Parentage assignment in hybrid abalones (Haliotis rufescens × Haliotis discus hannai) based on microsatellite DNA markers

Parentage analysis in aquaculture determines genealogical relationships between broodstock and progeny when the parents are unknown. Thus, parentage analysis is a useful tool to establish pedigree reports in molecular‐assisted selection programs. Here, we evaluated 10 heterologous microsatellite markers for parentage assignment in abalone hybrids produced from 43 abalone broodstocks of red abalone (Haliotis rufescens) and Japanese abalone (H. discus hannai). The allele frequencies, exclusion probabilities and broodstock contributions were calculated using CERVUS, PAPA and GERUD software. The polymorphic information content (PIC) values showed that most of the microsatellite loci were highly informative (>0.7) and more than 90% of parentage assignment was possible with a minimum of 5–6 microsatellite markers. Parentage assignment for hybrid and pure‐red progeny showed a better performance than pure‐Japanese progeny. This result could be due to the high level of allele loss in the parental genotypes. In addition, results indicated that only two sires contributed over 80% and 90% of red and hybrid progenies, respectively. This study gives a new molecular tool to support marker‐assisted selection in abalone hybrids produced in Chile.     

Karyotype analysis of interspecific hybrids between Haliotis rufescens and Haliotis discus HANNAI

Evidence of hybridization in Haliotis has been mainly supported by hatchery experiences and collection of wild hybrid abalones among several species from natural populations worldwide. However, despite the importance to understand the role of the hybridization process through Haliotidae evolution, and also its impact on the abalone aquaculture, genetic studies in hybrid abalones have been poorly developed. Herein, cytogenetic approach allows studying the genetic conformation in hybrid organisms at the chromosome level. This paper reports a quantitative karyotype analysis in Haliotis rufescens, Haliotis discus hannai and their interspecific hybrid. Thus, to characterize chromosome pairs and establish cytogenetic comparisons, chromosome banding with distamycin‐A/4,6‐diamidino‐2‐phenylindole fluorochromes and morphologic measurements were performed. The results showed that the hybrids are successfully viable and their karyotypes evidenced a conservative chromosome number of 2n=36. The karyo‐idiogram showed a high correspondence in chromosome pair morphology among the hybrids and their parental species, except for a single heteromorphic pair that corresponds to the chromosome 16 from H. rufescens andH. d. hannai respectively. The implications of the abalone hybrid viability derived from its chromosome composition are discussed.     

Genetic characterization and gonad development of artificially produced interspecific hybrids of the abalones, Haliotis discus discus Reeve, Haliotis gigantea Gmelin and Haliotis madaka Habe

Hybridization among abalone species has been suggested as a possible means to increase their growth rates for aquaculture. As a first step to test the usefulness of the hybrids of Japanese abalone species (Haliotis discus discus, Haliotis gigantea and Haliotis madaka) for aquaculture, we characterized the genetic background and gonad development of hybrids that were produced by artificial insemination. The hybrid status of the resulting offspring was confirmed by assaying 14 allozymes and by RFLP analysis of the 16s rRNA and cytochrome oxidase I (COI) regions of mtDNA using 13 restriction enzymes. Histological examination of the gonads of the hybrids was conducted in comparison with those of the parental species. Cross‐breeding among the three species was conducted successfully in all combinations although with lower fertilization rates (means of 1.3–60.8%) than the parental species (34.3–90%). Crosses between H. discus discus and H. madaka had higher fertilization rates (22.4–60.8%) than those involving H. gigantea (1.3–19.9%). The hybrids were ascertained by the presence of both parental genotypes at the LDH‐A, ME‐A, MDH‐A and GPI loci. The maternal origin of the hybrid mtDNA was confirmed by digestion with DdeI, TaqI, HpaII of the COI region. No polymorphism was observed in the 16S rRNA region. The hybrids had gonadal development and maturity stages similar to the parental species up to fully mature oocytes and sperm. They spawned upon stimulation and produced viable offspring with high fertilization rates and successful development to the juvenile stage in back‐ and homologous hybrid crosses.     

Hybridization between Haliotis rufescens and Haliotis discus hannai: evaluation of fertilization,larval development,growth and thermal tolerance

Two introduced abalone species are currently produced in Chile, red abalone Haliotis rufescens and Japanese abalone Haliotis discus hannai. However, red abalone accounts for 99% of total production, while the Japanese abalone has not adapted well to Chilean coastal waters. This study reports the hatching, growth and thermal tolerance performance in interspecific hybrids produced between red (R) and Japanese (J) abalone. Our results show that egg age and sperm concentration were critical factors to produce hybrids. The cross R♀ × J♂ showed a fertilization rate of 55.3 ± 3.5% using 20‐min‐old eggs and sperm concentrations of 14 × 10⁶ cells mL^?1, while the reciprocal cross (J♀ × R♂) was not successful. Further, larval development stages were similar in RR, JJ and RJ hybrid abalones. Among the experimental trials, settlement rate varied from 12.3% to 18.6% and final survival from 20.1% to 31.7%, being the RJ hybrid rates intermediate between parental species. The final shell lengths were similar between RR and RJ hybrids, but significantly higher in JJ abalones. In addition, thermal tolerance was ascertained due its pivotal role for the abalone physiology. Thus, RJ hybrids showed the highest HSP70 gene expression and offers new possibilities to expand Chilean abalone production in warm waters zones.     

Genetic variability of cultured populations of the Pacific abalone (Haliotis discus hannai Ino) in China based on microsatellites

The genetic diversity of cultured populations of the Pacific abalone (Haliotis discus hannai Ino) from northern China was analysed using seven microsatellite markers. The microsatellite loci were polymorphic for all the populations, with an average of 8.7 alleles per locus (range 8.0–9.4). The mean observed and expected heterozygosities were 0.547 (range 0.500–0.596) and 0.774 (range 0.754–0.787) respectively. The allelic diversity in terms of number of alleles per locus was considerably lower than that previously found in wild populations (range 21.8–23.0), indicating that bottleneck effects occurred when each population was founded. Significant genetic differentiation among the five cultured populations was shown using F_st and R_st values, and pairwise comparison based on allelic distribution. A neighbour‐joining analysis of the genetic distance did not show a consistent relationship between the geographic and the genetic distances, suggesting the existence of exchanges of breeds and eggs between the hatcheries. The results obtained in this study are useful for a number of areas of interest for fisheries management and the aquaculture industry, especially with regard to breeding programmes.     

皱纹盘鲍4个养殖群体杂交子代生长与存活状况比较

为利用杂种优势培育皱纹盘鲍(Haliotis discus hannai)优良新品种,本研究以4个不同养殖群体[黄岛(HD)、荣成(RC)、日本(JP)、大连(DL)]的皱纹盘鲍为亲本,设计4×4完全双列杂交,建立了4个自交家系和12个正反杂交家系,在利用微卫星标记进行亲子鉴定的基础上,对各家系的F1在1、5、13、17月龄时的生长性状、杂种优势率、生长速度和存活情况进行比较,分析杂交效应。结果表明,在各个生长阶段均有部分杂交家系与自交家系相比表现出显著的生长优势;HDRC、HDDL和JPDL家系的生长速度较高;HDDL、HDJP、RCDL、JPRC及RCHD家系有着较高的存活率;在杂种优势方面,HDRC、HDDL与DLHD家系在各生长参数与生长速度上有明显的杂种优势,HDDL、RCDL、DLHD家系在存活率上表现出明显的杂种优势。本研究筛选出的具有优势的交配组合,可作为皱纹盘鲍生产上种苗来源的参考,也为利用杂种优势培育皱纹盘鲍新品种提供了基础资料。     

The potential for utilizing fouling macroalgae as feed for abalone Haliotis discus hannai

In order to assess the potential of utilizing fouling macroalgae as feed for abalone Haliotis discus hannai, the species, biomass and total stock of fouling macroalgae attached on the aquaculture facility‐lines were investigated and assessed at Sungo Bay, China, in August 2007. The nutritional value of four fouling macroalgae (Codium fragile, Sargassum muticum, Ulva pertusa and Laminaria japonica) and one cultured macroalgae (Gracilaria lemaneiformis) as the diet of abalone was assessed by analysis of the chemical composition of the macroalgae and by measuring bioavailability and digestibility to abalone H. discus hannai under laboratory conditions in August 2007. The results showed that the biomass of fouling macroalgae at the two sites was 243.61 ± 26.23 and 1078.15 ± 50.28 g m^?1 respectively. The contents of protein of G. lemaneiformis and C. fragile were 18.30% and 17.60%, respectively, which were significantly higher than the others. The five macroalgae were accepted by the abalone, and growth energies (P) and growth efficiency (%) were positive. However, significant differences were found among the different macroalgae. The growth efficiencies of abalone show a negative relationship with the levels of dietary lipid and a positive relationship with the protein contents. These results suggest that fouling macroalgae have a great potential to be used as diets of abalone.     

Identification of quantitative trait loci for growth-related traits in the Pacific abalone Haliotis discus hannai Ino

The locations and effects of quantitative trait loci (QTL) were estimated for nine characters for growth‐related traits in the Pacific abalone (Haliotis discus hannai Ino) using a randomly amplified polymorphic DNA (RAPD), amplification fragment length polymorphism (AFLP) and SSR genetic linkage map. Twenty‐eight putatively significant QTLs (LOD>2.4) were detected for nine traits (shell length, shell width, total weight, shell weight, weight of soft part, muscle weight, gonad and digestive gland weight, mantle weight and gill weight). The percentage of phenotypic variation explained by a single QTL ranged from 8.0% to 35.9%. The significant correlations (P<0.001) were found among all the growth‐related traits, and Pearson's correlation coefficients were more than 0.81. For the female map, the QTL for growth were concentrated on groups 1 and 4 linkage maps. On the male map, the QTL that influenced growth‐related traits gathered on the groups 1 and 9 linkage maps. Genetic linkage map construction and QTL analysis for growth‐related traits are the basis for the marker‐assisted selection and will eventually improve production and quality of the Pacific abalone.     

Genetic variability revealed with microsatellite markers in an introduced population of the abalone Haliotis discus hannai Ino

One of the challenges for the culture of any species is to control the loss of genetic variability, which may result in a decrease in the quality of commercially important traits. The goal of this study is to assess the genetic diversity of a hatchery population of the Pacific abalone ( Haliotis discus hannai ) from the Center for Abalone Production of the Universidad Católica del Norte (CAP-UCN) that is maintained under a breeding programme. We used six polymorphic microsatellite markers within the cultivated population. The loci Awb033 and Awb079 had the highest number of alleles (11 and 10 respectively) and the loci Awb022 and Awb026 the lowest (two and four respectively). The mean number of alleles per locus was 6.83. The average observed and expected heterozygosities were 0.71 and 0.70, respectively, and the average F _IS ( f ) index was −0.023. We compared the population genetic parameters of the CAP-UCN population with previously published data of wild and hatchery populations of the same species. Results indicate lower genetic diversity estimated as allelic richness in the introduced population with a loss of 11–58% alleles per locus. Despite the high allelic loss, the estimated inbreeding coefficient suggests that the breeding programme carried out in the CAP-UCN has controlled and maintained heterozygosity levels successfully. A temporal study is necessary to determine whether the genetic diversity loss detected was caused during the initial introduction of breeders or to the breeding programme actually implemented.     

皱纹盘鲍与盘鲍杂交效果分析

从杂种优势的角度对皱纹盘鲍♀与盘鲍♂杂交子一代的经济性状进行分析。结果表明,4月龄杂交鲍子一代(F1)活体重比皱纹盘鲍增加36.87%,软体部干重增加33.21%,总干重增加29.20%,其它性状也显著优于皱纹盘鲍。杂交鲍F1的壳长、壳宽、活体重、软体部干重和总干重等主要性状的变异系数为12.94%~44.51%,其中软体部干重的变异系数最大,壳长的变异系数最小;且杂交鲍各性状的变异系数均大于皱纹盘鲍。杂交鲍子一代的变异有助于定向选择,提高杂交育种的效果。     

亲本数目对鲍养殖群体AFLP标记位点及其遗传结构的影响

为了探讨不同的交配亲本数对鲍养殖群体遗传结构的影响,实验中对两个皱纹盘鲍自交群体SS(亲本数少)和LS(亲本数多)及两个皱纹盘鲍(♀)×日本盘鲍(♂)的杂交群体SH(亲本数少)和LH(亲本数多)进行了位点频率和遗传结构的AFLP标记分析。研究表明,自交组中亲本数少的SS群体的总位点数少于LS群体,后者的一些低频位点在前者中丢失,同时群体的位点频率发生漂移,SS的低频位点数目减少而高频位点略有增加。另外统计了各群体的相似指数和杂合度,发现SS群体的相似指数低于LS,而杂合度高于LS群体。在杂交组两群体(SH,LH)中位点数目频率、相似指数和杂合度变化趋势与两自交群体相似。两组结果均表明群体的亲本数减少会引起一些低频位点丢失及位点频率发生漂移,同时亲本数过少会导致群体的杂合度暂时升高。     

Genetic certification of presumed hybrids of blue × red abalone (Haliotis fulgens Philippi and H. rufescens Swainson)

Presumed hybrids between Haliotis rufescens and H. fulgens abalones, produced in a commercial abalone laboratory, were evaluated for progeny viability, gonad characteristics and for their ‘true’ status as hybrids by means of allozyme and microsatellite analyses. Four out of 10 evaluated allozyme loci proved to be useful for the genetic certification of hybrids, Gpi, Mdh‐1, Mdh‐2 and Sod, as they had exclusive alleles for each of the abalone reference species from which the presumed hybrids were produced. Three microsatellite loci were also evaluated for their usefulness in abalone hybrid certification. Two were not useful for hybrid certification as the reference abalone species shared some of the same alleles. The third loci (Hka 56) also had shared alleles between the two species, but at very different frequencies. Therefore, this permitted a presumptive certification of hybrids, which, however, was not error‐proof as two allozyme certified hybrids were wrongly classified as H. rufescens rather than as hybrids. Progeny viability of the only female certified to be a hybrid was significantly lower compared with one viability of the other females spawned, which were certified as H. rufescens. However, the number of eggs spawned by the only certified hybrid female was as large as those spawned by females certified as H. rufescens. Gonads of abalones sampled for the macroscopic appearance of sterility indicated that in the absence of any gonad development, no distinctive difference allows for hybrid identification, as there were undifferentiated individuals that were both hybrid and non‐hybrid. On the other hand, two certified hybrid males were characterized by spermatocytes being the most abundant gamete type, although one had some spermatozoa present, indicating only partial sterility. Among the two female hybrids found, vitellogenesis was observed to proceed up to vitellogenic oocytes.     

Time to recover the upright posture in juvenile abalones (Haliotis discus discus Reeve, H. gigantea Gmelin and H. madaka Habe)

The adoption of an upright posture in abalones is essential to enable them to use their foot, and hence to be able to move and seek shelter, as well as to avoid exposure of soft parts and possible predation. In mass restocking programs for abalone, juveniles are released by divers near the seabed, but without control over their posture when they reach the bottom. Thus, the time to recover the upright posture is an important consideration in abalone restocking programs as the quicker they assume this posture the higher the likelihood of survival. This study reports significant differences in the speed of recovering the upright posture between juveniles of the abalones Haliotis discus discus, H. gigantea and H. madaka and between tests conducted under stagnant and flowing water conditions. Longer times were required for recovery in all species in stagnant than flowing water. On average, juveniles of H. discus discus (17.16 and 10.43 s) and H. gigantea (22.54 and 11.89 s) recovered faster than those of H. madaka (161.13 and 49.02 s) under stagnant and flowing water conditions respectively. These results suggest that different species require different levels of care and that water flow or current at the time of release may affect post‐release survival.     

Parentage testing for hatchery-produced abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> based on microsatellite markers: preliminary evaluation of early growth of selected strains in mixed family farming

This study dealt with the detection of parentage in a mixed family tank of abalone Haliotis discus hannai using microsatellite DNA markers. As a preliminary investigation, the early growth rate between selected and non-selected abalone families, which were reared together in the mixed family tank from settlement stage to juvenile stage (7 months of age), was also compared. The selected families were derived from seven parents (three females and four males) selected for larger size in shell length at about 1-year-old, and the non-selected families originated from five wild captives (three females and two males). Parentage analysis based on eight microsatellite markers unambiguously allocated the 170 juveniles sampled from the single tank to the 17 parental pairs. The family size was highly heterogeneous among families, as two males in the selected families and one male in the non-selected families dominated the contribution to the offspring pool (>80%). The mean shell length of the selected families was approximately 23% larger than that of the non-selected families (Student’s t-test, P<0.001). This study demonstrated that the use of microsatellite markers is effective for parentage determination in the mixed family farming, commonly used in abalone hatcheries, and selective operations for larger size could improve the growth of the next generation.     

Genetic difference between Ezo-awabi <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> and Kuro-awabi <Emphasis Type="Italic">H. discus discus</Emphasis> populations: Microsatellite-based population analysis in Japanese abalone

ABSTRACT:   This study documents the genetic population relationships between Ezo-awabi Haliotis discus hannai and Kuro-awabi H. discus discus by means of the microsatellite technique with an emphasis on the extent of genetic difference between Ezo- and Kuro-awabi populations. Eight markers were employed to screen five populations each collected from Ezo- and Kuro-awabi habitats. All eight loci showed polymorphisms in all populations (number of alleles per locus, 11.1–14.1; averaged expected heterozygosity, 0.64–0.70). The number of loci that yielded significant genetic heterogeneities (allele frequency distribution and/or pairwise θ ) between Ezo- and Kuro-awabi populations was larger than that generated in comparisons between populations within Ezo- and Kuro-awabi habitats. According to the bootstrap neighbor-joining trees constructed on the basis of two genetic distance measures ( D _A and D _ST), the 10 populations fell into two clusters of populations (Ezo-awabi and Kuro-awabi habitat groups), and the results of hierarchical amova testing also supported the neighbor-joining clustering. The outcomes presented here suggest that the microsatellite markers used in this study are potentially an efficient means to differentiate Ezo- and Kuro-awabi populations.     

Ecological and eco‐social models for the introduction of the abalone Haliotis discus hannai into benthic systems of north‐central Chile: sustainability assessment

• 1. Loop models of ecological and socio‐economic systems were developed to analyse and predict the impact of a possible accidental introduction (escapes) of the abalone Haliotis discus hannai into a benthic community of north‐central Chile.
• 2. Although the ‘new’ ecological system resulting from a successful invasion of abalone would be locally stable, the establishment of a self‐enhanced dynamic of recruits would transform this into an unstable system.
• 3. The harvest of the kelp, Lessonia trabeculata and other macroalgae is not recommended because this destabilizes the system. The harvest of abalone adults is only sustainable if they do not exert a negative effect upon other native invertebrates.
• 4. The eco‐social model showed three important results: (1) if the variables ‘Price’ and ‘Farming’ are in expansion and stationary dynamics, then the models were found unstable; (2) a self‐enhanced dynamic of abalone recruits tends toward instability; and (3) the harvest of the kelp L. trabeculata and other macroalgae would be non‐sustainable.
• 5. Based on our results, the sustainable development of extensive farming of H. discus hannai in the sea would be not reached. If it is done, an intensive monitoring of the community after introduction into the system is strongly recommended. Likewise, the farming of macroalgal species (source of food for abalone) should be promoted in order to avoid harvesting of macroalgae from natural systems.

Copyright © 2006 John Wiley & Sons, Ltd.     

Heat-shock protein70 gene expression in four hatchery Pacific Abalone Haliotis discus hannai Ino populations using for marker-assisted selection

Heat shock proteins (Hsps) are molecular chaperones that help organisms cope with stressful conditions. Here, we report on the growth rates and Hsp70 expressions in inbred and hybrid populations of abalone Haliotis discus hannai Ino. In abalone, inbred populations expressed more Hsp70 than hybrid populations at all temperatures, except at very high temperatures close to the physiological limit. At benign temperatures, there was a clear trend towards higher Hsp70 expression in inbred than hybrid populations, whereas at higher temperatures, a trend in the opposite direction was observed. The temperature of maximal Hsp70 expression (T_peak) varied with the population type. The T_peak of inbred populations (26°C) was lower than that of the hybrid populations (28°C). The maximal inducible Hsp70 of inbred populations was higher than that of hybrid populations. The results showed a trend towards higher expression in inbred population at a lower temperature. These results provide direct experimental evidence that hybrids can cope with the intrinsic stress even at non‐stressful temperatures. The constitutive Hsp70 may therefore be used for marker‐assisted selection in a breeding programme.     

四种鲍45S rDNA在染色体上的比较定位

为了研究皱纹盘鲍、西氏鲍、绿鲍和杂色鲍等4种鲍的核型特征,实验利用荧光原位杂交(fluorescence in situ hybridization, FISH)技术比较定位了上述4种鲍的45S rDNA位点。皱纹盘鲍中约83%的中期细胞均检出2对45S rDNA位点,分别位于13号和16号染色体的长臂端部。西氏鲍中约75%的中期细胞均检出3对45S rDNA位点,分别位于6号染色体短臂端部、14号和17号染色体长臂端部。绿鲍中约85%的中期细胞均检出3对45S rDNA位点,分别位于4号、6号和8号染色体长臂的端部。杂色鲍中约65%的中期细胞均检出3对45S r DNA,位点,分别位于3号、4号和12号染色体短臂的端部。此外,4种鲍均有少数中期相的45S rDNA位点数高于众数,这提示,除了明确的45S rDNA位点外,4种鲍可能均有若干个不稳定的45S rDNA位点。实验结果丰富了鲍细胞遗传学研究资料,同时为鲍的遗传育种研究提供了基础数据。     

Physiological metabolism of Haliotis discus hannai Ino under different light qualities and cycles

The goal of this experiment was to examine the effects of different light qualities (red, white, blue) and cycles (12L:12D, 8L:16D, 4L:20D, 0L:24D, 16L:8D) on the survival, growth, metabolism and antioxidant defence system of Haliotis discus hannai Ino. Under red and white light at 4L:20D, the body weight‐specific growth rate of abalones was significantly higher than that of the 0L:24D group (P < 0.05). At 16L:8D, succinate dehydrogenase activity decreased and lactate dehydrogenase and alanine aminotransferase activity and the lactic acid content significantly rose (P < 0.05) in the red and white light groups. Under blue light, reactive oxygen species and malondialdehyde content for each light cycle were significantly higher than those of the red and white groups (P < 0.05). Under blue, white and red light, total antioxidant capacity (T‐AOC), superoxide dismutase (SOD) activity, glutathione peroxidase (GP_X) activity and reduced glutathione (GSH) content gradually rose with increased duration of light exposure. Under red light, significant differences in these parameters were detected between the 4L:20D and 16L:8D groups (P < 0.05). Under blue and white light at 16L:8D, values of T‐AOC, SOD, GP_X activity and GSH content were significantly lower than those of the 12L:12D group (P < 0.05). Overall, these results indicate that red light at light cycles of 4L:20D and 8L:16D is optimal for the culture and production of H. discus hannai, as these conditions promoted food intake, growth and health maintenance as well as normal physiological metabolism of the organism.