A Second Generation Genetic Linkage Map for Silver Carp (Hypophthalmichehys Molitrix) Using Microsatellite Markers

Aquaculture 412-413 (2013) 97–106

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Aquaculture

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A second generation genetic linkage map for silver carp (Hypophthalmichehys molitrix) using microsatellite markers

Wenjie Guo a,c, Jingou Tong a,⁎, Xiaomu Yu a, Chuankun Zhu a,c, Xiu Feng a,c, Beide Fu b,c, Shunping He b, Fanzhen Zeng a,c, Xinhua Wang a,c, Haiyang Liu a,c, Lusha Liu a,c a State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, the Chinese Academy of Sciences, Wuhan 430072, China b Key Laboratory of Aquatic Biodiversity and Conservation of the CAS, Institute of Hydrobiology, the Chinese Academy of Sciences, Wuhan 430072, China c University of Chinese Academy of Sciences, Beijing 100049, China article info abstract

Article history: In the study, we constructed a second generation genetic linkage map for silver carp (Hypophthalmichthys Received 1 February 2013 molitrix) using anonymous and EST-derived microsatellite markers in a mapping panel containing 156 “pseudo Received in revised form 15 May 2013 BC” progenies from two interspeciﬁc crosses between silver carp and bighead carp (Aristichthys nobilis). A total of Accepted 25 June 2013 703 markers were ordered on 24 linkage groups (LGs) which are equal to chromosome numbers of the haploid Available online 10 July 2013 genome of the species. The consensus map spanned 1561.1 cM covering 93.1% of the silver carp genome with an

Keywords: average resolution of 2.2 cM/locus. Length of LGs ranged from 42.1 cM to 97.8 cM (mean 65.0 cM). Total number Silver carp (Hypophthalmichehys molitrix) of markers on individual LG varied from 13 to 56 (mean 29.3). Estimated total length of the female map Genetic linkage map (1809.0 cM) was 1.52 times longer than that of the male map (1188.5 cM), and the recombination ratio between Microsatellite (SSR) sexes (female vs. male) was 2.2, showing markedly higher recombination in the females. Percentage of distorted Pseudo-testcross loci in the male map was obviously higher than that in the female map, and 5 segregation distortion regions were Recombination rate identiﬁed in the male linkage groups. This second generation genetic linkage map evidently extends previous Segregation distortion genetic maps for silver carp, and provides a basis for such studies as quantitative trait locus mapping, comparative genomics and marker-assisted selection. © 2013 Elsevier B.V. All rights reserved.

1. Introduction (e.g., Coimbraa et al., 2003; Li et al., 2003; Li et al., 2005; Li et al., 2006), and now microsatellites are extensively applied in the prepa- Genetic linkage map is an essential tool to understand genome ration of second generation genetic linkage maps in some economi- organization, evolutionary relationships, and to verify DNA sequence cally important aquaculture fishes, such as tilapia (Lee et al., 2005), contig order and orientation for genome assembly (Botstein et al., rainbow trout (Palti et al., 2012; Rexroad et al., 2008), Japanese floun- 1980; Stapley et al., 2008; Wang et al., 2011; Xia et al., 2010). The der (Castaño-Sánchez et al., 2010; Song et al., 2012), channel catfish advance of molecular genetic techniques and sophisticated statistical (Kucuktas et al., 2009; Ninwichian et al., 2012), grass carp (Xia et tools for linkage analyses made genetic linkage map construction al., 2010) and common carp (Zhang et al., 2013; Zheng et al., 2011). possible for many aquaculture organisms. To date, dominant DNA The silver carp (Hypophthalmichthys molitrix)isafilter-feeding cyp- markers such as amplified fragment length polymorphism (AFLP) rinid fish and also one of the most important aquaculture fishes (Fu and and random amplified polymorphic DNA (RAPD) have been generally He, 2012). It has great values not only for food fish but also for biological replaced by co-dominant markers such as microsatellites (SSRs) and control of bloom-forming cyanobacteria in lakes, ponds and reservoirs single nucleotide polymorphisms (SNPs) in genome mapping (Xia (Ke et al., 2009). Due to fast growth rate, easy cultivation, high feed et al., 2010). Compared with other molecular markers, microsatellites efficiency ratio and high nutritional value, commercial harvest of silver have a number of advantages: they are PCR-based, hyperallelic, and carp has steadily increased in recent years, especially in China. widely distributed throughout eukaryotic genomes (Jewell et al., According to recent statistics (FAO, 2010), global annual production of 2006). Furthermore, the high information content and transferability silver carp has exceeded 4.0 million metric tons. However, in China, of microsatellite markers provide an important tool for integration of natural populations of silver carp have declined dramatically due to different maps and for comparative genomics studies (Xia et al., 2010; habitat fragmentation and over-fishing, and production traits are also Yu et al., 2004). So far, first generation genetic maps with low or declining due to long-term artificial reproduction and inappropriate medium resolution have been reported for many aquaculture animals management of broodstock resources. The initiation of breeding programs for silver carp and other Chinese major carps is thus required ⁎ Corresponding author. Tel.: +86 27 68780751; fax: +86 27 68780123. for genetic improvement of economically important traits such as E-mail address: [email protected] (J. Tong). growth, and disease and stress resistance.

Genetics and genomics studies on silver carp are in their infancy. sources of SSRs included those from three microsatellite-enriched geno- Approximately 600 SSR sequences for silver carp are available in mic libraries prepared by following the AFLP of sequences containing GenBank or other public databases. A few first-generation genetic repeats (FIASCO) method (Zane et al., 2002): one from silver carp maps have been previously reported for silver carp, primarily based with GT repeat (named with “HyGT” prefix), and two from bighead on AFLP markers and limited number of SSRs, resulting in relatively carp with GA and GT repeats (named with “ArGT” and “ArGA” prefixes, low resolution and more linkage groups than expected (Liao et al., respectively). In total, 1922 anonymous SSR and 260 EST-SSR markers 2007b; Zhang et al., 2010, 2011). A well-defined genetic map with were newly developed in this study using the software Primer premier co-dominant DNA markers is not available for this species. The objec- 5.0 (http://www.premierbiosoft.com/primerdesign/index. html) for tive of this study is to produce a second generation genetic linkage design of primers. Detailed information for those SSR markers mapped map for silver carp using anonymous and EST (expressed sequence on the silver carp consensus map is available in Appendix A. tag)-derived microsatellite markers to supply a framework for subse- In addition, a total of 584 SSRs previously published for silver carp quent genome-wide searches for quantitative trait loci (QTL) and for and bighead carp were also used for initial segregation screening in comparative genomics studies among Chinese carps. this study. These SSRs were named using their original nomenclatures with the prefixes of “Hym” (Zhang et al., 2010; Zhang et al., 2011), “Ar” (Cheng et al., 2008), “BL” (Liao et al., 2007a), “Hmo” (Gheyas et 2. Materials and methods al., 2006), and “Cid” (Guo et al., 2009). 2.1. Mapping family and DNA extraction 2.3. Microsatellite genotyping Two couples of bighead carp and silver carp were sampled from the middle reach of the Yangtze River, and a mapping panel including All candidate SSR markers were initially screened in the two par- two parents and 156 progenies from two F1 pseudo-testcross families ents of silver carp, and polymorphic loci segregated in either female (Fig. 1) were produced by reciprocal crossing between silver carp and or male parents were subsequently genotyped in the two mapping bighead carp at the Wuhan Donghu Fish Farm. 78 “pseudo BC” prog- families. Amplification of SSRs was carried out through PCR reaction enies at the age of two months were randomly sampled from each in a thermocycler (Veriti, ABI) with a total volume of 12.5 μl, contain- family and stored in 100% ethanol. Fin clips of parental fish were ing 50 ng of template DNA, 1.25 μl of 10× reaction buffer, 1 U of Taq also sampled. Genomic DNA was extracted from ethanol-preserved polymerase (TaKaRa, Japan), 0.5 μl of dNTP (2.5 mmol/l), 0.5 μlof fin tissues using a phenol–chloroform method (Taggart et al., 1992). forward and reverse primer mixture (2.5 μmol/l each) and water to DNA quality was checked using 1% agarose gel electrophoresis, and the final volume. The cycling profiles were as follows: an initial dena- the concentrations were estimated using NanoDrop 2000 spectropho- turation step at 94 °C for 5 min, followed by 35 cycles of 94 °C for tometer (Thermo Scientific, Wilmington, DE, USA). 35 s, optimal annealing temperature (see Appendix A and references) for 35 s, 72 °C for 40 s, and a final 72 °C extension for 10 min. The PCR products were separated on 10% polyacrylamide gels and visual- 2.2. Sources of microsatellites and marker nomenclature ized by staining with ethidium bromide. MajorSSRsinthisstudyweredevelopedfromscaffoldsequencesof the genome and transcriptome sequencing projects for silver carp and 2.4. Linkage analyses and genome coverage bighead carp (coordinated by Prof. S.P. He, Institute of Hydrobiology, the Chinese Academy of Sciences). These sources of SSR markers were The data from Family 1 was used for female map construction, and named with “Hysd”, “HysdE”,and“Arsd” prefixes, respectively. Other those from Family 2 for male map construction. Linkage analyses for

Fig. 1. A ﬂowchart for the production of two interspeciﬁc mapping families between silver carp and bighead carp. W. Guo et al. / Aquaculture 412-413 (2013) 97–106 99

M1 HY_LG1 F2 M2 HY_LG2 F13

0.0 Arsd444 0.0 Hysd1788-1 5.6 HysdE2857-1 Hysd1020-1 10.0 HysdE753-1 0.0 Hysd1020-1 HysdE2857-1 7.6 Hysd156-5 11.4 Hym334 4.1 Hysd1455-1 HysdE143-1 9.7 Hysd1455-1 HysdE143-1 14.0 Hysd708-1 cid04 7.0 Hym191 12.7 Hym191 16.5 Hysd1216-1 7.5 Hym208 13.1 Hym208 16.7 Hysd1436-1 0.0 Arsd444** 0.0 Hysd614-1 0.0 Hysd527-1 12.9 Arsd330* 13.7 Arsd675 17.2 Hym12 13.6 Hysd145-4 14.8 Arsd279 18.3 Arsd30 7.4 Hysd156-5 21.5 Hysd1214-1 15.5 Hysd223-3 6.3 Hysd1788-1 19.7 Hysd32-4 13.4 Arsd675 22.8 ArGT1001* 16.2 Hysd784-1 20.4 Arsd274 14.6 Arsd279 17.8 Hym334 23.7 Arsd578** 16.9 Arsd584 21.2 Hysd779-1 15.2 Hysd223-3 20.4 Hysd708-1 14.7 HysdE753-1 24.3 Arsd133 18.0 Arsd17 21.8 Hysd779-2 16.0 Hysd784-1 23.0 Hysd1436-1 25.0 Hysd994-1* 18.4 Arsd330 23.3 Arsd338 16.7 Arsd584 23.5 Hym12 19.9 cid04* 25.9 Arsd493* SDR1-1 19.8 Arsd65 23.5 Hysd749-1 17.8 Arsd17 24.6 Arsd30 23.5 Hysd1216-1 HL33* Hysd1253-1* 22.8 Arsd225 24.5 Arsd306 19.6 Arsd65 26.0 Hysd32-4 27.7 Hysd231-6* 23.2 Hysd410-1 27.4 Arsd427 22.7 Arsd225* 26.7 Arsd274 29.6 Hysd1235-1* 24.4 Hysd507-2 28.1 Hysd99-6 31.6 Arsd338 23.7 Hysd507-2 27.5 Hysd779-1 30.1 Arsd70 25.7 Hysd507-1 28.2 Hysd1611-1 32.9 Arsd306 24.8 Hysd507-1 28.1 Hysd779-2 Hysd131-1** HysdE77-1* 27.9 ArGT1001 29.4 HysdE2076-1 27.0 Arsd133 29.8 Hysd749-1 HysdE2076-1 30.5 Hysd244-1* Hym74* 28.2 Arsd133 29.8 Hym294 39.9 SDR1-2 30.3 Hysd1105-2 33.2 HysdE2344-1 HysdE919-1* ArGA431* 29.0 Arsd578 31.0 HysdE2344-1 42.5 Arsd698 31.7 Hysd1105-1 34.4 Arsd427 Hysd388-1 30.6 Hysd224-1* Hysd1507-1 30.0 Hysd994-1 Hysd1105-2 33.3 Arsd698 47.3 38.2 Hysd228-5 35.1 Hysd99-6 Arsd714 38.4 Hysd272-3 31.1 Arsd493 35.0 Hysd644-1 47.9 40.9 Arsd253 35.4 Hysd1611-1 Hysd410-1* 31.3 Hysd1105-1 38.1 Hysd388-1 49.5 Hysd1390-2 41.4 Hysd310-1 Hysd310-2 HysdE2076-1 32.9 Hysd1253-1 HL33 42.2 36.0 38.7 Arsd714 54.4 HysdE1320-1 Arsd258 Hym294 33.0 Hysd231-6 44.4 37.0 39.4 Hysd930-1 Arsd478 Hysd644-1 59.3 Hysd2314-1 35.3 Arsd70 47.8 41.6 40.3 Hysd1390-2 Hysd2511-1 Hysd930-1 60.9 Hysd1614-1 HysdE77-1 Hym74 53.2 46.1 45.2 HysdE1320-1 Hysd263-3 Hysd664-1 64.5 Hysd1590-1 35.6 Hysd244-1 ArGA431 56.4 53.0 46.0 Hysd664-1 Hysd2057-1 Hysd557-1 Hysd224-1 57.0 56.9 49.7 Hysd557-1 Hysd1549-2 Hysd527-1 35.7 Hysd1507-1 66.2 58.5 50.1 Hysd2314-1 Hysd527-1 Hysd192-4 Arsd346 36.5 Hysd131-1 73.8 64.7 51.6 Hysd1614-1 HysdE919-1 Hysd1188-2 36.6 HysdE919-1 75.3 65.4 53.3 Hysd614-1 Hysd1188-1 37.9 Hysd1235-1 66.8 55.2 Hysd1590-1 Hysd836-1 39.1 Arsd253 76.7 57.4 Arsd346 40.2 Hysd310-1 Hysd310-2 58.1 Hysd1188-2 Hysd126-5 42.1 Hysd1214-1 Arsd258 89.0 59.4 Hysd1188-1 44.2 Hysd228-5 69.6 Hysd836-1 44.4 Hysd272-3 45.9 Arsd478 50.0 Hysd2511-1 103.4 Hysd181-1 51.0 Hysd145-4 52.4 Hysd263-3 53.0 Hysd2057-1 54.6 Hysd192-4 57.2 Hysd1549-2 69.4 Hysd126-5 82.9 Hysd181-1

M3 HY_LG3 F9 M4 HY_LG4 F10

0.0 HysdE4129-1 0.0 Hysd176-1 0.0 Hym283 1.1 HysdE3348-1 6.7 Hysd1158-2 0.7 Hysd2112-1 1.2 Hym02 11.1 cid71 0.0 Hym283 5.1 Hysd1369-1 2.2 Hysd176-1 15.8 Hysd341-1 0.4 Hysd2112-1 8.0 Hysd711-1 8.7 Hysd1158-2 17.5 Hysd538-1 5.1 Hysd1369-1 9.1 Hysd711-2 13.3 cid71 20.2 Hym306 7.0 Hysd80-1 10.3 Arsd270 0.0 Hysd538-1** 0.0 Arsd480 15.5 Hym230 23.3 Hysd365-3 11.3 Arsd526 13.0 Hysd80-1 3.3 Arsd270 16.2 Hysd341-1 27.8 Hysd462-1 12.3 Hysd94-2 15.7 Arsd526 Hysd711-1 18.0 HysdE4671-1 27.9 Arsd659 13.3 Arsd482 16.3 Hysd94-2 6.5 21.2 Hysd538-1 29.8 Arsd452 17.0 Hysd94-1 14.5 Arsd767 14.3 Arsd526 22.2 Hym306 30.2 Arsd434 18.2 Arsd482 16.9 HysdE7454-1 15.2 Hysd94-1 26.5 Hysd41-2 31.3 Arsd120 19.3 Arsd767 19.1 Hysd305-1 19.7 HysdE3301-1 27.8 Arsd208 31.4 Arsd78 Hysd615-2 21.0 Arsd480 20.0 19.7 Hysd577-1 23.4 Hysd1855-1 Hysd150-1 Hysd1317-1 HysdE7454-1 29.0 32.6 21.3 Hysd615-1 20.1 Hym10 21.8 HysdE1356-1 HysdE8289-1 Hysd254-3 Hym176 Arsd592 29.9 33.5 21.1 Arsd480 23.9 24.3 HysdE213-1 HysdE1349-1 30.1 Hym176 33.8 Hym32 27.8 Arsd434 24.2 Hysd577-1 Ar182 Hysd585-1 24.4 HysdE2491-1 30.2 Hym32 34.1 Hysd254-3 21.2 Ar182 Hysd30-1 Hysd30-1 24.9 27.2 Arsd592 31.2 Hysd1317-1 35.3 Hysd150-1 Arsd97 32.7 Arsd296 Hysd585-1 21.3 Hysd635-1 Arsd122 28.9 HysdE1827-1 32.0 Arsd120 36.1 Arsd208 22.4 HysdE1827-1 25.0 Hysd635-1 38.8 Arsd97 31.9 ArGA46 32.1 Arsd78 37.9 HysdE8957-1 22.9 Arsd592 25.1 HysdE3301-1 41.8 Hysd412-1 37.9 Arsd196 32.3 Arsd659 38.4 Hysd412-1 24.3 Hysd711-2 25.3 Arsd122 33.0 Hysd462-1 38.8 Hysd41-2 28.0 ArGA46* 25.8 Hysd305-1 33.5 Hysd365-3 39.4 Arsd296 50.2 Arsd145 31.0 Arsd646 26.4 HysdE1827-1 35.8 Arsd434 42.7 Hysd615-1 54.9 Hysd448-1 36.1 Hysd674-1 26.9 Hym10 37.3 Arsd452 43.9 Hysd615-2 58.5 Hysd709-2 HysdE2491-1 HysdE1356-1 44.8 Hym427 29.4 38.7 HysdE8957-1 45.0 Hysd1095-1 58.6 Hysd302-1 HysdE8289-1 HysdE213-1 41.1 Hysd1095-1 46.4 Hysd211-2 61.0 Hysd1664-1 29.5 HysdE1349-1 47.9 HysdE4671-1 62.5 Hysd41-3 30.5 Hysd1855-1 50.1 Hym230 33.8 ArGA46 52.0 Hym08 70.2 Hym08 35.8 Arsd646 54.9 Hysd41-3 Hysd674-1 74.7 HysdE4671-1 38.3 57.5 Hysd1664-1 Arsd196 75.9 Hysd211-2 41.7 59.5 Hysd709-2 48.5 Hym427 59.6 Hysd302-1 62.4 Hysd448-1 63.7 Arsd145 66.3 Hym02 HysdE3348-1 67.6 HysdE4129-1

M5 HY_LG5 F12 M6 HY_LG6 F14

0.0 Hysd174-1 10.2 Arsd537 11.8 Hysd1471-1 0.0 Hysd293-1 12.3 Hysd881-1 1.7 Arsd309 0.0 Hysd174-1 15.1 Hysd180-3 3.2 HyGT370 Arsd537 0.0 Hysd78-5 0.0 Hysd78-5 10.2 15.5 Hysd536-1 0.0 Arsd614 4.2 Hysd35-1 12.3 Hysd881-1 16.5 ArGT56 5.1 Hysd774-2 15.2 Hysd180-3* 17.4 Hysd551-1 4.9 Ar18 7.7 Hysd539-2 16.4 Ar64 16.6 ArGT56* SDR5-1 18.4 HysdE7322-1 8.3 Hysd198-2 25.4 Hysd29-2 Hysd178-1 17.5 Hysd551-1** Hym366 HysdE5834-1 10.1 8.6 Arsd764 27.0 Hysd908-1 18.8 HysdE7322-1 19.3 HysdE6491-1 Hysd163-1 Hym136 Hysd558-2 27.6 Arsd33 Hysd212-1b 15.4 Ar64 Hym366 Hysd163-1 Hysd263-1 16.7 HysdE214-1 9.0 Hysd2095-1 Arsd157 30.6 HysdE6216-1 25.0 HyGT370 19.4 HysdE5834-1 Hysd263-1 19.5 Hysd805-2 19.7 HysdE367-1 Hysd408-2 31.7 Hysd558-1 25.1 Hysd29-2 HysdE6491-1 21.2 Hysd936-1 9.1 Arsd363 Hysd198-1 32.3 Arsd309 26.5 HysdE6216-1 19.6 Hysd805-2 22.4 Hysd572-1 10.0 Hysd16-1 33.6 Arsd414 27.7 Hysd558-1 21.3 Hysd936-1 24.3 Hysd122-1 28.6 HysdE769-1 10.4 Hysd29-1 33.7 HyGT370 28.9 Hysd558-2 22.5 Hysd572-1* 28.1 Hysd928-1 14.8 HysdE6216-1 35.1 Hysd35-1 31.6 Arsd414 23.4 Hysd122-1 Arsd292 Hysd774-2 28.9 36.0 Hysd1367-1 18.3 Arsd87 36.0 34.5 Hysd908-1 29.1 Arsd292* 32.7 Ar18 20.2 Arsd576 37.2 Hysd558-2 37.1 Arsd33* 36.5 Arsd614 34.1 Hysd551-2 29.4 Hym266 39.1 Arsd764 37.2 Hysd212-1b** 36.9 Hysd1351-1 35.5 Arsd614 38.0 Hysd1260-1 Arsd157 Hym136 47.8 Hysd1085-1 38.3 Hysd802-1 36.5 Hysd1351-1 47.5 Hysd551-2 39.4 Hysd2095-1 Hysd198-1 44.7 HyGT508 37.8 Hysd802-1 Hysd408-2 48.3 HyGT3** 39.4 Hysd178-1 40.0 Hysd198-2 53.8 Arsd363 41.2 HysdE867-1 40.3 Hysd29-1 57.0 HysdE7322-1 44.5 HyGT508 40.7 Arsd363 Hysd1471-1 45.9 ArGT711-2 60.6 42.0 Hysd539-2 63.0 Hysd16-1 46.3 HysdE214-1 43.9 Hysd1085-1 48.0 HyGT3 68.0 Hysd536-1 44.4 Hysd16-1 49.0 HysdE367-1 49.2 Arsd87 58.2 HysdE769-1 54.0 Arsd576 60.5 Hysd1367-1 76.3 Hysd928-1 57.2 Hysd293-1 65.9 Hym266 Hysd122-1 81.2 74.6 Hysd1260-1

91.3 Arsd309 91.3 ArGT711-2

97.9 HysdE867-1

M7 HY_LG7 F1 M8 HY_LG8 F15

0.0 Hysd49-1 1.1 Hysd342-4 HysdE2105-1 0.0 Hysd119-2 5.9 HysdE2386-1 1.1 HysdE2386-1 6.9 Hysd490-1 0.0 Hysd221-6 0.0 Hysd694-1 1.8 Hym180 10.2 Arsd305 Hysd49-1 4.1 Hysd358-1 3.9 Arsd233 6.3 Hysd856-1 10.9 0.0 Hysd119-2 Hysd342-4 10.8 Hysd419-1 9.2 Hysd795-1 0.0 Arsd233** 6.8 Hysd533-1 11.3 0.9 Hym180 HysdE2105-1 13.8 ArGA26 9.3 Arsd573 7.7 HysdE2066-1 11.9 4.4 ArGT755 HysdE2386-1 17.8 Hysd592-1 10.5 Hysd221-6 6.7 Arsd573 Hysd795-1 8.4 Hysd493-1 14.9 5.2 Arsd483 18.5 Arsd64 14.6 Hysd358-1 9.1 Arsd99 HyGT39 16.7 11.3 Hysd490-1 Arsd166 Hysd592-2 Arsd664 18.5 Hysd1245-1 9.2 Arsd8 Hym351 17.8 12.1 Arsd305 Hysd34-2 HysdE2066-1 18.6 Hysd212-1a HysdE4406-1 21.2 Hysd419-1 10.3 Hysd34-2 19.2 16.2 Hysd694-1 Hysd856-1 18.0 ArGT755 Hysd628-2 24.2 ArGA26 11.5 Arsd483 20.3 Arsd161 22.0 Arsd166 18.7 HysdE2145-2 27.7 Arsd170 20.6 Hysd1245-1 12.5 ArGT755 21.4 Hysd533-1 24.1 Arsd161 19.4 Hysd288-1 28.1 Hysd288-1 13.3 HysdE2105-1 21.9 Arsd8 HyGT39 26.9 Hysd308-5 22.2 Hysd349-1 28.7 Hysd628-2 18.2 Hysd112-1 23.3 Arsd99 Hym351 29.6 Arsd31 25.1 HysdE1235-1 28.9 HysdE2145-2 26.7 Hym456 Hym90 23.4 31.4 Arsd664 Hysd493-1 33.5 Arsd370 29.0 Hysd592-2 30.0 Arsd312 24.4 35.1 Arsd170 Hysd308-5 29.1 Hysd212-1a HysdE4406-1 32.1 ArGT171 25.0 37.0 Hysd955-1 36.7 Hysd628-2 25.8 Arsd31 39.0 Hysd268-2 29.2 Arsd64 34.0 Hysd1205-1 42.0 Hysd861-5 42.2 Hysd268-2 Hysd268-3 28.4 Hysd112-1 29.7 Arsd664 34.1 Arsd368 44.5 Hysd46-1 Hysd194-1 45.0 HysdE3775-1 30.4 Arsd370 30.0 Hysd592-1 45.6 Hysd36-1 Hysd1024-1 45.1 Arsd202 35.0 Hysd955-1 48.6 HyGT307 32.5 Hysd349-1 49.3 Hysd89-1 50.2 HyGT307 37.2 Hysd861-5 35.5 HysdE1235-1 38.8 Hym90 54.6 Arsd202 48.2 Hysd268-2 38.9 Hym456 56.7 Hysd127-3 49.3 Hysd268-3 40.5 Hysd46-1 Hysd194-1 60.6 Arsd683 52.5 HysdE3775-1 40.9 ArGT171 62.6 ArGT862 53.4 Arsd202 41.7 Hysd36-1 Hysd1024-1 65.8 ArGT636 59.8 HyGT307 63.1 Arsd683 43.9 Arsd368 70.0 Hysd443-2 Hysd443-1 44.5 Hysd1231-1 45.7 Hysd1205-1 45.9 Hysd89-1 78.9 Hysd440-4 46.8 Arsd312 48.6 Hysd278-1 85.2 Arsd312 48.9 Arsd184 90.8 Arsd184 52.8 Hysd127-3 91.1 Hysd1205-1 57.2 ArGT862 91.2 Hysd278-1 61.5 Hysd440-4 93.6 Hysd1231-1 63.7 Hysd443-2 100.4 ArGT171 65.2 Hysd443-1 71.0 ArGT636 Fig. 2. Consensus genetic linkage map of silver carp based on microsatellite markers. The male linkage group (left) is named as “M1–M24”; the female linkage group (right) is named as “F1–F25”; the consensus linkage group (middle) is named as “HY_LG1–HY_LG24”. Total lengths of linkage groups are expressed in Kosambi cM. Ampliﬁed duplicated loci indicated with lowercase letters (a) and (b) existed in Fig.2. One of the duplicated loci "Hysd212-1a" was located on the LGs of Hy_LG8 and M8; another locus was located on the LGs of Hy_LG6 and F14. Markers with segregation–distortion were indicated with stars, with P b 0.05 = *; P b 0.01 = **; P b 0.005 = *** or more stars. SDR: segregation–distortion region. 100 W. Guo et al. / Aquaculture 412-413 (2013) 97–106

M9 HY_LG9 F22 M10 HY_LG10 F6

0.0 Hym205 0.0 Hysd554-1 2.1 Arsd574 0.1 Hysd553-1 4.5 Arsd429 0.0 Hysd554-1 7.8 Hym34 0.0 Arsd125 0.0 Hysd1822-1 6.3 Arsd125 0.0 HysdE4333-1 0.1 Hysd553-1 11.0 Hysd608-1 2.9 HysdE4333-1 9.2 HysdE4333-1 13.6 Hysd1147-1 3.1 Hym182 9.4 Hym182 Hym205 7.8 Hym34 14.7 Hysd1822-1 7.1 22.5 Hysd1174-1 Arsd574 11.1 Hysd608-1 20.2 Hysd753-1 10.6 Hysd571-6 9.9 Hysd529-1 24.1 Arsd48 Arsd429 13.6 Hysd1147-1 22.0 Hysd753-2 18.2 12.6 Hysd397-2 24.6 Hysd529-1 24.9 Arsd709 21.2 20.2 Hysd753-1 22.9 Hysd346-1 27.5 Hysd397-2 25.0 Hysd571-6 Arsd361 22.1 Hysd753-2 21.9 23.8 Hysd471-1 29.3 Hysd346-1 28.3 Arsd48 28.6 Hysd149-3 Hysd786-2 Hym14 25.0 Arsd709 22.0 Hysd849-2 30.1 Hysd471-1 31.6 Hysd152-1 29.3 Hysd417-1 24.0 28.7 Hysd149-3 HysdE4353-1 30.3 Hysd849-2 Hysd470-1 BL133 29.4 Hysd417-2 26.9 34.0 29.5 Hysd417-1 30.8 Hysd718-1 Hysd500-1 Hym159 32.0 Arsd441 Hysd500-1 Hysd362-2 31.4 Hysd718-1 27.1 29.6 Hysd417-2 31.5 Hysd731-1 Arsd164 33.0 Hym193 34.1 HysdE4353-1 32.7 Hysd983-1 35.9 Hym14 Hym193 33.4 Hym159 Hysd470-1 34.5 Arsd164 36.0 Hysd16752-1 28.0 36.3 Hysd659-1 Hysd175-1 38.6 ArGT555-2 Hysd137-1 Hysd500-1 Hysd362-2 35.4 Arsd441 37.4 Hysd271-1 31.6 33.5 37.6 Hysd700-2 Arsd144 BL133 36.7 Hysd1237-6 37.9 Arsd361 Hysd786-2 34.0 41.0 Hysd271-1* Hysd1337-1 33.9 Arsd164 37.9 Hysd471-1 38.2 Hym14 39.8 42.8 Hysd16752-1 34.2 HysdE4353-1 40.0 Hysd1174-1 39.0 Hysd659-1 Hysd175-1 48.9 Hysd983-1* 36.0 Hysd152-1 40.1 Hysd700-2 Arsd226** 52.6 ArGT555-2 36.9 Hysd1237-6 53.0 42.7 Hysd731-1 55.7 ArGA68 38.0 Hysd137-1 51.6 ArGT555-2 59.2 Hysd1064-1 59.5 Hym40 40.4 Arsd144 46.3 Hysd1337-1 50.6 Arsd226 62.2 ArGA68 65.7 Hysd1064-1 71.4 Hysd837-5* 65.9 Hym40 69.1 Hysd837-5

M11 HY_LG11 F19 M12 HY_LG12 F24

0.0 Hysd6733-1 0.0 Hysd6733-1 1.3 Hysd57-1 0.0 Hysd381-5 0.0 Arsd721* 0.0 Hysd381-5 1.3 Arsd443 2.4 Arsd443 0.0 Hysd9-3 7.2 Hysd1561-1 4.5 Arsd375 1.8 Hysd311-1* 1.4 Hysd57-1 3.8 ArGT867 6.1 Hysd748-1 11.5 Hym06 5.6 Hysd311-1 8.5 HysdE10584-1 Hysd560-1 Hysd311-2* 8.4 7.5 Hysd1561-1 13.1 Arsd375 6.0 14.1 Hysd922-2 8.5 Arsd417 Hysd385-1 14.4 Hysd311-1 6.5 Hysd1-2* 11.9 Hym06** 12.7 HysdE10584-1 15.1 Hysd9-3 9.7 Hysd1425-2 15.7 Hym371 15.4 Hysd277-2** 21.1 Hysd748-1 17.8 Hysd1-2 16.9 Hysd922-2 17.7 Arsd418 23.6 Arsd417 Hysd385-1 Arsd418 18.6 Hysd197-2 Hysd735-1 18.5 24.0 Hysd833-2 18.5 Hysd735-1 28.0 Hysd833-2 Hysd88-1 19.2 24.3 Hysd1425-2 23.5 Hysd835-1 18.6 Hysd88-1 31.9 Hysd560-1 Hym371 19.4 25.8 Hysd560-1 26.7 Hysd835-2 19.1 Arsd721 32.5 Hysd1462-1 Hysd833-1 Hysd311-2* 19.9 Hysd1462-1 Hym73 25.5 Hysd277-2 32.6 Hym73 Hysd519-1 28.5 Arsd80** 20.8 Hysd833-1 29.7 Hysd311-2 33.2 Hysd835-1 Hysd660-1 24.7 28.6 Hysd519-1 33.5 Arsd80 35.4 Hym139 Hysd660-3 30.4 30.4 Hym139 38.8 Arsd544 36.5 Hysd197-2 40.6 Hysd395-5 Hysd1392-1* 31.2 31.6 Hysd835-1 40.6 Hysd660-1 41.4 Hym183 34.5 36.3 Hysd395-5 45.1 Hysd1026-1* Hysd660-3 43.3 Arsd26 37.3 37.1 Hym183 Hysd1392-1 44.8 Hym186 41.7 37.3 Hysd835-2 50.9 Hysd73-1 46.3 Hysd1026-1 39.0 Arsd26 52.3 Hysd73-1 40.6 Hym186 57.0 Arsd443 58.0 Arsd375** 44.3 Arsd544 57.1 ArGT867 60.9 ArGT686 56.6 ArGT686

M13 HY_LG13 F5 M14 HY_LG14 F17

0.0 Hysd335-1 2.4 Arsd512 Hysd290-1 0.0 Hysd1006-4 8.3 HysdE1262-1 9.6 Hysd744-1 10.1 0.0 Hysd1331-1 Hysd1190-1 0.0 Hysd744-1 13.8 Hysd828-2 0.0 Hysd1006-4 0.0 Hysd335-1 13.0 4.2 HysdE17-2 HysdE17-2 23.8 Hysd556-1 13.7 5.4 Hysd497-1 4.2 Hysd828-2 Hym155 33.1 ArGA196 10.1 HysdE1262-1 14.0 5.5 Arsd410 Arsd438 Hysd90-1 35.8 HysdE5961-1 13.0 Hysd1190-1 14.5 5.6 Arsd79 Arsd383 Arsd438 Arsd79 37.8 Hysd309-1 13.7 HysdE2120-1 10.6 Hysd290-1 14.7 Hysd497-1 Arsd383 39.1 Hym292 14.3 Hysd497-1 Arsd383 12.3 Arsd717 Arsd410 41.6 Hysd842-1 14.5 Hysd832-1 14.8 12.7 HysdE1238-1 23.7 ArGA196 Hysd832-1 45.5 Hysd309-2 14.6 Hysd28-2 14.9 15.4 Arsd639 26.2 HysdE5961-1 Hysd28-2 47.5 Arsd791 23.8 Hysd556-1 16.0 Hysd90-1 15.2 19.9 Arsd512 29.6 Hym292 HysdE2120-1 48.3 HysdE462-1 17.0 Hym155 16.0 31.9 Hysd842-1 Hysd236-6 F25 50.0 Hysd79-2 18.1 Hysd236-6 17.9 40.4 Hysd79-2 33.0 ArGA196 Arsd717 50.6 Hysd267-5 22.8 HysdE1238-1 22.1 41.8 HysdE3822-1 37.6 Hysd309-1 HysdE1238-1 51.1 Hysd258-4 26.3 HyGT452 22.7 42.4 Hysd74-2 Hysd461-1 45.3 Hysd309-2 Hysd461-2 31.3 Hysd858-2 26.1 HyGT452 Arsd761 HysdE1318-1 52.0 47.3 Arsd791 42.5 Arsd761 HysdE1318-1 36.7 HyGT399 28.6 Arsd639 Hysd461-2 Hym134 52.1 48.1 HysdE462-1 Hym134 44.3 Hysd868-1 29.7 Hysd1331-1 43.4 Arsd638 49.7 Arsd458 Hysd858-2 52.2 Hysd461-1 Hysd74-2 31.1 44.2 Hysd258-4 50.6 Arsd16 HyGT399 52.5 Hysd108-1 37.3 51.9 Hysd108-1 Hysd1350-1 52.6 Arsd16 40.4 0.0 Hysd1350-1 52.5 Hysd461-2 42.1 Hysd868-1 53.0 Arsd458 HysdE3822-1 53.0 Hysd461-1 1.3 HyGT399 Hysd868-1 Arsd541 59.0 53.6 Arsd638 55.6 Arsd29 56.1 Arsd29 55.8 Hysd74-1 56.2 Hysd74-1 56.5 Arsd706 57.1 Arsd706 59.0 Hysd230-1 58.6 Hysd230-1 61.5 Hysd267-5 68.6 Arsd541 71.3 Arsd7 71.4 Arsd7 75.7 Arsd298 74.1 Arsd298

M15 HY_LG15 F20 M16 HY_LG16 F11

0.0 ArGT532 5.6 Hysd61-5 7.3 ArGA257 0.0 Arsd595 8.7 Hysd56-1 2.0 Arsd89 9.3 Hysd465-2 4.3 Arsd376 0.0 Hysd851-1* 9.4 Arsd733 0.0 ArGT532 4.9 Hysd434-2 0.0 Hysd1179-1 0.0 Hysd1179-1 9.6 Hysd465-1 5.9 Hysd626-3 2.3 Arsd337 2.3 Arsd337 5.7 Hysd56-1 9.8 Hysd416-2 7.2 HL1 9.9 Hym282 10.6 Arsd280 8.5 Hysd759-1 15.1 Arsd534 18.8 ArGT532 10.8 Hym282 9.8 Hysd64-2 Hysd545-1 16.7 Hym85 13.1 ArGT161 13.1 ArGT161 23.9 Hysd1113-1 11.6 Ar222 11.1 Hysd692-2 17.8 Hysd2048-3 Hysd1113-1 26.7 Arsd506 12.4 Arsd506 12.0 Hysd25-1 17.9 Hym72 28.3 Arsd280* 15.7 Arsd534 13.6 Hysd275-1 21.1 Hysd2603-1 21.1 Hysd2603-1 18.0 Hysd662-2 Hysd465-1 Hysd465-2 17.6 Hysd1113-1 15.5 Hysd688-1 29.0 Hysd610-1 Hysd56-1 18.1 Hysd2048-3 22.0 27.1 Hysd432-1 Hysd432-1 Arsd509 27.2 29.1 Arsd733* 18.2 Hym72 Hysd662-2 27.9 29.8 Hysd495-1 28.5 Hysd495-1 29.6 Hysd416-2 18.9 Hym85 33.8 Arsd633 33.8 Arsd633 30.5 Ar222 19.0 Hym18 31.8 ArGA257 22.3 BL145 43.5 Arsd595 33.2 Hysd61-5 22.6 Hysd610-1 44.6 Hysd275-1 38.1 Hym18** 28.6 Arsd509 46.2 Hysd25-1 Hysd545-3 39.2 BL145** 46.5 Hysd545-3 46.6 48.7 Hysd851-1 47.2 Hysd692-2 49.1 Hysd759-1 48.2 Hysd64-2 50.6 HL1 48.3 Hysd545-1 55.8 Hysd110-4 Hysd944-1 48.9 Hysd688-1 58.4 Arsd89 49.7 Hysd759-1 51.0 HL1 53.0 Hysd434-2 53.6 Arsd376 71.9 Arsd466 54.8 Hysd626-3 55.3 Hysd944-1 Hysd110-4 56.4 Arsd89 79.9 Hysd1148-1 71.7 Arsd466 82.3 Arsd724 79.7 Hysd1148-1 82.1 Arsd724

Fig. 2 (continued).

sex-specific maps were performed with JoinMap 3.0 (Van Ooijen and using the Kosambi mapping function (Kosambi, 1943), with JoinMap Voorrips, 2001) under the BC1 algorithm. Genotyping data were ana- parameter settings as follows: a minimum LOD threshold of 4.0 and lyzed using the chi-square test to assess the goodness of fitto REC (recombination frequency) smaller than 0.4. A region with at expected 1:1 segregation ratio for each locus at the confidence level least three adjacent loci showing significant segregation distortion of 0.05. Subsequently, all loci including those with distorted segrega- (P b 0.05) was defined as the segregation distortion region (SDR). A tions were used for linkage analyses. Map construction was performed consensus linkage map for silver carp was constructed based on the W. Guo et al. / Aquaculture 412-413 (2013) 97–106 101

M17 HY_LG17 F18 M18 HY_LG18 F7

0.0 Arsd432 3.5 Hym30 0.0 Hysd1107-1 0.0 Hysd259-1* 0.0 Hysd259-1 4.5 Hysd516-2 0.0 Hysd651-3 7.5 Arsd27 0.0 Hysd1979-1 6.0 Hysd106-1 3.2 Hysd651-2 7.8 Hysd384-1 Arsd623 Hysd44-2 Hysd109-1 6.9 9.7 13.2 HysdE288-1 Hysd44-3 Arsd658 12.0 Arsd61 Hysd123-2 17.9 Hysd123-2* 18.4 7.4 Arsd682 20.8 HyGT443 Arsd439 17.5 Hysd817-1 19.3 HysdE288-1* 19.2 9.8 Arsd467 22.9 Arsd745 21.4 Hmo03 15.8 HyGT443 Arsd467** Hysd863-2** SDR17-1 20.5 BL76 Hysd863-2 12.2 Arsd439 27.8 Arsd690 20.4 22.7 Hysd85-5 Arsd745 BL76** 20.8 Arsd467 33.0 Hysd651-3 20.2 26.8 Hysd352-4 Arsd682** Hysd106-1** 21.8 Arsd71 36.3 Hysd651-2 Hysd39-2 21.4 22.6 Hysd106-1 27.2 Arsd180 Hym30* 27.0 HysdE288-1 39.4 Hysd1979-1 28.3 Arsd61 23.2 Arsd682 29.2 Arsd341 21.5 Arsd71** 52.1 Hmo03 29.8 Arsd27 cid03 31.7 Hysd587-1 22.4 Arsd439**** 23.6 53.3 Hysd85-5 Arsd658 Arsd623 32.3 Hysd312-1 34.0 Hysd384-1 24.1 cid03 53.5 Hysd817-1 23.8 Hysd44-3 Hysd44-2 35.3 HysdE1570-1 36.8 Hysd109-1 33.8 Hym55 54.5 Arsd790 37.8 Hysd987-1 24.8 Hym30 58.9 Hysd352-4 41.6 Hysd1107-1 40.0 Hysd234-1 25.8 Hysd516-2 59.4 Arsd180 42.9 Hysd852-1 47.9 Arsd112* 30.5 Arsd432 61.4 Arsd341 33.7 Hym55 64.3 HysdE1570-1 48.1 Arsd112 64.9 Hysd587-1 54.9 Arsd690 65.7 Hysd312-1 66.1 Arsd109 67.1 Arsd254 63.1 Hysd39-2 68.0 Hysd3655-1 68.8 Arsd92 69.3 Hysd987-1 72.9 Hysd234-1 75.1 Hysd852-1 81.4 Arsd790

93.3 Arsd254 94.1 Hysd987-1 94.2 Arsd92 97.8 Arsd182 95.2 Hysd3655-1 95.7 Arsd109 100.3 Hysd234-1

124.5 Arsd182

M19 HY_LG19 F8 M20 HY_LG20 F21

0.0 Hym253 HysdE18791-1 0.0 Hysd1033-1** 0.0 Arsd525 0.0 Arsd525******* 6.7 0.0 Arsd367 0.0 Arsd367 11.2 Hysd1423-1 4.2 Hysd1033-2 12.2 Arsd150 5.0 HyGT327 5.0 HyGT327* 16.2 Hysd264-1 16.7 Hysd1121-1 14.3 Hysd161-1 22.8 Hysd1033-1 19.9 Hysd782-2 16.8 Hysd348-4 27.0 Hysd1033-2 21.7 Hysd698-1 Hysd171-1 16.6 Hysd264-1 22.2 Hysd64-1 20.8 BL05 30.8 23.8 Hysd1474-1 Arsd353 22.3 Hysd1160-2 23.2 Hysd734-1 32.6 26.0 Hym253 Hysd1008-2 Hysd1008-1 22.9 Hysd782-1 27.1 HysdE1720-1 33.3 32.8 HysdE18791-1 23.9 Hysd1474-1** Hysd575-5 31.3 Hysd14-1 Hysd171-1 23.3 Hysd372-1 29.1 HysdE850-1 33.5 37.2 Hysd1423-1 Hym103 Hysd449-1 Arsd471 34.0 Arsd353 24.4 Hysd324-4 29.4 34.7 38.3 Arsd150 Hysd630-1 34.6 Hysd1008-2 Hysd1008-1 30.3 HysdE1230-1 42.9 Hysd1121-1 Hysd14-1 35.1 Hysd575-5 32.5 HysdE3690-1 35.3 45.3 Hysd372-1 36.8 Hysd161-1 Hysd449-1 Hysd630-1 36.4 47.3 Hysd324-4 37.1 Arsd472 Arsd471 Hysd441-1 48.7 Hysd782-1 42.6 Hysd348-4 38.2 Arsd472 39.2 49.4 Hysd64-1 45.0 Hysd372-1 44.1 HysdE3690-1 50.3 Hym103 49.5 Hysd1160-2 48.7 Arsd402 46.1 HysdE1720-1 51.0 BL05 50.4 Hysd698-1 51.0 Hysd782-1 52.4 Hysd931-1 47.4 HysdE850-1 52.2 Hysd257-1 50.7 Arsd402 Hysd667-4 47.9 Hym103 52.3 Arsd265 56.0 52.4 Hysd782-2 56.1 Arsd637 Hysd920-1 48.6 HysdE1230-1 Hysd667-4 Arsd637 50.1 Hysd734-1 55.7 Hysd920-1 50.5 Hysd257-1 50.7 Arsd265 51.4 BL05 71.4 Hysd19-4 61.7 Hysd441-1 69.9 Hysd19-4 71.4 Hysd931-1

M21 HY_LG21 F23 M22 HY_LG22 F4

0.0 HysdE6109-1 4.8 HysdE5132-1 6.8 HysdE4921-2 0.0 Hysd403-1 0.0 Hysd403-1 0.0 Hysd3112-1 0.0 Hysd18-2 0.0 Hysd589-2 8.5 Hysd18-2 6.9 Hysd3112-1 6.2 Hysd3112-1 13.6 Hysd159-1 7.9 Hysd392-2 7.9 Hysd392-2 14.8 Arsd199 8.0 Hysd392-1 8.0 Hysd392-1 9.8 Hysd589-2 15.3 Hysd143-1 Arsd137 8.1 Hysd1939-1 8.1 Hysd1939-1 16.2 Hysd589-2 15.6 Hysd98-1* 9.2 Hysd283-1 9.2 Hysd283-1 21.0 Hysd548-1 16.7 Hysd1022-1* 11.6 Arsd707 11.6 Arsd707 24.7 Hysd98-1 18.6 Hysd1022-1 21.6 Arsd268** 14.6 HysdE5239-1 15.7 HysdE5239-1 22.3 HysdE5239-1 27.8 Hym68 Hysd1022-1 25.3 Hysd1402-1** 22.7 Hysd45-5 22.7 Hysd45-5 30.7 Hym236 24.4 Hysd548-1 25.9 Arsd789*** 25.6 Hysd11-5 25.6 Hysd11-5 SDR22-1 31.9 Hysd65-1 27.2 Hym49 27.2 Hym49 Arsd268 32.2 33.5 HysdE6109-1 33.2 Arsd23 HysdE2519-1 36.3 38.4 HysdE4921-2 35.3 Hysd540-7 36.4 Hysd1402-1 39.8 Arsd23 39.1 Hysd144-5***** 43.4 Hysd159-1 36.4 Hysd27-6 37.5 Hysd209-2 41.8 Hysd540-7 40.9 Hysd548-1* 44.5 Arsd199 38.1 Arsd789 42.9 Hysd27-6 44.9 Arsd137 Hysd143-1 Hysd781-1 40.5 45.6 HysdE5132-1 42.4 Hysd582-1 48.5 Hysd996-1 Hysd297-1 49.9 Hysd1841-1 57.3 Hym68 ArGT445* 56.8 50.3 Hysd144-5 60.1 Hym236 61.2 Hysd65-1 63.4 Hysd513-1 65.1 Hysd209-2 65.9 HysdE2519-1 68.1 ArGT445 69.9 Hysd513-1 68.0 Arsd789 69.7 Hysd781-1 71.7 Hysd582-1* 77.7 Hysd996-1 Hysd297-1 79.1 Hysd1841-1

M23 HY_LG23 F3 M24 HY_LG24 F16

0.0 Hysd15-1 0.0 Arsd13 Hysd189-1 6.9 Hysd454-4 5.2 0.0 Arsd13 0.0 Hym397 0.0 Arsd93 0.0 Arsd93 Arsd731 16.1 Hysd671-1 6.0 Hysd454-4 1.3 Hysd455-2 Arsd169 Hysd1572-1 Hysd605-1 19.0 Arsd113 6.9 Hysd671-1 6.1 Hym135 26.7 Arsd612 16.3 19.4 Arsd113 8.5 Hysd1291-1 29.3 Hysd1384-1 29.4 Hysd7-1 9.8 Arsd455 30.2 Hysd7-1 12.9 Hysd188-4 12.9 Hysd188-4* 31.2 Hysd165-2 30.4 Hysd165-2 Arsd307 31.7 Arsd307 20.8 Hysd1384-1 32.4 19.8 Hysd423-2 19.8 Hysd423-2* 33.0 ArGA673 32.2 Hysd289-1 22.3 Hysd1852-1 Arsd107 Arsd107 33.4 Hysd289-1 32.8 ArGA673 26.8 26.8 Arsd631 Arsd631 34.1 Arsd610 33.4 Arsd610 29.0 29.0 Arsd21 Arsd21 34.4 Arsd641 33.8 Arsd641 30.2 30.2 Arsd245 Arsd245 35.1 Arsd194 34.6 Arsd194 33.9 33.9 HysdE3405-1 HysdE3405-1 36.0 Hysd364-3 35.2 Hysd364-3 34.7 34.7 HysdE6569-1 HysdE6569-1 37.4 Arsd634 36.9 Arsd634 35.1 35.1 38.5 Hysd255-9 38.0 Hysd255-9 40.6 Arsd455 41.6 Arsd612 41.9 Hysd1291-1 45.0 Hysd1572-1 44.1 Hysd189-1 45.6 Arsd731 44.6 Arsd731 50.4 Hysd460-5 Hysd1572-1 Hysd605-1 44.8 Hym135 49.6 Hysd460-5 HysdE819-1 HysdE819-1 51.2 Hysd15-1 65.6 ArGA377 65.1 65.1 68.2 HysdE920-1 63.4 Hysd1852-1 70.7 Hym397 70.7 Hym397 65.9 ArGA377 73.1 Hysd1852-1 72.0 Hysd455-2 Arsd169 72.1 Arsd169 70.2 HysdE920-1

Fig. 2 (continued). common markers located in both female and male maps using the same chromosome ends by adding two times the average spacing of frame- function. Graphical linkage maps were drawn using Mapchart software work markers to the length of each linkage group (Castaño-Sánchez (Voorrips, 2002). Genome estimation size was calculated to account for et al., 2010). 102 W. Guo et al. / Aquaculture 412-413 (2013) 97–106

Table 1 Characteristics of sex-speciﬁc and consensus linkage groups of silver carp.

LG Male map LG Female map LG Consensus map

No. of markers cM cM/marker No. of SDL No. of markers cM cM/marker No. of SDL No. of markers cM cM/marker

1 29 41.4 1.4 17 2 29 103.4 3.6 2 1 56 82.9 1.5 2 27 76.7 2.8 0 13 15 64.5 4.3 1 2 40 69.6 1.7 3 27 41.1 1.5 0 9 16 75.9 4.7 1 3 40 67.6 1.7 4 25 44.8 1.8 1 10 16 37.9 2.4 0 4 36 48.5 1.3 5 22 48.3 2.2 6 12 15 97.9 6.5 0 5 34 60.5 1.8 6 22 38.0 1.7 0 14 15 91.3 6.1 2 6 31 74.6 2.4 7 22 34.1 1.6 0 1 31 100.4 3.2 0 7 47 71.0 1.5 8 19 60.6 3.2 0 15 13 50.2 3.9 1 8 27 63.1 2.3 9 19 52.6 2.8 2 22 8 38.6 4.8 0 9 25 51.6 2.1 10 19 59.5 3.1 0 6 18 71.4 4.0 2 10 32 69.1 2.2 11 18 60.9 3.4 0 19 11 57.1 5.2 0 11 26 56.6 2.2 12 17 58.0 3.4 8 24 6 18.6 3.1 3 12 20 52.3 2.6 13 17 59.0 3.5 0 5 19 75.7 4.0 0 13 33 74.1 2.2 14 16 44.3 2.8 0 17 12 19.9 1.7 0 14 26 42.1 1.6 25 3 1.3 0.4 0 15 15 39.2 2.6 5 20 11 27.9 2.5 0 15 23 48.7 2.1 16 14 29.8 2.1 0 11 15 82.3 5.5 0 16 26 82.1 3.2 17 14 47.9 3.4 12 18 12 27.0 2.2 0 17 20 48.1 2.4 18 14 42.9 3.1 0 7 18 124.5 6.9 0 18 30 97.8 3.3 19 13 52.4 3.3 1 8 17 71.4 4.2 1 19 28 71.4 2.5 20 12 24.4 2.0 0 21 9 56.1 6.2 2 20 19 55.7 2.9 21 11 27.2 2.5 0 23 6 69.9 11.7 0 21 15 63.4 4.2 22 10 56.8 5.7 8 4 21 79.1 3.8 1 22 27 68.1 2.5 23 10 22.3 2.2 0 3 22 73.1 3.3 0 23 29 70.2 2.4 24 3 1.3 0.4 0 16 12 72.1 6.0 2 24 13 72.0 5.5

SDL: segregation–distortion loci.

3. Results map was 1.52 times that of male map. However, exceptions were observed in nine LGs (LGM2, LGM4, LGM8, LGM9, LGM11, LGM12, 3.1. Genetic linkage map and sex recombination ratio LGM14, LGM15, and LGM17), where male LGs were longer than female ones (Fig. 2; Table 1). Across the sex-specific maps, 82 Of all SSRs screened in male and female silver carp parents of the common markers were distributed on 25 female and 24 male LGs two mapping families, 782 reliably amplified polymorphic products (Appendix B). In most cases, each male LG corresponded to a female accounting for 28.3% of the total microsatellite markers. And loci LG, whereas LGM14 corresponded to two female LGs (LGF17 and Hysd212-1a/b were scored as duplicates. LGF25; Fig. 3). The recombination rate in the female parent was 2.2 In the construction of sex-specific maps with JoinMap 3.0, linkage times higher than that in the male parent (Table 3). The detailed analyses were applied with an LOD threshold of 4.0. For the male data characteristics of sex-specific maps were given in Table 1 and Table 2. set with 429 segregating markers, 415 were ordered on 24 linkage A consensus map for silver carp was constructed based on 82 groups (LGs) with 14 unmapped. Total length of the male map was common microsatellite markers shared between the sex-specific 1063.5 cM, with an average inter-marker distance of 2.6 cM. The maps. The total length of this consensus map was 1561.1 cM along length of LGs ranged from 1.3 cM in LGM24 to 76.7 cM in LGM2 with 703 SSRs ordered on 24 LGs (Fig. 2; Tables 1 & 2). The length (Fig. 2; Table 1). Using the same analysis procedure and threshold of LGs ranged from 42.1 cM in the HY_LG14 to 97.8 cM in the value for the female data set with 435 segregating markers, 370 HY_LG18, with an average size of 65.0 cM (Tables 1 & 2). The number were assigned onto 25 LGs with 65 unmapped. The female map of markers in each LG varied from 13 to 56 with an average of 29.3 covered 1587.5 cM of the silver carp genome, with an average (Tables 1 & 2). This consensus genetic linkage map covered 93.1% of inter-marker space of 4.3 cM. The length of LGs ranged from 1.3 cM the silver carp genome and had an average inter-marker distance of in LGF25 to 124.5 cM in LGF7 (Fig. 2; Table 1). Estimated genome 2.2 cM varying from 0.0 cM to 30.0 cM. Fig. 4 shows that most of sizes were 1188.5 cM for the male map and 1809.0 cM for the female intervals (77.9%) between markers over the consensus map were map. Accordingly, map coverages were 89.5% for male and 87.8% for less than 3 cM. female genomes (Table 2). It was evident that most LGs were longer in the females than in the males, and estimated total length of female 3.2. Segregation distortion

Segregation distortions were observed in both sex maps, but much Table 2 Summary of genetic linkage maps of silver carp. more in the male map than in the female map (60 vs. 18) (Fig. 2; Table 1). In the male map, 14.5% of the assigned loci showed segrega- Item Male Female Consensus tion distortion, and distributed on 9 of the 24 LGs (Fig. 2; Table 1). In Linkage groups (LGs) 24 25 24 the female map, however, only 4.9% of the assigned loci were Total number of markers 415 370 7.3 distorted, and distributed on 11 of the 25 LGs (Fig. 2; Table 1). In Average number of markers per LG 17.3 14.8 29.3 addition, distorted loci were unevenly distributed in the male map Average marker spacing (cM) 2.6 4.3 2.2 – Maximum marker spacing (cM) 18.8 30.0 30.0 with a frequency of 0 17 loci/LG. Most distorted loci were on LGM1, Average length of LG (cM) 44.3 63.5 65.0 LGM17, and LGM22, on which more than 50% loci were distorted Map length (cM) 1063.5 1587.5 1561.1 (Fig. 2; Table 1). A total of ﬁve SDRs were observed in the male Estimated map length (cM) 1188.5 1809.0 1677.5 map, with two on LGM1, and one on LGM5, LGM17 and LGM22, Coverage (%) 89.5 87.8 93.1 respectively (Fig. 2). W. Guo et al. / Aquaculture 412-413 (2013) 97–106 103 M14 F17

0.0 Hysd1331-1 0.0 Hysd335-1 4.2 HysdE17-2 10.1 HysdE1262-1 5.4 Hysd497-1 13.0 Hysd1190-1 5.5 Arsd410 Arsd438 13.7 HysdE2120-1 5.6 Arsd79 Arsd383 14.3 Hysd497-1 Arsd383 10.6 Hysd290-1 14.5 Hysd832-1 12.3 Arsd717 14.6 Hysd28-2 12.7 HysdE1238-1 16.0 Hysd90-1 15.4 Arsd639 17.0 Hym155 19.9 Arsd512 18.1 Hysd236-6 22.8 HysdE1238-1 F25 26.3 HyGT452 31.3 Hysd858-2 36.7 HyGT399 44.3 Hysd868-1

0.0 Hysd1350-1 1.3 HyGT399 Hysd868-1

Fig. 3. A striking example of recombination differences between two sexes of silver carp.

4. Discussion was suitable for map development like a classical backcross (Li et al., 2006; Liao et al., 2007a). Furthermore, an interspecificfamilycould 4.1. Mapping families produce maps for two species at the same time, and reduce the number of alleles common in the two parents and the occurrence of marker Many aquatic animals usually have long sexual maturation cycle, segregating at 3:1, a ratio at which codominant markers could not be such as silver carp and bighead carp (4–5 years), it is difficult to gener- included in a backcross (Liao et al., 2007a). Therefore, we used two ate commonly used mapping families (F2 and BC) for genetic map con- interspecific crosses between silver carp and bighead carp in order to struction. However, the natural populations of aquatic animals tend to locate markers as much as possible on new genetic maps for silver have higher polymorphism and provide more information of genetic di- carp. Using the same mapping panel, a second generation genetic map versity. Therefore, combining with pseudo-testcross strategy, most of for bighead carp was also produced by other members of our laboratory aquatic animals could use F1 population for map construction. So far, (Zhu et al., in press), and information of which was not shown in the it has been confirmed that pseudo-testcross strategy in F1 population study.

4.2. Genetic linkage maps and marker density Table 3 Comparative recombination rates between male and female silver carp. In this study, two interspecific crosses between silver carp Sex-specific No. of Cumulative Cumulative Female: and bighead carp were produced to construct the consensus and linkage groups shared distance in distance in male ratio sex-specific maps using genomic and EST-derived SSRs. A total of markers female (cM) male (cM) 782 SSRs were informative in two mapping families, and 370 and M1 & F2 2 48.3 6.2 7.8 415 of them were mapped in the female and male maps of silver M2 & F13 2 39.9 22.5 1.8 carp, respectively (Fig. 2; Table 2). Based on 82 common markers M3 & F9 3 149.4 35.6 4.2 between the sex-specific maps, a consensus map with a total length M4 & F10 5 156.8 70.4 2.2 of 1561.1 cM encompassed 703 SSR markers on 24 LGs (Fig. 2; M5 & F12 3 162.2 35.4 4.6 M6 & F14 6 465.9 86.0 5.4 Table 2), corresponding to the chromosome number of haploid M7 & F1 6 804.9 240.0 3.3 genome of silver carp (n = 24; Yao et al., 1994). A resolution of M8 & F15 5 92 204 0.5 2.2 cM/locus in this silver carp consensus map is one of the M9 & F22 2 16.6 16.7 1.0 well-defined genetic linkage maps based on microsatellites available M10 & F6 5 152.6 103.4 1.5 fi fi M11 & F19 3 97.2 63.8 1.5 among food sh species, offering suf cient marker density for QTL M12 & F24 3 3.0 112.4 NA studies and other genetic analyses. Chinese scientists are planning M13 & F5 3 40.0 37.6 1.1 to sequence several Chinese major carps for elucidation of genetic M14 & F17 3 14.6 17.0 0.9 and genomic bases of economically important traits. Our present M14 & F25 2 0.0 7.6 NA study would aid these research activities in silver carp, and may M15 & F20 3 35.6 20.4 1.7 M16 & F11 3 18.6 13.0 1.4 also provide useful genomic information for other closely-related M17 & F18 6 138.5 18.5 7.5 Chinese carps. M18 & F7 2 6.2 2.2 2.8 The length of majority of marker intervals in this study (b3cM)in- M19 & F8 2 0.7 8.6 0.1 dicated that microsatellite markers were generally evenly distributed M20 & F21 2 6.0 0.4 15.0 M21 & F23 2 22.3 7.7 2.9 on our consensus genetic map across the entire silver carp chromo- M22 & F4 4 209.8 102.5 2.0 somes. Nevertheless, a small number (3.5%) of gaps (N10 cM) were M23 & F3 3 56.2 32.6 1.7 also observed in this new genetic map for silver carp (Fig. 4). Large M24 & F16 2 1.4 1.3 1.1 gaps between markers are a relatively common phenomenon in genetic Total 82 2738.7 1265.8 2.2 linkage maps and it has been reported both in animals and plants 104 W. Guo et al. / Aquaculture 412-413 (2013) 97–106

350

300

250

200

150

100 Number of intervals

0 0—1cM 1—2cM 2—3cM 3—4cM 4—5cM 5—6cM 6—7cM 7—8cM 8—9cM9—10cM >10cM Distance between the markers (cM)

Fig. 4. Distribution of map distances between adjacent markers on consensus genetic map of silver carp.

(Bradley et al., 2011; Hwang et al., 2009; Yu et al., 2012; Zheng et al., 4.4. Sex-specific patterns of recombination 2011). These gaps may represent regions of increased recombination or may represent highly conserved regions where polymorphic markers Recombination rate in the female parent was about twice as high are fewer and therefore harder to identify (Gan et al., 2003). Addition of as in the male parent (Table 3), suggesting that males had an overall more microsatellites or other types of DNA markers (e.g., SNPs) should reduction in recombination rate relative to the females in silver carp. reduce large gaps on some LGs. Recombination rates varied between sexes in mammals (Dib et al., 1996; Dietrich et al., 1996; Neff et al., 1999). Although heteromorphic sex chromosomes were not observed in many fish species (Singer et 4.3. Map comparisons al., 2002), a similar phenomenon was reported in fish, for example, a ratio of 2.74:1 in zebrafish (Singer et al., 2002), 1.69:1 in Arctic Genetic maps with co-dominant markers (SSRs or SNPs) would be char (Woram et al., 2004), 3.25:1 in rainbow trout (Sakamoto et al., favorable for scientists to apply them for genetics and genomics stud- 2000), 1.60:1 in turbot (Bouza et al., 2007), 1.20:1 in gilthead sea ies. However, previous published genetic maps (Liao et al., 2007b; bream (Franch et al., 2006), and 2.00:1 in grass carp (Xia et al., Zhang et al., 2010; Zhang et al., 2011) for silver carp were mainly 2010). The sex-biased recombination in silver carp was at a moderate constructed using AFLPs. Although AFLP markers have many advan- level when compared with above fishes. A striking example of the tages, they are labor-intensive, technically challenging, and their recombination difference between two sexes of silver carp was that cross-applicability between laboratories can be impeded due to the several common markers on LGM14 were shared by two female LGs inherent difficulties associated with the identification of the corre- (LGF17 and LGF25) (Fig. 3). In humans, there is evidence that recombi- sponding AFLP fragment sizes (Cloutier et al., 2011). This would limit nation along the chromosomes depends on the chromosome structure the application of linkage maps for comparison and other genetic anal- (Lynn et al., 2004). Furthermore, cytogenetic evidence showed that yses. In this study, our consensus map contained more than 3 times as silver carp had the same karyotype formula (18 M + 22SM + 8ST) as many sequence-based codominant markers (SSRs) as did the previous grass carp (Yao et al., 1994; Zan and Song, 1979), and therefore similar maps, and those SSRs used in previous genetic maps were incorporated recombination rates were observed in silver carp (2.2, this study) and in our new genetic map if they were segregated in male or female par- grass carp (2.0) (Xia et al., 2010). ents of our mapping panel. These applications allowed us to perform With the development of high-density genetic maps, the recombina- simple map comparisons with previous studies, although SSRs in com- tion ratio may fall quite dramatically. Taking Atlantic salmon for example, mon were very limited between ours and previous genetic maps. there was a big difference between female and male recombination rates Of the three previously published maps for silver carp, Liao et al. in early low or medium genetic maps (8.26:1) (Johnson et al., 1987; (2007b) and Zhang et al. (2011) used the same mapping family. Moen et al., 2004). However, a high-density map was recently developed Therefore, our present map was mainly compared with two maps for Atlantic salmon using SNPs, and results indicated that the recombina- published by Zhang et al.(2010) and Zhang et al. (2011). As for the tion rate fell to 1.38:1 (Lien et al., 2011). Usually, female recombination number of linkage groups, ours and previous maps differed markedly rates around the centromere were much higher than those of males, in that we obtained 24 LGs, whereas Zhang et al. (2010) and Zhang et but male recombination rates appeared to be higher in telomeric regions al. (2011) had nine and two extra small LGs than expected chromo- (Lien et al., 2011; Lynn et al., 2004). The reason for the marked difference somes of haploid genome of silver carp (n = 24), respectively. 21 LGs in reported Atlantic salmon male and female recombination rates in in Zhang et al. (2010) were homologous to 20 of the 24 LGs in our different papers may be related to the marker coverage in telomeric map, with LG1 and LG16 of Zhang et al. (2010) being merged as regions. In our study, the female vs. male recombination ratio (2.2) was HY_LG2 in our map by three markers (Hym294, Hym12 and Hym334) similar to a previous report in silver carp (2.08) (Zhang et al., 2010), shared between two maps. Ten of the LGs in Zhang et al. (2011) showed suggesting that silver carp, maybe also grass carp, had a slight difference homologous to 10 LGs in our map based on 24 common SSRs shared be- in sex-specific recombination with a ratio around 2.0–2.2 when genetic tween two maps. When compared with the map of Zhang et al. (2010), maps were low or medium marker-density. With the number of markers our map significantly improved the genome coverage (from 86.4% to (SSRs or SNPs) increased on future genetic map(s) of silver carp, how 93.1%) and the resolution (from 3.2 cM to 2.2 cM). Although the map much the recombination ratio between sexes fell should be noticed. resolution was 2.2 cM in both this study and Zhang et al. (2011),our It is important to know the relative rate of recombinations in map extended total map length from 1049.6 cM to 1561.1 cM, with female and male animals. For both sexes of any species, a high rate the increase of genome coverage from 90.1% to 93.1%. of recombination is desirable to break up closely linked markers, W. 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