Novel means for variety breeding and sporeling production in the brown seaweed Undaria pinnatifida (Phaeophyceae): Crossing female gametophytes from parthenosporophytes with male gametophyte clones

The unialgal haploid gametophyte clones are frequently used for variety breeding and sporeling production in the brown seaweed Undaria pinnatifida because a single crossing of a pair of the selected male and female gametophyte clones can generate sporophytic offspring with identical genotype and phenotype. As the seeds to be sprayed on the collectors, the detachment rate of the filamentous gametophytes is high in comparison to the seeded spores. In this investigation, we report the use of parthenogenesis to achieve the same goal in variety selection and sporeling production but with higher efficiency. The selected female unialgal gametophyte clone (Code: 06‐8‐1F) was induced to produce parthenosporophytes. These sporophytes were grown up in a controlled system and used to release zoospores. All zoospores generated into female gametophytes. These female gametophytes were allowed to go through parthenogenesis for the second year to confirm the applicability of this means. In the third year, the zoospores released from the parthenosporophytes were seeded on collectors over summer in female gametophyte form. In the early autumn, a selected male unialgal gametophyte clone (code: 5#F1‐2‐5M) was used to cross the seeded female gametophytes on the collectors. When the sporelings reached a mean length of 780 μm, they were transplanted to open sea on longlines for growing up. At harvest, the average length, width and wet weight of the adult sporophytes were 211 cm, 48.8 cm and 373 g, respectively. The sporophytic blades were uniformly smooth without wrinkles on both sides of the midrib, indicating top quality of the products. Amplified fragment length polymorphism analyses confirmed the identical genotypes of sporophytic offspring. These results suggested that this novel variety breeding and sporeling production method could serve as an efficient alternative to the traditional breeding technique for U. pinnatifida and possibly other commercial kelps that have identical life cycles.     

A method to establish an “immortalized F2” sporophyte population in the economic brown alga Undaria pinnatifida (Laminariales: Alariaceae)

Studies of quantitative trait loci based on genetic linkage maps require the establishment of a mapping population. Permanent mapping populations are more ideal than temporary ones because they can be used repeatedly. However, there has been no reported permanent sporophyte population of economically important kelp species. Based on the characteristics of the kelp life cycle, we proposed a method to establish, and then constructed experimentally, an “immortalized F₂” (IF₂) population of Undaria pinnatifida. Doubled-haploid “female” and “male” sporophytes were obtained through the parthenogenesis of a female gametophyte clone and the selfing of a “monoicous” gametophyte clone (originally male), respectively, and they were used as the parents. The F₁ hybrid line was generated by crossing the female and male gametophytes derived from the respective female and male parents. Full-sibling F₂ gametophyte clones, consisting of 260 females and 260 males, were established from an F₁ hybrid sporophyte. Thirty-five females and 35 males were randomly selected and paired to give rise to an IF₂ population containing 35 crossing lines. A parentage analysis using 10 microsatellite markers confirmed the accuracy of the IF₂ population and indicated the feasibility of this method. This proposed method may be adapted for use in other kelp species, and thus, it will be useful for genetic studies of kelp.     

Sex‐linked microsatellite marker detected in the female gametophytes of Undaria pinnatifida (Phaeophyta)

In our microsatellite analysis of three male and three female gametophytes of Undaria pinnatifida (Harv.) Suringar, a microsatellite marker (part of the locus Up‐AC‐2A8, GenBank accession no. AY738602.1) was only polymerase chain reaction‐amplified in three female gametophytes. This putative female‐specific marker was further tested by the use of 32 male and 21 female gametophytes maintained in the Marine Biological Culture Collection Centre, China. In addition, three sporophytes were included for confirmation. Results showed that the marker was present in all of the female gametophytes and sporophyte cultures, but absent in all of the male gametophytes. To our knowledge, this is the first sex‐related marker ever reported in U. pinnatifida. The discovery of this marker will accelerate gender identification and shed light on our understanding of the mechanisms of sex determination at a molecular level in this commercially important seaweed.     

Ocean acidification and kelp development: Reduced pH has no negative effects on meiospore germination and gametophyte development of Macrocystis pyrifera and Undaria pinnatifida

The absorption of anthropogenic CO₂ by the oceans is causing a reduction in the pH of the surface waters termed ocean acidification (OA). This could have substantial effects on marine coastal environments where fleshy (non‐calcareous) macroalgae are dominant primary producers and ecosystem engineers. Few OA studies have focused on the early life stages of large macroalgae such as kelps. This study evaluated the effects of seawater pH on the ontogenic development of meiospores of the native kelp Macrocystis pyrifera and the invasive kelp Undaria pinnatifida, in south‐eastern New Zealand. Meiospores of both kelps were released into four seawater pH treatments (pH_T 7.20, extreme OA predicted for 2300; pH_T 7.65, OA predicted for 2100; pH_T 8.01, ambient pH; and pH_T 8.40, pre‐industrial pH) and cultured for 15 d. Meiospore germination, germling growth rate, and gametophyte size and sex ratio were monitored and measured. Exposure to reduced pH_T (7.20 and 7.65) had positive effects on germling growth rate and gametophyte size in both M. pyrifera and U. pinnatifida, whereas, higher pH_T (8.01 and 8.40) reduced the gametophyte size in both kelps. Sex ratio of gametophytes of both kelps was biased toward females under all pH_T treatments, except for U. pinnatifida at pH_T 7.65. Germling growth rate under OA was significantly higher in M. pyrifera compared to U. pinnatifida but gametophyte development was equal for both kelps under all seawater pH_T treatments, indicating that the microscopic stages of the native M. pyrifera and the invasive U. pinnatifida will respond similarly to OA.     

The genetic analysis and germplasm identification of the gametophytes of Undaria pinnatifida (Phaeophyceae) with RAPD method

Eleven pairs of Undaria pinnatifida (Harv.) Suringar gametophytes were identified with random amplified polymorphic DNA (RAPD) technique. After screening 100 primers, 20 ten-base primers were determined for the RAPD analysis. A total of 312 polymorphic loci were obtained, of which 97.7% were polymorphic. The primer S198 was found to distinguish all the selected Undaria pinnatifida gametophytes. The genetic distances between each two of the twenty-two U. pinnatifida gametophytes ranged from 0.080 to 0.428, while the distances to the Laminaria was 0.497 on average. After reexamination, two sequences characterized amplification region (SCAR) markers were successfully converted, which could be applied to U. pinnatifida germplasm identification. All these results demonstrated the feasibility of applying RAPD markers to germplasm characterization and identification of U. pinnatifida gametophytes, and to provide a molecular basis for Undaria breeding.     

Identification of a genomic region linked with sex determination of Undaria pinnatifida (Alariaceae) through genomic resequencing and genetic linkage analyses of a segregating gametophyte family

Different from the traditional knowledge about kelp, three sexual phenotypes (female, male, and monoecious) exist in the haploid gametophytes of Undaria pinnatifida. However, the sex-determining mechanisms remain unknown. Genetic linkage mapping is an efficient tool to identify sex-linked regions. In the present study, we resequenced a segregating gametophyte family based on the male genome of U. pinnatifida. A high-density genetic linkage map was constructed using 9887 SNPs, with an average distance of 0.41 cM between adjacent SNPs. On the basis of this genetic map and using the composite interval mapping method, we identified 62 SNPs significantly linked with the sexual phenotype. They were located at a position of 67.67 cM on the linkage group 23, corresponding to a physical range of 14.67 Mbp on the HiC_Scaffold_23 of the genome. Reanalysis of the previous specific length amplified fragment sequencing data according to the reference genome led to the identification of a sex-linked genomic region that encompassed the above-mentioned 14.67 Mbp region. Hence, this overlapped genomic range was likely the sex-determining region. Within this region, 129 genes were retrieved and 39 of them were annotated with explicit function, including the potential male sex-determining gene-encoding high mobility group (HMG) domain protein. Relative expression analysis of the HMG gene showed that its expression was higher in male gametophytes during the vegetative phase and monoecious gametophytes during both the vegetative and gametogenesis phases, but significantly lower in male gametophytes during the gametogenesis phase. These results provide a foundation for deciphering the sex-determining mechanism of U. pinnatifida.     

GENETIC LOAD IN OSMUNDA REGALIS POPULATIONS

Osmunda regalis sporophytes form haploid spores which develop into functionally hermaphroditic gametophytes. The self-fertilization of such gametophytes results in zygotes which are completely homozygous. Spore samples collected from sporophytes in natural populations were used to establish gametophyte cultures. The majority of these gametophytes were unable to form viable embryos when only self-fertilization was possible. Controlled selfing and crossing experiments revealed that the inability of these homozygous embryos to develop normally is attributable to the presence of recessive lethals. To account for this genetic load, an hypothesis is proposed integrating the morphology and ecology of the gametophyte generation with the polyploid genetic system of the sporophyte generation.     

Mutant induction in gametophytes of Undaria pinnatifida (Phaeophyceae) by heavy ion beam irradiation

Undaria pinnatifida, the brown macroalga, is a major commercial edible seaweed, and there is increasing interest in breeding new, improved cultivars for market expansion. In this study, we attempted to establish mutagenesis in U. pinnatifida using Ar and C ion beams as mutagens to meet future demands. To screen irradiated generations for mutants, U. pinnatifida zoospores irradiated with Ar and C ion beams were cultivated in plastic Petri dishes. Some gametophytes derived from the irradiated zoospores showed growth arrest or cell death at the initial developmental stage. Although the growth inhibition and lethal effects were observed at high doses of each ion irradiation, the Ar ion irradiation had high biological effects on cell division and growth. The gametophytes that showed a reduction in cell elongation were designated as an inhibited cell elongation mutant. A comparison of the mutant induction frequencies revealed that the C ion beam showed a higher frequency than the Ar ion beam. The highest frequency was 0.83% at 12.5 Gy of the C ion beam. We determined the total number of sporophytes and embryos per female gametophyte after sporophyte induction. High‐dose irradiation with the Ar ion beam decreased the embryo and sporophyte formation, suggesting that the Ar ion beam also has exhibited high biological effects on the fertilization or embryogenesis processes or both. The developed heavy ion mutagenesis and mutant screening methods would be useful for mutation breeding and constructing specific mutant libraries in brown algae, and not only in U. pinnatifida.     

Male gametophytic selection in maize

Summary There is evidence that male gametophyte selection is a widespread phenomenon in higher plants. The pollen tube growth rate is one of the main components of gametophyte selective value; genetic variability for this trait, due to the effect of single genes or to quantitative variation, has been described in maize. However, indication of gametophytic selection has been indirectly obtained; its effect was revealed by the positive relation observed between gametophyte competitive ability and sporophyte metrical traits.This paper considers the results of selection applied to gametophyte populations produced from single plants. The competitive ability of the lines was evaluated in comparison with that of a standard line by means of the pollen mixture technique. Sporophytic traits were measured in the hybrid progeny obtained by crossing selected S₃ and S₄ families with an unrelated single cross and an inbred line. Gametophyte selection produced inbred lines with high gametophyte competitive ability. In view of the selection procedure adopted, this result was interpreted as an indication of haploid expression of genes involved in the control of pollen tube growth. Moreover, this gametophytic trait was positively correlated with sporophytic traits (seedling weight, kernel weight and root tip growth in vitro), indicating that both groups of characters have a common genetic basis.     

Nuclear DNA level and life cycle of kelps: Evidence for sex‐specific polyteny in Macrocystis (Laminariales,Phaeophyceae)

Giant kelp, Macrocystis pyrifera (Linnaeus) C. Agardh, is the subject of intense breeding studies for marine biomass production and conservation of natural resources. In this context, six gametophyte pairs and a sporophyte offspring of Macrocystis from South America were analyzed by flow cytometry. Minimum relative DNA content per cell (1C) was found in five males. Unexpectedly, nuclei of all female gametophytes contained approximately double the DNA content (2C) of males; the male gametophyte from one locality also contained 2C, likely a spontaneous natural diploid variant. The results illustrate a sex‐specific difference in nuclear DNA content among Macrocystis gametophytes, with the chromosomes of the females in a polytenic condition. This correlates with significantly larger cell sizes in female gametophytes compared to males and resource allocation in oogamous reproduction. The results provide key information for the interpretation of DNA measurements in kelp life cycle stages and prompt further research on the regulation of the cell cycle, metabolic activity, sex determination, and sporophyte development.     

Temperature requirements for growth and maturation of the warm temperate kelp Eckloniopsis radicosa (Laminariales,Phaeophyta)

The annual kelp Eckloniopsis radicosa is distributed along Japanese coasts and occurs within the area with a February isotherm ranging 15–18°C and August isotherm ranging 25–28°C. In this study, the effects of temperature on the gametophyte growth and maturation, and the young sporophyte growth of E. radicosa were examined and the results are discussed in relation to the distribution of other warm‐adapted kelp species and the potential effects of climate change. The optimal temperature ranges for growth of male and female gametophytes were 23–27°C and 20–26°C, respectively. The upper survival temperature for gametophyte growth was 31°C for males and 30°C for females, respectively. The optimal temperature range for maturation of female gametophytes was ≤23°C. The optimal temperature range for growth of young sporophytes was 14–22°C. It was clarified that E. radicosa has the most warm‐adapted characteristics for growth and maturation of gametophytes among members of the Laminariales studied so far. The natural seawater temperature ranges during the growth and maturation seasons for gametophytes of E. radicosa, as well as the growth season for young sporophytes near to the northern and southern distribution limits (Izu‐Oshima: 14.9–24.5°C, Ichiki‐kushikino: 17.1–29.6°C), agreed with the experimentally determined temperature requirements. The warm‐adapted gametophyte stage and annual lifecycle are major factors enabling survival of E. radicosa in warm waters near tropical regions along the Japanese coast.     

Genetic diversity and relationships of the brown alga Undaria pinnatifida cultivated along the Dalian Coast as revealed by amplified fragment length polymorphism markers

In this study, the amplified fragment length polymorphism (AFLP) method was employed to estimate the genetic diversity of young sporophytes belonging to six Undaria pinnatifida populations collected from five seaweed cultivation farms along the Dalian Coast of China in 2008. A total of 397 loci were detected using ten combinations of selective primer pairs. Of these, 302 of which (76.07 %) were polymorphic, which was mainly caused by the 40 accessions of two populations collected from the LWT (44.08 %) and the JST (44.58 %) farms. According to the UPGMA dendrogram and biplot of principal component analysis, the 40 accessions represented high intra-population genetic diversity and far relationship from all of other populations. In contrast, the accessions from the other populations (HK, MS, LS, and QD) represented high intra-population similarity. The four populations may have originated from the same introduced U. pinnatifida strain. Analysis of molecular variance showed that a higher proportion of genetic variation resided among the populations (63.56 %) than that within populations (36.64 %). It is hypothesized that the differences in genetic traits, as well as the mixed mating system and reduced gene flow were the primary reasons that caused the high genetic variation among populations and the high similarity between accessions within the population. However, the genetic diversity of the partial U. pinnatifida cultivar populations in Dalian farms has been decreasing in the past years, which poses a potential risk of germplasm degradation in cultivation. In addition, the breeding techniques of cultivated U. pinnatifida in China and the application of AFLP in Undaria are discussed in this article. Based on the results, the identified polymorphic markers could be used for genetic improvement of the species through marker-assisted breeding and the results also provide excellent information for selecting indigenous gametophyte resources from Dalian farms using different breeding methods.     

Examining the reproductive success of bull kelp (Nereocystis luetkeana,Phaeophyceae, Laminariales) in climate change conditions

Climate change is affecting marine ecosystems in many ways, including raising temperatures and leading to ocean acidification. From 2014 to 2016, an extensive marine heat wave extended along the west coast of North America and had devastating effects on numerous species, including bull kelp (Nereocystis luetkeana). Bull kelp is an important foundation species in coastal ecosystems and can be affected by marine heat waves and ocean acidification; however, the impacts have not been investigated on sensitive early life stages. To determine the effects of changing temperatures and carbonate levels on Northern California's bull kelp populations, we collected sporophylls from mature bull kelp individuals in Point Arena, CA. At the Bodega Marine Laboratory, we released spores from field-collected bull kelp, and cultured microscopic gametophytes in a common garden experiment with a fully factorial design crossing modern conditions (11.63 ± 0.54°C and pH 7.93 ± 0.26) with observed extreme climate conditions (15.56 ± 0.83°C and 7.64 ± 0.32 pH). Our results indicated that both increased temperature and decreased pH influenced growth and egg production of bull kelp microscopic stages. Increased temperature resulted in decreased gametophyte survival and offspring production. In contrast, decreased pH had less of an effect but resulted in increased gametophyte survival and offspring production. Additionally, increased temperature significantly impacted reproductive timing by causing female gametophytes to produce offspring earlier than under ambient temperature conditions. Our findings can inform better predictions of the impacts of climate change on coastal ecosystems and provide key insights into environmental dynamics regulating the bull kelp lifecycle.     

Construction of integrated genetic linkage maps of the tiger shrimp (Penaeus monodon) using microsatellite and AFLP markers

The linkage maps of male and female tiger shrimp (P. monodon) were constructed based on 256 microsatellite and 85 amplified fragment length polymorphism (AFLP) markers. Microsatellite markers obtained from clone sequences of partial genomic libraries, tandem repeat sequences from databases and previous publications and fosmid end sequences were employed. Of 670 microsatellite and 158 AFLP markers tested for polymorphism, 341 (256 microsatellite and 85 AFLP markers) were used for genotyping with three F₁ mapping panels, each comprising two parents and more than 100 progeny. Chi‐square goodness‐of‐fit test (χ²) revealed that only 19 microsatellite and 28 AFLP markers showed a highly significant segregation distortion (P < 0.005). Linkage analysis with a LOD score of 4.5 revealed 43 and 46 linkage groups in male and female linkage maps respectively. The male map consisted of 176 microsatellite and 49 AFLP markers spaced every ～11.2 cM, with an observed genome length of 2033.4 cM. The female map consisted of 171 microsatellite and 36 AFLP markers spaced every ～13.8 cM, with an observed genome length of 2182 cM. Both maps shared 136 microsatellite markers, and the alignment between them indicated 38 homologous pairs of linkage groups including the linkage group representing the sex chromosome. The karyotype of P. monodon is also presented. The tentative assignment of the 44 pairs of P. monodon haploid chromosomes showed the composition of forty metacentric, one submetacentric and three acrocentric chromosomes. Our maps provided a solid foundation for gene and QTL mapping in the tiger shrimp.     

PATTERNS OF MATING,GENERATION OF DIVERSITY,AND FITNESS OF OFFSPRING IN A GEUM HYBRID SWARM

To understand the evolutionary consequences of hybridization between the outcrossing plant Geum rivale (Rosaceae) and the selfer Geum urbanum, we tested the predictions of two simple models that assume either (A) low or (B) high pollen fitness in hybrids. Model A predicts only four genotypic classes (G. rivale, G. rivale backcross [BC_R], F1, and Geum urbanum) and asymmetric introgression from inbreeding to outbreeding species. Model B predicts additional genotypic classes and potential generation of novel inbreeding lines in the hybrid swarm. Amplified fragment length polymorphism (AFLP) analysis of adults revealed only the four genotypes predicted by model A. However, microsatellite analysis of parent–progeny arrays demonstrated production of selfed offspring by F1 and BC_R maternal parents and contribution of these genotypes to outcross pollen pools, as predicted by model B. Moreover, AFLP and morphological analysis showed that the offspring generation comprised genotypes and phenotypes covering the entire spectrum of variation between the two parental species, in line with model B. A common garden experiment indicated no systematic reduction in fitness of offspring derived from hybrid parents. The genetic structure of the adults in the Geum hybrid swarm cannot be explained by restricted mating patterns but may result from ecological selection acting on a diverse offspring population.     

Historical influences dominate the population genetic structure of a sedentary marine fish,Atlantic wolffish (Anarhichas lupus), across the North Atlantic Ocean

Genetic variation was assessed in Atlantic wolffish, Anarhichas lupus, across the North Atlantic Ocean using microsatellite and amplified fragment length polymorphism (AFLP) markers. Despite unusual life history attributes such as large benthic eggs, large larvae, a limited pelagic stage and relatively sedentary adults, which suggest potential for strong population structure, range‐wide F_ST values were comparable to other marine fishes (≤0.035). Nevertheless, both significant genetic differentiation among regions and isolation by distance were observed, suggesting limited dispersal in this species. AFLP loci, evaluated on a subset of samples, revealed slightly higher F_ST values, but similar patterns of differentiation and isolation‐by‐distance estimates, compared to microsatellites. The genetic structure of Atlantic wolffish has likely been shaped by its post‐glacial history of recolonization, North Atlantic current patterns and continuity of habitat on continental shelves.     

Occurrence of the gametophyte of Agarum clathratum (Laminariales, Phaeophyceae) as an endophyte in Orculifilum denticulatum (Gigartinales, Rhodophyceae)

An endophytic filamentous brown alga, growing in the red alga Orculifilum denticulatum Lindstrom, was collected from the north‐east Pacific, near Juneau, Alaska. Within the host tissue, its branched filaments formed a network in the space between the filaments of the host tissues embedded in the host intercellular substances. Cells of the filaments contained many discoid chloroplasts without pyrenoids. Neither microscopic morphological observation nor culturing was sufficient to reveal the specific identity or even the familial affinity of the alga; in contrast, molecular phylogenetic analysis of its rbcL gene and rDNA ITS sequences showed that it was the gametophyte of Agarum clathratum Dumortier (Laminariales). There are few reports of laminarialean gametophytes in nature; this is the first report actually identifying the species of laminarialean gametophyte in a red alga.