Female Gametophyte Clones Research Articles

The Inheritable Characteristics of Monoecy and Parthenogenesis Provide A Means for Establishing A Doubled Haploid Population in the Economically Important Brown Alga Undaria pinnatifida (Laminariales, Alariaceae).

Monoecy and parthenogenesis exist in certain male and female gametophytes of the brown alga Undaria pinnatifida. The inheritance of these traits is not known. In this study, we made a cross between a male and a female gametophyte clone which could exhibit monoecy and parthenogenesis phenotypes, respectively, and obtained their next-generation gametophyte offspring. We found that under conditions suitable for gametogenesis, all of the male offspring (n=100) exhibited monoecy phenotype and all of the female offspring (n=100) only formed oogonia and underwent parthenogenesis, suggesting that monoecy and parthenogenesis phenotypes are inheritable. Then, we established a doubled haploid (DH) population through monoecious selfing and parthenogenesis, and evaluated the young sporophyte growth and the maximum quantum yield (Fv /Fm ) of 10 "male" and 10 "female" DH lines. On day 60, the average length of the "male" DH lines was significantly larger than that of the "female" DH lines, while their average Fv /Fm values were not significantly different. Monoecious selfing seemed superior to parthenogenesis as the sporophyte formation efficiency, and the young sporophyte growth was better in the former than in the latter. We also crossed two monoecious gametophytes with another male gametophyte, and a parentage analysis showed success of obtaining hybrid sporophytes, indicating that the female gametes released by the monoecious gametophyte can actually be fertilized by sperm. The approach of establishing a DH population proposed here will be useful in genetic breeding and quantitative trait loci mapping in U. pinnatifida and may be applicable to other kelp species.

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.

Breeding of an elite cultivar Haibao No. 1 of Undaria pinnatifida (Phaeophyceae) through gametophyte clone crossing and consecutive selection

As a commercially important kelp, Undaria pinnatifida has been farmed in northern China for more than 30 years. Farming of U. pinnatifida has been restrained historically by the lack of cultivars ever since the very beginning of its cultivation. The cultivation industry to a large extent relies on introducing seedlings or parental plants from other East Asian countries from time to time due to the degeneration of the sporophytes. In order to change this awkward situation, an elite cultivar named Haibao No. 1 was developed through gametophyte clone crossing and consecutive selection. Its original parents were selected from cultivation populations in Dalian. The most pronounced agronomic characteristic of this novel cultivar was its high yield. From F2 to F5, the average fresh weight per individual was all significantly higher than the control. In large-scale cultivation of F4 and F5 populations, the fresh weight reached 19–20 kg m−1 on cultivation line, increasing the yield by more than 40 %. F4 population of the cultivar showed significant heterozygosity deficiency (F is = 0.4728) as revealed by the microsatellite analysis at 20 loci, suggesting the effect of consecutive inbreeding. Coefficients of variations (CVs) of the blade width, the width of the undivided part of the blade, the stipe width, and the fresh weight ranged from 12.82 to 22.3 %, which seemed to be high; however, when compared with the crossing line derived from the crossing of a pair of male and female gametophyte clones, the CVs were close between them, which implied that these variations were attributed to environmental rather than genetic difference. The most suitable beginning time for harvesting of this cultivar was in middle April, when the average fresh weight per individual reached the highest value. Besides high yield, Haibao No. 1 was also characterized by possessing the wide stipe, long special-grade midrib, and large sporophylls. All these characteristics contributed to the much higher commercial value of this cultivar than the controls.

Intraspecific crossing of Saccharina japonica using distantly related unialgal gametophytes benefits kelp farming by improving blade quality and productivity at Sanggou Bay, China

When cultivating the kelp, Saccharina japonica, in China, one of the causes leading to degeneration in blade quality and productivity has been thought to be inbreeding caused by the use of limited numbers of sorus-bearing individuals that are continuously used over generations to produce large numbers of offspring. One of the effective solutions is to use intraspecific hybrid vigor to produce new cultivars or to recreate the original agronomic features of the older cultivars. This study produced a sporophytic B013 (cultivar code) by crossing a pair of distantly related unialgal male and female gametophyte clones. The male was isolated from an individual collected from a farmed population at Rongcheng, China, while the female came from a Korean wild individual sporophyte. In comparison with four controls, the B013 sporophytic offspring demonstrated excellent blade quality (entirely dark green in the dried product), high alginate (29.7-32.9 %) and iodine (0.29-0.31 mg g(-1)) contents in the dried product, the lowest fresh weight/sun-dried weight (FW/DW) ratio and the smallest coefficients of variation for total length (7.4 %), width (6.9 %), and biomass output (14.6 %). In addition, this cultivar remained in a vegetative state from June to August, greatly reducing the biomass loss during the hot summer season. In comparison with seeding zoospores for seedling production, seeding a selected pair of unialgal gametophytes had apparent advantages in effectively preventing inbreeding depression, which gave rise to a uniformed product and provided new cultivars for large-scale farming.

Breeding of an intraspecific kelp hybrid Dongfang no. 6 (Saccharina japonica, Phaeophyceae, Laminariales) for suitable processing products and evaluation of its culture performance

Dongfang no. 6, an intraspecific hybrid of kelp (Saccharina japonica) which was high in yield and had a high stress tolerance and was suitable for processing, was bred by crossing scientifically selected parental gametophyte clones. The sporophyte of the hybrid was raised by crossing a female gametophyte clone isolated from a Korean ecotype of S. japonica (Korean kelp) with a male gametophyte clone isolated from Lianza no. 1, a variety of S. japonica bred through continuous selection. The culture performance, stress tolerance, and suitability for processing of the hybrid and two commercial controls were compared for a period lasting 5 years (2009–2013). It was found that the hybrid was fast in growth, tolerant to high seawater temperature, resistant to strong solar irradiation, late in maturing time, and high in processed product yield. The percentage of salting processed product of the hybrid was 17.5 and 15.4 % higher than those of control 1 and 2, respectively. The yield of salting processed product of the hybrid increased by 24.2 and 38.8 % over control 1 and 2, respectively. The yield of air drying processed product of the hybrid increased by 109.4 and 88.1 % over control 1 and 2, respectively. The breeding of Dongfang no. 6 demonstrated that crossing gametophyte clones in combination with selecting desirable hybrids are highly effective for breeding kelp hybrids with high-culture performance and suitability for processing.

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

SummaryThe 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.

High genetic diversity in gametophyte clones of Undaria pinnatifida from Vladivostok, Dalian and Qingdao revealed using microsatellite analysis

Breeding practice for Undaria pinnatifida (Harvey) Suringar requires the screening of a large number of offspring from gametophyte crossings to obtain an elite variety for large-scale cultivation. To better understand the genetic relationships of different gametophyte cultures isolated from different sources, 20 microsatellite loci were screened and 53 gametophyte clone cultures analyzed for U. pinnatifida isolated from wild sporophytes in Vladivostok, Russia and from cultivated sporophytes from Dalian and Qingdao, China. One locus was abandoned because of poor amplification. At the sex-linked locus of Up-AC-2A8, 3 alleles were detected in 25 female gametophyte clones, with sizes ranging from 307 to 316 bp. At other loci, 3 to 7 alleles were detected with an average of 4.5 alleles per locus. The average number of alleles at each locus was 1.3 and 3.7 for Russian and Chinese gametophyte clones, respectively. The average gene diversity for Russian, Chinese, and for the combined total of gametophyte clones was 0.1, 0.4, and 0.5, respectively. Russian gametophyte clones had unique alleles at 7 out of the 19 loci. In cluster analysis, Russian and Chinese gametophyte clones were separated into two different groups according to genetic distance. Overall, high genetic diversity was detected in gametophyte clones isolated from the two countries. These gametophyte cultures were believed to be appropriate parental materials for conducting breeding programs in the future.

CONSTRUCTION AND CHARACTERIZATION OF A TENTATIVE AMPLIFIED FRAGMENT LENGTH POLYMORPHISM-SIMPLE SEQUENCE REPEAT LINKAGE MAP OF LAMINARIA (LAMINARIALES, PHAEOPHYTA)(1).

A tentative amplified fragment length polymorphism-simple sequence repeat (AFLP-SSR) linkage map of Laminaria was constructed using a haploid population of 40 gametophyte clones isolated from an individual of Dongfang No. 2, the first commercially cultured hybrid of a female gametophyte clone of L. japonica Aresch. [=Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders] and a male one of L. longissima Miyabe [=Saccharina longissima (Miyabe) C. E. Lane, C. Mayes et G. W. Saunders]. To the map, 263 markers (255 AFLP, seven SSR, and the gametophyte sex) were assigned. The map consisted of 25 linkage groups (LGs) with ≥ four markers, five triplets, and 15 doublets, which is 1,629.0 centiMorgans (cM) in length, covering 66% of Laminaria genome. The maximum space between loci is 24.63 cM. A putative sex-determining region was identified in LG2 , which was characterized by a dense marker distribution around the gametophyte sex locus. The linkage map itself and the methodology associated with its construction will facilitate the genetic study and further trials of the linkage map construction of Laminaria.

Assessing genetic identity of sporophytic offspring of the brown alga Undaria pinnatifida derived from mono‐crossing of gametophyte clones by use of amplified fragment length polymorphism and microsatellite markers

SUMMARYThe haploid stage of gametophytes of the subtidal brown alga Undaria pinnatifida can be vegetatively propagated under favorable conditions. This unique characteristic makes it possible to establish independent gametophyte cell lines that are zoospore‐derived. Sporophytic offspring can be generated through hybridizing the male and female gametophytes, which are derived from different cell lines. Accumulated experiences in this and other species in Laminariales demonstrated the applicability of this novel way to breed desired strains for open‐sea cultivation. Sporophytic offspring originated from mono‐crossing of male and female gametophyte clones were shown to have similar morphological characteristics under identical ambient conditions. However, there has been no report to relate this similarity on molecular levels. In this report, amplified fragment length polymorphism (AFLP) and microsatellite markers were used to analyze the genetic identity of sporophytic offspring of U. pinnatifida originated from two mono‐crossing lines (M1 and M2), two self‐breeding lines (S1 and S2) and one wild population (W). Totally 318 AFLP loci were revealed by use of 11 primer sets, of which 4.7%, 0.3%, 17.9%, 16.4% and 36.5% were polymorphic in M1, M2, S1, S2 and W, respectively. The pairwise genetic identity among the individuals of the same line was assessed. It was shown that offspring from mono‐crossing lines had a higher degree of identity (95.6–100%) than self‐breeding lines (87.7–98.4%) and the wild population (81.5–92.1%). Analysis by use of six microsatellite loci also revealed a higher genetic identity among individuals of the mono‐crossing line, further confirming the results of AFLP analysis. Results from this investigation support, on molecular levels, the novel way to produce and maintain strains in U. pinnatifida by use of different gametophyte cell lines.

The response of the early developmental stages of Laminaria japonica to enhanced ultraviolet-B radiation.

The responses of the early development of Laminaria japonica collected from Kiaochow Bay in China to enhanced ultraviolet-B radiation (UV-B, 280-320 nm) were studied in the laboratory. The low UV-B radiations (11.7-23.4 J x m(-2) x d(-1)) had no significant effects on zoospores attachment, but when the UV-B dose > 35.1 J x m(-2) x d(-1) the attachment decreased significantly compared with the control. Germination of embryospores was >93% under the low (11.7-35.1 J x m(-2) x d(-1)) doses, and in the range of 78.5%-88.5% under the high (46.8-70.2 J x m(-2) x d(-1)) UV-B doses, indicating a significant radiation effect. Under the higher UV-B exposure (35.1-70.2 J x m(-2) x d(-1)), all of the few gametophytes formed from embryospores died 120 h post-release. After exposure to the low UV-B radiation (11.7-23.4 J x m(-2) x d(-1)), the formation of sporophytes decreased and the female gametophyte clones increased compared with the control. However, the sex ratio and the relative growth of female gametophytes/sporophytes had not significantly changed. According to the results, enhanced UV-B radiation has a significant effect on the early development of L. japonica under laboratory conditions, suggesting that the UV-B radiation could not be overlooked as one of the important environmental factors influencing the ontogeny of macroalgae living in marine ecosystems.

Breeding and trial cultivation of Dongfang No. 3, a hybrid of Laminaria gametophyte clones with a more than intraspecific but less than interspecific relationship

Dongfang No. 3, a Laminaria hybrid, was bred by hybridizing a female gametophyte clone isolated from Line 7 of L. japonica with a male one isolated from Line LZZ which was developed through hybridizing female gametophyte clones of L. lonngissima and male gametophyte clones of L. japonica followed by continuous self-crossing and tolerances of strong irradiance and high seawater temperature targeted selection. The parental gametophyte clones of Dongfang No. 3 have a more than intraspecific but less than interspecific relationship. The seedlings of the hybrid were raised with the techniques of gametophyte clone asexual reproduction and hybridization and cultivated on trial. The dark brown hybrid has a wide, uniform, ripple-less and very tough blade and a very robust holdfast. A deep vertical groove and a half-round joint between stripe and blade distinguished also the hybrid from the varieties of L. japonica. The hybrid is tolerant of strong irradiance and high seawater temperature and resistant to the tissue rottenness of blade tip and edge. The hybrid matured later than the varieties of L. japonica widely used currently. The trial cultivation of this Laminaria hybrid has reached >406 ha in total. It increased the yield by 60.5% and 54.8% in average on that of Benniu (Control I) and Zaohoucheng (Control II), respectively, and by 62.4% and 75.8% on that of Line LZZ and Line 7, respectively, as were observed in Weihai, Shandong Province, China, in 3 continuous years, and by 57.0% and 32.1% in average on that of Control I and II, respectively, across 10 sites on the coast of north China in 2 continuous years. The breeding and application of Laminaria hybrids have paved an avenue of the direct use of heterosis, the easy integration of elite agronomical traits and fast renewing of Laminaria varieties.

Parentage analysis of Dongfang No.2, a hybrid of a female gametophyte clone of Laminaria japonica (Laminariales, Phaeophyta) and a male clone of L. longissima

The cultivation of the first filial generation of monoploid gametophyte clones of different Laminaria species (hybrid Laminaria) is an effective way of utilizing heterozygous vigor (heterosis). A female gametophyte clone of L. japonica and a male gametophyte clone of L. longissima were hybridized, generating Dongfang No.2 hybrid Laminaria. The parentage of this hybrid Laminaria was determined using AFLP of total DNA, SNP of the ITS region of ribosomal RNA transcription unit and microsatellite DNA variation at two loci. In addition to 167 AFLP bands shared by Dongfang No.2, L. japonica and L. longissima, Dongfang No.2 hybrid Laminaria shared another 70 and 55 bands with L. japonica and L. longissima, respectively, which were obviously more than 11 bands shared by L. japonica and L. longissima. Dongfang No.2 held both ‘T’ and ‘C’ at position 847 of the ITS region, while ‘T’ at this position was specific for L. japonica and ‘C’ for L. longissima, respectively. Dongfang No.2 also held the microsatellite DNA alleles of two parents together at two microsatellite DNA marker loci. These observations clearly proved that Dongfang No.2 is a true hybrid of L. japonica and L. longissiuma. Unfortunately, the origin of the chloroplast of Dongfang No.2 was not determined based on the variation of RuBisCo spacer. More sequence variants of both ITS region and RuBisCo spacer were identified in Dongfang No.2 and most of them did not exist in either L. japonica or L. longissima. The unexpected variants may be due to the mutation of gametophyte clones occurring during their vegetative amplification.

High throughput culture and gametogenesis induction of Laminaria japonica gametophyte clones

In anticipation of the application of a new sporeling-raising method using gametophyte clones to Laminaria commercial cultivation in China, techniques of mass culture and gametogenesis induction of L. japonica gametophyte clones were developed, as a mass of fertile gametophytes is a prerequisite for sporeling-raising with the new method. Gametophyte clones which were subjected to fed-batch culture exhibited a classical logistic growth curve. Growth rates decreased gradually after 2 months of culture, and were negatively correlated to cell density. UNOVA also showed that only cell density has a significant effect on the growth of gametophyte clones under the experimental conditions. Based on the dynamics models revealed, a culture strategy only directed at the control of cell density was adopted. By this strategy, a total of 36 kg wet weight from an initial weight of 0.75 kg was achieved after 3 months culture in 100 20-L bottles. The final average density reached 24 g L−1. For the subsequent gametogenesis induction, amplificatory male and female gametophyte clones were cut, mixed and cultured in bottles under the same conditions used in amplification except for a change of photoperiod from continuous irradiance to 10 h light: 14 h dark cycle. Egg discharge occurred 10 days after the mixed culture and increased gradually with the culture duration. Most gametophytes gave rise to sporophytes 20 days after induction. Large-scale culture of gametophyte clones and gametogenesis induction for commercial cultivation in 2003–2005 have been conducted successfully.

Breeding of an elite Laminaria variety 90-1 through inter-specific gametophyte crossing

Laminaria longissima and Zaohoucheng No.1 (a commercial variety selected from Laminaria japonica) differ to a certain extent in their morphological characteristics and biological habits. It was assumed that varieties bred through their hybridization should exhibit high yield potential and tolerate relatively high seawater temperatures. Female gametophyte clones isolated from L. longissima were crossed with male clones isolated from Zaohoucheng No.1. Laminaria variety 90-1 was obtained after gametophyte crossing, continuous self-crossing and selection. This variety was genetically homozygous; the indices of variation of blade length, width and thickness of the final two selection cycles were 7–8%; i.e., not different significantly. Variety 90-1 grew faster, lost less tissue and had higher yield potential than two widely used commercial varieties of L. japonica (all commercial varieties currently used in China originate from this latter species). The blade of variety 90-1 increased 3.71 cm day−1 on average during the whole period of cultivation, almost two-fold that of two controls, and growth was maintained even when seawater temperature was higher than 18°C–3°C higher than the temperature tolerated by other Laminaria varieties. Variety 90-1 increased yield by more than 70% over two controls and also synthesized desirable amounts of iodine, mannitol and algin. In blade length, variety 90-1 was more similar to L. longissima than to L. japonica, but more similar to L. japonica in blade width and thickness. Since the adoption of variety 90-1 in 1999, its culturing area has increased each year to reach its current area of 7,000 ha, i.e., almost one-third of the total cultivation acreage of Laminaria in China. Breeding of variety 90-1 has demonstrated that it is feasible to develop elite Laminaria varieties by crossing gametophytes from different Laminaria species in combination with successive self-crossing and selection.

Study on gametophyte vegetative growth ofUndaria pinnatifida and its applications

When cultured under certain environmental conditions (25°C, light intensity 80 μmol/m2·s, LD 12/12, in enriched seawater medium with 7×10−4 mol/L NO3−N, 1.56×10−4 mol/L, PO4−P and supplements of other elements like Mn, Fe, I, etc.), male and female gametophytes ofU. pinnatifida kept growing vegetatively and propagated fast at average daily fresh weight increase rate of about 20%. The empirical formulaGm=Go·3m was established to estimate the output of vegetative gametophytes. Vigorous vegetative gametophyte cells began to form reproductive structures (oogonium and spermatangium, when the temperature was lower than 25°C and other environmental factors were kept optimal. The sufficient supply of gametophyte cells provided enough seeds for raisingUndaria sporelings on production scale. Controlled cross-breeding experiments using selected male and female gametophyte clones which increase their cell number by mitosis instead of meiosis were also carried out in vitro. Juvenile sporophytes from the cross-breeding had almost the same length and width increase rates as those of the control. The fact that vegetative gametophytes can be purposely selected, propagated quickly, cross-bred, with the resulting sporophytes having almost the same characteristics leads to a new way to select desiredUndaria strains for long time use without losing the desired economic characteristics.