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Breeding, economic traits evaluation, and commercialcultivation of a new Saccharina variety‘‘Huangguan No. 1’’
Fuli Liu • Xiutao Sun • Feijiu Wang • Wenjun Wang •
Zhourui Liang • Zhelong Lin • Zhian Dong
Received: 20 October 2013 / Accepted: 26 March 2014� Springer International Publishing Switzerland 2014
Abstract New variety breeding is very significant for Saccharina japonica cultivation
industry. In this paper, we reported the breeding process, evaluation of economic traits, and
commercial cultivation of a new Saccharina variety ‘‘Huangguan No. 1’’ (variety approval
number: GS-01-006-2011). The sporophytes with best performance were screened out
from varieties cultivated throughout Fujian Province, China, and mixed together as the
parent population. The ‘‘Huangguan No. 1’’ variety was bred successfully from 5 years of
targeted selection of objective traits and continuous progeny selfing/inbreeding. This new
variety has a much longer, wider, and thicker blade; faster growth rate; heavier individual
weight; and stronger resistance to high temperatures compared to the control variety
(p \ 0.05). Cultivation tests showed that the economically important traits remained steady
in different years and sea areas, implying that ‘‘Huangguan No. 1’’ has the stable genetic
basis for its excellent performance. Compared to the control variety, the yield and final
product rate of ‘‘Huangguan No. 1’’ increased 30 and 25 %, respectively. Nutritional
constituent analysis indicated that ‘‘Huangguan No. 1’’ could be used as a healthy food
because of its higher protein (6.85 %) but lower fat content (0.2 %). Heavy metal (As, Cd,
Pb) content was lower than the standard content for food safety. The suitability of ‘‘Hu-
angguan No. 1’’ as a food source rather than a raw material for the chemical industry was
increased due to the low algin (10.2 %) and iodine content (0.32 %). ‘‘Huangguan No. 1’’
variety was welcomed by farmers and has been commercially cultivated about 13,293 ha.
Fuli Liu and Xiutao Sun have contributed equally to this paper.
F. Liu (&) � X. Sun � F. Wang � W. Wang � Z. LiangYellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,No. 106 Nanjing Road, Qingdao 266071, Chinae-mail: [email protected]
F. Wange-mail: [email protected]
Z. Lin � Z. DongGuanwu Ocean Development Co., Ltd, Lianjiang 352051, Fujian Province, China
123
Aquacult IntDOI 10.1007/s10499-014-9772-8
Keywords Saccharina japonica � ‘‘Huangguan No. 1’’ � Variety breeding �Economic traits assessment
Introduction
Saccharina (Laminaria) japonica is one of the most economically and ecologically important
seaweeds in China. It is used as a food, fertilizer, feed for aquatic animals, and raw material for
algin, mannitol, and iodine extraction, as well as benefiting offshore environmental resto-
ration. This kelp has been cultivated as early as the 1930s in China. The rapid evolution of the
seaweed industry is due to technological developments such as artificial seedling-rearing
(summer sporeling-rearing), floating raft cultivation, and southward transplant (Tseng 2001;
Zemke-White and Ohno 1999). In 2012, the production of S. japonica in China was 4,895,030
tons in wet weight, accounting for about 86 % of the Asian production (FAO: http://www.fao.
org/fishery/statistics/global-aquaculture-production).
Several varieties played a crucial role in the development of the Saccharina cultivation
industry. Since the 1960s, selective breeding has been applied to Saccharina breeding, with
which the first variety ‘‘Haiqing No. 1’’ was bred (Fang et al. 1962). In the 1970s, physical
mutagenesis was used along with selective breeding to obtain the elite varieties ‘‘860’’ and
‘‘1170’’ (Algal Breeding Team of Qingdao Maricultural Institute 1976). At the end of the
1970s, gametophyte cloning and hybridization methods were developed, with which a
number of varieties such as ‘‘Yuanza No. 10’’ and ‘‘901’’ were bred (Fang et al. 1985;
Zhang et al. 2007). In recent years, heterosis has been utilized in Saccharina breeding and
two varieties ‘‘Dongfang No. 2’’ and ‘‘Dongfang No. 3’’ have been bred successfully (Li
et al. 2007, 2008). These varieties have improved economic characters (higher yield,
higher quality, and stronger resistance) and have been commercially cultivated, promoting
the continued development of the Saccharina cultivation industry.
However, with the development of the Saccharina cultivation industry and its pro-
cessing industry, problems with the varieties have emerged. Firstly, an extended period of
breeding and intensive selection has reduced the genetic diversity and narrowed the
germplasm base of the varieties. The reduced genetic diversity and the narrowed germ-
plasm base decrease the adaptability of the varieties to environmental conditions and
jeopardize the performance of the economic traits (Bi et al. 2011; Liu et al. 2012a, b).
Secondly, during the process of summer sporeling-rearing, recirculated seawater causes
cross-hybridization among different varieties, resulting in the degeneration of economic
traits of varieties (Shan et al. 2011). Lastly, in order to supply enough raw materials for the
seaweed chemical industry, high yield has been the main objective of the Saccharina
breeding program during the last few decades. However, with the diversified development
of the Saccharina processing industry, it is urgent to breed different varieties with specific
characters to satisfy the different demands. Nowadays, most Saccharina (about 80 %) are
used as food for humans rather than raw material (personal communication). Hence, higher
quality (such as nutritional value and lower heavy metal content) and ease for food pro-
cessing need to be included in the breeding objectives.
With these breeding objectives in mind, we bred a variety named ‘‘Huangguan No. 1’’
using the method of continuous selfing/inbreeding and targeted selection. Owing to the
excellent performance, this new variety has been approved by the ‘‘Chinese Approving
Committee of Aquacultural Stock Seeds and Elite Varieties’’ with an approval number GS-
01-006-2011 in 2011. In this paper, we report on the breeding process, evaluation of
economic traits, and commercial cultivation of ‘‘Huangguan No. 1.’’
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Materials and methods
Breeding process of ‘‘Huangguan No. 1’’
The selection of parent population and cultivation of seedlings were carried out in GuanWu
nursery, Fujian Province, China. From later May to early June 2005, a total of 300 Sac-
charina sporophytes were screened from different varieties cultivated throughout the
Fujian Province, China, according to the following criteria: the sporophytes were healthy
and not mature (without any sporangia); the middle belt along the blade was long, wide and
thick; the edge along the blade was narrow, thick, and less crinkled; the sporophytes were
dark brown with robust stipe and rhizoid. The distal end of the blade was removed and the
chosen sporophytes were re-cultivated on a floating raft in an area with clear seawater and
a low water temperature. In early July, when the seawater temperature reached 26 �C,
sporophytes that had decayed or matured were discarded. The distal end of the blade was
again removed and the remaining 150 sporophytes measuring approximately 70 cm long
and 20 cm wide were rinsed and cultivated in an indoor pond with circulating seawater.
Sporophyte maturity was regulated by controlling water temperature and illumination. In
later September, seedlings were produced artificially using the summer sporeling-rearing
method (Tseng et al. 1984) and cultivated at sites in the Fujian, Shandong, and Liaoning
Provinces using the floating raft technique (Tseng et al. 1984).
Using the method described above, 1,200 sporophytes screened from the F1 population
cultivated in Fujian and Liaoning Province, respectively, were mixed together to be used as
the parents for F2 seedling production. Over the next 3 years, the same method was utilized
to reproduce the F3, F4, and F5 generation seedlings.
Assessment of economically important traits of ‘‘Huangguan No. 1’’
To assess the effect of breeding (targeted selection and continuous selfing/inbreeding),
several economically important traits, such as blade length (BL), blade width (BW), and
individual fresh weight (IFW), were selected as the indices to monitor the growth and
development of Saccharina from the F1 to F5 generation. The ‘‘Huangguan No. 1’’ and the
control variety were cultivated synchronously in Fujian and Liaoning Provinces. A total of
30 individuals were randomly sampled from both the ‘‘Huangguan No. 1’’ and control
varieties. The phenotypic characters were measured and obtained using methods reported
by Li et al. (2007). The effect of different cultivation sites was examined using a t test, and
the difference between generations was examined using F tests and multiple comparisons
(SPSS software).
To evaluate the nutritional value of ‘‘Huangguan No. 1,’’ the main nutritional constit-
uents were analyzed. Crude protein (CP), crude fat (CF), crude fiber (CFI), crude ash (CA),
and iodine were determined according to GB/T 5009.5-2003, GB/T 5009.6-2003, GB/T
5009.10-2003, GB/T 5009.4-2003, and SC/T 3010-2001, respectively. To assess the
potential as raw industry material, the algin and mannitol contents were determined using
the reported method (Sheng et al. 2011).
When Saccharina is produced as food, heavy mental content has been the focus of
attention. The contents of total arsenic (TAs), inorganic arsenic (IAs), cadmium (Cd), and
lead (Pb) were determined according to GB/T 5009.11-200, GB/T 5009.11-2003, GB/T
5009.15-2003, and GB/T 5009.12-2003, respectively.
Rate of final product (RFP) was an economically important index when Saccharina was
shredded to be used as food. The formula of RFP was weight of final products/weight of
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raw material 9 100 %. The RFP of ‘‘Huangguan No. 1’’ was determined and compared
with the control variety.
Genetic diversity assessment and parentage analysis
Six simple sequence repeat (SSR) loci were used to assess the genetic diversity of ‘‘Hu-
angguan No. 1’’ and to clarify the relationship between ‘‘Huangguan No. 1’’ and other
varieties, such ‘‘Rongfu’’ (RF), ‘‘Dongfang No. 2’’ (DF2), ‘‘Dongfang No. 3’’ (DF3),
‘‘901’’ (901) as well as a wild population from Japan. The SSR loci CX943071 and
CX943061 were developed by Liu et al. (2010), while LD1, LD11, LD12, and LD13 were
developed by Liu et al. (2012b). A total of 30 sporophytes were sampled for each variety
and population. The genomic DNA was extracted using a Plant genomic DNA kit (Tiangen
Biotech Co., Ltd, Beijing, China) according to the manufacturer’s instructions, and the
SSR analysis was conducted according to Liu et al. (2012b).
Hardy–Weinberg equilibrium at each locus and linkage disequilibrium among loci were
tested with software POPGENE version 1.3 (Yeh et al. 1999) and Arlequin 3.11 (Excoffier
et al. 2005), respectively. The parameters of the observed number of alleles (Na), the
effective number of allele (Ne), the Shannon’s information index (I), the mean observed
heterozygosity (Ho), and the mean expected heterozygosity (He) were calculated by
genetic analysis package POPGENE version 1.3 (Yeh et al. 1999) to assess the genetic
diversity level. To clarify the relationship between ‘‘Huangguan No. 1’’ and the other
varieties, a pairwise matrix of Nei’s unbiased genetic distances (Nei 1978) among different
varieties was calculated, on which the dendrogram was constructed using the unweighted
pair group method with the arithmetic averages (UPGMA) method with software TFPGA
1.3 (Miller 1997). Confidence levels for the dendrogram were calculated by bootstrapping
the original data 1,000 times with replacement over all loci.
Commercial cultivation
From 2008, the F3 generation of ‘‘Huangguan No. 1’’ was cultivated in large scale in Fujian
Province, to assess its performance. Cultivation was then continued northward from Fujian
to Shandong and Liaoning Provinces. The sporelings of ‘‘Huangguan No. 1’’ were pro-
duced in large numbers in Fujian Province using the summer sporeling-rearing method.
Sporelings were then transported to Shandong and Liaoning Provinces. The economically
important traits of ‘‘Huangguan No. 1’’ were comparatively evaluated with an endemic
cultivar.
Results
Morphological characteristics of ‘‘Huangguan No. 1’’
During the breeding process of ‘‘Huangguan No. 1,’’ morphological characteristics were
the primary objective. Individuals meeting the morphological requirements were chosen as
the parents to reproduce the next generation. The F5 generation formed unique morpho-
logical characteristics compared to the control cultivar (Fig. 1): the blade, especially the
middle belt was wider and thicker; the edge along the blade was narrower, thicker, and less
crinkled; the color was dark brown; the stipe was shorter but stronger; and the rhizoid was
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well developed and robust. These morphological characteristics ensured that ‘‘Huangguan
No. 1’’ had a higher yield and was easier to process as a food product.
Assessment of economically important traits of ‘‘Huangguan No. 1’’
For the trait of BL, the difference between the control variety and the selected parent
population was significant, with the progenies (F1–F5) of the parent population having a
significantly longer blade than the control population (p \ 0.05). In comparison with the
parent population, the BL increase in F1 and F2 generations was not significant (p [ 0.05).
However, BL of F3 to F5 generations was significantly different to the parent population
(Table 1). The BL of F5 generation increased 20.5 and 37.9 % compared to the parent
population and the control variety, respectively. For the traits of BW and IFW, the dif-
ference between parent and control population as well as the difference between progeny
generations and control variety was significant (p \ 0.05). The effect of targeted selection
on these two traits was stronger than that on the trait of BL. Beginning from the F2
generation, the increase in BW and IFW was significant compared to parent and control
population (Tables 2, 3). For all of the three traits, the selection effect first increased and
then decreased as the breeding program proceeded. As an example, the increase rate of BW
increased from parents to the F3 generation, but declined between the F3 and F5 genera-
tions. In addition, both the SD and the coefficient of variance (CV) decreased gradually
between the F1 and F5 generations (Fig. 2), indicating that ‘‘Huangguan No. 1’’ gradually
Fig. 1 Comparison of the morphological characteristic between ‘‘Huangguan No. 1’’ and the controlvariety. a Control variety, b ‘‘Huangguan No.1.’’ Scale bar 1 cm
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shaped the steady phenotype of the economically important traits under the continuous
selfing/inbreeding and targeted selection.
To investigate whether the economically important traits of ‘‘Huangguan No. 1’’
remained steady in different cultivation environments, ‘‘Huangguan No. 1’’ and an ende-
mic variety were both cultivated in Liaoning and Fujian Provinces synchronously. While
the endemic variety had different (p \ 0.05) traits performance when it was cultivated,
respectively, in northern (Liaoning Province) and southern (Fujian Province) sea area,
Table 1 Charactering and multiple comparison of BL of parent population, different generation (F1–F5),and control variety
Mean SD CV (%) Parents F1 F2 F3 F4 F5
Parents 328.50 38.01 10.02
F1 341.60 55.67 16.30 13.10
F2 356.00 31.23 8.77 27.50 14.40
F3 377.40 18.90 5.01 48.90* 35.80* 21.40
F4 388.70 15.63 5.01 60.20** 47.10* 32.70 11.30
F5 396.00 11.38 2.87 67.50** 54.40** 40.00* 18.60 7.30
Control 287.00 41.28 12.57 41.50* 54.60* 69.00** 90.40** 101.70** 109.00**
* The difference was significant (p \ 0.1); ** the difference was very significant (p \ 0.05)
Table 2 Charactering and multiple comparison of BW of parent population, different generation (F1–F5),and control variety
Mean SD CV (%) Parents F1 F2 F3 F4 F5
Parents 32.00 2.21 8.50
F1 35.60 6.08 17.07 3.60
F2 47.00 4.11 8.74 15.00* 11.40*
F3 54.10 2.96 5.47 22.10* 18.50* 7.10
F4 58.90 3.31 5.63 26.90** 23.30* 11.90* 4.80
F5 60.10 2.88 4.68 28.10** 25.90* 14.50* 7.40* 2.60
Control 27.00 8.50 25.90 5.00* 8.60* 20.00** 27.10** 31.90** 34.50**
* The difference was significant (p \ 0.1); ** the difference was very significant (p \ 0.05)
Table 3 Charactering and multiple comparison of IFW of parent population, different generation (F1–F5),and control variety
Mean (kg) SD CV (%) Parent F1 F2 F3 F4 F5
Parent 1.85 0.23 8.92
F1 2.07 0.27 12.89 0.22
F2 2.41 0.25 9.47 0.56* 0.34*
F3 2.87 0.22 8.22 1.02* 0.80** 0.46*
F4 3.01 0.22 6.90 1.16** 0.94** 0.60* 0.14
F5 3.16 0.18 5.62 1.31** 1.09** 0.60* 0.29* 0.15
Control 1.51 0.25 14.79 0.34* 0.56* 0.90** 1.36** 1.50** 1.65**
* The difference was significant (p \ 0.1); ** the difference was very significant (p \ 0.05)
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‘‘Huangguan No. 1’’ had no traits performance difference (p \ 0.05) when it was culti-
vated in different sea area. This implied that ‘‘Huangguan No. 1’’ has formed a stable
genetic basis for the phenotypic characteristics; that is to say, the elite alleles for the
economic traits have been selected, accumulated, and homogenized by the targeted
selection and continuous selfing/inbreeding.
Nutritional constituent analysis showed that the CP content and CA of ‘‘Huangguan No.
1’’ were higher than that of control variety, whereas the CF content and CFI content were
lower (Table 4). This implies that ‘‘Huangguan No. 1’’ was more suitable to be used as
healthy food. The algin and iodine content of ‘‘Huangguan No. 1’’ was lower than that of
control variety, indicating that ‘‘Huangguan No. 1’’ was not suitable to be as raw industry
material for algin and iodine extraction. However, the lower algin content made ‘‘Hu-
angguan No. 1’’ much crisper and better tasting. Heavy metal assays showed that the
content of TAs, IAs, Cd, and Pb in ‘‘Huangguan No. 1’’ was all lower than the national
standard content for food safety (Table 4). In addition, RFP of ‘‘Huangguan No. 1’’ was
Fig. 2 Trend of coefficient of variation of the objective traits in the parent population, different generations,and the control variety. BL blade length; BW blade width; IFW individual fresh weight
Table 4 Comparison of nutrient contents, heavy mental contents, and RFPs between ‘‘Huangguan No. 1’’and control variety
CP (%) CF (%) CFI (%) CA (%) Algin (%) Mannitol (%)
Huangguan No. 1 6.85 0.2 6.8 36 10.2 15.7
Control 6.4 0.4 7.3 26.8 20 11
Iodine (%) TAs (mg/kg) IAs (mg/kg) Cd (mg/kg) Pb (mg/kg) RFP (%)
Huangguan No. 1 0.32 38 \0.1 0.54 0.22 81
Control 0.47 46 0.15 0.56 0.32 65
CP crude protein, CF crude fat, CFI crude fiber, CA crude ash, TAs total arsenic, IAs inorganic arsenic, Cdcadmium, Pb lead, RFP rate of final product
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higher than that of control variety when this new variety was processed as food (Table 4).
All of the above results indicate the new variety was more suitable to be used as food rather
than raw industry materials.
Genetic diversity assessment and parentage analysis
The six SSR markers amplified 21 alleles in total, with an average of 3.5 alleles per
marker. All the six SSR markers were polymorphic in different varieties. The genotype
data of all accessions were used to calculate the parameters of Na, Ne, I, Ho, and He for
genetic diversity assessment (Table 5). The genetic diversity indexes showed that the
genetic diversity of these varieties was low to moderate. Among the five varieties,
‘‘Huangguan No. 1’’ had the lowest genetic diversity while ‘‘Dongfang No. 3’’ had the
highest. These varieties all had lower genetic diversity than the wild population. The
dendrogram (Fig. 3) showed that ‘‘Huangguan No. 1’’ had the closest genetic relationship
with the RF variety, but greatest distance in the genetic relationship with the wild pop-
ulation from Japan.
Table 5 Estimated genetic diversity level of different Saccharina varieties and a wild population bydifferent genetic parameters
Variety Na Ne I Ho He
HG 1.920 ± 0.719 1.531 ± 0.457 0.459 ± 0.412 0.431 ± 0.399 0.304 ± 0.211
RF 2.275 ± 0.870 1.781 ± 0.437 0.615 ± 0.356 0.541 ± 0.421 0.399 ± 0.178
DF2 2.127 ± 0.792 1.695 ± 0.392 0.509 ± 0.331 0.569 ± 0.401 0.352 ± 0.238
DF3 2.312 ± 0.795 1.698 ± 0.432 0.611 ± 0.271 0.559 ± 0.312 0.401 ± 0.159
‘‘901’’ 1.933 ± 0.257 1.649 ± 0.388 0.521 ± 0.239 0.578 ± 0.359 0.361 ± 0.191
WP 2.352 ± 0.967 1.973 ± 0.397 0.619 ± 0.324 0.577 ± 0.350 0.412 ± 0.163
HG ‘‘Huangguan No. 1,’’ RF ‘‘Rongfu,’’ DF2 ‘‘Dongfang No. 2,’’ DF3 ‘‘Dongfang No. 3,’’ WP wildpopulation
Fig. 3 Relationship of ‘‘Huangguan No. 1’’ with other varieties and a wild population demonstrated by theUPGMA dendrogram. HG ‘‘Huangguan No. 1’’ variety; RF variety, 901 ‘‘901’’ variety; DF3 ‘‘Dongfang No.3’’ variety; DF2 ‘‘Dongfang No. 2’’ variety; and WP a wild population from Japan
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Commercial cultivation
The F3 generation of ‘‘Huangguan No. 1,’’ which had the best performance of the desirable
traits, was cultivated in large scale in the Fujian Province, and was then cultivated
northward from Fujian to Shandong and Liaoning Provinces. In the next 2 years, the
cultivation of ‘‘Huangguan No. 1’’ has increased markedly in southern and northern China.
Although this new variety was bred in southern China, ‘‘Huangguan No. 1’’ did better in
northern China. The longer, wider, and thicker blades increased the total yield in northern
sites. The average yield of ‘‘Huangguan No. 1’’ was 41.1 ton/ha in Dalian, Liaoning
Province, while the yield was 38.9 ton/ha in Lianjing, Fujian Province. On the whole, the
yield of ‘‘Huangguan No. 1’’ increased 30–35 % compared to the control variety. In
addition, because the blade was much wider and less crinkled, ‘‘Huangguan No. 1’’ was
suitable to be processed for food as it had a higher final product rate. Consequently, the
new variety was welcomed by farmers and has been cultivated in large scale. The accu-
mulative cultivation area in southern and northern China was approximately 13,293 ha.
Discussion
Targeted population selection combined with progeny continuous selfing/inbreeding was
the first method used for Saccharina variety breeding (Fang et al. 1962). This method was
based on three hypotheses: Saccharina population has high heterozygosity and contains
elite alleles for economically important traits; targeted selection of objective traits can
select the alleles that control the desired traits; continuous selfing/inbreeding can accu-
mulate and homogenize the selected elite alleles (Fang et al. 1962; Wu and Lin, 1987).
Several varieties such as ‘‘Haiqing No. 1,’’ ‘‘Haiqing No. 2,’’ and ‘‘Zaohoucheng No. 1’’
have been successfully bred using this method, proving that this method was efficient for
Saccharina variety breeding (Fang et al. 1962; Tian and Yuan 1989). The parent popu-
lation of ‘‘Huangguan No. 1’’ was sampled from the Saccharina cultivation site in Fujian
Province, where a number of Saccharina varieties are cultivated together. These individ-
uals with the best performance were screened from different varieties cultivated in dif-
ferent sea areas and were mixed together to be used as the parent population. This sample
strategy ensured that the parent population contained enough abundance of genetic
diversity and elite alleles for economic traits.
Through 5 years of targeted selection of objective traits and continuous selfing/
inbreeding of progeny, the variety ‘‘Huangguan No. 1’’ was bred successfully. This new
variety has much longer, wider, and thicker blade, faster growth rate; and heavier indi-
vidual weight, all of which ensured the yield of ‘‘Huangguan No. 1’’ increased 30–35 %
compared to the control variety. The smooth, straight, wide blade middle made it easier
when ‘‘Huangguan No. 1’’ to be processed as a food, which increases the final product rate
about 25 % in contrast to the control variety. Particularly, the high temperature resistance
of ‘‘Huangguan No. 1’’ was stronger than that of the control variety. During cultivation
practice, stronger temperature resistance helps Saccharina to reduce disease and decay,
prolong cultivation period and harvest time. Nutritional constituent analysis and heavy
metal assay showed that ‘‘Huangguan No. 1’’ was more suitable as food rather than raw
industry material for algin and iodine extraction.
During the late 1950s, the Chinese psychologists began to breed varieties from wild
Saccharina populations. Over the past several decades, traditional methods of population
selection, mutagenesis, hybridization, and heterosis utilization were developed and applied
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in breeding practice separately or jointly (Tseng 2001; Wu and Lin 1987). With the
development of cultivation industry and process industry of S. japonica, the objective of
variety breeding also changed. Except for higher yield, higher quality and easier processing
for diversified foods are becoming the main breeding objectives. To realize these breeding
objectives, new breeding methods and theory should be continuously developed. Based on
the traditional methods, the fast developing molecular breeding methods and theories
including molecular marker-assisted selection, transgenic breeding, and molecular design
breeding should be applied in Saccharina variety breeding system in the future.
Acknowledgments This work was funded by Public Service industry (Agriculture) Special Research Fund(200902030), National High Technology Research and Development program (2012AA10A406), NationalNatural Science Foundation of China (41306176), and Shandong Natural Science Foundation(ZR2012CQ030). We thank Dr. Sheryl Miller for English language revise and also thank the anonymousreviewers for their suggestions to this paper.
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