Production of somatic hybrids between Diospyros glandulosa and D. kaki by protoplast fusion

Interspecific somatic hybrids between Diospyros glandulosa (2n=2x=30) and D. kaki cv. Jiro (2n=6x=90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using a modified KM8p medium. Flow cytometry revealed that the nuclear DNA content was the sum of those of D. glandulosa and D. kaki cv. Jiro in 149 of the 166 calli obtained. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and further confirmed that they were interspecific somatic hybrid calluses. Shoots were regenerated from 63 of the 149 interspecific hybrid calluses. Chloroplast DNA analysis by PCR-RFLP, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained the nuclear genomes from both parents but only the chloroplast genome from D. glandulosa. Microscopic observation of root tip cells demonstrated that somatic chromosome number of the interspecific hybrids was 2n=8x=120. This revised version was published online in June 2006 with corrections to the Cover Date.     

A systematic revision of Potentilla lineata and allied species (Rosaceae) in the Himalaya and adjacent regions

IKEDA, H. & OHBA, H., 1993. A systematic revision of Potentilla lineata and allied species (Rosaceae) in the Himalaya and adjacent regions . Potentilla lineata and allied species (Rosaceae) in the Himalaya and adjacent regions are revised. Potentilla festiva Soják, P. josephiana H. Ikeda & H. Ohba, P. lineata Trev., P. fallens Card, and P. polyphylla Wall, ex Lehm. are recognized. Potentilla josephiana is a new name for P.fulgens Wall, ex Hook. var. intermedia Hook. f. Four varieties are recognized in P. polyphylla: var. polyphylla; var. himalaka H. Ikeda & H. Ohba, var. nov.; var. interrupta (Yü & Li) H. Ikeda & H. Ohba, stat. & comb, nov.; and var. barbata Lehm. A polyploid series is found in this group. Four putative hybrids between the species are also recognized.     

Multiplex single nucleotide polymorphism (SNP)-based genotyping in allohexaploid wheat using padlock probes

Single nucleotide polymorphisms are the most common polymorphism in plant and animal genomes and, as such, are the logical choice for marker-assisted selection. However, many plants are also polyploid, and marker-assisted selection can be complicated by the presence of highly similar, but non-allelic, homoeologous sequences. Despite this, there is practical and academic demand for high-throughput genotyping in several polyploid crop species, such as allohexaploid wheat. In this paper, we present such a system, which utilizes public single nucleotide polymorphisms previously identified in both agronomically important genes and in randomly selected, mapped, expressed sequence tags developed by the wheat community. To achieve relatively high levels of multiplexing, we used non-amplified genomic DNA and padlock probe pairs, together with high annealing temperatures, to differentiate between similar sequences in the wheat genome. Our results suggest that padlock probes are capable of discriminating between homoeologous sequences and hence can be used to efficiently genotype wheat varieties.     

The high‐quality genome of Brassica napus cultivar ‘ZS11’ reveals the introgression history in semi‐winter morphotype

Allotetraploid oilseed rape (Brassica napus L.) is an agriculturally important crop. Cultivation and breeding of B. napus by humans has resulted in numerous genetically diverse morphotypes with optimized agronomic traits and ecophysiological adaptation. To further understand the genetic basis of diversification and adaptation, we report a draft genome of an Asian semi‐winter oilseed rape cultivar ‘ZS11’ and its comprehensive genomic comparison with the genomes of the winter‐type cultivar ‘Darmor‐bzh’ as well as two progenitors. The integrated BAC‐to‐BAC and whole‐genome shotgun sequencing strategies were effective in the assembly of repetitive regions (especially young long terminal repeats) and resulted in a high‐quality genome assembly of B. napus ‘ZS11’. Within a short evolutionary period (~6700 years ago), semi‐winter‐type ‘ZS11’ and the winter‐type ‘Darmor‐bzh’ maintained highly genomic collinearity. Even so, certain genetic differences were also detected in two morphotypes. Relative to ‘Darmor‐bzh’, both two subgenomes of ‘ZS11’ are closely related to its progenitors, and the ‘ZS11’ genome harbored several specific segmental homoeologous exchanges (HEs). Furthermore, the semi‐winter‐type ‘ZS11’ underwent potential genomic introgressions with B. rapa (A_r). Some of these genetic differences were associated with key agronomic traits. A key gene of A03.FLC3 regulating vernalization‐responsive flowering time in ‘ZS11’ was first experienced HE, and then underwent genomic introgression event with A_r, which potentially has led to genetic differences in controlling vernalization in the semi‐winter types. Our observations improved our understanding of the genetic diversity of different B. napus morphotypes and the cultivation history of semi‐winter oilseed rape in Asia.     

Detection of individual ploidy levels with genotyping‐by‐sequencing (GBS) analysis

Ploidy levels sometimes vary among individuals or populations, particularly in plants. When such variation exists, accurate determination of cytotype can inform studies of ecology or trait variation and is required for population genetic analyses. Here, we propose and evaluate a statistical approach for distinguishing low‐level ploidy variants (e.g. diploids, triploids and tetraploids) based on genotyping‐by‐sequencing (GBS) data. The method infers cytotypes based on observed heterozygosity and the ratio of DNA sequences containing different alleles at thousands of heterozygous SNPs (i.e. allelic ratios). Whereas the method does not require prior information on ploidy, a reference set of samples with known ploidy can be included in the analysis if it is available. We explore the power and limitations of this method using simulated data sets and GBS data from natural populations of aspen (Populus tremuloides) known to include both diploid and triploid individuals. The proposed method was able to reliably discriminate among diploids, triploids and tetraploids in simulated data sets, and this was true for different levels of genetic diversity, inbreeding and population structure. Power and accuracy were minimally affected by low coverage (i.e. 2×), but did sometimes suffer when simulated mixtures of diploids, autotetraploids and allotetraploids were analysed. Cytotype assignments based on the proposed method closely matched those from previous microsatellite and flow cytometry data when applied to GBS data from aspen. An R package (gbs2ploidy) implementing the proposed method is available from CRAN.     

秋水仙素对蚕豆胚根生长的影响及多倍体诱导效应分析

筛选秋水仙素诱导蚕豆胚根多倍体的最适诱导处理组合并分析其诱导效应。以秋水仙素5个浓度(0.025%、0.050%、0.100%、0.150%、0.200%)和4个诱导时间(12、24、48、60h)正交组合(蒸馏水处理为对照)分别处理蚕豆胚根。以根尖诱导率和胚根膨大率的显著性差异确定蚕豆胚根多倍体的最适诱导处理组合,并以胚根细胞染色体数目和幼叶气孔数目的变化对诱导效果进行鉴定,分析其幼苗期的胚根数目和幼苗长度。结果表明:秋水仙素最适诱导时间为48h和浓度为0.100%;气孔数量在诱导时间和诱导浓度之间均有显著差异,但幼苗期的胚根数量和幼苗长度在不同的诱导时间之间无显著差异,诱导浓度之间差异显著。     

Inheritance in tetraploid yeast revisited: segregation patterns and statistical power under different inheritance models

In their recent article, Albertin et al. (2009) suggest an autotetraploid origin of 10 tetraploid strains of baker’s yeast (Saccharomyces cerevisiae), supported by the frequent observation of double reduction meiospores. However, the presented inheritance results were puzzling and seemed to contradict the authors’ interpretation that segregation ratios support a tetrasomic model of inheritance. Here, we provide an overview of the expected segregation ratios at the tetrad and meiospore level given scenarios of strict disomic and tetrasomic inheritance, for cases with and without recombination between locus and centromere. We also use a power analysis to derive adequate sample sizes to distinguish alternative models. Closer inspection of the Albertin et al. data reveals that strict disomy can be rejected in most cases. However, disomic inheritance with strong but imperfect preferential pairing could not be excluded with the sample sizes used. The possibility of tetrad analysis in tetraploid yeast offers a valuable opportunity to improve our understanding of meiosis and inheritance of tetraploids.     

Different patterns of colonization of Oxalis alpina in the Sky Islands of the Sonoran desert via pollen and seed flow

Historical factors such as climatic oscillations during the Pleistocene epoch have dramatically impacted species distributions. Studies of the patterns of genetic structure in angiosperm species using molecular markers with different modes of inheritance contribute to a better understanding of potential differences in colonization and patterns of gene flow via pollen and seeds. These markers may also provide insights into the evolution of reproductive systems in plants. Oxalis alpina is a tetraploid, herbaceous species inhabiting the Sky Island region of the southwestern United States and northern Mexico. Our main objective in this study was to analyze the influence of climatic oscillations on the genetic structure of O. alpina and the impact of these oscillations on the evolutionary transition from tristylous to distylous reproductive systems. We used microsatellite markers and compared our results to a previous study using chloroplast genetic markers. The phylogeographic structure inferred by both markers was different, suggesting that intrinsic characteristics including the pollination system and seed dispersal have influenced patterns of gene flow. Microsatellites exhibited low genetic structure, showed no significant association between geographic and genetic distances, and all individual genotypes were assigned to two main groups. In contrast, chloroplast markers exhibited a strong association between geographic and genetic distance, had higher levels of genetic differentiation, and were assigned to five groups. Both types of DNA markers showed evidence of a northward expansion as a consequence of climate warming occurring in the last 10,000 years. The data from both types of markers support the hypothesis for several independent transitions from tristyly to distyly.