Relative fitness of females and hermaphrodites in a natural gynodioecious population of wild radish,Raphanus sativus L. (Brassicaceae): comparison based on molecular genotyping

In many gynodioecious species, sex determination involves both cytoplasmic male‐sterility (CMS) genes and nuclear genes that restore male function. Differences in fitness among genotypes affect the dynamics of those genes, and thus that of gynodioecy. We used a molecular marker to discriminate between hermaphrodites with and without a CMS gene in gynodioecious Raphanus sativus. We compared fitness through female function among the three genotypes: females, hermaphrodites with the CMS gene and those without it. Although there was no significant difference among the genotypes in seed size, hermaphrodites without the CMS gene produced significantly more seeds, and seeds with a higher germination rate than the other genotypes, suggesting no fitness advantage for females and no benefit to bearing the CMS gene. Despite the lack of fitness advantage for females in the parameter values we estimated, a theoretical model of gynodioecy shows it can be maintained if restorer genes impose a cost paid in pollen production. In addition, we found that females invest more resources into female reproduction than hermaphrodites when they become larger. If environmental conditions enable females to grow larger this would facilitate the dynamics of CMS genes.     

Patterns of genetic variability within and among populations of wild radish,Raphanus raphanistrum (Brassicaceae)

The genetic structure of populations is an important determinant of the evolutionary potential of a species. Colonizing plants tend to be characterized by low within- and high among-population variability. Genetic differentiation of both floral traits and isozymes was studied in six populations of wild radish (Raphanus raphanistrum). Evidence for differentiation in both sets of traits was found, but patterns of differentiation of floral traits did not coincide with isozyme differentiation. Contrary to most colonizing species, wild radish showed high within- and only moderate among-population variability at isozyme loci. In addition, levels of differentiation did not correspond to geographic distance between the populations. These results are likely due at least in part to the self-incompatibility system of this species, long-distance movement of large numbers of wild radish seeds by humans, and introgression from cultivated radish (R. sativus).     

萝卜CMS不育系与保持系小孢子发生的细胞学研究

研究了萝卜胞质雄性不育系A2、A4及其相应保持系B2、B4的小孢子发生与花药壁发育的细胞学特征.结果表明,不育系A2的绒毡层细胞在四分体时期出现异常,小液泡增多,至单核期汇合形成大液泡,绒毡层细胞异常膨大;小孢子外壁染色浅,细胞壁受到破坏,最后与绒毡层一同降解.不育系A4在减数分裂期即表现出异常,绒毡层异常肥大;花药发育后期,小孢子外壁亦染色较浅;绒毡层细胞融合形成细胞团块侵入药室挤压小孢子,两者一同降解.     

Management of Alternaria blight of radish seed crop (Raphanus sativus L.)

Radish (Raphanus sativus L. var. Tasaki Mula) was used in this investigation. On foliar spray, among the three fungicides (Iprodione, Mancozeb and Carbendazim), Iprodione (0.25%) followed by Mancozeb (0.3%) provided the maximum control of Alternaria blight and increased seed yield during the first cropping season (2004–2005). The results in second cropping season (2005–2006), 10 December (early) transplanting with three sprays of Iprodione resulted lowest leaf area diseased, whereas plants in 25 December (normal) transplanting had the lower pod area diseased. In third cropping season (2006–2007), 25 October transplantation with BAU-Biofungicide (2.0%) and Iprodione (0.25%) foliar application showed significant effect in reducing Alternaria blight and increased seed yield by 120.45% and 97.73%, respectively over non-sprayed control. Bio-Biofungicide ranked second in reducing the disease, but ranked first in increasing seed yield. The cost benefit analysis of management practices for radish seed crop during 2005–2006 revealed that stickling transplanting in 25 December (normal) with two sprays of Iprodione (0.25%) yielded the highest return (BCR 3.27), whereas negative result (BCR ? 0.89) has been estimated in case of non-sprayed control plots in late transplanting (10 January). However, cost benefit analysis of cropping season 2006–2007 revealed that BAU-Biofungicide (2.0%) six sprays yielded the highest return (BCR 4.41) followed by six sprays of Iprodione (BCR 3.90). Finally, it may be concluded that Integration of early transplanting (25 October) with foliar sprays ofBAU-Biofungicide (2.0%) was found best for eco-friendly management of Alternaria blight of radish seed crop with significantly higher seed yield.     

Identification and expression of the kosena radish (Raphanus sativus cv. Kosena) homologue of the ogura radish CMS-associated gene,orf138

A CMS-associated gene, orf125, present in the Japanese radish cultivar Kosena, has a sequence homologous to that of the ogura CMS-associated gene, orf138, except for two amino acid substitutions and a 39 bp deletion in the orf138 coding region. In Kosena radish, orf125 is linked with orfB, whereas the orf125 locus differs in a Brassica napus CMS cybrid derived from protoplast fusion between Kosena radish and B. napus. A novel mtDNA sequence is present in the 3-flanking region of orf125 in the B. napus kosena CMS cybrid. The orf125 is expressed both in the radish and the B. napus kosena CMS cybrid. Its accumulation is strongly associated with the CMS phenotype in B. napus. Fertility restoration was accompanied by a decrease in the amount of ORF125 in B. napus.     

Draft Sequences of the Radish (Raphanus sativus L.) Genome

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.     

Effects of pollen load size and composition on pollen donor performance in wild radish, Raphanus sativus (Brassicaceae)

A critical concern in the debate over the importance of sexual selection in plants is whether the nonrandom mating demonstrable in greenhouse crosses can occur in the field. Field populations likely experience smaller and more variable pollen load sizes than those that have been used in many greenhouse experiments. Therefore, we performed a greenhouse experiment in which we varied both pollen load size and composition in wild radish, Raphanus sativus, and examined the paternity of seeds. We used five maternal plants and four pairs of pollen donors. We were able to produce pollen loads of 40, 118, and 258 grains per stigma. The smallest of the pollen loads was scant enough to result in a slight, but significant reduction in seed number per fruit. While variation in pollen load composition significantly affected the proportions of seeds fathered by different donors, variation in pollen load size did not. The relative performance of different donors was constant across pollen load sizes, suggesting that, for this species, differential performance of pollen donors can occur at pollen load sizes that are likely to occur in field populations.     

Dynamics of maternal and paternal effects on embryo and seed development in wild radish (Raphanus sativus)

Background and Aims

Variability in embryo development can influence the rate of seed maturation and seed size, which may have an impact on offspring fitness. While it is expected that embryo development will be under maternal control, more controversial hypotheses suggest that the pollen donor and the embryo itself may influence development. These latter possibilities are, however, poorly studied. Characteristics of 10-d-old embryos and seeds of wild radish (Raphanus sativus) were examined to address: (a) the effects of maternal plant and pollen donor on development; (b) the effects of earlier reproductive events (pollen tube growth and fertilization) on embryos and seeds, and the influence of embryo size on mature seed mass; (c) the effect of water stress on embryos and seeds; (d) the effect of stress on correlations of embryo and seed characteristics with earlier and later reproductive events and stages; and (e) changes in maternal and paternal effects on embryo and seed characteristics during development.

Methods

Eight maternal plants (two each from four families) and four pollen donors were crossed and developing gynoecia were collected at 10 d post-pollination. Half of the maternal plants experienced water stress. Characteristics of embryos and seeds were summarized and also compared with earlier and later developmental stages.

Key Results

In addition to the expected effects of the maternal plants, all embryo characters differed among pollen donors. Paternal effects varied over time, suggesting that there are windows of opportunity for pollen donors to influence embryo development. Water-stress treatment altered embryo characteristics; embryos were smaller and less developed. In addition, correlations of embryo characteristics with earlier and later stages changed dramatically with water stress.

Conclusions

The expected maternal effects on embryo development were observed, but there was also evidence for an early paternal role. The relative effects of these controls may change over time. Thus, there may be times in development when selection on the maternal, paternal or embryo contributions to development are more and less likely.     

Fitness of backcross six of hybrids between transgenic oilseed rape (Brassica napus) and wild radish (Raphanus raphanistrum)

The process of introgression between a transgenic crop modified for better agronomic characters and a wild relative could lead potentially to increased weediness and adaptation to the environment of the wild species. However, the formation of hybrid and hybrid progeny could be associated with functional imbalance and low fitness, which reduces the risk of gene escape and establishment of the wild species in the field. Our work compares the fitness components of parents and different types of backcross in the sixth generation of hybrids between transgenic oilseed rape (Brassica napus, AACC, 2n = 38) resistant to the herbicide glufosinate and wild radish (Raphanus raphanistrum, RrRr, 2n = 18). The backcross with oilseed rape cytoplasm (OBC) has a fitness value 100 times lower than that of the backcross with wild radish cytoplasm (RBC). The herbicide-resistant RBC has similar growth to the susceptible RBC, but final male and female fitness values are two times lower. In turn, susceptible RBC exhibit similar fitness to the control wild radishes. The relative fitnesses of the different types are the same whether or not they grow under competitive conditions. The consequence on fitness of the chromosome location of the transgene conferring resistance and the relevance of these results to the impact of gene flow on the environment are discussed.     

Fitness effects of mutation accumulation in a natural outbred population of wild radish (Raphanus raphanistrum): comparison of field and greenhouse environments

Spontaneous deleterious mutation has been measured in a handful of organisms, always under laboratory conditions and usually employing inbred species or genotypes. We report the results of a mutation accumulation experiment with an outbred annual plant, Raphanus raphanistrum, with lifetime fitness measured in both the field and the greenhouse. This is the first study to report the effects of spontaneous mutation measured under field conditions. Two large replicate populations (N(e) approximately 600) were maintained with random mating in the greenhouse under relaxed selection for nine generations before the field assay was performed and ten generations before the greenhouse assay. Each generation, every individual was mated twice, once as a pollen donor and once as a pollen recipient, and a single seed from each plant was chosen randomly to create the next generation. The ancestral population was maintained as seeds at 4 degrees C. Declines in lifetime fitness were observed in both the field (1.7% per generation; P= 0.27) and the greenhouse (0.6% per generation; P= 0.07). Significant increases in additive genetic variance for fitness were found for stems per day, flowers per stem, fruits per flower and seeds per fruit in the field as well as for fruits per flower in the greenhouse. Lack of significance of the fitness decline may be due to the short period of mutation accumulation, the use of outbred populations, or both. The percent declines in fitness are at the high end of the range observed in other mutation accumulation experiments and give some support to the idea that mutational effects may be magnified under harsher field conditions. Thus, measurement of mutational parameters under laboratory conditions may underestimate the effects of mutations in natural populations.     

Calcium and calmodulin inhibitor effects on the growth of carrot (Daucus carota L.) and radish (Raphanus sativus L.) in nutrient culture

Growth of carrot and radish seedlings in nutrient culture was inhibited by pretreatment with three calmodulin inhibitors. There was little selective effect on specific organs, shoots, tap root and fibrous roots over a range of concentrations. Although pretreatment with CaCl₂ (0.5 mM) did not affect growth of untreated seedlings, it partially reduced the inhibitory effects of trifluoperazine over the concentration range 0.01–0.05 mM. Trifluoperazine reduced the growth of GA₃-treated seedlings but did not overcome the modifying effect of GA₃ in favouring shoot/root ratio; ABA exacerbated its inhibitory effect on overall seedling growth and particularly on tap root development.Abbreviations GA₃ gibberellic acid - ABA abscisic acid - CaCl₂ calcium chloride - GAs gibberellins - Tfp trifluoperazine     

心里美萝卜花青素合成酶基因RsANS克隆及花青素生物合成相关基因表达分析

花青素合成酶(ANS,anthocyanidin synthase)是植物花青素合成的关键酶,催化无色花色素转变成有色花色素。本研究从心里美萝卜HX12Q-49中克隆获得了花青素合成酶基因Rs ANS(Gen Bank登录号:KR262954)。该基因全长1137 bp,包含2个外显子和1个内含子;开放阅读框1071 bp,编码356个氨基酸。同源性分析显示Rs ANS蛋白与大白菜、甘蓝和芥菜ANS蛋白同源性较高。qRT-PCR表明Rs ANS在心里美萝卜5个不同发育时期均有表达,且在破肚期表达量最高。通过对花青素合成途径其他8个结构基因和3个调控基因表达进行分析,结果显示心里美萝卜中b HLH转录因子在不同发育时期的表达模式与Rs ANS、CHI、DFR和UFGT基本一致,即在破肚期的表达量最高;WD40转录因子的表达与CHS类似,其表达量在芽期、破肚期、膨大前期和膨大盛期逐渐上升,随后在成熟期下降。     

Characterization of Annexin gene family and functional analysis of RsANN1a involved in heat tolerance in radish (Raphanus sativus L.)

Plant annexins are a kind of conserved Ca²⁺-dependent phospholipid-binding proteins which are involved in plant growth, development and stress tolerance. Radish is an economically important annual or biennial root vegetable crop worldwide. However, the genome-wide characterization of annexin (RsANN) gene family remain largely unexplored in radish. In this study, a comprehensive identification of annexin gene family was performed at the whole genome level in radish. In total, ten RsANN genes were identified, and these putative RsANN proteins shared typical characteristics of the annexin family proteins. Phylogenetic analysis showed that the RsANNs together with annexin from Arabidopsis and rice were clustered into five groups with shared similar motif patterns. Chromosomal localization showed that these ten RsANN genes were distributed on six chromosomes (R3-R8) of radish. Several cis-elements involved in abiotic stress response were identified in the promoter regions of RsANN genes. Expression profile analysis indicated that the RsANN genes exhibited tissue-specific patterns at different growth stages and tissues. The Real-time quantitative PCR (RT-qPCR) revealed that the expression of most RsANN genes was induced under various abiotic stresses including heat, drought, salinity, oxidization and ABA stress. In addition, stress assays showed that overexpression of RsANN1a improved plant’s growth and heat tolerance, while artificial microRNAs (amiRNA)-mediated knockdown of RsANN1a caused dramatically decreased survival ratio of Arabidopsis plants. These findings not only demonstrate that RsANN1a might play a critical role in the heat stress response of radish, but also facilitate clarifying the molecular mechanism of RsANN genes in regulating the biological process governing plant growth and development.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01056-5.     

Identification of RAPD markers linked to a fertility restorer gene for theOgura radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.     

Pollen donor performance can be consistent acrossmaternal plants in wild radish (Raphanus sativus,Brassicaceae): a necessary condition for the action of sexualselection

The possibility that sexual selection has played a role in theevolution of plant reproductive characters remains interesting, butcontroversial. One reason is that clear demonstration of the necessaryconditions for sexual selection is lacking. For sexual selection tooccur, differences in pollen donor performance must be consistent acrossmaternal plants and not be due to mechanisms such asself-incompatibility that produce interactions between maternal plantsand pollen donors. Here, I performed two experiments with wild radish totest whether differences in pollen donor performance are consistentacross maternal plants and not due to subtle effects of theincompatibility system. In the first, all maternal and paternal lineageshad different S-alleles. There were 16 maternal plants, four in each offour lineages and four pollen donors, one in each of four lineages. Rankorder of pollen donor performance, in terms of number of seeds siredafter mixed pollination, was highly consistent across maternal plantsand maternal lineages. In addition, maternal stress treatment had aneffect on mating success of pollen donors, but the effect was subtle anddid not affect the rank order of seeds sired by the four pollen donors.In the second experiment, pollinations were performed on both mature andimmature stigmas. Immature stigmas allowed some self seed set, so theincompatibility system was compromised. There was some nonrandom seedpaternity on both mature and immature stigmas. However, the amount ofnonrandom mating was less on immature stigmas. Taken together, theseexperiments show that the kind of consistent nonrandom mating necessaryfor sexual selection occurs in wild radish, but that the incompatibilitysystem and the mechanisms for sorting among compatible mates may overlapin time of development or in somepathways.     

Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.)

Hypocotyl segments of 2- to 3-week-old radish (Raphanus sativus L. cv. F₁ Handsome Fall) seedlings produced yellowish compact calli when cultured on Murashige and Skoog's (MS) medium supplemented with 1 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and -naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. When subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced white, compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on medium containing 0.1 to 3 mgl^-1 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto medium containing 0.1 mgl^-1 2,4-D and 1 mgl^-1 abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - GA₃ gibberellin A₃ - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid     

Complete mitochondrial genome sequence and identification of a candidate gene responsible for cytoplasmic male sterility in radish (Raphanus sativus L.) containing DCGMS cytoplasm

A novel cytoplasmic male sterility (CMS) conferred by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its restorer-of-fertility gene (Rfd1) was previously reported in radish (Raphanus sativus L.). Its inheritance of fertility restoration and profiles of mitochondrial DNA (mtDNA)-based molecular markers were reported to be different from those of Ogura CMS, the first reported CMS in radish. The complete mitochondrial genome sequence (239,186 bp; GenBank accession No. KC193578) of DCGMS mitotype is reported in this study. Thirty-four protein-coding genes and three ribosomal RNA genes were identified. Comparative analysis of a mitochondrial genome sequence of DCGMS and previously reported complete sequences of normal and Ogura CMS mitotypes revealed various recombined structures of seventeen syntenic sequence blocks. Short-repeat sequences were identified in almost all junctions between syntenic sequence blocks. Phylogenetic analysis of three radish mitotypes showed that DCGMS was more closely related to the normal mitotype than to the Ogura mitotype. A single 1,551-bp unique region was identified in DCGMS mtDNA sequences and a novel chimeric gene, designated orf463, consisting of 128-bp partial sequences of cox1 gene and 1,261-bp unidentified sequences were found in the unique region. No other genes with a chimeric structure, a major feature of most characterized CMS-associated genes in other plant species, were found in rearranged junctions of syntenic sequence blocks. Like other known CMS-associated mitochondrial genes, the predicted gene product of orf463 contained 12 transmembrane domains. Thus, this gene product might be integrated into the mitochondrial membrane. In total, the results indicate that orf463 is likely to be a casual factor for CMS induction in radish containing the DCGMS cytoplasm.     

Pollinator preferences and the persistence of crop genes in wild radish populations (Raphanus raphanistrum, Brassicaceae)

Crop-weed hybridization can potentially influence the evolutionary ecology of wild populations. Many crops are known to hybridize with wild relatives, but few studies have looked at the long-term persistence of crop genes in the wild. This study investigated one factor in the hybridization process in radish: differential pollinator visitation to wild radish (Raphanus raphanistrum) vs. crop-wild F1 hybrids (R. sativus x R. raphanistrum). Wild genotypes had yellow flowers, a recessive single-locus trait, whereas hybrids always had white or pale pink flowers. In experimental arrays in northern Michigan, total pollinator visitation was significantly biased toward wild plants when the frequencies of wild and hybrid plants were equal. Syrphid flies, the most frequent visitors, preferred wild plants while bumble bees showed no preference. This pattern was also observed when hybrid plants were overrepresented in the array (12 hybrid:2 wild). In contrast, when hybrid plants were rare (2 hybrid:12 wild), neither morph was preferred by any pollinator group. Later in the summer, pollinators were also observed in a large experimental garden with nearly equal frequencies of wild and hybrid plants. Cabbage butterflies (Pieris rapae) strongly overvisited wild plants, while bumble bees showed a slight preference for hybrids. Taken together, these studies suggest that F1 hybrids may not be at a disadvantage with regard to pollinator visits when they occur at low frequencies or when bumble bees are frequent flower visitors. Thus, variation in the proportion of white-flowered morphs among wild radish populations could be influenced by different histories of crop-to-wild hybridization, as well as by variation in the composition of local pollinator taxa.