheat (F. esculentum ssp. esculentum Moench) from out-crossing to self-pollinating. Despite its importance as a genetic resource in buckwheat breeding, the genetic variation of F. homotropicum is poorly understood. In this study, we investigated the genetic variation and phylogenetic relationships of the diploid and tetraploid forms of F. homotropicum based on the nucleotide sequences of a nuclear gene, AGAMOUS (AG). Neighbor-joining analysis revealed that representative individuals clustered into three large groups (Group I, II and III). Each group contained diploid and tetraploid forms of F. homotropicum. We identified tetraploid plants that had two diverged AG sequences; one belonging to Group I and the other belonging to Group II, or one belonging to Group II and the other belonging to Group III. These results suggest that the tetraploid form originated from at least two hybridization events between deeply differentiated diploids. The results also imply that the genetic diversity contributed by tetraploidization of differentiated diploids may have allowed the distribution range of F. homotropicum to expand to the northern areas of China.     

DNA profiling of pineapple cultivars in Japan discriminated by SSR markers

We developed 18 polymorphic simple sequence repeat (SSR) markers in pineapple (Ananas comosus) by using genomic libraries enriched for GA and CA motifs. The markers were used to genotype 31 pineapple accessions, including seven cultivars and 11 breeding lines from Okinawa Prefecture, 12 foreign accessions and one from a related species. These SSR loci were highly polymorphic: the 31 accessions contained three to seven alleles per locus, with an average of 4.1. The values of expected heterozygosity ranged from 0.09 to 0.76, with an average of 0.52. All 31 accessions could be successfully differentiated by the 18 SSR markers, with the exception of ‘N67-10’ and ‘Hawaiian Smooth Cayenne’. A single combination of three markers TsuAC004, TsuAC010 and TsuAC041, was enough to distinguish all accessions with one exception. A phenogram based on the SSR genotypes did not show any distinct groups, but it suggested that pineapples bred in Japan are genetically diversed. We reconfirmed the parentage of 14 pineapple accessions by comparing the SSR alleles at 17 SSR loci in each accession and its reported parents. The obtained information will contribute substantially to protecting plant breeders’ rights.     

Evaluation of genetic variation among wild rice populations in Cambodia

A total of 448 samples in five natural populations of wild rice (Oryza rufipogon) were collected in Cambodia. They were examined using 12 SSR and two chloroplast markers to evaluate the degree of variation among populations and the genetic structure within populations. In the two annual populations, the number of plants with homozygous alleles at all 12 SSR loci were high (66.3% and 79.5%), suggesting that these plants propagate mainly through self-pollination. In the three perennial populations, no individuals had all homozygous genotypes, but redundant genotypes resulted from clonal propagation were observed. Percentages of the redundant genotypes were highly varied (3.6%, 29.2% and 86.0%). This may be due to the different stable levels of environmental conditions. As for chloroplast genome, most of the wild plants showed the same chloroplast types as most Indica-type cultivars have. However, plants with different chloroplast types were maintained, even in the same population. In tropical Asian countries, many wild rice populations were observed under similar ecological conditions examined in this study. Therefore, the present results concerning population structure will be important to further elucidate genetic features of wild rice, and will also give strong clues to utilize and conserve wild natural genetic resources.     

Enhancement of aluminum tolerance in wheat by addition of chromosomes from the wild relative Leymus racemosus

Aluminum (Al) toxicity is the key factor limiting wheat production in acid soils. Soil liming has been used widely to increase the soil pH, but due to its high cost, breeding tolerant cultivars is more cost-effective mean to mitigate the problem. Tolerant cultivars could be developed by traditional breeding, genetic transformation or introgression of genes from wild relatives. We used 30 wheat alien chromosome addition lines to identify new genetic resources to improve wheat tolerance to Al and to identify the chromosomes harboring the tolerance genes. We evaluated these lines and their wheat background Chinese Spring for Al tolerance in hydroponic culture at various Al concentrations. We also investigated Al uptake, oxidative stress and cell membrane integrity. The L. racemosus chromosomes A and E significantly enhanced the Al tolerance of the wheat in term of relative root growth. At the highest Al concentration tested (200 μM), line E had the greatest tolerance. The introgressed chromosomes did not affect Al uptake of the tolerant lines. We attribute the improved tolerance conferred by chromosome E to improved cell membrane integrity. Chromosome engineering with these two lines could produce Al-tolerant wheat cultivars.     

Evolutionary and comparative analyses of the soybean genome

The soybean genome assembly has been available since the end of 2008. Significant features of the genome include large, gene-poor, repeat-dense pericentromeric regions, spanning roughly 57% of the genome sequence; a relatively large genome size of ~1.15 billion bases; remnants of a genome duplication that occurred ~13 million years ago (Mya); and fainter remnants of older polyploidies that occurred ~58 Mya and >130 Mya. The genome sequence has been used to identify the genetic basis for numerous traits, including disease resistance, nutritional characteristics, and developmental features. The genome sequence has provided a scaffold for placement of many genomic feature elements, both from within soybean and from related species. These may be accessed at several websites, including http://www.phytozome.net, http://soybase.org, http://comparative-legumes.org, and http://www.legumebase.brc.miyazaki-u.ac.jp. The taxonomic position of soybean in the Phaseoleae tribe of the legumes means that there are approximately two dozen other beans and relatives that have undergone independent domestication, and which may have traits that will be useful for transfer to soybean. Methods of translating information between species in the Phaseoleae range from design of markers for marker assisted selection, to transformation with Agrobacterium or with other experimental transformation methods.     

Diversity of seed cesium accumulation in soybean mini-core collections

Radiocesium is an extremely harmful radionuclide because of its long half-life; it is important to reduce its transfer from contaminated soil into crops. Here we surveyed genetic variation for seed cesium (Cs) concentration in soybean mini-core collections representing large genetic diversity. The collections grown over 3 years in rotational paddy fields exhibited varying seed Cs concentrations with significant year-to-year correlations, although the phenotypic stability of Cs concentration was lower than that of the congeners potassium (K) and rubidium (Rb). Although Cs is supposedly accumulated in plants via the K transport system, there was no apparent relationship between Cs and K concentrations, whereas a clear positive correlation was observed between Cs and Rb concentrations. Cs and K concentrations in seed showed slightly positive and negative correlations, respectively, with days to flowering. We selected several high or low Cs accumulator candidates on the basis of the 3 years of seed concentration data. These two groups showed significantly different seed Cs concentrations in another field. The differences could not be explained by flowering time alone. These results suggest that genetic variation for seed Cs concentration is present in soybean germplasm and would be useful for breeding low Cs-accumulating varieties.     

Characterization and genetic mapping of eceriferum-ym (cer-ym), a cutin deficient barley mutant with impaired leaf water retention capacity

The cuticle covers the aerial parts of land plants, where it serves many important functions, including water retention. Here, a recessive cuticle mutant, eceriferum-ym (cer-ym), of Hordeum vulgare L. (barley) showed abnormally glossy spikes, sheaths, and leaves. The cer-ym mutant plant detached from its root system was hypersensitive to desiccation treatment compared with wild type plants, and detached leaves of mutant lost 41.8% of their initial weight after 1 h of dehydration under laboratory conditions, while that of the wild type plants lost only 7.1%. Stomata function was not affected by the mutation, but the mutant leaves showed increased cuticular permeability to water, suggesting a defective leaf cuticle, which was confirmed by toluidine blue staining. The mutant leaves showed a substantial reduction in the amounts of the major cutin monomers and a slight increase in the main wax component, suggesting that the enhanced cuticle permeability was a consequence of cutin deficiency. cer-ym was mapped within a 0.8 cM interval between EST marker {"type":"entrez-nucleotide","attrs":{"text":"AK370363","term_id":"326489160","term_text":"AK370363"}}AK370363 and {"type":"entrez-nucleotide","attrs":{"text":"AK251484","term_id":"151420132","term_text":"AK251484"}}AK251484, a pericentromeric region on chromosome 4H. The results indicate that the desiccation sensitivity of cer-ym is caused by a defect in leaf cutin, and that cer-ym is located in a chromosome 4H pericentromeric region.     

A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker for the identification of Amaranthus cruentus species

A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker was developed to identify the Amaranthus cruentus species by comparing sequences of the starch branching enzyme (SBE) locus among the three cultivated grain amaranths. We determined the partial SBE genomic sequence in 72 accessions collected from diverse locations around the world by direct sequence analysis. Then, we aligned the gene sequences and searched for restriction enzyme cleavage sites specific to each species for use in the PCR-RFLP analysis. The result indicated that MseI would recognize the sequence 5′-T/TAA-3′ in intron 11 from A. cruentus SBE. A restriction analysis of the amplified 278-bp portion of the SBE gene using the MseI restriction enzyme resulted in species-specific RFLP patterns among A. cruentus, Amaranthus caudatus and Amaranthus hypochondriacus. Two different bands, 174-bp and 104-bp, were generated in A. cruentus, while A. caudatus and A. hypochondriacus remained undigested (278-bp). Thus, we propose that the PCR-RFLP analysis of the amaranth SBE gene provides a sensitive, rapid, simple and useful technique for identifying the A. cruentus species among the cultivated grain amaranths.     

Genetic diversity within commercial populations of watercress (Rorippa nasturtium-aquaticum), and between allied Brassicaceae inferred from RAPD-PCR

Commercial productivity of watercress (Rorippa nasturtium-aquaticum) can be adversely affected by the pathogenic crook-root fungus, Spongospora subterranea f.sp. nasturti, and watercress viruses. As there are no effective control measures for these diseases, attempts have been made to breed varieties resistant to the crook-root pathogen. This work has been hindered by a lack of knowledge of the genetic base of commercial watercress, and the genetic distance between watercress and allied Brassicaceae which have been identified as candidates for hybridisation programmes. We measured the diversity within these two groups using the RAPD-PCR fingerprinting technique and analysed the data by both distance methods and principal co-ordinate analysis. Little genetic diversity was found within commercial watercress populations. However, watercress formed a unique cluster genetically distinct from other Rorippa species, but equidistant to Cardamine species. It was placed closer to Barbarea verna. This revised version was published online in August 2006 with corrections to the Cover Date.     

Production and characterization of an alloplasmic and monosomic addition line of Brassica rapa carrying the cytoplasm and one chromosome of Moricandia arvensis

Intergeneric hybridization was performed between Moricandia arvensis and four inbred lines of Brassica rapa following embryo rescue. Three F₁ hybrid plants were developed from three cross combinations of M. arvensis × B. rapa, and amphidiploids were synthesized by colchicine treatment. Six BC₁ plants were generated from a single cross combination of amphidipolid × B. rapa ‘Ko1-303’ through embryo rescue. One BC₂ and three BC₃ plants were obtained from successive backcrossing with B. rapa ‘Ko1-303’ employing embryo rescue. Alloplasmic and monosomic addition lines of B. rapa (Allo-MALs, 2n = 21) were obtained from backcrossed progeny of three BC₃ plants (2n = 21, 22 and 23) without embryo rescue. An alloplasmic line of B. rapa (2n = 20) degenerated before floliation on 1/2 MS medium due to severe chlorosis. Allo-MALs of B. rapa (2n = 21) showed stable male sterility without any abnormal traits in vegetative growth and female fertility. Molecular analyses revealed that the same chromosome and cytoplasm of M. arvensis had been added to each Allo-MAL of B. rapa. This Allo-MAL of B. rapa may be useful material for producing cytoplasmic male sterile lines of B. rapa.     

Expressed sequence tags from organ-specific cDNA libraries of tea (Camellia sinensis) and polymorphisms and transferability of EST-SSRs across Camellia species

Tea is one of the most popular beverages in the world and the tea plant, Camellia sinensis (L.) O. Kuntze, is an important crop in many countries. To increase the amount of genomic information available for C. sinensis, we constructed seven cDNA libraries from various organs and used these to generate expressed sequence tags (ESTs). A total of 17,458 ESTs were generated and assembled into 5,262 unigenes. About 50% of the unigenes were assigned annotations by Gene Ontology. Some were homologous to genes involved in important biological processes, such as nitrogen assimilation, aluminum response, and biosynthesis of caffeine and catechins. Digital northern analysis showed that 67 unigenes were expressed differentially among the seven organs. Simple sequence repeat (SSR) motif searches among the unigenes identified 1,835 unigenes (34.9%) harboring SSR motifs of more than six repeat units. A subset of 100 EST-SSR primer sets was tested for amplification and polymorphism in 16 tea accessions. Seventy-one primer sets successfully amplified EST-SSRs and 70 EST-SSR loci were polymorphic. Furthermore, these 70 EST-SSR markers were transferable to 14 other Camellia species. The ESTs and EST-SSR markers will enhance the study of important traits and the molecular genetics of tea plants and other Camellia species.     

Distorted genetic segregation of the transposon mPing at the long arm of chromosome 12 in rice

A class II transposable element, mPing exists in the rice genome ubiquitously and can transpose even in ordinary cultivation conditions. A copy of mPing was identified at the long arm of chromosome 12. In reciprocal backcrossed F₁s between a heterozygote and a homozygote without mPing, the male gametes with this mPing from heterozygotes were transmitted to the next generation at a lower frequency than those without mPing, resulting in distorted genetic segregation in self-fertilized progenies, as well as in F₁s after backcrossing. Pollens with mPing tended to germinate on stigma less than those without mPing. These results, however, could not explain the lower transmission of male gametes with mPing. In addition, no excision of mPing was observed in a homozygote. Thus, it was suggested that male gametes with mPing were eliminated partly from pollination to fertilization by negative competition against male gametes without mPing. Less formation of microspores with mPing in meiosis could also be a cause for the distorted segregation, although this could not be examined. At least two ORFs, whose functions have not been identified, are located near this mPing. It is plausible that either of these ORFs or both are necessary for the normal functioning of male gametes.