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Selection and characterization of an improved strain produced by inter-species hybridization between Pyropia sp. from India and Pyropia haitanensis from China
Aquaculture 491 (2018) 177–183
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Short communication
Selection and characterization of an improved strain produced by interspecies hybridization between Pyropia sp. from India and Pyropia haitanensis from China Hongchang Dinga,b,c, Feng Lvd, Hongxiao Wua, Xinghong Yana,b,c,
T
⁎
a
Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, Shanghai 201306, China National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China c Shanghai Engineering Research Center of Aquaculture, Shanghai 201306, China d Nantong Science and Technology College, 226007, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Pyropia haitanensis Inter-species hybridization Recombinant strain Growth rate Photosynthetic pigment
A crossing-experiment between Pyropia sp. from India and P. haitanensis from China was carried out for breeding improved strains. The male parent was the wild-type strain (Ps-WT01) of Pyropia sp., which was characterized by thick blade, slow growing, good toughness and more production of conchospores, and the female parent was the mutation strain (Ph-HMC5) of P. haitanensis, which was characterized by thin blade, fast growing, poor toughness and low production of conchospores. At first, four improved strains with obvious recombinant advantages were obtained from the F1 color-sectored blades, and finally the most improved strain (HR-5) was selected from them. The maximum of absolute growth rate of the male and female parents were 0.39 and 5.24 cm/d, respectively, while that of HR-5 strain was 10.11 cm/d and its absolute growth rate was greater than the maximum growth rate of the female parent during 41–55 days of culture. After 60 days, the mean blade length of HR-5 was 168.67 cm, which was 12.80-fold of that (13.18 cm) of its male parent and 1.97-fold of that (85.67 cm) of its female parent, respectively. The chlorophyll a content in the 35-day-old blades of HR-5 strain was 10.57 mg/g, which increased by 68% and 35% compared with that of its male and female parents, respectively. The phycobiliprotein content of HR-5 blades was 114.88 mg/g, which was 2.51- and 2.01-fold of that of its male and female parents, respectively. The mean thickness of the 35-day-old blades of HR-5 was 25.06 μm, which was 60% and 124% of that of its male and female parents, respectively. The total number of conchospores per shell released from HR-5 was 404.14 × 104, which was 2.73- and 6.38-fold of that of its male and female parents, respectively. The above results confirmed that HR-5 strain was characterized by fast growth, high quality as well as large amount of conchospores, and may offer an alternative for the nori industry.
1. Introduction Pyropia haitanensis is an important economic algae. Its total output accounts for approximately 75% of the total Pyropia production in China (Ma and Cai, 1996). In the 1960s, artificial breeding and cultivation of P. haitanensis was succeeded, leading to the establishment and development of culture industry of P. haitanensis (Fujian Fisheries Bureau, 1979). However, the wild-type strain of P. haitanensis has been cultivated without any selection or improvements for over 50 years, thus, breeding new cultivars has been placed in the important agenda (You, 1999). Until today, numerous stable artificial mutants with inheritable and valuable characteristics, such as heat tolerance, low-salt resistance and low-nitrogen and phosphorus resistance have been
⁎
isolated (Yan and Ma, 2007; Yan and Chen, 2008; Liu et al., 2009; Yan et al., 2010; Tan et al., 2014). Meanwhile, several improved strains with high quality and yield have been obtained and received the certification of new cultivar from Chinese government (Wang et al., 2011). In addition, through the intra-species hybridization of artificial pigment mutants of P. haitanensis, some strains with better characteristics than their parents have been obtained (Xu et al., 2008; Wang et al., 2010), but their cultivation in large-scale has not been reported. Crossbreeding of Pyropia mainly utilize the hybrid recombination advantage, that is, the hybrid progeny produced by interbreeding between individuals with different characters is superior to parents in growth and biochemical characteristics (He et al., 2007). One of the important reasons for generated hybrid recombination advantage in
Corresponding author at: Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University), Ministry of Education, Shanghai 201306, China. E-mail address: [email protected] (X. Yan).
https://doi.org/10.1016/j.aquaculture.2018.03.015 Received 27 October 2017; Received in revised form 1 March 2018; Accepted 8 March 2018 Available online 09 March 2018 0044-8486/ © 2018 Elsevier B.V. All rights reserved.
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the most obvious hybrid recombinant advantages was selected, namely the improved strain.
intraspecific cross is the significantly different characteristics of parental strains (Xu et al., 2008). The Pyropia sp. was derived from the India peninsula, we have previously (Zhang and Yan, 2014) reported that the morphological structure, chromosome counts, sex and 5.8S rDNA-ITS region of this species were very similar to those of P. haitanensis from China. This indicated that the genetic relationship between them is very close although they were isolated so long distance from China to India, But, there are still no enough evidences to identify they are the same species. Therefore, we think these two species are different species in this research. In addition, we have previously (Liu et al., 2013) reported that the gametophytic blades of heterozygous conchocelis produced by crossing Pyropia sp. from India and P. haitanensis from China were fertile. The wild-type Pyropia sp. (Ps-WT01) was characterized by thick blades, slow growing, good toughness in elasticity, maroon thallus with greenish basal part and reddish apical part, and many marginal denticles. The mutation strain of P. haitanensis (PhHMC5) was characterized by thin blade, fast growing, poor toughness in elasticity and thallus with uniform pale red brown color, and no marginal denticles. In this paper, we further screened the improved hybrids strains with recombinant advantages through the hybridization between Ps-WT01 and Ph-HMC5 strains.
2.4. Characteristics of the improved strain 2.4.1. Blade growth After cultured for 30 days, twenty blades of each strain were randomly selected and cultured in three aerated flasks (1 L), respectively. The blade length, width and wet weight were measured every 5 days in a 30-day period. The specific and absolute growth rates of the blades were calculated as previously reported by Stein (1973). 2.4.2. In vivo absorption spectra and contents of chlorophyll a as well as the contents of phycobiliproteins After being cultured for 35 days, the in vivo absorption spectra and contents of chlorophyll a (Chl. a) of the F1 blades were measured (Yan and Aruga, 1997). The contents of phycoerythrin (RPE), phycocyanin (RPC) and allophycocyanin (APC) were measured according to the methods described by Gao (1993). Specifically, the fresh blades were dried at 80 °C for 6 h, and ground after three freezing and thawing cycles (between −20 °C and room temperature). The extract was centrifuged at 15,000 ×g for 20 min, and the supernatant was used to determine the OD562, OD615, OD650, and OD652. The absorbance and OD were measured by using an ultraviolet spectrophotometer (UV2600, Shimadzu). The contents of Chl. a, PE and PC were calculated using the following formulae:
2. Materials and methods 2.1. Materials The male parental stain was wild-type Pyropia sp. (Ps-WT01) originated from the coast of Indian peninsula. In detail, its free-living conchocelis were developed from carpospores released by a gametophytic blade (Zhang and Yan, 2014) and cultured in our laboratory to obtain conchospores and blades. These blades were prepared as single somatic cell suspensions using enzymatic hydrolysis technology and then cultured and developed into regenerative blades (Wang et al., 1986). After cultured for a period of time, single regenerative blades underwent parthenogenesis (Yan et al., 2007) and produced homozygous conchocelis (strains). The female parent was a mutation strain (Ph-HMC5) of P. haitanensis. Its homozygous conchocelis were also obtained through blade parthenogenesis. The conchocelis filaments were stored at 18 °C in the laboratory.
Chl. a = (11.6 × OD665–1.31 × OD645–0.14 × OD630) × V/W
(1)
PE = (0.123 × OD562–0.07 × OD615 + 0.015 × OD652) × V/W
(2)
RPC = (0.162 × OD615–0.099 × OD650–0.001 × OD562) × V/W
(3)
APC = (0.171 × OD650–0.0006 × OD562–0.004 × OD615) × V/W
(4)
PC = RPC + APC
(5)
where V (in mL) is the volume of grinding fluid and W (in mg) is dry weight of sample. 2.4.3. Blade thickness After being cultured for 35 days, the blades were cut into three parts with bare-handed, double-edges blades. Blade thickness of every part was measured with an optical microscope (OLYMPUS-BH) and expressed as the mean thickness of thirty slices.
2.2. Inter-species crossing experiment, heterozygous conchocelis isolation and F1 gametophytic blades culture Crossing experiments, heterozygous conchocelis isolation and F1 gametophytic blades culture were conducted as previously described by Liu et al. (2013) with slight modification. In brief, 3–5 vinylon monofilaments (3 cm long) were placed in a flask for attachment of conchospores released from conchocelis. Then, the conchospores were cultured with aeration to develop into gametophytic blades. When the blades grew to 1 cm, they were detached from the monofilaments and sequentially cultured with aeration at 23 °C in an incubator (250 mL) with 50 μmol photons m−2 s−1 and 10:14 h light/dark cycle (10 L:14D). The culture medium was rich in MES nutrient (Wang et al., 1986) and refreshed every 5 days.
2.4.4. Amount of released conchospores One clam shell was inoculated with free conchocelis weighing 0.5 mg. When conchosporangias were formed, one conchocelis shell of each strain was placed in a 250 mL flask containing 50 mL culture media to collect conchospores and conchospores per strain were collected and inoculated in three Petri dishes (9 cm in diameter) to measure the amount of released conchospores. As the releasing peak of conchospores of P. haitanensis usually appeared at 7–12 am (Fujian Fisheries Bureau, 1979), we collected the conchospores released from each strain at 1 pm every day and inoculated them into three Petri dishes containing culture media. At 24 h post-incubation, the amount of conchospores of 20 fields (10×) selected randomly in cross along the dish center was counted under light microscope and calculated as mean amount per field. According to the areas of the single field and Petri dish, the amount of conchospores in one Petri dish was calculated and used to calculate the daily amount of conchospores from one conchocelis shell. The number of conchospores was counted for twenty days and the number of conchospores released in twenty days was calculated as the total amount of one conchocelis shell. The experiments at each strain were repeated three times.
2.3. Selection of the improved strains After cultured for 5–7 weeks, the color-sectored F1 gametophytic blades were developed from the conchospores of heterozygous conchocelis. Four sectors with fast growth rate and good color were collected and treated with sea snail enzyme to obtain single somatic cells, respectively (Yan and Wang, 1989). These cells were respectively cultured to regenerate into blades and obtain the homozygous free-living conchocelis (strain) by blade parthenogenesis. By comparing the morphology, color, growth rate, pigment content of their F1 gametophytic blades of each strain with those of their parental strains, one strain with 178
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Fig. 1. The gametophytic blades of parental strains (Ps-WT01 of Pyropia sp. from India and Ph-HMC5 of P. haitanensis from China), the carposporangium formed on the blade of Ph-HMC5 strain, F1 sectored blades and the F1 gametophytic blades of the improved strain (HR-5) selected from the crossing between Ps-WT01 and Ph-HMC5 strains. The F1 gametophytic blade of the male parental strain (Ps-WT01) after cultured for 55 days; b. The F1 gametophytic blade of the female parental strain (Ph-HMC5) after cultured for 55 days; c. The carposporangiam formed on the female parental blade about 7 days after fertilization; d. The F1 gametophytic blades of the improved strain (HR-5) after cultured for 55 days; e–h. The F1 sectored blades produced in the cross between Ps-WT01 and Ph-HMC5 after cultured for 20 days; e–f. Two sectors; g. Three sectors; h. Four sectors. The junction between sectors (red arrows). Scale bar was 50 μm in c, 100 μm in e–h and 5 cm in a, b and d, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
sectors with good characteristics were selected and continuously cultured singly for about 70 days. Then the homozygous free-living conchocelis (strain) were obtained by parthenogenesis of the blades and an improved strain HR-5 with obvious hybrid recombinant advantages was selected from four strains after compared with parental strains in morphology, color, growth speed and maturation period.
2.5. Statistic analysis of data Data were given as mean ± standard deviation and analyzed in SPSS (version 19.0 for Windows) using paired-sample t-test. P < 0.05 and P < 0.01 were considered as significantly different and highly significantly different between the improved strain and its parental strains.
3.3. Blade growth and maturity of HR-5 strain
3. Results
The blades of HR-5 strain were long and thin and characterized by dark reddish color, softness, good toughness, many marginal spines, uniform width, and no significant difference in thickness between apical and basal parts (Fig.1d). Fig. 2 shows the growth of F1 blades of HR-5 strain. The growth trend of HR-5 strain was similar to its female parent. At 30–40 days old, the blades of HR-5 strain grew slowly, but after 40 days, the blades entered into fast growing period as indicated by the rapidly increased mean blade length and significantly faster growth rate than its female parental strain. At 60 days old, the mean blade length of HR-5 strain was 168.67 cm, which was 12.80- and 1.97-fold of that (13.18 cm) of its male parental strain and that (85.67 cm) of its female parental strain, respectively. The wet weights per blade of HR-5 strain and its parental strains are shown in Fig. 3. When the blades were at age of 30–40 days, the wet weight of HR-5 strain increased slowly, showing no obvious advantages. However, when cultured for over 40 days, the wet weight of HR-5 strain as well as the wet weight per blade increased rapidly and much faster than its parental strains. At 60 days old, the wet weight per blade of HR-5 strain was 4.17 mg, which was 37.97 and 1.72 times
3.1. Results of crossing experiment The gametophytic blades of the male parental strain (Fig. 1a) with mature spermatangium, and the gametophytic blades of the female parental strain (Fig. 1b) with mature carpogonium were mixed at ratio of 1:1 and cultured. After cultured for 5 days, many carposporangia appeared on the female parental gametophytic blades. Then, the male parental gametophytic blades were removed and the remaining female parental gametophytic blades were cultured for another 3–5 days. After the matured carposporangia (Fig. 1c) on the female parental blades were naturally dried for about 4 h, the carpospores were released and germinated into heterozygous free-living conchocelis. 3.2. Selection of the improved strain Linear segregation of colors occurred in the F1 gametophytic blades developed from conchospores of the heterozygous conchocelis produced in the crossing experiment between Ps-WT01 and Ph-HMC5 strains, forming color-sectored blades with 2–4 sectors (Fig. 1e-h). Four 179
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Fig. 2. The mean length of F1 gametophytic blades of the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5).
Fig. 4. The length to width ratios of the F1 gametophytic blades of the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5).
greater than the maximum absolute growth rate of its female parental strain. The length to width ratios of the F1 gametophytic blades of HR-5 strain and its parental strains are shown in Fig. 4. During 30–55 days of culture, the blade length to width ratio of HR-5 strain showed a trend of rapid increase before decline, while that of its female parental strain increased slowly and its male parental strain increased the last among the three strains and began to decline after 40 days of culture. The blade maturity of HR-5 strain and its parental strains is shown in Fig. 5. The growth period of the male parent strain was shortest, showing a few individuals matured at 31–35 days, 95% of individuals matured at 45 days, and all individuals matured in 50 days old. The growth period of the female parental strain was the longest, showing a few individuals matured at 51–55 day, 85% of individuals matured at 75 days and all individuals matured at 80 days. The growth period of HR-5 strain was in between, showing a few individuals matured at 46–50 days, 85% of individuals matured at 65 days and all individuals matured at 70 days. Fig. 3. The wet weight per F1 gametophytic blade of the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5).
3.4. In vivo absorption spectrum and contents of photosynthetic pigments in the blades of HR-5 strain
greater than those of its male and female parental strains, respectively. As shown in Table 1, the blades of the male parental strain grew slowly, showing maximum absolute growth rate of 0.39 cm/d at 31–35 days. The maximum absolute growth rate of the female parental strain was 5.24 cm/d appearing at 51–55 days. Compared with its parental strains, the maximum absolute growth rate of HR-5 strain was as high as 10.11 cm/d appearing at 46–50 days. In addition, during 41–55 days of culture, the absolute growth rate of HR-5 strain was
As shown in Fig. 6, in vivo absorption spectrum of the blades of HR-5 and its parental strains all had five distinct absorption peaks between wavelength of 350–750 nm, but all the absorption peaks of HR-5 strain were higher than its parental strains. The contents of three major photosynthetic pigments Chl. a, RPE and RPC of HR-5 and its parental strains are shown in Table 2. The content of Chl. a was 10.57, 6.29 and 7.83 mg/g in the 35-day-old
Table 1 Growth rates of the F1 gametophytic blades of the improved strain (HR-5) and its parental strains (Ps-WT01 and Ph-HMC5). Culture days (d)
31–35 36–40 41–45 46–50 51–55 56–60
Absolute growth rate (cm/d)
Specific growth rate (%/d)
Ps-WT01
Ph-HMC5
HR-5
Ps-WT01
Ph-HMC5
HR-5
0.39 0.33 0.29 0.37 0.27 0.20
1.15 1.66 2.14 3.81 5.24 2.37
1.45 (3.72a, 1.26b) 3.27 (9.91, 1.97) 7.27 (25.08, 3.40) 10.11 (27.34, 2.65) 6.53 (1.25, 24.20) 3.13 (15.65, 1.32)
12.35 6.71 4.48 4.54 2.78 4.41
18.26 12.47 9.41 10.22 8.77 2.97
15.03 (1.22c, 0.82d) 15.66 (2.33, 1.26) 15.85 (3.54, 1.68) 11.32 (2.49, 1.11) 4.93 (1.77, 0.56) 2.39 (0.54, 0.80)
Notes: a, c: The ratio of the growth rate of the improved strain (HR-5) and the male parental strain (Ps-WT01); b, d: The ratio of the growth rate of the improved strain (HR-5) and the female parental strain (Ph-HMC5).
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Fig. 5. Percentage of the mature blades of the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5).
3.5. Blade thickness of HR-5 strain Similar to its parental strains, the blades of HR-5 strain at different parts had different thickness. The basal blades had the highest mean thickness, followed in turn by middle blades and apital blades. But the difference in blade thickness in HR-5 was different from that in its parental strains. Male parental strain had the greatest difference in thickness of blades at different parts, followed in turn by HR-5 strain and female parental strain, which had the smallest difference in thickness of blades at different parts. As shown in Table 3, the average blade thickness was 41.58, 20.17 and 25.06 μm for the male parental, female parental and HR-5 strains, respectively. The mean blade thickness of HR-5 strain was closer to that of its female parental strain.
3.6. Numbers of conchospores released from HR-5 strain There was obvious difference in HR-5 and its parental strains at the difficulty degree of releasing conchospores. The male and female parental strains began to release conchospores on the 16th and 25th day after cooling and aeration stimulation, respectively, while HR-5 strain began to release conchospores on the 5th day after the same stimulation. As shown in Fig. 7, the amount of conchospores released daily had two peaks in the first 7 days of culture, reaching first peak of 92.18 × 104 on the 2nd day for HR-5 strain, 28.78 × 104 at the 3rd day for the male parent and 16.44 × 104 on the 4th day for the female parent. As shown in Fig. 8, the total amount of conchospores released from HR-5 strain within 20 days of conchospores-releasing was 404.14 × 104, which was 2.73 and 6.38 times of that of its male and female parental strains (P < 0.01).
Fig. 6. In vivo absorption spectra of 35-day-old F1 gametophytic blades of the improved strain (HR-5) and parental strains (Ps-WT01 and Ph-HMC5).
blades of HR-5 strain, its male and female parental strains, respectively. The content of RPE was 75.24 mg/g in the 35-day-old blades of HR-5 strain, increased by 205% and 78% compared with that of its male and female parental strains, respectively (P < 0.01). The contents of RPC and APC in the 35-day-old blades of HR-5 strain were much higher than those of its parental strains (P < 0.01). Phycobiliprotein content was 114.88 mg/g in HR-5 strain, 2.51 and 2.01 times of that in its male and female parental strains, respectively (P < 0.01).
Table 2 Contents of chlorophyll a (Chl. a), phycoerythrin (RPE) and phycocyanin (RPC and APC) in F1 gametophytic blades of the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5) after cultured for 35 days (n = 3; x ± SD). Strains
Chl. a ± SD Ps-WT01 Ph-HMC5 HR-5 a ⁎⁎
Phycobiliproteina
Contents (mg/g, DW) RPE ± SD ⁎⁎
6.29 ± 0.12 7.83 ± 0.07⁎⁎ 10.57 ± 0.46
RPC ± SD ⁎⁎
APC ± SD ⁎⁎
24.65 ± 1.94 42.33 ± 2.69⁎⁎ 75.24 ± 2.25
11.56 ± 1.08 7.04 ± 0.89⁎⁎ 24.34 ± 1.09
Phycobiliprotein consisted of RPE, RPC and APC. Highly significant difference (P < 0.01, t-test).
181
9.54 ± 0.90⁎⁎ 7.81 ± 0.67⁎⁎ 15.30 ± 1.02
45.75⁎⁎ 57.18⁎⁎ 114.88
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Table 3 Mean thickness at different parts of F1 gametophytic blades of the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5) after cultured for 35 days (n = 30; x ± SD). Strains
Mean thickness of different parts of blades/μm Apical
Ps-WT01 Ph-HMC5 HR-5 ⁎⁎
Middle ⁎⁎
32.00 ± 2.10 18.64 ± 0.88⁎⁎ 20.24 ± 1.22
Mean thickness/μm
Basal ⁎⁎
41.56 ± 1.53 19.82 ± 0.93⁎⁎ 24.40 ± 1.10
51.20 ± 1.41⁎⁎ 22.05 ± 0.95⁎⁎ 30.54 ± 1.86
41.58⁎⁎ 20.17⁎⁎ 25.06
Highly significant difference (P < 0.01, t-test).
4. Discussion To date, some algal scientists have attempted to perform interspecies hybrids of Pyropia (Shin et al., 1997; Niwa et al., 2010; Kim, 2011; Gu et al., 2018), and the inter-species hybridizations between P. dentata and P. pseudolinearis were proved to be successful and fertile (Kim, 2011), however, hybrid progeny from P. yezoensis and P. tenera, P. haitanensis and P. dentata occurred serious cell breakdown (Shin et al., 1997; Niwa et al., 2010; Gu et al., 2018). In the study, the gametophytic blades of heterozygous conchocelis produced by crossing Pyropia sp. from India and P. haitanensis from China were fertile, indicating that the genetic relationship between them is very close, however, there are still no enough evidences to identify they are the same species. Therefore, we think these two species are different species in this research. We selected an improved strain (HR-5) with hybrid recombinant advantages from inter-species hybridization between Pyropia sp. from India and P. haitanensis from China. The growth rate, photosynthetic pigments contents, blade thickness, as well as blade toughness and amount of conchospores in HR-5 strain were all greater than its parental strains, indicating it is feasible to breed new strains through inter-species hybridization between these two species of Pyropia. Some characteristics of HR-5 strain were inherited from one parent. For example, its greater growth rate and high pigments contents were from its female parent and its greater blade thickness and conchospores amount were from its male parent, but with much advantage than its parents. The greater blade thickness made HR-5 strain able to be cultured in the sea areas with big waves. Besides HR-5 strain, we also obtained some other improved strains (data not shown). Based on the results, we can draw a preliminary conclusion that inter-species hybridization of Pyropia is feasible to obtain new strains with many advantages. Wright (1968) pointed out that each character was regulated by
Fig. 8. Total numbers of conchospores released from the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5) during 20 days after conchospores-releasing. Notes: ** Highly significant difference (P < 0.01, t-test).
many genes and one gene mutation would affect many characteristics. Ji et al. (2008) used the ISSR method to analyze the hybrid progeny from P. haitanensis intra-species hybridization, indicated that the hybrid can enhance the genetic diversity of P. haitanensis. The following reasons may explain the hybrid restructuring advantages of HR-5 strain. Firstly, the two parental strains had far away distribution area and distinct ecological environment factors and characteristics, which significantly increased the probabilities of gene recombination and achieving hybrid recombination advantages. Secondly, the greater complementary characteristics of the parents allowed many characteristics to reconstruct and optimize, forming better characteristics. The molecular mechanisms on formation of reconstructing advantages need to be further researched in the future. The quality of Pyropia is related to the contents of photosynthetic pigments. It has been reported that chlorophyll a (Chl. a) content was a factor closely related to the blade growth rate (Ji et al., 2011) and phycobiliprotein content was also one of the important indices for measuring nutritional value of Pyropia (Ma et al., 1998). During the whole culture period, HR-5 strain had dark reddish color and showed obvious growth advantage, which were related to its high Chl. a and phycobiliprotein contents. The length to width ratio is also one of the important characteristics of Pyropia. The maximum length to width ratio of HR-5 strain was 64.65, thus its blade shape was long and thin,
Fig. 7. Numbers of conchospores released daily from the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5) during 20 days after conchospores-releasing.
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which reflected its rapid growth in the length and was beneficial to increasing harvest times. The mean thickness of wild-type Pyropia yezoensis was 34.49 μm (Zhang, 2009), while that of HR-5 strain was only 25.06 μm, showing a potential advantage for preparing the blades of HR-5 strain into qualified square sheet. In order to eliminate or prevent degradation of the excellent characteristics of new improved strains, we generally used free-living conchocelis transplant technology to culture conchocelis shell. However, the amount of conchospores released from the conchocelis shell inoculated with free-living conchocelis was fewer than that inoculated with carpospores, leading to increase conchocelis shell to get enough conchospores to net-seeding for cultivation in the sea and increased netseeding cost (Wang et al., 2011). Therefore, the amount of conchospores is the most important limiting factor next to other excellent characteristics to determine whether an improved strain could be widely promoted in the nori production. HR-5 strain could release a large amount of conchospores and its total number of conchospores was 404.14 × 104 during 20 successive conchospore-releasing days, which could greatly meet requirements of net-seeding for large scale cultivation. Overall, HR-5 strain was characterized by fast growth, high quality as well as large amount of conchospores, and may offer an alternative for the nori industry.
Kim, N.G., 2011. Culture study on the hybrid by interspecific crossing between Porphyra pesudolinearis and P. dentata (Bangiales, Rhodophyta), two dioecious species in culture. Algae 26, 79–86. Liu, P.J., Ji, D.H., Xie, C.T., Xu, Y., Chen, C.S., Shi, X.Z., Liang, Y., Wang, F.X., 2009. Study on Selection of Low N and P Resistant Strains in Porphyra haitanensis. 14. J Jimei Univ., pp. 109–114 (in Chinese with English abstract). Liu, H.Y., Li, L., Yan, X.H., 2013. Study on the Inter-species Cross Between Pyropia haitanensis and Pyropia radi. 22. J Shanghai Ocean Univ., pp. 882–887 (in Chinese with English abstract). Ma, J.H., Cai, S.Q., 1996. Cultivation and Process of Porphyra yezoensis. Science Press, Beijing, pp. 1–58 (in Chinese). Ma, J.H., Zhang, L.M., Ji, C.L., Yuan, M., Chen, S.G., Wang, H.Q., Shen, W., Shen, H.S., Sun, G.M., Gu, S.B., Sha, J.R., Chen, C.Y., 1998. On the refrigerated nets of Porphyra yezoensis and quality analysis of produce. J. Fish. China 22, 65–71 (in Chinese). Niwa, K., Kobiyama, A., Sakamoto, T., 2010. Interspecific hybridization in the haploid blade-forming marine crop Porphyra (Bangiales, Rhodophyta): occurrence of allodiploidy in surviving F1 gametophytic blades. J. Phycol. 46, 693–702. Shin, J.A., Miura, A., Fu, P.F., 1997. Hybrid breakdown and breakthrough in interspecific crosses between Porphyra yezoensis and P. tenera. Nat. Hist. Res. 3, 65–70. Stein, J.R., 1973. Handbook of Phycological Methods: Culture Methods and Growth Measurements. Cambridge University Press, London, pp. 289–311. Tan, Y.H., Huang, L.B., Yan, X.H., 2014. Selection and characterization of a low-salinity tolerant strain in Pyropia haitanensis (Bangiales, Rhodophyta). Oceanol. Limnol. Sinica 45, 504–512 (in Chinese with English abstract). Wang, S.J., Zhang, X.P., Xu, Z.D., Sun, Y.L., 1986. A study on the cultivation of the vegetative cells and protoplasts of P. haitanensis I. Oceanol. et Limnol. Sin. 17, 217–221 (in Chinese with English abstract). Wang, F.X., Xie, C.T., Chen, C.S., Ji, D.H., Xu, Y., Shi, X.Z., Liang, Y., Zhang, Y., 2010. Study on Chimeras and Breeding from Hybridization of the Breen-type and the Wildtype of Porphyra haitanensis. 15. J. Jimei Univ., pp. 1–6 (in Chinese with English abstract). Wang, C.Q., Yan, X.H., Huang, L.B., Liu, C.J., 2011. Characterization of an improved strain (SF-2) of Porphyra haitanensis (Bangiales, Rhodophyta) and its pilot cultivation in mariculture farm. J. Fish. China 35, 1658–1667 (in Chinese with English abstract). Wright, S., 1968. Evolution and the Genetics of Populations: Genetic and Biometric Foundations. Vol. 1. University of Chicago Press, Chicago, pp. 55–107. Xu, Y., Chen, C.S., Xie, C.T., Ji, D.H., Liang, Y., Liu, P.J., Wang, F.X., Shi, X.Z., 2008. Preliminary evaluation of hybridized strains of Porphyra haitanensis. Mar. Fish. Res. 29, 62–69 (in Chinese with English abstract). Yan, X.H., Aruga, Y., 1997. Induction of pigmentation mutants by treatment of monospore germlings with MNNG in Porphyra yezoensis Ueda (Bangiales, Rhodophyta). Algae 12, 39–54. Yan, X.H., Chen, M., 2008. Selection of low-salinity resistant improved varieties in Porphyra haitanensis (Bangiales, Rhodophyta). J. Shanghai Fish. Univ. 17, 316–320 (in Chinese with English abstract). Yan, X.H., Ma, S.Y., 2007. Selection of a high-temperature resistant strain of Porphyra haitanensis (Rhodophyta). J. Fish. China 31, 112–119 (in Chinese with English abstract). Yan, X.H., Wang, S.J., 1989. Studies on development and differentiation of somatic cells in Porphyra spp. (Rhodophyta). Mar. Sci. 6, 28–32 (in Chinese with English abstract). Yan, X.H., Li, L., Chen, J.H., Aruga, Y., 2007. Parthenogenesis and isolation of genetic pure strains in Porphyra haitanensis (Bangiales, Rhodophyta). High Technol. Lett. 17, 205–210 (in Chinese with English abstract). Yan, X.H., Lv, F., Liu, C.J., Zheng, Y.F., 2010. Selection and characterization of a hightemperature tolerant strain of Porphyra haitanensis Chang et Zheng (Bangiales, Rhodophyta). J. Appl. Phycol. 22, 511–516. You, H., 1999. The present situation of breeding and development countermeasures in Porphyra haitanensis of Pingtan County. Fish Sci & Tech. Infor. 26, 272–273 (in Chinese). Zhang, B.L., 2009. Isolation and Characterization of Heat-resistant Strains in Porphyra yezoensis Ueda (Bangiales, Rhodophyta) M.S. thesis. Shanghai Ocean University, Shanghai, China, pp. 35 (in Chinese). Zhang, Y.Y., Yan, X.H., 2014. Biological characteristics and a part life-cycle of Pyropia radi (Bangiales, Rhodophyta) from India. Oceanol. Limnol. Sinica 45, 51–57 (in Chinese with English abstract).
Acknowledgments The study was supported in part by the National High Technology Research & Development Program of China (“863” Program) (2012AA10A411), the National Natural Science Foundation of China (31072208), and the Major Science and Technology Special Fund of Agriculture (Fisheries) New Variety Breeding of Zhejiang (2016C02055-6), Large-scale Cultivation and Industrialization of Important Economic Red Algae with an Annual Output of 2000 tons (2014FJ19). References Fujian Fisheries Bureau, 1979. The Artificial Cultivation of Porphyra haitanensis. Fujian People Press, Fuzhou, pp. 1–101 (in Chinese). Gao, H.F., 1993. The variation in the contents of phycobiliproteins from Porphyra haitanensis collected in different growing stages. Oceanol. Limnol. Sinica 24, 645–648 (in Chinese with English abstract). Gu, L.Z., Ding, H.C., Yan, X.H., 2018. Phenomenon of cell breakdown and phenotypic observation on surviving blades in interspecific hybridization progeny between Pyropia haitanensis and Pyropia dentata. J. Fish. China 42, 60–71 (in Chinese with English abstract). He, P.M., Qin, S., Yan, X.J., Wu, W.N., 2007. Seaweed Biological Technology and its Application. Chemical Industry Press, Beijing, pp. 86–98 (in Chinese). Ji, D.H., Xie, C.T., Xu, Y., Chen, C.S., 2008. ISSR analysis on the heterosis in hybrids of Porphyra haitanensis. Acta Oceanol. Sin. 30, 147–153 (in Chinese with English abstract). Ji, D.H., Xie, C.T., Shi, X.Z., Xu, Y., Zhang, Y., 2011. Analysis of the Major Quality Traits in Wild Porphyra haitanensis of Fujian Coast. 16. J. Jimei Univ., pp. 401–406 (in Chinese with English abstract).
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Short communication
Selection and characterization of an improved strain produced by interspecies hybridization between Pyropia sp. from India and Pyropia haitanensis from China Hongchang Dinga,b,c, Feng Lvd, Hongxiao Wua, Xinghong Yana,b,c,
T
⁎
a
Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, Shanghai 201306, China National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China c Shanghai Engineering Research Center of Aquaculture, Shanghai 201306, China d Nantong Science and Technology College, 226007, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Pyropia haitanensis Inter-species hybridization Recombinant strain Growth rate Photosynthetic pigment
A crossing-experiment between Pyropia sp. from India and P. haitanensis from China was carried out for breeding improved strains. The male parent was the wild-type strain (Ps-WT01) of Pyropia sp., which was characterized by thick blade, slow growing, good toughness and more production of conchospores, and the female parent was the mutation strain (Ph-HMC5) of P. haitanensis, which was characterized by thin blade, fast growing, poor toughness and low production of conchospores. At first, four improved strains with obvious recombinant advantages were obtained from the F1 color-sectored blades, and finally the most improved strain (HR-5) was selected from them. The maximum of absolute growth rate of the male and female parents were 0.39 and 5.24 cm/d, respectively, while that of HR-5 strain was 10.11 cm/d and its absolute growth rate was greater than the maximum growth rate of the female parent during 41–55 days of culture. After 60 days, the mean blade length of HR-5 was 168.67 cm, which was 12.80-fold of that (13.18 cm) of its male parent and 1.97-fold of that (85.67 cm) of its female parent, respectively. The chlorophyll a content in the 35-day-old blades of HR-5 strain was 10.57 mg/g, which increased by 68% and 35% compared with that of its male and female parents, respectively. The phycobiliprotein content of HR-5 blades was 114.88 mg/g, which was 2.51- and 2.01-fold of that of its male and female parents, respectively. The mean thickness of the 35-day-old blades of HR-5 was 25.06 μm, which was 60% and 124% of that of its male and female parents, respectively. The total number of conchospores per shell released from HR-5 was 404.14 × 104, which was 2.73- and 6.38-fold of that of its male and female parents, respectively. The above results confirmed that HR-5 strain was characterized by fast growth, high quality as well as large amount of conchospores, and may offer an alternative for the nori industry.
1. Introduction Pyropia haitanensis is an important economic algae. Its total output accounts for approximately 75% of the total Pyropia production in China (Ma and Cai, 1996). In the 1960s, artificial breeding and cultivation of P. haitanensis was succeeded, leading to the establishment and development of culture industry of P. haitanensis (Fujian Fisheries Bureau, 1979). However, the wild-type strain of P. haitanensis has been cultivated without any selection or improvements for over 50 years, thus, breeding new cultivars has been placed in the important agenda (You, 1999). Until today, numerous stable artificial mutants with inheritable and valuable characteristics, such as heat tolerance, low-salt resistance and low-nitrogen and phosphorus resistance have been
⁎
isolated (Yan and Ma, 2007; Yan and Chen, 2008; Liu et al., 2009; Yan et al., 2010; Tan et al., 2014). Meanwhile, several improved strains with high quality and yield have been obtained and received the certification of new cultivar from Chinese government (Wang et al., 2011). In addition, through the intra-species hybridization of artificial pigment mutants of P. haitanensis, some strains with better characteristics than their parents have been obtained (Xu et al., 2008; Wang et al., 2010), but their cultivation in large-scale has not been reported. Crossbreeding of Pyropia mainly utilize the hybrid recombination advantage, that is, the hybrid progeny produced by interbreeding between individuals with different characters is superior to parents in growth and biochemical characteristics (He et al., 2007). One of the important reasons for generated hybrid recombination advantage in
Corresponding author at: Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University), Ministry of Education, Shanghai 201306, China. E-mail address: [email protected] (X. Yan).
https://doi.org/10.1016/j.aquaculture.2018.03.015 Received 27 October 2017; Received in revised form 1 March 2018; Accepted 8 March 2018 Available online 09 March 2018 0044-8486/ © 2018 Elsevier B.V. All rights reserved.
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the most obvious hybrid recombinant advantages was selected, namely the improved strain.
intraspecific cross is the significantly different characteristics of parental strains (Xu et al., 2008). The Pyropia sp. was derived from the India peninsula, we have previously (Zhang and Yan, 2014) reported that the morphological structure, chromosome counts, sex and 5.8S rDNA-ITS region of this species were very similar to those of P. haitanensis from China. This indicated that the genetic relationship between them is very close although they were isolated so long distance from China to India, But, there are still no enough evidences to identify they are the same species. Therefore, we think these two species are different species in this research. In addition, we have previously (Liu et al., 2013) reported that the gametophytic blades of heterozygous conchocelis produced by crossing Pyropia sp. from India and P. haitanensis from China were fertile. The wild-type Pyropia sp. (Ps-WT01) was characterized by thick blades, slow growing, good toughness in elasticity, maroon thallus with greenish basal part and reddish apical part, and many marginal denticles. The mutation strain of P. haitanensis (PhHMC5) was characterized by thin blade, fast growing, poor toughness in elasticity and thallus with uniform pale red brown color, and no marginal denticles. In this paper, we further screened the improved hybrids strains with recombinant advantages through the hybridization between Ps-WT01 and Ph-HMC5 strains.
2.4. Characteristics of the improved strain 2.4.1. Blade growth After cultured for 30 days, twenty blades of each strain were randomly selected and cultured in three aerated flasks (1 L), respectively. The blade length, width and wet weight were measured every 5 days in a 30-day period. The specific and absolute growth rates of the blades were calculated as previously reported by Stein (1973). 2.4.2. In vivo absorption spectra and contents of chlorophyll a as well as the contents of phycobiliproteins After being cultured for 35 days, the in vivo absorption spectra and contents of chlorophyll a (Chl. a) of the F1 blades were measured (Yan and Aruga, 1997). The contents of phycoerythrin (RPE), phycocyanin (RPC) and allophycocyanin (APC) were measured according to the methods described by Gao (1993). Specifically, the fresh blades were dried at 80 °C for 6 h, and ground after three freezing and thawing cycles (between −20 °C and room temperature). The extract was centrifuged at 15,000 ×g for 20 min, and the supernatant was used to determine the OD562, OD615, OD650, and OD652. The absorbance and OD were measured by using an ultraviolet spectrophotometer (UV2600, Shimadzu). The contents of Chl. a, PE and PC were calculated using the following formulae:
2. Materials and methods 2.1. Materials The male parental stain was wild-type Pyropia sp. (Ps-WT01) originated from the coast of Indian peninsula. In detail, its free-living conchocelis were developed from carpospores released by a gametophytic blade (Zhang and Yan, 2014) and cultured in our laboratory to obtain conchospores and blades. These blades were prepared as single somatic cell suspensions using enzymatic hydrolysis technology and then cultured and developed into regenerative blades (Wang et al., 1986). After cultured for a period of time, single regenerative blades underwent parthenogenesis (Yan et al., 2007) and produced homozygous conchocelis (strains). The female parent was a mutation strain (Ph-HMC5) of P. haitanensis. Its homozygous conchocelis were also obtained through blade parthenogenesis. The conchocelis filaments were stored at 18 °C in the laboratory.
Chl. a = (11.6 × OD665–1.31 × OD645–0.14 × OD630) × V/W
(1)
PE = (0.123 × OD562–0.07 × OD615 + 0.015 × OD652) × V/W
(2)
RPC = (0.162 × OD615–0.099 × OD650–0.001 × OD562) × V/W
(3)
APC = (0.171 × OD650–0.0006 × OD562–0.004 × OD615) × V/W
(4)
PC = RPC + APC
(5)
where V (in mL) is the volume of grinding fluid and W (in mg) is dry weight of sample. 2.4.3. Blade thickness After being cultured for 35 days, the blades were cut into three parts with bare-handed, double-edges blades. Blade thickness of every part was measured with an optical microscope (OLYMPUS-BH) and expressed as the mean thickness of thirty slices.
2.2. Inter-species crossing experiment, heterozygous conchocelis isolation and F1 gametophytic blades culture Crossing experiments, heterozygous conchocelis isolation and F1 gametophytic blades culture were conducted as previously described by Liu et al. (2013) with slight modification. In brief, 3–5 vinylon monofilaments (3 cm long) were placed in a flask for attachment of conchospores released from conchocelis. Then, the conchospores were cultured with aeration to develop into gametophytic blades. When the blades grew to 1 cm, they were detached from the monofilaments and sequentially cultured with aeration at 23 °C in an incubator (250 mL) with 50 μmol photons m−2 s−1 and 10:14 h light/dark cycle (10 L:14D). The culture medium was rich in MES nutrient (Wang et al., 1986) and refreshed every 5 days.
2.4.4. Amount of released conchospores One clam shell was inoculated with free conchocelis weighing 0.5 mg. When conchosporangias were formed, one conchocelis shell of each strain was placed in a 250 mL flask containing 50 mL culture media to collect conchospores and conchospores per strain were collected and inoculated in three Petri dishes (9 cm in diameter) to measure the amount of released conchospores. As the releasing peak of conchospores of P. haitanensis usually appeared at 7–12 am (Fujian Fisheries Bureau, 1979), we collected the conchospores released from each strain at 1 pm every day and inoculated them into three Petri dishes containing culture media. At 24 h post-incubation, the amount of conchospores of 20 fields (10×) selected randomly in cross along the dish center was counted under light microscope and calculated as mean amount per field. According to the areas of the single field and Petri dish, the amount of conchospores in one Petri dish was calculated and used to calculate the daily amount of conchospores from one conchocelis shell. The number of conchospores was counted for twenty days and the number of conchospores released in twenty days was calculated as the total amount of one conchocelis shell. The experiments at each strain were repeated three times.
2.3. Selection of the improved strains After cultured for 5–7 weeks, the color-sectored F1 gametophytic blades were developed from the conchospores of heterozygous conchocelis. Four sectors with fast growth rate and good color were collected and treated with sea snail enzyme to obtain single somatic cells, respectively (Yan and Wang, 1989). These cells were respectively cultured to regenerate into blades and obtain the homozygous free-living conchocelis (strain) by blade parthenogenesis. By comparing the morphology, color, growth rate, pigment content of their F1 gametophytic blades of each strain with those of their parental strains, one strain with 178
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Fig. 1. The gametophytic blades of parental strains (Ps-WT01 of Pyropia sp. from India and Ph-HMC5 of P. haitanensis from China), the carposporangium formed on the blade of Ph-HMC5 strain, F1 sectored blades and the F1 gametophytic blades of the improved strain (HR-5) selected from the crossing between Ps-WT01 and Ph-HMC5 strains. The F1 gametophytic blade of the male parental strain (Ps-WT01) after cultured for 55 days; b. The F1 gametophytic blade of the female parental strain (Ph-HMC5) after cultured for 55 days; c. The carposporangiam formed on the female parental blade about 7 days after fertilization; d. The F1 gametophytic blades of the improved strain (HR-5) after cultured for 55 days; e–h. The F1 sectored blades produced in the cross between Ps-WT01 and Ph-HMC5 after cultured for 20 days; e–f. Two sectors; g. Three sectors; h. Four sectors. The junction between sectors (red arrows). Scale bar was 50 μm in c, 100 μm in e–h and 5 cm in a, b and d, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
sectors with good characteristics were selected and continuously cultured singly for about 70 days. Then the homozygous free-living conchocelis (strain) were obtained by parthenogenesis of the blades and an improved strain HR-5 with obvious hybrid recombinant advantages was selected from four strains after compared with parental strains in morphology, color, growth speed and maturation period.
2.5. Statistic analysis of data Data were given as mean ± standard deviation and analyzed in SPSS (version 19.0 for Windows) using paired-sample t-test. P < 0.05 and P < 0.01 were considered as significantly different and highly significantly different between the improved strain and its parental strains.
3.3. Blade growth and maturity of HR-5 strain
3. Results
The blades of HR-5 strain were long and thin and characterized by dark reddish color, softness, good toughness, many marginal spines, uniform width, and no significant difference in thickness between apical and basal parts (Fig.1d). Fig. 2 shows the growth of F1 blades of HR-5 strain. The growth trend of HR-5 strain was similar to its female parent. At 30–40 days old, the blades of HR-5 strain grew slowly, but after 40 days, the blades entered into fast growing period as indicated by the rapidly increased mean blade length and significantly faster growth rate than its female parental strain. At 60 days old, the mean blade length of HR-5 strain was 168.67 cm, which was 12.80- and 1.97-fold of that (13.18 cm) of its male parental strain and that (85.67 cm) of its female parental strain, respectively. The wet weights per blade of HR-5 strain and its parental strains are shown in Fig. 3. When the blades were at age of 30–40 days, the wet weight of HR-5 strain increased slowly, showing no obvious advantages. However, when cultured for over 40 days, the wet weight of HR-5 strain as well as the wet weight per blade increased rapidly and much faster than its parental strains. At 60 days old, the wet weight per blade of HR-5 strain was 4.17 mg, which was 37.97 and 1.72 times
3.1. Results of crossing experiment The gametophytic blades of the male parental strain (Fig. 1a) with mature spermatangium, and the gametophytic blades of the female parental strain (Fig. 1b) with mature carpogonium were mixed at ratio of 1:1 and cultured. After cultured for 5 days, many carposporangia appeared on the female parental gametophytic blades. Then, the male parental gametophytic blades were removed and the remaining female parental gametophytic blades were cultured for another 3–5 days. After the matured carposporangia (Fig. 1c) on the female parental blades were naturally dried for about 4 h, the carpospores were released and germinated into heterozygous free-living conchocelis. 3.2. Selection of the improved strain Linear segregation of colors occurred in the F1 gametophytic blades developed from conchospores of the heterozygous conchocelis produced in the crossing experiment between Ps-WT01 and Ph-HMC5 strains, forming color-sectored blades with 2–4 sectors (Fig. 1e-h). Four 179
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Fig. 2. The mean length of F1 gametophytic blades of the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5).
Fig. 4. The length to width ratios of the F1 gametophytic blades of the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5).
greater than the maximum absolute growth rate of its female parental strain. The length to width ratios of the F1 gametophytic blades of HR-5 strain and its parental strains are shown in Fig. 4. During 30–55 days of culture, the blade length to width ratio of HR-5 strain showed a trend of rapid increase before decline, while that of its female parental strain increased slowly and its male parental strain increased the last among the three strains and began to decline after 40 days of culture. The blade maturity of HR-5 strain and its parental strains is shown in Fig. 5. The growth period of the male parent strain was shortest, showing a few individuals matured at 31–35 days, 95% of individuals matured at 45 days, and all individuals matured in 50 days old. The growth period of the female parental strain was the longest, showing a few individuals matured at 51–55 day, 85% of individuals matured at 75 days and all individuals matured at 80 days. The growth period of HR-5 strain was in between, showing a few individuals matured at 46–50 days, 85% of individuals matured at 65 days and all individuals matured at 70 days. Fig. 3. The wet weight per F1 gametophytic blade of the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5).
3.4. In vivo absorption spectrum and contents of photosynthetic pigments in the blades of HR-5 strain
greater than those of its male and female parental strains, respectively. As shown in Table 1, the blades of the male parental strain grew slowly, showing maximum absolute growth rate of 0.39 cm/d at 31–35 days. The maximum absolute growth rate of the female parental strain was 5.24 cm/d appearing at 51–55 days. Compared with its parental strains, the maximum absolute growth rate of HR-5 strain was as high as 10.11 cm/d appearing at 46–50 days. In addition, during 41–55 days of culture, the absolute growth rate of HR-5 strain was
As shown in Fig. 6, in vivo absorption spectrum of the blades of HR-5 and its parental strains all had five distinct absorption peaks between wavelength of 350–750 nm, but all the absorption peaks of HR-5 strain were higher than its parental strains. The contents of three major photosynthetic pigments Chl. a, RPE and RPC of HR-5 and its parental strains are shown in Table 2. The content of Chl. a was 10.57, 6.29 and 7.83 mg/g in the 35-day-old
Table 1 Growth rates of the F1 gametophytic blades of the improved strain (HR-5) and its parental strains (Ps-WT01 and Ph-HMC5). Culture days (d)
31–35 36–40 41–45 46–50 51–55 56–60
Absolute growth rate (cm/d)
Specific growth rate (%/d)
Ps-WT01
Ph-HMC5
HR-5
Ps-WT01
Ph-HMC5
HR-5
0.39 0.33 0.29 0.37 0.27 0.20
1.15 1.66 2.14 3.81 5.24 2.37
1.45 (3.72a, 1.26b) 3.27 (9.91, 1.97) 7.27 (25.08, 3.40) 10.11 (27.34, 2.65) 6.53 (1.25, 24.20) 3.13 (15.65, 1.32)
12.35 6.71 4.48 4.54 2.78 4.41
18.26 12.47 9.41 10.22 8.77 2.97
15.03 (1.22c, 0.82d) 15.66 (2.33, 1.26) 15.85 (3.54, 1.68) 11.32 (2.49, 1.11) 4.93 (1.77, 0.56) 2.39 (0.54, 0.80)
Notes: a, c: The ratio of the growth rate of the improved strain (HR-5) and the male parental strain (Ps-WT01); b, d: The ratio of the growth rate of the improved strain (HR-5) and the female parental strain (Ph-HMC5).
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Fig. 5. Percentage of the mature blades of the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5).
3.5. Blade thickness of HR-5 strain Similar to its parental strains, the blades of HR-5 strain at different parts had different thickness. The basal blades had the highest mean thickness, followed in turn by middle blades and apital blades. But the difference in blade thickness in HR-5 was different from that in its parental strains. Male parental strain had the greatest difference in thickness of blades at different parts, followed in turn by HR-5 strain and female parental strain, which had the smallest difference in thickness of blades at different parts. As shown in Table 3, the average blade thickness was 41.58, 20.17 and 25.06 μm for the male parental, female parental and HR-5 strains, respectively. The mean blade thickness of HR-5 strain was closer to that of its female parental strain.
3.6. Numbers of conchospores released from HR-5 strain There was obvious difference in HR-5 and its parental strains at the difficulty degree of releasing conchospores. The male and female parental strains began to release conchospores on the 16th and 25th day after cooling and aeration stimulation, respectively, while HR-5 strain began to release conchospores on the 5th day after the same stimulation. As shown in Fig. 7, the amount of conchospores released daily had two peaks in the first 7 days of culture, reaching first peak of 92.18 × 104 on the 2nd day for HR-5 strain, 28.78 × 104 at the 3rd day for the male parent and 16.44 × 104 on the 4th day for the female parent. As shown in Fig. 8, the total amount of conchospores released from HR-5 strain within 20 days of conchospores-releasing was 404.14 × 104, which was 2.73 and 6.38 times of that of its male and female parental strains (P < 0.01).
Fig. 6. In vivo absorption spectra of 35-day-old F1 gametophytic blades of the improved strain (HR-5) and parental strains (Ps-WT01 and Ph-HMC5).
blades of HR-5 strain, its male and female parental strains, respectively. The content of RPE was 75.24 mg/g in the 35-day-old blades of HR-5 strain, increased by 205% and 78% compared with that of its male and female parental strains, respectively (P < 0.01). The contents of RPC and APC in the 35-day-old blades of HR-5 strain were much higher than those of its parental strains (P < 0.01). Phycobiliprotein content was 114.88 mg/g in HR-5 strain, 2.51 and 2.01 times of that in its male and female parental strains, respectively (P < 0.01).
Table 2 Contents of chlorophyll a (Chl. a), phycoerythrin (RPE) and phycocyanin (RPC and APC) in F1 gametophytic blades of the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5) after cultured for 35 days (n = 3; x ± SD). Strains
Chl. a ± SD Ps-WT01 Ph-HMC5 HR-5 a ⁎⁎
Phycobiliproteina
Contents (mg/g, DW) RPE ± SD ⁎⁎
6.29 ± 0.12 7.83 ± 0.07⁎⁎ 10.57 ± 0.46
RPC ± SD ⁎⁎
APC ± SD ⁎⁎
24.65 ± 1.94 42.33 ± 2.69⁎⁎ 75.24 ± 2.25
11.56 ± 1.08 7.04 ± 0.89⁎⁎ 24.34 ± 1.09
Phycobiliprotein consisted of RPE, RPC and APC. Highly significant difference (P < 0.01, t-test).
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9.54 ± 0.90⁎⁎ 7.81 ± 0.67⁎⁎ 15.30 ± 1.02
45.75⁎⁎ 57.18⁎⁎ 114.88
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Table 3 Mean thickness at different parts of F1 gametophytic blades of the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5) after cultured for 35 days (n = 30; x ± SD). Strains
Mean thickness of different parts of blades/μm Apical
Ps-WT01 Ph-HMC5 HR-5 ⁎⁎
Middle ⁎⁎
32.00 ± 2.10 18.64 ± 0.88⁎⁎ 20.24 ± 1.22
Mean thickness/μm
Basal ⁎⁎
41.56 ± 1.53 19.82 ± 0.93⁎⁎ 24.40 ± 1.10
51.20 ± 1.41⁎⁎ 22.05 ± 0.95⁎⁎ 30.54 ± 1.86
41.58⁎⁎ 20.17⁎⁎ 25.06
Highly significant difference (P < 0.01, t-test).
4. Discussion To date, some algal scientists have attempted to perform interspecies hybrids of Pyropia (Shin et al., 1997; Niwa et al., 2010; Kim, 2011; Gu et al., 2018), and the inter-species hybridizations between P. dentata and P. pseudolinearis were proved to be successful and fertile (Kim, 2011), however, hybrid progeny from P. yezoensis and P. tenera, P. haitanensis and P. dentata occurred serious cell breakdown (Shin et al., 1997; Niwa et al., 2010; Gu et al., 2018). In the study, the gametophytic blades of heterozygous conchocelis produced by crossing Pyropia sp. from India and P. haitanensis from China were fertile, indicating that the genetic relationship between them is very close, however, there are still no enough evidences to identify they are the same species. Therefore, we think these two species are different species in this research. We selected an improved strain (HR-5) with hybrid recombinant advantages from inter-species hybridization between Pyropia sp. from India and P. haitanensis from China. The growth rate, photosynthetic pigments contents, blade thickness, as well as blade toughness and amount of conchospores in HR-5 strain were all greater than its parental strains, indicating it is feasible to breed new strains through inter-species hybridization between these two species of Pyropia. Some characteristics of HR-5 strain were inherited from one parent. For example, its greater growth rate and high pigments contents were from its female parent and its greater blade thickness and conchospores amount were from its male parent, but with much advantage than its parents. The greater blade thickness made HR-5 strain able to be cultured in the sea areas with big waves. Besides HR-5 strain, we also obtained some other improved strains (data not shown). Based on the results, we can draw a preliminary conclusion that inter-species hybridization of Pyropia is feasible to obtain new strains with many advantages. Wright (1968) pointed out that each character was regulated by
Fig. 8. Total numbers of conchospores released from the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5) during 20 days after conchospores-releasing. Notes: ** Highly significant difference (P < 0.01, t-test).
many genes and one gene mutation would affect many characteristics. Ji et al. (2008) used the ISSR method to analyze the hybrid progeny from P. haitanensis intra-species hybridization, indicated that the hybrid can enhance the genetic diversity of P. haitanensis. The following reasons may explain the hybrid restructuring advantages of HR-5 strain. Firstly, the two parental strains had far away distribution area and distinct ecological environment factors and characteristics, which significantly increased the probabilities of gene recombination and achieving hybrid recombination advantages. Secondly, the greater complementary characteristics of the parents allowed many characteristics to reconstruct and optimize, forming better characteristics. The molecular mechanisms on formation of reconstructing advantages need to be further researched in the future. The quality of Pyropia is related to the contents of photosynthetic pigments. It has been reported that chlorophyll a (Chl. a) content was a factor closely related to the blade growth rate (Ji et al., 2011) and phycobiliprotein content was also one of the important indices for measuring nutritional value of Pyropia (Ma et al., 1998). During the whole culture period, HR-5 strain had dark reddish color and showed obvious growth advantage, which were related to its high Chl. a and phycobiliprotein contents. The length to width ratio is also one of the important characteristics of Pyropia. The maximum length to width ratio of HR-5 strain was 64.65, thus its blade shape was long and thin,
Fig. 7. Numbers of conchospores released daily from the improved strain (HR-5) and the parental strains (Ps-WT01 and Ph-HMC5) during 20 days after conchospores-releasing.
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which reflected its rapid growth in the length and was beneficial to increasing harvest times. The mean thickness of wild-type Pyropia yezoensis was 34.49 μm (Zhang, 2009), while that of HR-5 strain was only 25.06 μm, showing a potential advantage for preparing the blades of HR-5 strain into qualified square sheet. In order to eliminate or prevent degradation of the excellent characteristics of new improved strains, we generally used free-living conchocelis transplant technology to culture conchocelis shell. However, the amount of conchospores released from the conchocelis shell inoculated with free-living conchocelis was fewer than that inoculated with carpospores, leading to increase conchocelis shell to get enough conchospores to net-seeding for cultivation in the sea and increased netseeding cost (Wang et al., 2011). Therefore, the amount of conchospores is the most important limiting factor next to other excellent characteristics to determine whether an improved strain could be widely promoted in the nori production. HR-5 strain could release a large amount of conchospores and its total number of conchospores was 404.14 × 104 during 20 successive conchospore-releasing days, which could greatly meet requirements of net-seeding for large scale cultivation. Overall, HR-5 strain was characterized by fast growth, high quality as well as large amount of conchospores, and may offer an alternative for the nori industry.
Kim, N.G., 2011. Culture study on the hybrid by interspecific crossing between Porphyra pesudolinearis and P. dentata (Bangiales, Rhodophyta), two dioecious species in culture. Algae 26, 79–86. Liu, P.J., Ji, D.H., Xie, C.T., Xu, Y., Chen, C.S., Shi, X.Z., Liang, Y., Wang, F.X., 2009. Study on Selection of Low N and P Resistant Strains in Porphyra haitanensis. 14. J Jimei Univ., pp. 109–114 (in Chinese with English abstract). Liu, H.Y., Li, L., Yan, X.H., 2013. Study on the Inter-species Cross Between Pyropia haitanensis and Pyropia radi. 22. J Shanghai Ocean Univ., pp. 882–887 (in Chinese with English abstract). Ma, J.H., Cai, S.Q., 1996. Cultivation and Process of Porphyra yezoensis. Science Press, Beijing, pp. 1–58 (in Chinese). Ma, J.H., Zhang, L.M., Ji, C.L., Yuan, M., Chen, S.G., Wang, H.Q., Shen, W., Shen, H.S., Sun, G.M., Gu, S.B., Sha, J.R., Chen, C.Y., 1998. On the refrigerated nets of Porphyra yezoensis and quality analysis of produce. J. Fish. China 22, 65–71 (in Chinese). Niwa, K., Kobiyama, A., Sakamoto, T., 2010. Interspecific hybridization in the haploid blade-forming marine crop Porphyra (Bangiales, Rhodophyta): occurrence of allodiploidy in surviving F1 gametophytic blades. J. Phycol. 46, 693–702. Shin, J.A., Miura, A., Fu, P.F., 1997. Hybrid breakdown and breakthrough in interspecific crosses between Porphyra yezoensis and P. tenera. Nat. Hist. Res. 3, 65–70. Stein, J.R., 1973. Handbook of Phycological Methods: Culture Methods and Growth Measurements. Cambridge University Press, London, pp. 289–311. Tan, Y.H., Huang, L.B., Yan, X.H., 2014. Selection and characterization of a low-salinity tolerant strain in Pyropia haitanensis (Bangiales, Rhodophyta). Oceanol. Limnol. Sinica 45, 504–512 (in Chinese with English abstract). Wang, S.J., Zhang, X.P., Xu, Z.D., Sun, Y.L., 1986. A study on the cultivation of the vegetative cells and protoplasts of P. haitanensis I. Oceanol. et Limnol. Sin. 17, 217–221 (in Chinese with English abstract). Wang, F.X., Xie, C.T., Chen, C.S., Ji, D.H., Xu, Y., Shi, X.Z., Liang, Y., Zhang, Y., 2010. Study on Chimeras and Breeding from Hybridization of the Breen-type and the Wildtype of Porphyra haitanensis. 15. J. Jimei Univ., pp. 1–6 (in Chinese with English abstract). Wang, C.Q., Yan, X.H., Huang, L.B., Liu, C.J., 2011. Characterization of an improved strain (SF-2) of Porphyra haitanensis (Bangiales, Rhodophyta) and its pilot cultivation in mariculture farm. J. Fish. China 35, 1658–1667 (in Chinese with English abstract). Wright, S., 1968. Evolution and the Genetics of Populations: Genetic and Biometric Foundations. Vol. 1. University of Chicago Press, Chicago, pp. 55–107. Xu, Y., Chen, C.S., Xie, C.T., Ji, D.H., Liang, Y., Liu, P.J., Wang, F.X., Shi, X.Z., 2008. Preliminary evaluation of hybridized strains of Porphyra haitanensis. Mar. Fish. Res. 29, 62–69 (in Chinese with English abstract). Yan, X.H., Aruga, Y., 1997. Induction of pigmentation mutants by treatment of monospore germlings with MNNG in Porphyra yezoensis Ueda (Bangiales, Rhodophyta). Algae 12, 39–54. Yan, X.H., Chen, M., 2008. Selection of low-salinity resistant improved varieties in Porphyra haitanensis (Bangiales, Rhodophyta). J. Shanghai Fish. Univ. 17, 316–320 (in Chinese with English abstract). Yan, X.H., Ma, S.Y., 2007. Selection of a high-temperature resistant strain of Porphyra haitanensis (Rhodophyta). J. Fish. China 31, 112–119 (in Chinese with English abstract). Yan, X.H., Wang, S.J., 1989. Studies on development and differentiation of somatic cells in Porphyra spp. (Rhodophyta). Mar. Sci. 6, 28–32 (in Chinese with English abstract). Yan, X.H., Li, L., Chen, J.H., Aruga, Y., 2007. Parthenogenesis and isolation of genetic pure strains in Porphyra haitanensis (Bangiales, Rhodophyta). High Technol. Lett. 17, 205–210 (in Chinese with English abstract). Yan, X.H., Lv, F., Liu, C.J., Zheng, Y.F., 2010. Selection and characterization of a hightemperature tolerant strain of Porphyra haitanensis Chang et Zheng (Bangiales, Rhodophyta). J. Appl. Phycol. 22, 511–516. You, H., 1999. The present situation of breeding and development countermeasures in Porphyra haitanensis of Pingtan County. Fish Sci & Tech. Infor. 26, 272–273 (in Chinese). Zhang, B.L., 2009. Isolation and Characterization of Heat-resistant Strains in Porphyra yezoensis Ueda (Bangiales, Rhodophyta) M.S. thesis. Shanghai Ocean University, Shanghai, China, pp. 35 (in Chinese). Zhang, Y.Y., Yan, X.H., 2014. Biological characteristics and a part life-cycle of Pyropia radi (Bangiales, Rhodophyta) from India. Oceanol. Limnol. Sinica 45, 51–57 (in Chinese with English abstract).
Acknowledgments The study was supported in part by the National High Technology Research & Development Program of China (“863” Program) (2012AA10A411), the National Natural Science Foundation of China (31072208), and the Major Science and Technology Special Fund of Agriculture (Fisheries) New Variety Breeding of Zhejiang (2016C02055-6), Large-scale Cultivation and Industrialization of Important Economic Red Algae with an Annual Output of 2000 tons (2014FJ19). References Fujian Fisheries Bureau, 1979. The Artificial Cultivation of Porphyra haitanensis. Fujian People Press, Fuzhou, pp. 1–101 (in Chinese). Gao, H.F., 1993. The variation in the contents of phycobiliproteins from Porphyra haitanensis collected in different growing stages. Oceanol. Limnol. Sinica 24, 645–648 (in Chinese with English abstract). Gu, L.Z., Ding, H.C., Yan, X.H., 2018. Phenomenon of cell breakdown and phenotypic observation on surviving blades in interspecific hybridization progeny between Pyropia haitanensis and Pyropia dentata. J. Fish. China 42, 60–71 (in Chinese with English abstract). He, P.M., Qin, S., Yan, X.J., Wu, W.N., 2007. Seaweed Biological Technology and its Application. Chemical Industry Press, Beijing, pp. 86–98 (in Chinese). Ji, D.H., Xie, C.T., Xu, Y., Chen, C.S., 2008. ISSR analysis on the heterosis in hybrids of Porphyra haitanensis. Acta Oceanol. Sin. 30, 147–153 (in Chinese with English abstract). Ji, D.H., Xie, C.T., Shi, X.Z., Xu, Y., Zhang, Y., 2011. Analysis of the Major Quality Traits in Wild Porphyra haitanensis of Fujian Coast. 16. J. Jimei Univ., pp. 401–406 (in Chinese with English abstract).
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