Germplasm Resources of Largemouth Bass

CHAPTER 1

Germplasm Resources of Largemouth Bass Contents 1.1 Introduction 1.2 Morphological Characteristics of Largemouth Bass Subspecies 1.2.1 Materials and Methods 1.2.2 Results and Analysis 1.3 To Identify Largemouth Bass Subspecies Using Microsatellite Markers 1.3.1 Materials and Methods 1.3.2 Results and Analysis 1.4 Difference in mtDNA Between Largemouth Bass Subspecies 1.4.1 Materials and Methods 1.4.2 Results and Analysis 1.5 DNA Fingerprint Chromatogram of Microsatellite DNA in Largemouth Bass 1.5.1 Materials and Methods 1.5.2 Results and Analysis 1.6 Microsatellite Analysis of Genetic Diversity of Largemouth Bass in China 1.6.1 Materials and Methods 1.6.2 Results and Analysis 1.7 Genetic Diversities of Largemouth Bass in China and America 1.7.1 Materials and Methods 1.7.2 Results and Analysis References

1 2 3 3 6 6 8 9 9 10 13 14 18 24 24 25 32 33 36 39

1.1 INTRODUCTION Largemouth bass Micropterus salmoides (Perciformes, Centrarchidae) is native to freshwater lakes and rivers in North America. It is generally considered to comprise two subspecies (Bailey and Hubbs, 1949): the Florida largemouth bass (M. salmoides floridanus) found on the Florida peninsula, and the Northern largemouth bass (M. salmoides salmoides), distributed in most central and eastern parts of America, northeast Mexico, and southeast areas of Canada (Maceina and Murphy, 1992). Largemouth bass were introduced to Taiwan (China) at the end of the 1970s and successfully reproduced there Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass DOI: https://doi.org/10.1016/B978-0-12-816473-0.00001-3 Copyright © 2019 China Science Publishing & Media Ltd. Published by Elsevier Inc.

1

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Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

in 1983. In the same year, the species was introduced to mainland China in Guangdong province. It is a popular species with farmers because it is adaptable, fast-growing, and easy to harvest. Moreover, its meat is considered to be delicious, with few bones, making it attractive to consumers. The largemouth bass is now cultivated throughout China, and has become an important species for freshwater aquaculture. The largemouth bass has a generalized fusiform, compressed shape. Its mouth is oblique, with a wide gape and a retractable jaw. The dorsal muscle is oval-shaped in transverse section. The teeth are small but sharp. The fish are olive green on the back, with an off-white abdomen, and are coated with small ctenoid scales. A line of black spots runs from the rostrum to the base of the tail fin, and there are three radial black spots on the branchiostegite. The dorsal fin is not fully connected, with a small gap between the hard and soft sections. The lateral line does not reach the bottom of the tail fin, which has a rounded apex and a central depression. The gill raker of the first gill arch is well developed and ossified, with a sickle-shape. Apart from the back of the gill raker, the other three sides are fully ossified with inverted jagged bumps. The fifth gill arch is reduced to a short stick with no gills or gill rakers. The quantifiable external characteristics of the largemouth bass include: dorsal fin rays: D IX-1
13
15; anal fin rays: A III-9; pectoral fin rays: 1
12
13; pelvic fin rays: 1
15; number of lateral-line scales: 62
63; scales above the lateral line 7
8; scales below the lateral line 15; number of gill rakers 6
7; number of vertebrae 26
32. The internal characteristics include a long, cylindrical swim bladder, white peritoneum, bulky coiled intestines, with a total length 0.54
0.73 times the length of the body. The maximum body weight of largemouth bass in their native North America is up to 10 kg, and maximum body length is 970 mm. Largemouth bass generally grow faster in the first or second year. However, the growth rate reduces during the third year. In South China, largemouth bass can reach 500
750 g during their first year. Fish normally reach sexual maturity after about 1 year, after which spawning occurs several times during February to July, producing adhesive eggs.

1.2 MORPHOLOGICAL CHARACTERISTICS OF LARGEMOUTH BASS SUBSPECIES The Florida and Northern subspecies of largemouth bass differ in various aspects of their morphology, physiology, and ecology. Bailey and

Germplasm Resources of Largemouth Bass

3

Hubbs (1949) found different numbers of ribs and lateral-line scales in these two subspecies. Although most studies have demonstrated that the growth performance of the Northern largemouth bass is better than that of the Florida subspecies, David and Hugh (1979) and Bottroff and Lembeck (1978) found that the Florida largemouth bass grew faster than the Northern subspecies under the same conditions. Fields et al. (1987) demonstrated that the Northern largemouth bass was better able to adapt to different temperatures. The largemouth bass has become an important freshwater species in Chinese aquaculture, and is widely bred throughout the country. However, the subspecies of largemouth bass in China has not yet been identified, given that little information on subspecies classification and the origin of the fish were available when they were first introduced to China. Here the morphological methods were used to identify the subspecies of largemouth bass in China, to provide basic information for the improvement of the species and to facilitate its introduction in the future.

1.2.1 Materials and Methods A total of 124 8-month-old healthy largemouth bass were removed from breeding ponds at the Pearl River Fisheries Research Institute. Eleven measurable characteristics, including body weight, full length, body length, body height, body width, head length, lip length, interorbital distance, anteroposterior and rectilinear eyeball axes, caudal peduncle length, and caudal peduncle height, were measured using electronic scales, measuring plates, and vernier calipers, according to Li (1998). The weight and length were accurate to 0.1 g and 0.1 cm, respectively. A further 30 fish were randomly selected for counting characters including dorsal fin, anal fin, pectoral fin, pelvic fin, lateral line scales, vertebrae, and ribs. The ratios of body length/body height, body length/head length, and caudal peduncle length/caudal peduncle height were calculated to derive the body index. The data were analyzed using Excel 2007 and SPSS 15.0. The average, standard deviation, range of variation, and standard error were calculated for each parameter.

1.2.2 Results and Analysis 1.2.2.1 Morphological Characteristics The largemouth bass has a generalized fusiform, compressed shape, and a medium-sized head, with small eyes and a large mouth. The maxilla

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Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

extends beyond the eye. The lateral line is completely visible, and extends from the top of the operculum to the base of the caudal fin. The two dorsal fins are joined by a fin membrane and are slightly higher than the caudal peduncle. The pelvic fins are not joined by a membrane. The caudal fin is small and rounded. The body of the fish is green-to-olive dorsally and milky-white-to-yellow ventrally, with a black band running from the operculum to the base of the caudal fin. The gill rakers are comb-type. 1.2.2.2 Quantifiable and Measurable Characteristics The quantifiable and measurable characteristics of the largemouth bass in China are shown in Tables 1.1 and 1.2, respectively. The fin type of the largemouth bass in China was D IX-13
15, A III-10
12, V I-4
5, and P 12
13. The scale formula was (58
68)[(6
9)/(12
17)]. The gill raker formula was 2 1 6. Other quantifiable characteristics included 26
32 vertebrae, 15 pairs of ribs, and 58
68 lateral line scales. As shown in Table 1.2, the average body weight and full length of the largemouth bass in the present study were 468.27 6 194.54 g and 29.05 6 3.38 cm, respectively. The ratios of body length/body height, body length/head length, and caudal peduncle length/caudal peduncle height were 3.08 6 0.18, 3.10 6 0.23, and 1.58 6 0.21, respectively. These morphological results are coincident with those of Zhang and He (1994) and Li and Yang (2001) regarding largemouth bass in China, and suggest that Table 1.1 Quantifiable characteristics of largemouth bass in China Characteristic Range Mean SD Range of variation

Dorsal fin Anal fin Pectoral fin Pelvic fin Vertebrae Lateral line scales Scales above lateral line Scales below lateral line Gill rakers Ribs

SE

IX-13
15 III-10
12 12
13 I-4
5 26
32 58
68 6
9

14.20 11.00 12.30 4.70 30.40 61.66 7.83

0.63 0.67 0.48 0.48 2.46 2.64 0.60

14.2 6 0.63 11.0 6 0.67 12.3 6 0.48 4.70 6 0.48 30.40 6 2.46 61.66 6 2.64 7.83 6 0.60

0.2 0.211 0.153 0.153 0.777 0.489 0.112

12
17

15.69

1.04

15.69 6 1.04

0.193

216 15 pairs

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Germplasm Resources of Largemouth Bass

Table 1.2 Measurable characteristics of largemouth bass in China Characteristic

Range

Mean

SD

Range of variation

SE

Body weight (g) Full length (cm) Body length (cm) Body height (cm) Head length (cm) Lips length (cm) Body width (cm) Eyeball diam (cm) Interorbital distance (cm) Tail length (cm) Caudal peduncle length (cm) Caudal peduncle height (cm) Body length/body height Body length/head length Caudal peduncle length/caudal peduncle height

103.5
967.5 18.95
37.30 16.30
33.13 4.81
12.00 5.35
29.00 0.97
2.17 2.5
6.0 0.90
1.80 1.20
2.90

468.27 29.05 25.50 8.34 8.35 1.46 4.38 1.18 2.17

194.54 3.38 3.13 1.42 2.11 0.23 0.76 0.13 0.31

468.27 6 194.54 29.05 6 3.38 25.50 6 3.13 8.34 6 1.42 8.35 6 2.11 1.46 6 0.23 4.38 6 0.76 1.18 6 0.13 2.17 6 0.31

17.47 0.30 0.28 0.13 0.19 0.02 0.07 0.01 0.03

5.42
33.79 3.16
7.69

8.42 5.10

2.51 0.74

8.42 6 2.51 5.10 6 0.74

0.23 0.07

1.93
7.67

3.28

0.62

3.28 6 0.62

0.06

2.57
3.48

3.08

0.18

3.08 6 0.18

0.02

0.88
3.75

3.10

0.23

3.10 6 0.23

0.02

0.62
2.86

1.58

0.21

1.58 6 0.21

0.02

the quantifiable and measurable characteristics of the species in China have remained relatively stable. In the present study, the dorsal fin type of largemouth bass in China was D IX-13
15, and the number of vertebrae ranged from 26 to 32. These results differed slightly from those of Ramsey (1975), who found a dorsal fin type of D IX-12
13. However, the present results were similar to those of other Chinese researchers, who reported D IX-13
15, and 29
30 vertebrae, as well as similar numbers of pectoral, pelvic, and anal fin rays, and scales above and below the lateral line. Based on differences in quantifiable characteristics, Bailey and Hubbs (Bailey and Hubbs, 1949) and Bryan (1969) classified the largemouth bass into two subspecies, the Florida largemouth bass and Northern largemouth bass, which mainly differ in the number of lateral line scales and ribs. The Northern subspecies has 59
64 lateral line scales and 15 pairs of ribs, while the Florida subspecies has 69
73 lateral line scales, and 14 pairs of ribs. According to the present study, largemouth bass in China

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Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

had 59
64 lateral line scales and 15 pairs of ribs, identifying them as Northern largemouth bass.

1.3 TO IDENTIFY LARGEMOUTH BASS SUBSPECIES USING MICROSATELLITE MARKERS The largemouth bass was widely introduced into many areas of the world in the 1970s, thus ending the geographical isolation of this species. The lack of management of the introduction process has resulted in the creation of a “mid-subspecies” (Northern largemouth bass 3 Florida largemouth bass) in many areas, with morphological characteristics midway between the two original subspecies, making identification of the origin of largemouth bass difficult on the basis of morphology alone (Rogers et al., 2006). However, molecular biological techniques have become available for identifying the two subspecies. Nedbal and Philipp (1994) found that the amplification products of mitochondrial DNA (mtDNA) differed between the two subspecies according to restriction fragment length polymorphism (RFLP) analysis, and Williams et al. (1998) reported that the “mid-subspecies” could be distinguished from the original two subspecies by random amplified polymorphic DNA (RAPD) analysis. Philipp et al. (1983) identified specific sites of the isoenzyme in the two subspecies. Lutz-Carrillo et al. (2006) used microsatellite molecular markers to distinguish between the two subspecies, and found specific microsatellite primers for polymerase chain reaction (PCR). Microsatellite markers were therefore used to identify the subspecies of largemouth bass in China, to clarify its classification status.

1.3.1 Materials and Methods A total of 124 healthy fishes (M. salmoides) were obtained from the breeding base at the Pearl River Fisheries Research Institute, CAFS. Blood was sampled via the caudal vein and kept at 220˚C. Microsatellite markers were analyzed in 24 largemouth bass from America (data was donated by Dr. Lutz-Carrillo, Texas Parks & Wildlife Department, A. E. Wood Lab, TX, USA), including 14 Northern and 10 Florida samples. Genomic DNA-extraction kits were purchased from Tiangen and Omega Biotechnology Companies. The microsatellite primers were synthesized by Invitrogen (Shanghai, China), Taq-DNA polymerase was from Biocolors (Shanghai, China), and the SSR DNA Marker II was from

Germplasm Resources of Largemouth Bass

7

Beijing Dingguo Biotechnology Co. Ltd. (Beijing, China). Other reagents were of analytically pure grade produced in China. Genomic DNA extraction: Blood samples were fixed in anticoagulant citrate dextrose (ACD) and DNA was extracted using a whole blood genomic DNA isolation kit (Tiangen) according to the manufacturer’s instructions. Genomic DNA was extracted from the fin ray samples from America using a whole blood genomic DNA isolation kit (spin column, Omega), according to the manufacturer’s instructions. DNA samples were kept at 220˚C until use. The quality and concentration of the genomic DNA was detected by agarose (0.8%) gel electrophoresis. The specific microsatellite primers Mdo6 and Msal21, which are able to distinguish between the Florida and Northern largemouth bass, were used to identify the subspecies of largemouth bass in China. The total volume of the PCR reaction was 20 µL, including 10 3 buffer 2.0 µL, MgCl2 (25 mmol/L) 0.8 µL, 4 3 dNTP (10 µmol/L) 0.3 µL, forward and reverse primers (20 µmol/L) 0.5 µL each, genomic DNA 40 ng, and Taq polymerase 1 U, with ddH2O added to 20 µL. The reaction was performed using a PTC-200 PCR Amplifier. Following the first heating (94˚C, 4 min), 25 cycles of PCR (94˚C, 30 s; 55˚C/Mdo6, 48˚C/Msal21 30 s; 72˚C, 30 s) and 7 min at 72˚C were performed, resulting in good amplification of the target regions of Mdo6 and Msal21. The PCR products were isolated by nondenaturing polyacrylamide gradient gel electrophoresis (8%), followed by silver staining and scanning. The sequences of the primers are shown in Table 1.3. Table 1.3 Specific microsatellite molecular markers and primers for largemouth bass Primers Primer sequence Length of the PCR products (bp) (50
30 ) Northern Florida Largemouth largemouth largemouth bass in China bass bass

Mdo6

Msal21

F:TGAAATGTA CGCCAGAGCAG R:TGTGTGGGTG TTTATGTGGG F:CACTGTAAATG GCACCTGTGG R:GTTGTCAAGT CGTAGTCCGC

146/151

153/153

146/151

196/196

205/210

196/196

F, forward primer; R, reverse primer; PCR, polymerase chain reaction.

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Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

1.3.2 Results and Analysis Genomic DNA from largemouth bass in China, and from Florida and Northern largemouth bass in America, was amplified by PCR using the primers Mdo6 and Msal21, which are specific for the different subspecies of largemouth bass. The PCR products of largemouth bass in China were identical to the products for the Florida and Northern largemouth bass (Table 1.3). It had previously been reported that Mdo6 (153 bp) was only found in the Florida subspecies, while Msal21 (196 bp) was only found in the Northern subspecies. Our results demonstrated that Mdo6 and Msal21 were found in the Florida and Northern largemouth bass, respectively. Moreover, as shown in Figs. 1.1 and 1.2, the PCR products for the largemouth bass in China were the same as those for the American Northern largemouth bass. As reported by Rogers et al. (2006), the “mid-subspecies” of largemouth bass has been found in areas of North Florida and some other places with introduced largemouth bass. The morphological characteristics of the

Figure 1.1 Nondenaturing polyacrylamide gel electrophoresis (8%) of PCR products using Mdo6 primers in Northern, Florida, and largemouth bass cultured in China. PCR, polymerase chain reaction. Note: M, DL2000 marker; C1
C12, largemouth bass in China; N1
N14, Northern largemouth bass; S1
S10, Florida largemouth bass.

Figure 1.2 Nondenaturing polyacrylamide gel electrophoresis (8%) of PCR products using Msal21 primers in Northern, Florida, and largemouth bass cultured in China. PCR, polymerase chain reaction. Note: M, DL2000 marker; C1
C12, largemouth bass cultured in China; N1
N14, Northern largemouth bass; S1
S4, Florida largemouth bass.

Germplasm Resources of Largemouth Bass

9

“mid-subspecies” are midway between the two subspecies, making it impossible to identify the subspecies in China on the basis of morphology alone. We therefore used specific microsatellite primers (Mdo6 and Msal21) for largemouth bass subspecies to identify the subspecies cultured in China. The PCR products of largemouth bass from China were identical to those from the Northern largemouth bass, thus identifying the Chinese fish as Northern largemouth bass.

1.4 DIFFERENCE IN MTDNA BETWEEN LARGEMOUTH BASS SUBSPECIES As shown above, both morphological and microsatellite analyses identified largemouth bass cultured in China as belonging to the Northern subspecies. We further investigated differences in the sequences of the displacement-loop (D-loop) regions of mtDNA among the Northern, Florida, and largemouth bass cultured in China.

1.4.1 Materials and Methods 1.4.1.1 Sample Collection Largemouth bass cultured in China (G) (n 5 5) were collected from breeding ponds at the Pearl River Fisheries Research Institute, CAFS. Eighteen samples of American largemouth bass caudal fin were donated by Dr. LutzCarrillo (Texas Parks & Wildlife Department, A. E. Wood Lab, TX, USA), including 11 Northern (N), and seven Florida largemouth bass (F). 1.4.1.2 Preparation of Genomic DNA From Largemouth Bass The caudal fin rays were sampled from all 23 largemouth bass, and 30 mg per fish was prepared for DNA extraction. Genomic DNA was extracted from the caudal fin rays using a genomic DNA isolation kit (spin column, Omega), according to the manufacturer’s instructions. DNA samples were kept at 220˚C until use. The quality and concentration of the genomic DNA was detected by agarose (0.8%) gel electrophoresis. 1.4.1.3 Primers for D-loop Regions, PCR Amplification, and Electrophoresis A pair of primers was designed to amplify the mtDNA D-loop regions (GenBank, DQ536425) of the largemouth bass, as follows: F, 50 -TCCCAAAGCTAGGATTCTAAAC-30 ; R, 50 -TCTTAACATCT TCAGTGTCATGC-30 . After synthesis by Invitrogen (Shanghai, China),

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Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

the primers were dissolved in ddH2O to a concentration of 20 pmol/L. The total volume of the PCR reaction was 50 µL, including 10 3 buffer (containing Mg21) 5.0 µL, dNTPs (10 mmol/L) 1 µL, forward and reverse primers (20 pmol/L) 1 µL each, Taq polymerase 3 U, and genomic DNA 60 ng. The PCR procedure was 94˚C for 4 min, followed by 32 cycles of 94˚C for 30 s, 53˚C for 30 s, 72˚C for 1 min, and finally 7 min at 72˚C. The PCR products were isolated by 1.3% agarose gel electrophoresis, and then recovered and purified using commercial kits. The recycled PCR products were bi-directionally sequenced at Sangon Biotech (Shanghai, China) to ensure accuracy. 1.4.1.4 Data Analysis The results of bi-directional sequencing were subjected to basic local alignment search tool (BLAST) analysis using Vector NTI 8.0, manually when necessary. The haplotype and variation sites, haplotype diversity (h) and nucleotide diversity (π), and levels of genetic diversity were analyzed using DnaSP 4.0 software. Analysis of molecular variance (AMOVA) among groups was performed using Arlequin 3.1 software by calculating the squared Euclidean distance of the matrices (Excoffier and Schneider, 2005). Evolutionary distances, base composition, and nucleotide substitutions were analyzed using MEGA 4.0 software. Evolutionary distances were calculated using the Kimura 2-parameter model, and clustered by the unweighted pair group method using arithmetic average (UPGMA) to construct a cluster analysis tree.

1.4.2 Results and Analysis 1.4.2.1 PCR Amplification of mtDNA D-loop Regions in Largemouth Bass The 23 DNA samples from the three groups of largemouth bass were used as DNA templates for mtDNA D-loop region amplification. The lengths of the PCR products were similar (about 860 bp), as demonstrated by agarose gel electrophoresis (Fig. 1.3). 1.4.2.2 mtDNA D-loop Region Sequences and Haplotypes in Largemouth Bass The mtDNA D-loop regions of 23 largemouth bass from three groups were sequenced. Detailed analysis of an 810-bp sequence was performed after BLAST analysis, which found 73 variation sites, 15 transition sites (Ts), and 6 transposition sites (Tv). The total aberration rate was 9.0%, and mean Ts/Tv ratio was 2.4. The average codon contents were

11

Germplasm Resources of Largemouth Bass

Figure 1.3 PCR amplification results on the mtDNA D-loop regions 1
4: Northern largemouth bass; 5
8: Florida largemouth bass; 9
11: largemouth bass in China; M, DNA marker. PCR, polymerase chain reaction; mtDNA, mitochondrial DNA; D-loop, displacement-loop.

Table 1.4 Origin, abbreviation, and genetic diversities of the three largemouth bass groups Group Origin Sample Haplotype Nucleotide size diversity diversity

N

Northern largemouth bass Florida largemouth bass Chinese largemouth bass

F G

11

0.946

0.0082

7

1.000

0.013

5

0.400

0.0005

Table 1.5 Distribution of haplotypes in the three largemouth bass groups Group

N F G

Haplotype 1

2

3

4

5

6

7

8

9

3

1

1

1

1

1

1

1

1

10

11

12

13

14

15

16

1

1

1

1

1

1

1

17

18

4

1

29.7% A, 31.7% T, 21.6% C, and 17.0% G. The A 1T content (61.4%) was apparently higher than the C 1 G content (38.6%). A total of 18 haplotypes were found, with no haplotypes shared among the three groups: seven haplotypes were found in group F, nine in group N, and two in group G (Tables 1.4 and 1.5). 1.4.2.3 Taxonomic Status of Largemouth Bass in China MEGA 4.0 software was used to construct a molecular phylogenetic tree by the UPGMA method, according to differences in mtDNA D-loop

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Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

Figure 1.4 Molecular phylogenetic tree of mtDNA D-loop regions by UPGMA method 1
9: Northern largemouth bass; 10
16: Florida largemouth bass; 19
20: largemouth bass in China. mtDNA, mitochondrial DNA; D-loop, displacement-loop; UPGMA, unweighted pair group method using arithmetic average.

regions (Fig. 1.4). The molecular phylogenetic tree was divided into two branches, representing the Florida largemouth bass and the Northern and largemouth bass in China, respectively. These results confirmed that the largemouth bass in China belong to the Northern subspecies. 1.4.2.4 Population Genetic Diversity and Genetic Differentiation Genetic diversity analysis showed nucleotide diversity indexes (π) for the groups N, F, and G of 0.0082, 0.013, and 0.0005, respectively. These results indicate that the genetic diversity of the largemouth bass in China was dramatically lower than that of the wild population in America. Moreover, the haplotype diversity index (h) in groups N, F, and G were 0.946, 1.000, and 0.400, respectively, also suggesting a decline in genetic diversity in largemouth bass in China. Together, these results indicate that the genetic diversity of largemouth bass in China has declined during its long, artificial-breeding history, probably as a result of the small base population, inbreeding, and genetic drift. The results of AMOVA showed that the intragroup heritable variation was relatively high (83.44%), while the intergroup genetic differentiation was low (Fst 5 16.56, Table 1.6). Pairwise comparison of the genetic differentiation indexes (Fst) in the three groups showed the highest genetic

Germplasm Resources of Largemouth Bass

Table 1.6 AMOVA results for largemouth bass groups Source of genetic Degree Heritable variation difference

Percentage of total variation

Intergroup Intragroup Total

16.56 83.44 100

2 20 22

0.085 0.426 0.511

13

Table 1.7 Genetic differentiation index (lower left) and genetic distance (upper right) of largemouth bass groups Groups N F G

N F G


0.798 0.419

0.052
0.843

0.009 0.053


differentiation between groups G and F (Fst 5 0.843), and the lowest between groups N and G (Fst 5 0.419). These results were in accord with the fact that there was greater genetic differentiation between than within subspecies. The genetic distances between the groups (Table 1.7) were in agreement with the genetic differentiation index results. Wright (1951) reported that the degree of differentiation would be high if Fst was .0.25. According to Wright’s explanation, largemouth bass in China met this criterion, suggesting that their long history of artificial breeding had affected their genetic constitution.

1.5 DNA FINGERPRINT CHROMATOGRAM OF MICROSATELLITE DNA IN LARGEMOUTH BASS Largemouth bass in China have been artificially bred by Pearl River Fisheries Research Institute, CAFS, since 2005. To date, they have introduced Northern and Florida largemouth bass from America as germplasms for breeding. However, in order to utilize these germplasm resources, it is necessary to analyze their hereditary constitutions, determine the differences between them, and establish methods to differentiate them. DNA fingerprinting provides an effective means of identifying the relationships within this species. This technique can accurately distinguish species or individuals closely related to each other by analyzing differences in genomic sequences, with great individual specificity and environmental stability (McConnell et al., 1995; Scribner et al., 1996; Dewoody et al., 2000). The main markers for DNA fingerprinting

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Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

include RFLP (Xuan and Qu, 1994), RAPD (Du et al., 2002), microsatellites (Gao et al., 2005; Li et al., 2005; Song et al., 2009), and single nucleotide polymorphisms (Primmer et al., 2002), among which microsatellite markers are randomly distributed in the whole genome. They are preferable to other markers, and are widely used for constructing DNA fingerprints because of their diversity and ability to reveal the overall characteristics of the whole genome (Zietkiewicz et al., 1994). In this section, a DNA fingerprint database was established by amplifying 43 microsatellite DNA markers in largemouth bass from different areas, to provide basic information for the preservation of germplasm resources, hybrid identification, and seed-selecting and breeding.

1.5.1 Materials and Methods 1.5.1.1 Largemouth Bass Groups Four populations of largemouth bass were selected for the microsatellite DNA fingerprint database: largemouth bass from China (CH), Florida largemouth bass from America in 2009 and 2010, respectively (FL-09 and FL-10), and Northern largemouth bass from America in 2010 (NT-10). A total of 186 fish were selected, including 32 from each of the FL-09, FL-10, and NT-10 populations. The CH population comprised 30 fish that were randomly selected from each of three fisheries including Guangzhou, Shunde, and Zhongshan in Guangdong province. Blood was sampled via the caudal vein and put into tubes containing ACD anticoagulant, at a ratio of 1:6 (blood: ACD anticoagulant), and stored at 220˚C. 1.5.1.2 Microsatellite Primers Forty-three microsatellite DNA markers were selected, including 21 produced in our lab via magnetic bead enrichment (Liang et al., 2008), and 22 from Lutz-Carrillo et al. (2006). The polymorphism of the primers was verified. The PCR products revealed clear and stable bands by electrophoresis. All primers in the present study were synthesized by Sangon Biotech. Information on the primers is shown in Table 1.8. 1.5.1.3 Preparation of Genomic DNA Blood samples were extracted using a whole blood genomic DNA isolation kit (spin column, Tiangen), according to the manufacturer’s instructions. The quality and concentration of the genomic DNA were determined by agarose (0.8%) gel electrophoresis. DNA samples were kept at 220˚C until use.

Table 1.8 Microsatellite primers for largemouth bass No.

Sites

Repetitive sequence

Alleles

Annealing temperature (°C)

Primer sequence (50
30 )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

JZL31 JZL37 JZL43 JZL48 JZL60 JZL67 JZL68 JZL71 JZL72 JZL83 JZL84 JZL85 JZL105 JZL106 JZL108 JZL111 JZL114 JZL124 JZL126 JZL127 JZL131 MiSaTPW01 MiSaTPW11 MiSaTPW12 MiSaTPW25

(CA)25 (CA)24 (CA)21 (GT)13 (CA)21 (CA)16 (CA)20 (GT)21 (CA)21 (CA)23 (CA)20 (CA)17 (GT)13 (GT)35 (CA)17 (GT)27 (GA)11(GT)17 (CA)28(CT)25 (AC)24 (CA)15 (CA)8 (AC)16 (AGAT)13 (AGAT)21 (AGAT)11

5 4 3 3 4 4 7 7 7 5 7 3 2 5 2 7 5 11 5 7 2 2 7 9 5

60 56 58 55 60 60 58 60 58 55 55 56 58 54 55 52 55 50 55 52 55 56 55 48 55

F:TGGACTGAGGCTACAGCAGA F:TCCAGCCTTCTTGATTCCTC F:GCTGCGAGTGCGTGTAACTA F:TCGACGATCAATGGACTGAA F:AGTTAACCCGCTTTGTGCTG F:CCGCTAATGAGAGGGAGACA F:AGGCACCGTCTTCTCTTCA F:GCAGCTTCAGGTGTGTGTT F:AGGGTTCATGTTCATGGTAG F:TGTGGCAAAGACTGAGTGGA F:GAAAACAGCCTCGGGTGTAA F:GGGGCTCACTCACTGTGTTT F:GTGTCCCTGACTGTATGGC F:GCAGGCAGTGAACCCAGATT F:GTGACAGATGAGCGGAGAA F:TGTCTCAACTCCACCTACG F:CTACAGGTTAGGGAGTTACACG F:GCATTCATACACCATCATTG F:CAGGTAGCAGCGGTTAGGATG F:CAGAGAGATAGTGTCAACCA F:CAAATGCCCGGTCCACAATAAC F: AGTAAAGGACCACCCTTGTCCA F: CAACATGGACGCTACTAT F: CGGTTGCAAATTAGTCATGGCT F: CCAAGGTCAGGTTTAAC

R:CCAAGAGAGTCCCAAATGGA R:CCCGTTTAGCCAGAGAAGTG R:GGGAAGCGAGAGTCAGAGTG R:TCTGGACAACACAGGTGAGG R:GAAGGCGAAGAAGGGAGAGT R:ACAGACTAGCGTCAGCAGCA R:CATTGTGGGTGCATTCTCC R:TCGGTGAACTCCTGTCAGG R:ACACAGTGGCAAATGGAGGT R:ATTTCTCAACGTGCCAGGTC R:CACTTGTTGCTGCGTCTGTT R:GTGCGCAGACAGCTAGACAG R:TCTGATGAGGCTGTGAAAT R:TATGTATTGACGAGCGAGCAG R:GATGCTTGAGATACGACTA R:CACCCTGGCTTCATCTGC R:TGCTGAGGACACAACGAGGT R:AGCATTTTGTCAGACCACC R:TCTGAAACACGGACTCACGAC R:ACCACGGAGAAAGCCATT R:GTATTTGAGCCGGATGATAAGTG R:GCCTGGTCATTAGGTTTCGGAG R:CAACCATCACATGCTTCT R:CAGGGTGCTCGCTGTCT R:ACCTTTGTGCTGTTCTGTC (Continued)

Table 1.8 (Continued) No.

Sites

Repetitive sequence

Alleles

Annealing temperature (°C)

Primer sequence (50
30 )

26

MiSaTPW51

(AGAT)31

5

55

27 28 29 30 31

MiSaTPW70 MiSaTPW76 MiSaTPW90 MiSaTPW96 MiSaTPW117

(AGAT)43 (AGAT)22(AGAC)10 (ACAT)6 (AGAT)15(AGAC)6 (AC)24

13 7 8 7 6

55 48 48 55 55

32 33

MiSaTPW123 MiSaTPW157

(AC)22 (AC)21

4 11

55 55

F:CACAGAGACATTGCAGCTGA CCCT F: ACTTCGCAAAGGTATAAC F:ACACAGTGTCAGTTCTGCA F:TGCCAGAGATCCTGAGCTAC F:CTTCTAAATGTGTGTAGGGTTGC F:TGTGAAAGGCACAACACAGC CTGC F:GCTAACTTAATCTGCTGGATGGTG F:GACCTCAATGCGGATACTGTGACC

34

MiSaTPW165

(AC)16

4

55

35 36 37 38 39 40 41 42 43

MiSaTPW173 MiSaTPW184 Lar7 Lma10 Lma21 Lma120 Mdo6 Mdo7 Msal21

(AC)15 (AC)14(CT)10 (AC)15 (TG)10(TATGTG)4 TC）19AC）11 (GT)28 (CA)7(TA)4 (CA)12 (AC)15

10 5 12 3 7 5 4 2 5

55 55 48 55 53 54 55 55 55

F:GTTCGCATCTGAATGCATGT GGTG F:CCACACAGTGACACAAACTGTGC F:TTGTATACCAAGTGACCTGTGG F:GTGCTAATAAAGGCTACTGTC F:GTCTGTAAGTGTGTTTGCTG F:CAGCTCAATAGTTCTGTCAGG F:TGTCCACCCAAACTTAAGCC F:TGAAATGTACGCCAGAGCAG F:TCAAACGCACCTTCACTGAC F:CACTGTAAATGGCACCTGTGG

R:TGACGTATAGTACCAGCT GTGGTT R:CCTCATGCAGAAGATGTAA R: GTGAATACCTCAGCAAGCAT R:CACTTACCTGAATAACCAGAGACA R:AGCTTAGCATAAAGACTGGGAAC R:ATCGACCTGCAGACCAGCAACACT R:TGAACCTTCATAGGACAGCC R:AGGCACTCATCTGAATTGTCC ATGT R:TGAAGGTATTAGCCTCAGCCTACA R:GCCATTGTGCTGCTGCAGAG R:GGGAGTGCATCTTTCTGAAGTGCC R:TGTTCCCTTAATTGTTTTGA R:GAAACCCGAAACTTGTCTAG R:ACTACTGCTGAAGATATTGTAG R:TAAGCCCATTCCCAATTCTCC R:TGTGTGGGTGTTTATGTGGG R:GTCACTCCCATCATGCTCCT R:GTTGTCAAGTCGTAGTCCGC

Germplasm Resources of Largemouth Bass

17

1.5.1.4 PCR Reaction and Amplification The total volume of the PCR reaction was 20 µL, including 10 3 buffer 2.0 µL, MgCl2 (25 mmol/L) 0.8 µL, dNTPs (10 mmol/L) 0.3 µL, forward and reverse primers (20 µmol/L) 0.5 µL each, Taq polymerase 1 U, and genomic DNA 40 ng. The PCR reaction was performed using a PTC-200 amplifier. The procedure was carried out at 94˚C for 4 min, followed by 25 cycles of 94˚C for 30 s, 48
60˚C (according to the primers) for 30 s, 72˚C for 30 s, and finally 7 min at 72˚C. The PCR products were kept at 4˚C. 1.5.1.5 Electrophoresis Four microliters of PCR products (sample/buffer, v/v, 3/1) and 0.5 µL DNA marker were isolated on an 80 mg/mL nondenaturing polyacrylamide gradient gel (8%). The gels were stained with silver, as described by Huo et al. (2005), and scanned. 1.5.1.6 Establishment of Microsatellite DNA Fingerprint Database Based on the results of electrophoresis, the 43 alleles amplified by microsatellites in all the individuals in each group were combined to create a microsatellite DNA fingerprint using Excel. 1.5.1.7 Establishment of Digital DNA Fingerprint Database Microsatellite markers amplified in the largemouth bass groups were recorded as “1” if a distinct stripe was present on the gel, or “0” if there was no stripe. For example, in the case of five alleles with lengths of 202, 190, 182, 166, and 150 bp, if the 202 and 182 bp stripes were distinct on the gels, the DNA fingerprint would read as 10100. 1.5.1.8 Data Analysis The sizes of the bands were analyzed using AlphaEase FC Gel Image Analysis Software. The genotype was determined by the location of the bands for each individual. Popgene (version 1.32) software was used to calculate the number of alleles (A) and the expected heterozygosity (He) of each microsatellite site in the four groups. The genetic identity and genetic distance in Nei’s groups were also calculated and a UPGMA tree dendrogram was constructed based on the results for the four largemouth bass groups. The polymorphism information content (PIC) for each microsatellite site was calculated by Botstein’s (1980) method:

18

Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

PIC 5 1 2

n X i51

Pi2 2

n21 X n X

2ðPiPjÞ2

i51 j5i11

where Pi and Pj are the frequency of the ith and jth alleles in the group, respectively, and n is the number of alleles.

1.5.2 Results and Analysis 1.5.2.1 DNA Amplification Forty-three microsatellite DNA markers were selected to analyze the alleles in 186 fish from four groups of largemouth bass. A total of 246 alleles were detected. The mean number of the alleles at each microsatellite site was 5.72 (range 2
13). The average numbers of alleles in CH, FL-09, FL-10, and NT-10 were 2.58, 3.74, 3.70, and 4.21, the average He values were 0.4549, 0.4896, 0.5010, and 0.6138, and the average PICs were 0.3786, 0.4443, 0.4566, and 0.5546, respectively (Table 1.9). In China, largemouth bass larvae are mainly produced in Guangdong province because of the climate and early breeding, and are then introduced to most areas of China. Larvae from these three farmers can thus be considered to represent the largemouth bass population in China. RAPD (Zhu et al., 2006; Liang et al., 2007), amplified fragment length polymorphism (Lu et al., 2010), and microsatellite DNA (Liang et al., 2008; Bai et al., 2008) methods have recently been used to analyze the genetic diversity of largemouth bass in China. They found moderate genetic diversity, consistent with our results of a PIC in the CH group of 0.3786, compared with .0.4443 in the other three groups. This difference may be attributable to the limited introduction times and scales, suggesting that the largemouth bass population in China should be supplemented by new introductions to enrich its genetic diversity. 1.5.2.2 DNA Fingerprints of Largemouth Bass Groups The DNA fingerprints of the CH, FL-09, FL-10, and NT-10 populations, drawn in Excel based on the amplification results for 43 microsatellite sites in each population, are shown in Fig. 1.5. A total of 246 alleles were detected in these populations. The probability of identical bands at all 246 sites was (1/2)246, that is 8.84 3 10275, making it effectively impossible for any two individuals to have identical bands. The fingerprints can thus be used to identify individual fish, and to label largemouth bass individuals during genetic breeding.

Table 1.9 Number of alleles, expected heterozygosity values, and polymorphism information contents of microsatellite sites in largemouth bass No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Sites

JZL31 JZL37 JZL43 JZL48 JZL60 JZL67 JZL68 JZL71 JZL72 JZL83 JZL84 JZL85 JZL105 JZL106 JZL108 JZL111 JZL114 JZL124 JZL126 JZL127 JZL131 MiSaTPW01 MiSaTPW11 MiSaTPW12 MiSaTPW25 MiSaTPW51 MiSaTPW70

Fragment size

187
223 177
201 217
227 209
223 207
227 253
266 148
181 192
258 171
215 140
172 179
209 199
225 284
320 250
286 276
283 118
189 192
230 197
268 182
214 148
173 180
188 288
298 166
357 171
332 270
300 571
654 282
609

Number of alleles (A)

Expected heterozygosity (He)

Polymorphism information contents (PIC)

FL-09

FL-10

NT-10

CH

FL-09

FL-10

NT-10

CH

FL-09

FL-10

NT-10

CH

4 2 1 1 4 4 3 7 5 4 4 3 4 3 4 3 1 8 3 4 1 1 4 4 5 3 6

4 2 1 1 4 4 3 7 5 4 4 3 4 3 4 3 1 9 3 4 1 2 4 4 5 3 8

1 3 3 3 4 4 4 7 5 3 4 3 5 5 5 6 4 6 3 7 2 2 4 6 3 5 7

3 2 2 2 3 4 2 2 3 3 3 3 2 3 2 3 2 3 2 3 2 2 2 2 2 2 5

0.6647 0.2222 0.0000 0.0000 0.7212 0.6974 0.5734 0.8611 0.7510 0.7406 0.6007 0.5337 0.6511 0.5947 0.5969 0.4291 0.0000 0.8611 0.3775 0.6612 0.0000 0.0000 0.6503 0.4608 0.7525 0.4320 0.7237

0.6969 0.4955 0.0000 0.0000 0.7555 0.6682 0.6567 0.8274 0.7644 0.7178 0.7495 0.5397 0.6455 0.6429 0.5847 0.4539 0.0000 0.8819 0.5580 0.6126 0.0000 0.0615 0.6592 0.3765 0.687 0.5456 0.8075

0.0000 0.5362 0.4782 0.6344 0.7361 0.7436 0.7436 0.7981 0.7445 0.6766 0.7118 0.5709 0.7000 0.7436 0.6788 0.7798 0.4926 0.7778 0.4479 0.8333 0.5 0.0615 0.7436 0.7847 0.6587 0.6741 0.7212

0.5629 0.2837 0.4034 0.3830 0.6489 0.6924 0.4965 0.1560 0.6250 0.3821 0.5426 0.3874 0.2234 0.3856 0.2544 0.3511 0.5027 0.6746 0.2837 0.5496 0.5071 0.156 0.4539 0.4965 0.4885 0.422 0.6365

0.6019 0.1948 0.0000 0.0000 0.6573 0.6288 0.5025 0.8286 0.6921 0.6788 0.5393 0.4165 0.5393 0.4995 0.5813 0.3806 0.0000 0.8289 0.3220 0.5813 0.0000 0.0000 0.5703 0.4223 0.6988 0.3865 0.6662

0.6388 0.3688 0.0000 0.0000 0.6960 0.5868 0.5708 0.7902 0.7117 0.6552 0.6900 0.4683 0.6900 0.5556 0.5238 0.3882 0.0000 0.8543 0.4696 0.5238 0.0000 0.0586 0.5882 0.3473 0.6263 0.4783 0.7645

0.0000 0.4178 0.4164 0.5463 0.6731 0.6817 0.6817 0.7542 0.6868 0.5919 0.6435 0.4899 0.6435 0.6883 0.7953 0.7324 0.4439 0.7290 0.3767 0.7953 0.3711 0.0586 0.6826 0.7392 0.5749 0.6038 0.6761

0.4874 0.2392 0.3170 0.3047 0.5609 0.6247 0.3680 0.1411 0.5423 0.3363 0.4278 0.3476 0.1948 0.3438 0.2181 0.3067 0.3711 0.5863 0.2392 0.4802 0.3733 0.1411 0.3457 0.3680 0.3639 0.3279 0.5828 (Continued)

Table 1.9 (Continued) No.

28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Mean

Sites

MiSaTPW76 MiSaTPW90 MiSaTPW96 MiSaTPW117 MiSaTPW123 MiSaTPW157 MiSaTPW165 MiSaTPW173 MiSaTPW184 Lar7 Lma10 Lma21 Lma120 Mdo6 Mdo7 Msal21

Fragment size

257
303 137
196 372
405 209
242 148
166 166
301 236
258 195
271 219
253 124
210 108
124 158
211 192
210 146
164 156
172 213
222

Number of alleles (A)

Expected heterozygosity (He)

Polymorphism information contents (PIC)

FL-09

FL-10

NT-10

CH

FL-09

FL-10

NT-10

CH

FL-09

FL-10

NT-10

CH

4 4 7 5 2 6 3 6 4 10 1 7 1 1 1 3

4 5 7 3 1 7 3 3 4 9 1 6 1 1 1 3

7 6 6 3 3 4 4 4 4 5 3 6 5 4 1 2

3 2 2 3 2 5 3 4 3 2 3 3 2 2 2 1

0.7287 0.6850 0.8388 0.5278 0.0615 0.8249 0.4683 0.6825 0.6513 0.8284 0.0000 0.7872 0.0000 0.0000 0.0000 0.4107

0.7421 0.4618 0.8165 0.6344 0.0000 0.7897 0.4509 0.5184 0.6354 0.8834 0.0000 0.7783 0.0000 0.0000 0.0000 0.4419

0.8209 0.7372 0.7009 0.6448 0.561 0.6066 0.7212 0.5471 0.6245 0.2579 0.5818 0.8418 0.7465 0.5809 0.0000 0.4479

0.6463 0.5071 0.4388 0.6002 0.4885 0.6534 0.5098 0.7482 0.3289 0.383 0.6002 0.5842 0.5027 0.3369 0.2837 0.0000

0.6664 0.6180 0.8028 0.4382 0.0586 0.7847 0.3977 0.6232 0.5943 0.7923 0.0000 0.7463 0.0000 0.0000 0.0000 0.3665

0.6809 0.4243 0.7763 0.5463 0.0000 0.7455 0.4022 0.4577 0.5702 0.8552 0.0000 0.7337 0.0000 0.0000 0.0000 0.3984

0.7807 0.6666 0.6378 0.5615 0.4541 0.5264 0.6582 0.4822 0.5380 0.2427 0.4843 0.8047 0.6926 0.4788 0.0000 0.3437

0.5556 0.3733 0.3374 0.4986 0.3639 0.5982 0.4475 0.6834 0.2984 0.3047 0.5137 0.4783 0.3711 0.2754 0.2392 0.0000

3.74

3.70

4.21

2.58

0.4896

0.5010

0.6138

0.4549

0.4443

0.4566

0.5546

0.3786

Germplasm Resources of Largemouth Bass

Figure 1.5 DNA fingerprints of largemouth bass populations, created using Excel. Top to bottom: CH, FL-09, FL-10, and NT-10 populations.

21

22

Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

Table 1.10 Digital DNA fingerprints of largemouth bass groups Marker FL NT-10 CH Identification groups

JZL114 MiSaTPW11 Lma120 Mdo6 Msal21

00100 0111100 00010 1000 11100

11110 1101100 11111 1111 00011

10001 0000011 01001 0011 00010

FL versus CH CH versus FL or NT-10 FL versus CH FL versus CH FL versus NT-10 or CH

1.5.2.3 Identification of Largemouth Bass Groups Using Microsatellite DNA Markers Analysis of the 43 microsatellite DNA markers in the PCR bands in the four populations found no specific markers able to distinguish between FL-09 and FL-10, suggesting that these populations had similar genetic structures. These two populations were therefore collectively named “Population FL.” Five specific microsatellite markers (JZL114, MiSaTPW11, Lma120, Mdo6, and Msal21) were able to identify groups CH, FL, and NT-10. Each marker could be amplified in at least one group, allowing it to be distinguished from the other groups, while the combination of MiSaTPW11 and Msal21 could differentiate among all three populations. Digital DNA fingerprints were generated based on using “1” to represent the presence and “0” to represent the absence of a band (Table 1.10). The 26 polymorphic sites were ordered by the combination of the five microsatellite DNA markers. These results provide a valuable reference for the identification of largemouth bass germplasm. The above five specific microsatellite markers would allow the genetic origins of unknown fish samples to be determined. DNA fingerprints may thus be used to identify individuals resulting from crossbreeding, given that the microsatellite markers were codominant and the offspring would receive an allele from each parent. Individuals resulting from crossbreeding could be distinguished by alignment of the digital DNA fingerprints. This provides an effective method for identifying species and cenospecies in largemouth bass, as well as for determining the purity of the germplasm in the different groups. 1.5.2.4 Genetic Distance and Gene Ontogeny Analysis in Largemouth Bass Groups Nei’s genetic distance in the four largemouth bass groups, calculated by Popgene (version 1.32), is shown in Table 1.11. The results of gene ontology analysis by UPGMA is shown in Fig. 1.6. These results

Germplasm Resources of Largemouth Bass

Table 1.11 Genetic distance of largemouth bass groups Group FL-09 FL-10

NT-10

FL-09 FL-10 NT-10 CH

0.4244

0.0506 0.4481 1.0867

0.4349 1.0054

23

CH

CH NT-10 FL-09 FL-10

5

Figure 1.6 UPGMA dendrogram of the largemouth bass groups. UPGMA, unweighted pair group method using arithmetic average.

indicated that FL-09 and FL-10 were clustered together, with a very close genetic distance of 0.0506. CH and NT-10 clustered together with a genetic distance of 0.4244. These data suggested that FL-09 and FL-10 were from the same area of Florida, while CH and NT-10 both belonged to the Northern largemouth bass subspecies, but were from different areas. Genetic relationships among populations are generally estimated by genetic distances calculated from gene frequencies. Genetic distance is the basic parameter used for genetic-diversity research, and reflects the phyletic evolution of the groups. It is generally acknowledged that the less time a population has differentiated for, the closer the genetic distance will be (Dong et al., 2007). The present study found that the genetic distance between FL-09 and FL-10 was small, with identical amplification bands in both groups. This suggested that FL-09 and FL-10 were from the same area, or that the distribution of the Florida largemouth bass is centralized to Florida (MacCrimmon and Robbins, 1975), making germplasm exchange and genetic differentiation among populations insignificant. In contrast, although largemouth bass in China and NH-10 both belong to the Northern subspecies, they had a relatively large genetic distance. This may be attributable to the wide distribution of the Northern subspecies in its native country (MacCrimmon and Robbins, 1975), where it can occur in the mid-east United States, northeast Mexico, and southeast Canada. Philipp et al. (Nedbal and Philipp, 1994; Philipp et al., 1983) indicated

24

Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

that this subspecies had differentiated genetically between north and south, suggesting that largemouth bass in China and NT-10 were likely to have come from different areas.

1.6 MICROSATELLITE ANALYSIS OF GENETIC DIVERSITY OF LARGEMOUTH BASS IN CHINA Microsatellite markers are preferable to other molecular markers because of their good repeatability, and abundant hereditary information, including polymorphic sites, and gene location heterozygosity and homozygosity. They thus provide an effective method for analyzing and evaluating population structures and polymorphisms (Zhao et al., 2006). Here we analyzed the population genetic structure of largemouth bass in China using microsatellite markers to provide a basis for future germplasm conservation and improvement.

1.6.1 Materials and Methods 1.6.1.1 Largemouth Bass Samples and Reagents Three groups of largemouth bass were selected for microsatellite testing, including G group (30) from Guanzhou, D group (28) from Daliang, Shunde, and N group (30) from Nanshui, Shunde. The reagents, including dNTPs, Taq DNA polymerase, and dNTPs were from Biocolors, genomic DNA extraction kits purchased from Tiangen, and the SSR DNA Marker II was from Beijing Dingguo Biotechnology Co. Ltd. Other reagents were of analytical quality from within China. The microsatellite primers were synthesized by Sangon Biotech. 1.6.1.2 Genomic DNA Extraction and Microsatellite Marker Selection Genome DNA was extracted from largemouth bass blood using a commercial kit (Tiangen), according to the manufacturer’s instructions. The microsatellite markers were selected by magnetic-capture methods. Positive clones were sequenced by SinoGenomax Co., Ltd. (Beijing, China). The primers were designed by Primer 3.0, according to the results of sequencing. 1.6.1.3 Templates and Microsatellite Polymorphism Analysis Mixed genomic DNA from the three groups was used as templates for primer verification by PCR. Primers that were stably amplified with clear bands were selected for microsatellite polymorphism analysis. The total

Germplasm Resources of Largemouth Bass

25

volume of the PCR reaction was 20 µL, including 10 3 buffer 2 µL, MgCl2 (25 mmol/L) 0.8 µL, dNTPs (10 mmol/L) 0.3 µL, forward and reverse primers (20 µmol/L) 0.5 µL each, Taq polymerase 1 U, and genomic DNA 40 ng, made up to 20 µL with ddH2O. The PCR reaction was performed using a PTC-200 amplifier. The procedure was carried out at 94˚C for 5 min, followed by 25 cycles of 94˚C for 30 s, annealing for 30 s at 72˚C, and finally 7 min at 72˚C. The primers and their annealing temperatures are summarized in Table 1.12. The PCR products were isolated by polyacrylamide gel electrophoresis (8%). The gels were stained with silver and scanned. 1.6.1.4 Genetic Analysis The selected microsatellite primers were used for genetic analysis of the three breeding groups of largemouth bass. The number of alleles for each microsatellite site was counted, and the gene frequency and other genetic parameters were calculated. PIC was calculated, as described by Botstein (1980): PIC 5 1 2

n X i51

Pi2 2

n21 X n X

2ðPiPjÞ2

i51 j5i11

where Pi and Pj are the frequency of the ith and jth alleles in the group, respectively, and n is the number of alleles. The observed polymorphic site heterozygosity (Ho) 5 the ratio of heterozygote numbers to the total number of individuals P 2 observed. Expected polymorphic site heterozygosity ðHe Þ 5 1 2 pi , where Pi is the frequency of the ith allele. The Hardy
Weinberg genetic deviation index (d) 5 (Ho 2 He)/He.

1.6.2 Results and Analysis A total of 288 positive clones were sequenced and 276 sequences were obtained, of which 267 contained microsatellites, accounting for 96.7% of the total. The repeated counts for most of the microsatellites were between 20 and 50, with a maximum count of 174. According to Weber’s standard, microsatellite sequences can be classified as perfect, imperfect, and mixed, accounting for 175 (65.5%), 79 (29.6%), and 13 (4.9%), respectively, in the current study. Eighteen pairs of microsatellite primers were used to analyze the genetic structure of the largemouth bass in the three breeding areas. The PCR amplification results are partly shown in Fig. 1.7. Two to three

Table 1.12 Microsatellite DNA markers and primers in largemouth bass Site Primer sequence (50
30 ) Length of products (bp)

Repetitive sequence

Number of alleles

Annealing temperature (°C)

GenBank number

JZL12

202

(CA)40

2

50

EF055991

397

3

50

EF055992

202

(TG)29 (AG)7 (CA)25

2

60

EF055993

214

(CA)15

2

56

EF055994

200

(CA)24

2

56

EF055995

214

(CA)15

2

58

EF055996

215

(CA)21

2

58

EF055997

207

(GT)13

2

56

EF055998

200

(GT)13

2

54

EF055999

183

(CA)21

2

55

EF056000

205

(CA)21

2

60

EF056001

248

(CA)16

2

60

EF056002

JZL23 JZL31 JZL36 JZL37 JZL40 JZL43 JZL48 JZL53 JZL59 JZL60 JZL67

F:ACTCAGAGCCTCACATTC R:CAGGTGGACTCAAGACAG F:GTCCGCTGCTTAGTTTAT R:TCCTTTATCCTTCCCTCT F:TGGACTGAGGCTACAGCAGA R:CCAAGAGAGTCCCAAATGGA F:GCTGAGAGCCTGAAGACCAG R:ATGGAGGACAGCAGGAACAT F:TCCAGCCTTCTTGATTCCTC R:CCCGTTTAGCCAGAGAAGTG F:GCTGAGAGCCTGAAGACCAG R:ATGGAGGACAGCAGGAACAT F:GCTGCGAGTGCGTGTAACTA R:GGGAAGCGAGAGTCAGAGTG F:TCGACGATCAATGGACTGAA R:TCTGGACAACACAGGTGAGG F:AGCCAATTTCAGCCAAGGT R:TCGACGATCAATGGACTGAA F:CACAAGGCAAACAGAACGTC R:TTGGCTACCCAGTGATGACA F:AGTTAACCCGCTTTGTGCTG R:GAAGGCGAAGAAGGGAGAGT F:CCGCTAATGAGAGGGAGACA R:ACAGACTAGCGTCAGCAGCA

JZL68 JZL71 JZL72 JZL83 JZL84 JZL85 Lma21 Mdo7 Lar7

F:AGGCACCGTCTTCTCTTCA R:CATTGTGGGTGCATTCTCC F:GCAGCTTCAGGTGTGTGTT R:TCGGTGAACTCCTGTCAGG F:AGGGTTCATGTTCATGGTAG R:ACACAGTGGCAAATGGAGGT F:TGTGGCAAAGACTGAGTGGA R:ATTTCTCAACGTGCCAGGTC F:GAAAACAGCCTCGGGTGTAA R:CACTTGTTGCTGCGTCTGTT F:GGGGCTCACTCACTGTGTTT R:GTGCGCAGACAGCTAGACAG F:CAGCTCAATAGTTCTGTCAGG R:ACTACTGCTGAAGATATTGTG F:TCAAACGCACCTTCACTGAC R:GTCACTCCCATCATGCTCCT F:GTGCTAATAAAGGCTACTGTC R:TGTTCCCTTAATTGTTTTGA

166

(CA)20

3

58

EF056003

202

(GT)21

2

60

EF056004

170

(CA)21

2

59

EF056005

157

(CA)23

3

56

EF056006

197

(CA)20

3

56

EF056007

213

(CA)17

3

58

EF056008

158
183

3

47.5

Colbourne et al. [6]

156
172

2

53

Malloy et al. (2000)

127
155

2

47

DeWoody et al. (2000)

28

Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

Figure 1.7 Amplification results of JZL23 in the three largemouth bass groups. Note: M, SD011 marker; G1
G10, from Guangzhou group; D1
D10 from Daliang Shunde Group; N1
N10 from Nanshui Shunde Group.

alleles were obtained from the different primers; the microsatellite sites JZL23, JZL68, JZL83, JZL84, JZL85, and Lma21 produced three alleles, while the other microsatellite sites produced two alleles. The PIC values for JZL23, JZL85, and Lma213 were .0.5, indicating a high degree of polymorphism at these sites. The other sites showed moderate or low polymorphism. The gene frequency and PIC for each site are shown in Table 1.13. The selected microsatellite primers were used for genetic analysis of the three breeding groups of largemouth bass. The genetic parameters, including A, Ho, He, and PIC, which reflect the genetic diversity of the largemouth bass population in China, are shown in Tables 1.13 and 1.14. The average numbers of alleles in the three groups ranged from 2.14 to 2.28, Ho ranged from 0.356 to 0.396, He ranged from 0.368 to 0.403, and PIC ranged from 0.0906 to 0.7480. The levels of polymorphism of the microsatellite sites can be evaluated by the PIC, which reflects the capacity of the genetic information provided by the genetic markers. In general, a PIC .0.5 indicates that the genetic marker could provide abundant genetic information, a PIC .0.25 and ,0.5 indicates reasonable genetic information, and a PIC ,0.25 indicates poor genetic information. JZL23, Lma21, and JZL85 were highly polymorphic, JZL31, JZL36, JZL37, JZL43, JZL48, JZL53, JZL59, JZL60, JZL68, JZL72, JZL83, JZL84, and Lar7 were moderately polymorphic, and JZL12, JZL40, JZL67, JZL71, and Mdo7 were lowly polymorphic in largemouth bass (Table 1.13). Heterozygosity represents the ratio of heterozygous microsatellite sites, and can reflect the richness and evenness of the alleles, and the levels of heritable variation (Nei et al., 1975). Genetic diversity can be quantified using the parameters Ho and He. The balance between these is reflected by the parameter d; the closer d is to “0,” the more balanced the genotype distribution, while d . 0 indicates excessive heterozygosity, and d , 0

Table 1.13 Gene frequency and polymorphism information content of 21 microsatellite sites in three largemouth bass groups Site Alleles Gene frequency Polymorphism information content

JZL12 JZL23

JZL31 JZL36 JZL37 JZL40 JZL43 JZL48 JZL53 JZL59 JZL60 JZL67

a b a b c a b a b a b a b a b a b a b a b a b a b

Mean

G group

D group

N group

G group

D group

N group

0.0500 0.9500 0.2167 0.2167 0.4833 0.3500 0.6500 0.1667 0.8333 0.3833 0.6167 0.1333 0.8667 0.7167 0.2833 0.3000 0.7000 0.7667 0.2333 0.8667 0.1333 0.2833 0.7167 0.9000 0.1000

0.0714 0.9286 0.3571 0.3929 0.2500 0.6071 0.3929 0.2500 0.7500 0.1429 0.8571 0.2500 0.7500 0.6964 0.3036 0.1786 0.8214 0.8036 0.1964 0.7857 0.2143 0.3214 0.6786 0.8214 0.1786

0.1333 0.8667 0.3500 0.2667 0.3833 0.5000 0.5000 0.1667 0.8333 0.3500 0.6500 0.1500 0.8500 0.7667 0.2333 0.1500 0.8500 0.7833 0.2167 0.7667 0.2333 0.2667 0.7333 0.9667 0.0333

0.0905

0.1238

0.2044

0.1396

0.6462

0.5969

0.7436

0.6622

0.3515

0.3633

0.375

0.3632

0.2392

0.3047

0.2392

0.2610

0.3610

0.2150

0.3515

0.3092

0.2044

0.3047

0.2225

0.2439

0.3236

0.3334

0.2938

0.3169

0.3318

0.2504

0.2225

0.2682

0.2938

0.2658

0.2829

0.2808

0.2044

0.2802

0.2938

0.2595

0.3234

0.3411

0.3146

0.3264

0.1638

0.2504

0.0623

0.1588 (Continued)

Table 1.13 (Continued) Site Alleles

JZL68

JZL71 JZL72 JZL83

JZL84

JZL85

Lma21

Mdo7 Lar7

a b c a b a b a b c a b c a b c a b c a b a b

Gene frequency

Polymorphism information content

Mean

G group

D group

N group

G group

D group

N group

0.6667 0.3167 0.0167 0.9333 0.0667 0.2667 0.7333 0.6333 0.3333 0.0333 0.6333 0.3500 0.0167 0.6000 0.1500 0.2500 0.3500 0.5167 0.1333 0.1333 0.8667 0.1667 0.8333

0.6429 0.2321 0.125 0.9286 0.0714 0.3571 0.6429 0.5000 0.4464 0.0536 0.6429 0.2500 0.1071 0.5893 0.107 0.3036 0.5000 0.4107 0.0893 0.1786 0.8214 0.2500 0.7500

0.5167 0.4833

0.3657

0.4710

0.3747

0.4038

0.9833 0.0167 0.3000 0.7000 0.6167 0.3833

0.1158

0.1238

0.0323

0.0906

0.3147

0.3537

0.3318

0.3334

0.3974

0.4470

0.3610

0.4018

0.5167 0.4833

0.3778

0.4696

0.3747

0.4074

0.7167 0.0667 0.2167 0.6000 0.2500 0.1500 0.0333 0.9667 0.3833 0.6167

0.5360

0.5390

0.9436

0.6729

0.6404

0.7363

0.8672

0.7480

0.2444

0.2504

0.0623

0.1723

0.2392

0.3047

0.3610

0.3016

Table 1.14 Genetic parameters of the microsatellite sites Sites G group

JZL12 JZL23 JZL31 JZL36 JZL37 JZL40 JZL43 JZL48 JZL53 JZL59 JZL60 JZL67 JZL68 JZL71 JZL72 JZL83 JZL84 JZL85 Lma21 Mdo7 Lar7 Mean

D group

N group

A(a)

Ho

He

d

A(a)

Ho

He

d

A(a)

Ho

He

d

2(1.1) 3(2.7) 2(1.8) 2(1.4) 2(1.9) 2(1.3) 2(1.7) 2(1.7) 2(1.6) 2(1.3) 2(1.7) 2(1.2) 3(1.8) 2(1.1) 2(1.6) 3(1.9) 3(1.9) 3(2.2) 3(2.5) 2(1.3) 2(1.4) 2.28(1.67)

0.100 0.700 0.433 0.333 0.633 0.267 0.433 0.400 0.267 0.267 0.433 0.133 0.467 0.067 0.467 0.500 0.467 0.567 0.667 0.200 0.267 0.384

0.097 0.640 0.463 0.283 0.481 0.235 0.413 0.427 0.364 0.235 0.413 0.183 0.463 0.127 0.398 0.495 0.484 0.564 0.603 0.235 0.283 0.375

0.031 0.093 0.065 0.177 0.316 0.136 0.048 0.063 0.266 0.136 0.048 0.273 0.008 0.472 0.173 0.010 0.035 0.005 0.106 0.149 0.056 0.004

2(1.2) 3(2.9) 2(1.9) 2(1.6) 2(1.3) 2(1.6) 2(1.7) 2(1.4) 2(1.4) 2(1.5) 2(1.7) 2(1.4) 3(2.1) 2(1.2) 2(1.8) 3(2.2) 3(2.1) 3(2.2) 3(2.3) 2(1.4) 2(1.6) 2.28(1.75)

0.143 0.536 0.429 0.357 0.286 0.357 0.536 0.357 0.321 0.357 0.500 0.071 0.571 0.143 0.357 0.571 0.571 0.607 0.607 0.357 0.286 0.396

0.135 0.668 0.486 0.382 0.249 0.382 0.431 0.299 0.321 0.343 0.444 0.299 0.527 0.135 0.468 0.558 0.522 0.559 0.584 0.299 0.382 0.403

0.059 0.198 0.117 0.065 0.149 0.065 0.244 0.134 0.000 0.041 0.126 0.763 0.083 0.059 0.199 0.023 0.094 0.086 0.039 0.194 0.251 0.008

2(1.3) 3(2.9) 2(2) 2(1.4) 2(1.8) 2(1.3) 2(1.5) 2(1.3) 2(1.5) 2(1.5) 2(1.6) 2(1.1) 2(1.9) 2(1) 2(1.7) 2(1.8) 2(1.9) 3(1.7) 3(2.2) 2(1.1) 2(1.9) 2.14(1.67)

0.133 0.667 0.533 0.333 0.367 0.300 0.400 0.233 0.433 0.333 0.333 0.067 0.500 0.033 0.467 0.367 0.500 0.267 0.567 0.067 0.567 0.356

0.235 0.671 0.509 0.283 0.463 0.259 0.364 0.259 0.345 0.364 0.398 0.066 0.508 0.033 0.427 0.481 0.508 0.442 0.564 0.065 0.481 0.368

0.434 0.006 0.472 0.177 0.207 0.158 0.099 0.100 0.255 0.085 0.163 0.015 0.016 0.000 0.094 0.237 0.016 0.396 0.004 0.031 0.179 0.008

32

Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

indicates a deficiency of heterozygotes. In the present study, the Ho values in Guangzhou, Daliang Shunde, and Nanshui Shunde were 0.384, 0.396, and 0.356, and the He values were 0.375, 0.403, and 0.356, respectively. Among the 21 microsatellite sites, only three were highly polymorphic, 13 were moderately polymorphic, and five were lowly polymorphic. The genetic parameters such as allele numbers, heterozygosity, and polymorphic information content indicated that the genetic diversity of largemouth bass in China was relatively low. This may be because the effective parent size was small, and because increased artificial selection for certain characteristics may have reduced the genetic diversity. A bottleneck effect and inbreeding depression occur under artificial breeding conditions, accelerating homogenization of the germplasm and leading to reduced genetic diversity. Lutz-Carrillo et al. (2006) analyzed the genetic diversity of largemouth bass in North America and Florida using 11 microsatellite sites, and found average heterozygosities of 0.52 and 0.41, and average sites were 4.57 and 4.51, respectively. This degree of genetic diversity was greater than that shown for largemouth bass in China in the current study, suggesting a reduction in genetic diversity compared with native populations, and a relative lack of germplasm resources in China. The low genetic diversity in China may also have been influenced by the source of the introduced fish, which is unknown, and it is possible that all the introduced fish may have come from a single source. Artificialbreeding practices for largemouth bass in China should thus take account of potential inbreeding depression, and should select individuals with high heritable variation in order to increase the genetic variation of the population and reduce the inbreeding coefficient. Updating the germplasm and improving breeding variety should be implemented promptly to prevent further depression of the germplasm and ensure the healthy and sustainable development of largemouth bass aquaculture.

1.7 GENETIC DIVERSITIES OF LARGEMOUTH BASS IN CHINA AND AMERICA In its native areas in America, the largemouth bass is generally considered to be composed of two subspecies (Bailey and Hubbs, 1949), the Florida subspecies found in the Florida peninsula, and the Northern subspecies, distributed in most central and eastern parts of the United States, northeast Mexico, and southeast Canada (MacCrimmon and Robbins, 1975). These subspecies and their fertile hybrids are adapted to the temperatures

Germplasm Resources of Largemouth Bass

33

in their native areas (Fields et al., 1987). These two subspecies have different spawning periods (Rogers et al., 2006) and growth rates (Isely et al., 1987). To make full use of the characteristics of the subspecies, it is necessary to understand the genetic relationships and differences in genetic diversity between cultured and wild populations in China and America. We therefore used six microsatellite sites to analyze the heritable variation of the largemouth bass population in China, and compared these results with data from the wild populations of largemouth bass in their native habitats.

1.7.1 Materials and Methods 1.7.1.1 Sample Collection A total of 136 largemouth bass were collected from four aquafarms in Guangdong province: Guangzhou (G; n 5 30), Daliang (D; n 5 28), Xinshiji (X; n 5 48), and Nanshui (N; n 5 30). The genotype of the wild population was provided by Lutz-Carrillo et al. (2006), based on five populations of Florida largemouth bass (n 5 175) and eight populations of Northern largemouth bass (n 5 249). The details are shown in Table 1.15. 1.7.1.2 DNA Extraction Genomic DNA was extracted from 20 µL blood from adult fish using a genomic DNA isolation kit (Dp304; Tiangen), or according to the improved method of Miller et al. (1988), replacing NaCl with ammonium acetate to denature the cell proteins. The genomic DNA was diluted in water and kept at 220˚C until use. The quality and concentration of the DNA were detected by agarose (0.8%) gel electrophoresis and spectrophotometry. 1.7.1.3 Microsatellite Markers Six microsatellite markers (Lma10, Lma21, Lma120, Mdo7, Msa25, and Lar7) were used to evaluate the heritable variation in the cultured population of largemouth bass in China. These markers were selected from the 11 markers used by Lutz-Carrillo et al. (2006) to evaluate the genetic structure of largemouth bass in North America. Amplification of these markers produced reliably clear bands in the present samples. The genotype data from the wild population were compared with those for the cultured population.

34

Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

Table 1.15 Largemouth bass samples from cultured and wild populations Populations Source Abbreviation Sample Classification size

Guangzhou, Guangdong, China Daliang, Guangdong, China New century, Guangdong, China Nanshui, Guangdong, China Lake Dora, Florida, USAa East Lake Tohopekaliga, Florida, USAa Lake Kissimmee, Florida, USAa Hillsborough River, Florida, USAa Medard Reservoir, Florida, USAa Twin Oaks Reservoir, Texas, USAa Lake Fryer, Texas, USAa Lake Kickapoo, Texas, USAa Devils, River, Texas, USAa Lake Charlotte, Oklahoma, USAa Lake Minnetonka, Minnesota, USAa Pepin Lake, Minnesota, USAa Pike Lake, Wisconsin, USAa

G

30

D X

28 48

Cultured population in South China (CS) CS CS

N

30

CS

Dora Toho

27 51

FLMB FLMB

Kiss

28

FLMB

Hill

35

FLMB

Meda

34

FLMB

Twin

31

NLMB-S

Fryr Kick

30 27

NLMB-S NLMB-S

Devr Char

37 50

NLMB-S NLMB-S

Minn

27

NLMB-N

Pepn

23

NLMB-N

Pike

24

NLMB-N

FLMB, Florida largemouth bass; NLMB-S, Northern largemouth bass in South; NLMB-N, Northern largemouth bass in North. a Data from Lutz-Carrillo et al. (2006).

The total volume of the PCR reaction was 20 µL, including genomic DNA 50
100 ng, forward and reverse primers (20 mmol/L) 0.5 µL each, MgCl2 (25 mmol/L) 0.8 µL, dNTPs (10 mmol/L) 0.3 µL, 10 3 buffer 2 µL, and Taq polymerase 1 U (Biocolors). The PCR reaction was performed using a thermal cycler (Bio-Rad). The procedure was carried out at 94˚C for 5 min, followed by 25 cycles of 94˚C for 30 s, 50
54˚C for

Germplasm Resources of Largemouth Bass

35

30 s, 72˚C for 30 s, and finally 7 min at 72˚C. The primers and their annealing temperatures are summarized in Table 1.12. The PCR products were isolated by nondenaturing polyacrylamide gradient gel electrophoresis (8%, 1 3 TBE buffer 6
7 V/cm, 4
5 h). The gels were stained with silver and scanned. The type of alleles was identified based on the length of the PCR products, analyzed using a Gel-Pro Analyzer. 1.7.1.4 Data Analysis The number of alleles at each site was analyzed using GenAlEx Software (v.6; Peakall and Smouse, 2006). Bottleneck Software (Cornuet and Luikart, 1996; Piry et al., 1999) was used to determine if the effective population decreased in the cultured populations. The data were analyzed using Infinite Alleles Mutation Model (IAM) and Stepwise Mutation Model (SMM). The significance of differences was detected by the sign test, standardized difference test, Wilcoxon’s sign-rank test, and the mode-shift test (Luikart et al., 1999). The Markov-chain method was used for Hardy
Weinberg-equilibrium (HWE) analysis. The Markovchain extension to Fisher’s exact test for R 3 C contingency tables (Slatkin, 1994) was used to analyze each allele nonrandomly. The genetic difference in the cultured populations was calculated using Rstcalc Software (Goodman, 1997) with the Rst parameter, and the variation was analyzed using the Rho statistic. The estimated value of Slatkin’s Rst was obtained via a 1000-times bootstrap repeated sampling (Slatkin, 1995). The significance of differences in alleles among populations was calculated by two-factor analysis of variance (ANOVA). The significance of differences in Ho at specific sites or as a whole between cultured and wild populations were also analyzed by ANOVA using SYSTAT11 software (Systat Software, Inc.). Pairwise comparisons were performed by Tukey’s post-hoc tests. The populations information was as follows: four populations of largemouth bass in China, five populations of wild Florida largemouth bass, and five populations of Northern largemouth bass in North America. Heritable variation in the populations was assessed according to the Cavalli-Sforza and Edwards (1967) distance. An Neibor-joining (NJ) cluster tree was constructed using PHYLIP 3.63 software via 1000-times bootstrap repeated sampling. The cluster tree was read using TreeView (1.6.6) software.

36

Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

1.7.2 Results and Analysis The microsatellite sites in the present study were successfully amplified in the four populations of cultured largemouth bass. There were three alleles at the microsatellite sites Lma10 and Lma21, one at Msa25, and two at each of the other microsatellite sites. The average number of alleles in the four populations was 2.17 6 0.00 (Table 1.16). No population-specific sites were found in the four cultured populations. Regarding the same microsatellite sites in the wild populations, the lowest average number of alleles was 3.56 6 0.26 (in the Northern largemouth bass populations from North America), and the highest was 6.30 6 0.92 (in the Northern largemouth bass populations from South America). The heterozygosity of largemouth bass in China ranged from 0.34 6 0.24 (Group X) to 0.41 6 0.27 (Group D). The average heterozygosity was 0.37 6 0.03. The IAM and SMM models were used to evaluate bottleneck effects in the groups. The results indicated that the effective population of the largemouth bass in China was depressed (Table 1.17). The allele frequency was reduced in all groups, suggesting a genetic bottleneck effect. The cultured population of largemouth bass in China was in HWE for all populations and sites. The parameter Rst was used to assess the heritable variation in the cultured population. The results indicated a small (0.032) to moderate (0.208) variation in the groups. All pairwise comparisons revealed a significant difference (Table 1.18). Genetic-distance analysis confirmed that largemouth bass in China were more closely related to the Northern than to the Florida subspecies of largemouth bass (Fig. 1.8). Thirty samples each of groups D and X Table 1.16 Average allele number and heterozygosity in cultured and wild largemouth bass Population AN HO

Cultured populations in China G D X N Wild populations in America FLMB NLMB-S NLMB-N

2.167 6 0.000 2.167 6 0.753 2.167 6 0.753 2.167 6 0.753 2.167 6 0.753 5.551 6 1.351 6.000 6 0.808 6.300 6 0.923 3.556 6 0.255

AN, average allele number; Ho, observed heterozygosity.

0.370 6 0.032 0.350 6 0.255 0.405 6 0.267 0.389 6 0.396 0.337 6 0.238 0.508 6 0.150 0.371 6 0.053 0.674 6 0.056 0.458 6 0.057

Germplasm Resources of Largemouth Bass

Table 1.17 Hypothesis-testing results of IAM/SMM model Population Sign test Standardized Wilcoxona difference

Modeshift

G D X N

Yes Yes Yes Yes

0.021/0.38 0.020/0.038 0.015/0.034 0.140/0.216

0.021/0.111 0.004/0.025 0.011/0.059 0.023/0.120

0.016/0.016 0.016/0.016 0.016/0.016 0.031/0.109

37

a One-tailed test of redundant heterozygote. Analyzed by Bottleneck methods, as described by Piry, S., Luikart, G., Cornuet, J., 1999. Computer note. BOTTLENECK: a computer program for detecting recent reductions in the effective size using allele frequency data. J. Hered. 90 (4), 502
503.

Table 1.18 Heritable variation in the cultured largemouth bass population in China, assessed by Rst parameter G D X N

G D X N



0.00001 6 0.0034

Values above the

0.00001 6 0.0038 0.00002 6 0.0048



0.032 0.093 0.066



0.176 0.037 

0.00002 6 0.0045 0.00001 6 0.0034 0.00003 6 0.0058 

0.208

are P values; values below the  are Rho values; α 5 0.05.

X

100

N

53 65

D G

100

Char Minn

100 83 84 100

Pike Pepn

75

Fryr

51

Kick Devr

Twin

92

Meda

100

Hill

58

Dora

37 48

Kiss Toho

Figure 1.8 Molecular phylogenetic tree of four cultured populations and 13 wild populations of largemouth bass, according to the NJ method. The abbreviations refer to Table 1.15

38

Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

were analyzed at the AE Wood Lab (San Marcos, TX, USA). Mdo6 and Msa21, which are specific for the Northern largemouth bass, were found in the samples from populations D and X, further verifying that the largemouth bass in China belonged to the Northern subspecies. However, phylogenetically, the cultured populations were located in an individual branch. Records of the sources of the largemouth bass in mainland China are limited. The fish were introduced to Taiwan (China) in the mid-1970s, followed by their successful artificial reproduction in 1983 (Liao, 2000). Largemouth bass were introduced to Guangdong (Ma et al., 2003; Zhang and He, 1994) in the same year. However, there are no detailed records of the origin, number, or classification of the introduced fish. The results of the current study revealed that the four populations of largemouth bass in China are derived from the Northern subspecies of largemouth bass, with no genomic introgression from the Florida subspecies in the analyzed Chinese populations. Further analysis of the genetic diversity of largemouth bass in China was therefore based on the Northern subspecies. Compared with the results of Lutz-Carrillo et al. (2006) for the same sites, the numbers of alleles in the cultured population (2.17 6 0.00) were reduced by 39% and 64%, compared with the Northern largemouth bass in Northern USA (3.56 6 0.26) and Southern USA (6.30 6 0.92), respectively. Similarly, the Ho in the cultured population (0.37 6 0.03) was reduced by 19% and 45%, compared with the Northern subspecies in Northern USA (0.46 6 0.06) and Southern USA (0.67 6 0.06), respectively. However, only the NLMB-S populations was significantly different from the cultured populations. These results indicate that the levels of heritable variation had declined in the cultured populations, with the decline in allele number being more serious than the decline in heterozygosity. There was no significant difference in HWE and linkage equilibrium (LE) deviation in any of the microsatellite sites in the cultured populations, indicating that inbreeding had not had any remarkable effect on genetic diversity. However, a genetic bottleneck developed as the number of alleles and heterozygosity declined, and the effective population number decreased in the four cultured populations. It is therefore important to introduce more individuals from other populations to address this problem. Analysis of phylogenetic differentiation revealed that all the cultured populations clustered in a single branch, suggesting that they may all have been derived from the same population, given that no allele specific to any individual population was detected. In summary, analysis of genetic variation verified that the four tested cultured populations of largemouth bass in China belonged to the Northern

Germplasm Resources of Largemouth Bass

39

subspecies. Genetic diversity is important not only for selective breeding, but also in relation to the increased susceptibility to epidemic diseases when diversity is reduced (Springbett et al., 2003). We therefore suggest that new wild populations of Northern largemouth bass should be introduced to increase the genetic diversity of the present population in China, while introduction of the Florida subspecies would allow crossbreeding.

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Genetic Breeding and Molecular Marker-Assisted Selective Breeding of Largemouth Bass

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885. Huo, J., Zeng, R., Pan, W., et al., 2005. Analysis and study of influence factors about microsatellite PCR-polyacrylamide gel-silver staining. J. Yunnan Agric. Univ. 20 (1), 67
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