Construction of microsatellite-based linkage map and mapping of ...

c Indian Academy of Sciences

RESEARCH ARTICLE

Construction of microsatellite-based linkage map and mapping of nectarilessness and hairiness genes in Gossypium tomentosum MEIYING HOU, CAIPING CAI, SHUWEN ZHANG, WANGZHEN GUO, TIANZHEN ZHANG and BAOLIANG ZHOU∗ State Key Laboratory of Crop Genetics and Germplasm Enhancement, Cotton Research Institute, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

Abstract Gossypium tomentosum, a wild tetraploid cotton species with AD genomes, possesses genes conferring strong fibers and high heat tolerance. To effectively transfer these genes into Gossypium hirsutum, an entire microsatellite (simple sequence repeat, SSR)-based genetic map was constructed using the interspecific cross of G. hirsutum × G. tomentosum (HT). We detected 1800 loci from 1347 pairs of polymorphic primers. Of these, 1204 loci were grouped into 35 linkage groups at LOD ≥ 4. The map covers 3320.8 cM, with a mean density of 2.76 cM per locus. We detected 420 common loci (186 in the At subgenome and 234 in Dt) between the HT map and the map of TM-1 (G. hirsutum) and Hai 7124 (G. barbadense; HB map). The linkage groups were assigned chromosome numbers based on location of common loci and the HB map as reference. A comparison of common markers revealed that no significant chromosomal rearrangement exist between G. tomentosum and G. barbadense. Interestingly, however, we detected numerous (33.7%) segregation loci deviating from 3:1 ratio (P < 0.05) in HT, mostly clustering on eight chromosomes in the Dt subgenome, with some on three chromosomes in At. Two morphological traits, leaf hairiness and leaf nectarilessness were mapped on chromosomes 6 (A6) and 26 (D12), respectively. The SSR-based map constructed in this study will be useful for further genetic studies on cotton breeding, including mapping loci controlling quantitative traits associated with fiber quality, stress tolerance and developing chromosome segment specific introgression lines from G. tomentosum into G. hirsutum using marker-assisted selection. [Hou M., Cai C., Zhang S., Guo W., Zhang T. and Zhou B. 2013 Construction of microsatellite-based linkage map and mapping of nectarilessness and hairiness genes in Gossypium tomentosum. J. Genet. 92, 445–459]

Introduction Cotton (Gossypium spp.) is one of the world’s most important economic crops, with world-wide cotton production valued at approximately US$27–29 billion annually in 2005– 2007 (Campbell et al. 2010). Gossypium hirsutum L. and G. barbadense L., which are commercially important cultivated species belonging to the (AD)1 and (AD)2 genome groups, respectively, are very susceptible to insect pests such as white flies, aphids, jassids and bollworms. G. tomentosum Nutt ex Seem, a wild tetraploid species with (AD)3 genome, is closely related to G. hirsutum but quite different from the tetraploid cultivated species G. hirsutum and G. barbadense in terms of phenotype, isozymes and markers (Fryxell 1979; Saha and Zipf 1997). G. tomentosum has hairy

∗ For correspondence. E-mail: [email protected].

leaves and stems but no nectary on its leaves or bracteoles, but has an extra floral nectary that attracts pests (Hutchinson et al. 1947). It also has strong fibers (Meyer and Meredith 1978) and is the most heat-tolerant species of Gossypium. These traits from G. tomentosum can be introgressed into G. hirsutum by wide-crossing and are important for cotton breeding. However, it is quite difficult to transfer these traits directly into cultivated cotton by conventional breeding due to segregation distortion (Jiang et al. 2000), suppression of recombination (Paterson et al. 1990) and linkage drag (Young and Tanksley 1989). Constructing a molecular map provides the foundation for the genetic dissection of important traits and will facilitate utilization of G. tomentosum in breeding by marker-assisted selection (MAS) and map-based cloning. To date, several high density genetic molecular maps have been constructed using diverse DNA molecular markers and mapping populations (Ulloa et al. 2002; Nguyen et al. 2004; Rong et al.

Keywords. microsatellite; leaf hairiness; leaf nectarilessness; genetic linkage map; Gossypium tomentosum. Journal of Genetics, Vol. 92, No. 3, December 2013

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Meiying Hou et al. 2004; Guo et al. 2007; Zhang et al. 2009, 2012; Yu et al. 2011), but most of these are HB maps (from a cross between G. hirsutum L. and G. barbadense L.). To date, only one HT map (from a cross between G. hirsutum and G. tomentosum) has been reported (Waghmare et al. 2005). Using this map, Zhang et al. (2011) identified 28 QTLs controlling fiber quality traits. However, this HT map is difficult to use for high throughput cotton breeding because the markers used in the map are hybridization-based (i.e., restriction fragment length polymorphism, RFLPs). RFLP analysis is inefficient due to large amount of highquality DNA required for this time-consuming process. In recent years, many new sets of molecular markers have been generated to facilitate the development of a high-resolution integrated genetic map of cotton. Among the different types of molecular markers, microsatellites or simple sequence repeats (SSRs) are becoming the markers of choice for tagging genes and assessing genetic diversity. This is mainly because SSR analysis requires only a small amount of DNA. Moreover, SSRs are easily detectable by PCR, amenable to high-throughput analysis, codominantly inherited, multiallelic, highly polymorphic, abundant and evenly distributed in the genome. SSRs exist throughout the entire genome of an organism in both noncoding and coding regions. These traits allow the complexity of the multilocus SSR fingerprint to be customized; SSRs are therefore ideal for the analysis of large genomes. The unique mechanism responsible for generating SSR allelic diversity arose through replication slippage. The codominant nature of SSR markers also permits the detection of a large number of alleles per locus and contributes to higher levels of expected heterozygosity. Manosh et al. (2011) found that SSRs generate the highest percentage of mappable loci among the techniques examined, indicating that SSR markers are more suitable for mapping in citrus. To effectively transfer desirable genes conferring strong fibers and high heat tolerance, entirely PCR-based linkage mapping of the cross between G. hirsutum and G. tomentosum is a prerequisite for its utilization in cotton breeding. In this study, we produced and reported the first SSR (PCR-based) genetic map (HT) covering a large region of the cotton genome and compared with the HB map (constructed by Dr Zhang’s group of Nanjing Agricultural University, Guo et al. 2007). Moreover, the genes conferring leaf trichome and leaf nectary were mapped as discrete markers. The results of this study will contribute to the alignment of morphological and molecular maps identification of DNA markers diagnostic of phenotypic variation for these traits and also improve insect resistance in cotton.

Materials and methods Plant materials and DNA extraction

Ninety-three individuals of the F2 generation were derived from a single cross between G. hirsutum L. acc 08N2162 and 446

G. tomentosum Nutt ex Seem grown under field conditions at Jiangpu Experiment Station of Nanjing Agricultural University (NAU), China. The former is an upland cotton cultivar with high yield and moderate fiber quality, whereas the latter, a wild tetraploid species, possesses many desirable traits, such as morphological resistance to insect pest (heavy leaf hairs and nectariless leaves), heat and drought tolerance and good fiber quality. Total genomic DNA was extracted from young leaves of the two parents, F1 and each F2 individual as described by Paterson et al. (1993) with some modifications.

SSR analysis, PCR amplification and electrophoresis

A total of 8488 SSR primer pairs were evaluated for detecting polymorphism between the two parents. All SSR primer information used in this study can be obtained from http://www.cottonmarker.org. SSR-PCR amplifications were performed using a Peltier Thermal Cycler-EDC-810 (Eastwin, Hongkong) and electrophoresis of the products was performed as described by Zhang et al. (2000, 2002).

Phenotypic analysis

Plants were scored for each of the following traits: F2 individuals were investigated for leaf pubescence and nectaries; trichomes on the surface of leaves were scored as smooth (very similar to the maternal parent, G. hirsutum L. acc 08N2162) or heavy hairy leaves (very similar to the paternal parent, G. tomentosum); nectaries on the abaxial midribs of leaves were scored as present or absent.

Genotyping and testing for segregation distortion

All 8488 SSR primer pairs were first used to screen polymorphisms between the two parents. Markers found to be polymorphic were then used to survey 93 individuals of the F2 mapping population. All distinctive and unambiguous polymorphic bands were scored as 1 (present) or 0 (absent). Missing data were noted as ‘–’. Each marker system identified both monomorphic and polymorphic markers. At each locus, the allele from G. hirsutum was denoted as A, whereas the allele from G. tomentosum was denoted as B. The expected allelic ratio for F2 was 1:1 (A:B). The expected genotypic ratio was 1:2:1 (AA:AB:BB) for codominant markers or 3 : 1 (dominant : recessive) for dominant markers in the F2 population. The observed ratios for each marker were tested for deviation from the expected values with a χ 2 goodness-of-fit test (P < 0.05). A region with at least three adjacent loci showing significant segregation distortion was defined as the segregation distorted region (SDR) (Paillard et al. 2003). A chi-square test was used to compute segregation distortion by bi-parent genotypes to ascertain whether they skewed

Journal of Genetics, Vol. 92, No. 3, December 2013

Genetic mapping of a cross between G. hirsutum and G. tomentosum towards the female genotype or male genotype. For codominant markers, allele frequency (p = q) and the distribution of different genotype frequencies in the F2 population (p2 :2pq:q2 ) were analysed to characterize factors resulting in distortion. Construction of genetic linkage map

A molecular map was constructed using JoinMap ver. 3.0 (Van Ooijen and Voorrips 2001), and the Kosambi mapping

A1(Chr.1) 0.0 5.9 8.8 11.4 14.6 17.2 19.6 19.9 22.2 22.3 25.1 27.0 29.7 31.3 32.1 35.6 35.9 37.6 38.1 38.8 40.7 42.3 44.8 45.0 46.2 46.8 48.0 49.3 49.5 50.6 51.9 54.3 59.1 61.6 65.4 68.3 72.1 75.6 0.0

NAU4073-170 NAU2798-250 HAU2425-230 NAU1399-320 BNL3580-230 NAU5107-220 NAU6939-200 JESPR221-180 NAU1363-150 HAU0940-200 CIR049-250 cgr5579-100 NAU3135-280 BNL3090-250 cgr5417-170 cgr5301-200 TMD03-240 NAU902-260 NAU3861-400 NAU4891-380 NAU5085-800 JESPR56-140 NAU3022-250 CIR018-230 HAU2307-310 NAU3384-210 BNL2921-180 NAU4044-210 BNL1355-600 NAU1312-210 NAU5085-450 cgr6356-150 NAU6951-270 HAU2056-390 NAU2095-180 Gh649-130 NAU1263-310 NAU7049-250 dc40052-180

11.7

cgr5572-210

29.4

NAU7393-700

36.8

dPL0687-300

69.2

NAU6716-430

81.4

dPL0687-100

function (Kosambi 1944) was used to convert recombination frequency to genetic map distance (centimorgen, cM). All pairs of linked markers were identified using the ‘group’ command at log of odds (LOD) scores ≥ 4 and a maximum recombination fraction of 0.4. Linkage groups were assigned to corresponding chromosomes by mapped SSRs (Guo et al. 2007; Yu et al. 2011); the order of groups on the same chromosomes was linked by dotted lines. The resulting linkage map was drawn using MapChart ver. 2.2 software (Voorrips 2002).

D1(Chr.15) 0.0 10.6 19.3 26.8 33.3 34.9 38.7 41.1 43.4 44.8 49.8 51.8 52.5 55.9 58.4 59.1 60.8 62.5 65.9 67.8 70.3 71.5 73.3 73.9 75.1 78.4 79.7 80.4 80.6 83.7 84.6 86.7 89.0 90.5 93.1 94.7 97.6 111.4 0.0 5.2 9.2 10.2

24.5

NAU1566-200 HAU1740-250 cgr6129-180 NAU1356-250 NAU6468-230 NAU6269-500 NAU4073-180 HAU3297-350 NAU1499-200 NAU6269-580 NAU3694-220 HAU1619-350 NAU1556-170 Gh350-130 HAU1427-140 CIR089-380 HAU2490-130 NAU5843-170 BNL1355-170 HAU3351-320 NAU4930-180 MUSB1079-130 TMN20-230 NAU2116-450 BNL2646-130 NAU3671-150 JESPR205-100 JESPR298-100 NAU3603-700 MUCS322-270 NAU2523-390 NAU3975-390 NAU6951-300 NAU2741-250 NAU5163-210 NAU6952-150 NAU7096-180 NAU2523-300 NAU1481-100 HAU3132-170 NAU1495-150 HAU077-200

A2(Chr.2) 0.0

23.7 30.1 35.6 37.9 40.4 42.0 44.0 44.7 46.5 49.5 50.2 51.1 54.1 57.4 64.3 67.7 0.0 0.6 11.6 11.7 16.7 19.8 20.2 22.0 23.5 25.5 28.8 37.6 38.3

dPL0261-210

cgr5876-160 cgr5029-175 HAU2537-360 dc40265-160 HAU1980-250 HAU2923-900 MUCS620-350 NAU3957-800 cgr5571-150 NAU1018-500 HAU2643-210 BNL3590-180 NAU3761-1000 NAU6146-1300 cgr5385-130 cgr5688-160 NAU2896-400 NAU1246-250 NAU895-350 NAU805-500 NAU895-210 NAU2265-220 NAU3292-230 NAU1246-200 NAU3419-220 NAU895-80 NAU7087-180 NAU5383-460 NAU5383-430

BNL2440-230

D2(Chr.14) 0.0 22.8 31.5 34.4 38.1 46.0 46.9 47.4 48.3 49.3 51.6 52.1 53.0 53.9 57.5 60.1 60.7 61.9 62.3 62.5 63.1 63.6 63.8 64.0 64.3 65.1 65.9 67.2 70.9 74.1 75.4 79.0 79.9 81.5 82.5 84.4 86.9 88.7 89.7 91.4 95.9 100.7 103.9 125.0

NAU7026-700 NAU2190-400 NAU6623-320 NAU5465-220 HAU2337-500 NAU3913-380 NAU6692-230 NAU6474-300 NAU3242-200 NAU803-180 NAU3120-200 NAU3913-420 NAU5035-250 HAU1741-380 TMJ09-400 NAU2929-200 NAU2987-300 NAU3120-610 NAU3691-250 NAU514-200 NAU3312-250 Gh462-150 NAU2155-250 NAU3816-300 NAU2845-250 NAU4025-172 NAU3308-220 NAU4088-300 NAU1265-170 NAU485-410 HAU1888-290 HAU2046-140 Gh669-100 cgr5876-170 HAU133-330 HAU2917-140 NAU6626-240 Gh669-130 CIR332-240 NAU3189-520 HAU3238-180 NAU5421-210 NAU5467-230 HAU1485-220 HAU3236-245 NAU823-450

Figure 1. Comparison of interspecific cotton linkage maps of G. hirsutum and G. tomentosum. Homologous loci in At/Dt are indicated with solid lines. Distorted loci are underlined and SDRs are framed. Linkage groups are connected with dotted lines. Journal of Genetics, Vol. 92, No. 3, December 2013

447

Meiying Hou et al.

Results Polymorphism rates of SSR markers

A total of 5108 eSSR (derived from expressed sequence tag SSR, EST-SSR) and 3380 gSSR (derived from genomic DNA SSR) primer pairs/combinations were used to screen polymorphisms between G. hirsutum and G. tomentosum. Approximately 15.8% (807/5, 108) of eSSRs and 16.0% (540/3, 380) of gSSRs showed polymorphism and generated 1045 and 755 polymorphic loci, respectively. Each eSSR primer and gSSR produced 1.29 and 1.40 loci, respectively.

24.9 25.9 42.9 46.7 48.3 54.5 64.9 70.2 74.3 76.7 78.5 83.4 84.0 87.0 90.8 91.7 92.1 93.6 95.0 95.9 97.5 98.1 99.2 102.6 103.3 106.8 111.3 112.7 116.9 119.6 124.0

CIR133-120

NAU862-180 NAU1081-180 NAU483-250 NAU759-290 NAU1498-420 HAU1322-680 HAU2511-320 NAU972-220 NAU3595-500 NAU1248-350 NAU5035-240 NAU4886-850 NAU3884-190 HAU0604-250 NAU884-210 NAU3680-250 NAU2954-150 BNL3441-200 NAU3884-250 NAU3309-190 CIR288-255 BNL2443-140 NAU5443-180 NAU3639-180 NAU3680-450 NAU3016-200 NAU889-350 NAU5233-200 NAU3995-200 NAU1167-200 NAU5444-390

A4(Chr.4)

D3(Chr.17)

A3(Chr.3) 0.0

The ratios of polymorphic primer pairs/combinations between G. hirsutum and G. tomentosum were far higher than those between G. hirsutum and G. hirsutum (obtained by Shen et al. 2005; Lin et al. 2009; Zhang et al. 2009, 2012; Lopez-Lavalle et al. 2012), but lower than those between G. hirsutum and G. barbadense (by Nguyen et al. 2004; Frelichowski et al. 2006; Han et al. 2006; Guo et al. 2007; Yu et al. 2011). Generally, the lower the ratio of polymorphic primer pairs, the closer the relationship between parents. Of the 1800 polymorphic markers, 682 (37.89%) loci were codominant, whereas 458 (25.44%) were dominant to

21.3 23.7 24.4 24.9 26.1 27.7 30.2 33.5 36.0 38.3 45.2 50.5 56.9 65.5 71.7 84.5 0.0

NAU7328-1400 NAU2907-410 NAU2649-180 NAU3700-200 NAU2908-600 NAU6234-170 dPL0200-210 NAU2663-570 NAU3626-1100 NAU2994-220 NAU2909-180 NAU2662-230 NAU7328-400 NAU1290-500 HAU0306-1500 NAU3700-1400 BNL3590-185 BNL3955-170 NAU3884-200 NAU5443-160 NAU889-190 NAU3309-170 NAU3037-650 CIR023-2503 HAU1022-300 NAU3884-240 BNL2443-135 NAU3800-150 NAU6542-200 NAU795-210 HAU197-230 NAU3309-300 NAU1157-257 BNL2496-130 NAU5260-160 NAU6104-200 NAU855-240 NAU2836-220 NAU2898-1300

28.2 30.3

NAU5780-200 NAU5111-230

41.7

NAU805-200

78.0

NAU2898-1100

0.0 3.1 3.7 6.7 8.1 10.3 11.0 11.5 12.1 12.7 13.2 13.4 14.3 15.4 15.8 16.5 17.5 18.4 18.9 19.7 20.5

0.0 6.2 16.0 20.4 24.7 25.4 28.5 30.5 30.9 32.0 33.1 33.8 34.2 35.1 37.8 39.8 43.8 44.7 49.0 50.8 54.3 57.1 61.7 65.0 65.1 67.6 72.8 77.1 78.2 81.5 83.7 90.1

BNL4047-100 cgr5819-240 cgr5733-140 cgr5819-280 HAU3120-300 JESPR223-150 HAU1300-240 HAU2016-750 Gh124-180 NAU1577-230 NAU1346-310 NAU5180-200 NAU3825-220 cgr5252-170 Gh117-250 NAU6672-150 HAU1332-270 cgr6743-140 HAU2016-430 HAU0938-600 NAU3009-400 NAU2477-230 MUCS440-500 NAU1151-480 NAU3514-480 MUSS506-400 NAU6992-240 NAU6993-100 HAU0751-300 NAU2235-170 NAU1325-170 NAU2235-220

Figure 1. (contd)

448


D4(Chr.22) 0.0 3.0 5.3 9.8 13.0 13.5 14.4 15.4 16.8 18.4 20.7 21.2 21.5 22.5 24.0 24.5 25.9 28.2 30.0 31.7 32.3 36.3 37.3 46.3 53.7 55.1 58.5 59.4 61.4 61.6 67.4 69.1 72.1 74.6 78.5 85.6 91.7

NAU2376-380 NAU2302-170 NAU4062-200 NAU1577-210 NAU5046-450 NAU7472-800 cgr5150-140 NAU2601-260 NAU6286-250 NAU3323-250 BNL3994-100 NAU3942-150 NAU3103-250 NAU6578-240 JESPR184-170 Gh166-190 NAU1158-170 NAU5099-265 Gh330-120 MUSB1050-200 NAU2089-580 NAU2783-200 HAU0558-310 BNL358-170 HAU0558-500 NAU7576-270 NAU2162-200 NAU913-210 NAU2477-250 NAU2782-240 HAU3302-600 NAU1325-150 NAU6992-220 NAU2235-150 HAU3302-700 HAU1895-590 NAU3868-180

Genetic mapping of a cross between G. hirsutum and G. tomentosum A5(Chr.5) 0.0 6.6 8.9 13.8 20.2 26.1 30.6 40.6 48.7 52.9 56.0 57.6 61.6 63.9 64.3 67.8 69.8 72.9 75.2 79.3 82.7 86.0 87.3 89.9 90.4 93.7 96.1 97.8 98.8 105.1 107.1 108.1 108.6 110.1 111.0 111.5 112.6 113.8 114.0 114.5 116.0 119.9 123.5 127.1 128.4 134.9 135.3 138.7 139.2 141.3 143.4 144.9 146.7 147.8 149.6 150.3 152.0 152.8 153.5 153.9 155.0 156.2 156.9 157.0 157.5 158.8 160.0 160.7 162.0 164.3 166.3 168.4 171.7 173.5 174.6 178.6 181.2 185.8 187.0 187.1 190.5 191.0 195.0 198.3 207.0 208.5 209.9 215.2 219.3

NAU2865-500 NAU3737-420 NAU3826-550 NAU3828-300 NAU2884-300 dPL0063-220 NAU2000-1200 cgr6756-180 NAU1132-500 cgr5539-140 cgr6756-120 NAU5273-250 NAU6968-200 NAU2630-240 NAU1109-240 NAU3212-180 Gh381-100 NAU1221-235 NAU3418-210 NAU5273-240 NAU5160-140 HAU1952-240 NAU2800-120 NAU3014-250 NAU2978-400 NAU6657-250 NAU6207-310 NAU1015-500 NAU1266-200 NAU4106-380 Gh83-140 NAU1003-220 NAU3620-230 NAU3001-260 NAU6993-140 dPL0155-200 HAU2313-130 NAU2978-380 BNL2448-140 Gh691-200 NAU4106-350 NAU5088-250 HAU1976-250 HAU0506-210 HAU0506-270 NAU2140-135 STV191-350 dPL0384-480 NAU6186-320 NAU2376-1200 NAU6494-1000 Gh211-130 NAU3636-480 NAU2376-310 JESPR184-190 Gh594-110 Gh691-100 HAU2500-260 dPL0384-230 cgr6733-210 NAU1406-400 NAU4040-290 JESPR197-60 NAU3569-300 NAU2001-190 NAU1127-200 NAU5752-200 NAU5149-190 Gh166-200 NAU6494-480 NAU6109-170 NAU5400-230 Gh260-80 JESPR50-130 NAU5522-180 Gh641-100 cgr6733-230 JESPR50-170 BNL448-230 NAU2120-250 NAU4058-240 NAU5522-160 NAU961-200 NAU3402-470 HAU2691-450 NAU3781-370 NAU6966-270 HAU0576-260 MUSS219-280

D5(Chr.19) 0.0 6.8 7.0 8.9 12.7 14.9 18.3 19.6 22.0 23.6 24.9 26.2 27.0 27.3 28.0 29.3 31.4 32.8 33.2 34.2 35.7 38.3 39.3 41.3 41.7 42.7 45.0 47.5 56.2 57.8 59.2 59.5 61.4 63.1 64.3 67.3 68.1 70.6 71.1 74.8 75.5 76.5 77.8 81.4 83.2 87.2 89.5 93.9 94.7 96.4 99.5 113.2 120.7

NAU1087-350 cgr5732-310 CIR280-230 NAU828-350 NAU797-350 HAU1034-390 NAU828-180 NAU1187-170 NAU1255-320 HAU0979-245 HAU1952-250 NAU1006-400 NAU1518-420 HAU2008-170 NAU1230-350 NAU3674-260 NAU1221-310 NAU3949-220 NAU911-500 HAU3109-250 HAU2783-310 Gh229-120 NAU1042-270 NAU2584-360 NAU1590-230 CIR219-135 BNL1611-200 NAU3729-270 NAU4896-150 NAU3096-220 NAU986-210 NAU2380-220 NAU2062-510 NAU2894-180 NAU2274-130 HAU3069-240 NAU3761-900 NAU6105-230 NAU3652-250 BNL1671-130 JESPR236-130 BNL3347-130 NAU879-200 NAU3110-260 CIR222-300 HAU1785-250 MUSB1056-240 NAU3405-180 NAU4907-400 NAU3096-170 BNL632-310 NAU2503-350 STV103-380

A6(Chr.6) 0.0

cer0077-170

13.6

NAU3427-500

20.3

NAU3427-230

29.6

cer0077-260

48.3 0.0 2.5 9.6 15.6 17.2 19.7 21.6 22.3 23.7 24.9 25.9 26.9 28.6 29.8 30.4 31.6 32.1 36.0 36.9 37.5 39.0 42.6 42.8 44.2 46.3 48.8 52.1 53.5 54.7 57.6 58.3 61.5 64.5 67.6 70.3 75.2 76.3 78.5 80.5 88.5

NAU3677-160 cgr6019-450 NAU2679-470 cgr5801-160 BNL2884-180 BNL3650-360 NAU2968-260 NAU1385-310 HAU1143-370 NAU1218-160 NAU2417-400 NAU5434-260 NAU3524-260 MUSB0919-380 NAU6387-220 NAU6110-170 NAU3715-310 cgr5562-150 NAU3374-700 NAU5759-300 T1 HAU0940-590 Gh82-160 NAU1496-200 NAU2156-140 cgr5801-130 Gh441-130 NAU3352-360 NAU1027-550 cgr6019-200 NAU3243-220 CIR280-250 NAU5270-180 CIR280-270 Gh449-110 BNL1061-170 Gh32-120 Gh39-140 TMD02-300 CIR203-240 NAU2967-180

D6(Chr.25) 0.0 8.3 15.8 16.7 17.9 19.6 21.7 21.9 23.7 25.7 27.3 27.5 28.3 28.7 29.4 29.8 29.9 30.1 30.4 30.7 30.8 31.3 33.3 34.3 36.3 38.4 38.7 39.5 41.0 44.4 45.4 48.0 50.3 52.0 53.9 57.2 58.5 60.0 61.3 61.8 64.3 76.7 86.1 0.0

JESPR308-270 HAU3258-600 NAU4868-320 dPL0243-510 HAU1931-255 NAU2580-800 HAU3121-260 CIR267-100 NAU433-190 NAU1192-400 NAU6240-170 NAU2714-200 BNL3103-200 NAU2838-130 NAU2963-230 Gh515-120 NAU6398-450 NAU2963-180 NAU2565-180 BNL3103-190 NAU2186-240 JESPR6-270 NAU1219-360 NAU2388-260 NAU5514-250 NAU2119-240 NAU2717-220 NAU2714-220 NAU2397-250 dPL0124-170 BNL3806-170 HAU2367-250 NAU6573-250 NAU5748-160 NAU3935-280 MUCS098-230 NAU3298-400 NAU3715-370 CIR280-260 MUCS098-200 CIR220-230 NAU1606-250 BNL3436-220 NAU7498-450

11.3

cgr5525-320

17.6

cgr5667-170

23.8

NAU7266-300

40.0

cot012-170

45.7 50.9

cot058-190 dPL0375-220

56.3 60.8

cgr6932-170 cgr5525-140

76.5

cgr5525-450

Figure 1. (contd)


449

Meiying Hou et al. the G. hirsutum allele and 660 (36.67%) dominant to the G. tomentosum allele. Construction of linkage map

In this study, polymorphic markers were employed to construct a genetic linkage map using JoinMap 3.0 and 1204 loci, including two phenotypic traits. Seven hundred and forty-five eSSR and 457 gSSR loci were mapped onto 35 linkage groups at LOD ≥ 4 which were assigned to 26 chromosomes of the cotton AD genome. The total recombination length of the present map is 3320.8 cM (figure 1; table 1); however, this obviously underestimates the true

A7(Chr.7) 0.0

NAU2863-230

12.5 16.5 23.8 26.5 29.2 30.7 32.7 36.1 39.1 40.4 44.5 50.5 51.2 55.2 60.1

NAU933-180 NAU4030-170 NAU1043-240 NAU2686-230 NAU1043-220 NAU474-340 BNL1597-200 NAU2820-170 NAU5120-165 cgr6512-170 cgr5175-210 NAU3280-350 cgr6512-210 NAU3919-200 NAU2772-270

72.2 0.0 3.8 5.0 6.6 8.1 11.7

NAU2104-170 NAU6205-390 Gh527-190 cgr6815-140 Gh506-150 cgr5880-130 HAU1172-520

22.3

NAU1186-170

29.7

NAU1086-160

recombination length of the HT map due to the presence of gaps and unlinked markers. In fact, nine linkage groups located on their respective corresponding chromosomes were still separated, including six linkage groups in the At subgenome and three in the Dt subgenome. Adding 30 cM for each known gap (Waghmare et al. 2005), the estimated length of the At subgenome increased to 1830.9 cM of the cotton genome and the average distance of adjacent markers was 3.30 cM while Dt subgenome covered 1759.9 cM, and the average distance of adjacent markers was 2.71 cM. The HT map increased to 3590.8 cM covering approximately 80.7% of the total recombination length of the cotton genome, based on estimates of the map distance of the

D7(Chr.16) 0.0 4.9 10.6 12.6 13.2 16.0 17.6 19.1 21.2 21.5 22.9 24.9 27.6 31.7 35.5 39.7 40.7 41.3 42.3 45.8 47.8 49.4 49.9 51.7 57.2 57.8 64.6 68.6 70.6 72.0 74.3 75.4 76.7 77.7 78.2 79.1 80.5 81.5 83.1 85.2 88.5 91.8 94.3 95.4 102.4 105.4 106.7 111.1 114.0 118.0 121.7 126.6 133.6 140.8 159.6

A8(Chr.8)

HAU1129-280 NAU3053-170 cgr5611-230 NAU4956-220 dPL0364-90 NAU4030-180 NAU3459-180 NAU4029-400 NAU1043-250 NAU474-260 NAU1020-300 HAU3082-180 NAU862-270 NAU3280-400 cgr6012-150 NAU3068-410 NAU1183-320 Gh684-100 NAU450-220 NAU2108-480 NAU493-230 NAU5838-300 NAU1524-160 MUSB1181-270 NAU747-1100 NAU2432-180 MUSB0641-100 NAU6326-460 NAU6430-400 NAU6136-500 NAU6375-240 cgr5594-150 NAU5702-240 cgr5516-200 NAU6722-80 NAU6664-200 NAU6326-350 NAU4017-160 BNL1122-170 NAU1305-190 HAU137-260 NAU839-210 HAU2481-320 dc40255-300 NAU2680-400 NAU7405-300 NAU3911-190 HAU1399-170 NAU2734-230 NAU4082-310 NAU2680-280 NAU3608-240 NAU3279-490 NAU5325-200 NAU766-200 NAU3678-160

0.0 7.3 14.0 14.3 22.7 23.0 31.4 36.5 37.9 41.7 43.3 47.2 49.8 50.9 52.5 53.6 54.3 55.3 56.5 56.9 57.9 58.7 59.6 60.9 62.5 64.7 66.4 77.0 78.0 88.2 93.9

NAU789-210 CIR055-200 NAU6956-200 NAU6959-230 HAU1846-510 MUSS544-150 NAU780-610 NAU2214-255 HAU1846-140 NAU1336-160 NAU1164-170 CIR082-240 NAU1165-400 Gh634-180 Gh669-850 NAU2202-250 TMB04-200 HAU2086-250 NAU3954-500 NAU1531-140 CIR017-450 JESPR127-900 NAU1262-450 Gh532-170 NAU5418-600 HAU1632-400 NAU2731-170 NAU2293-170 NAU1209-210 NAU3904-180 NAU6977-150

Figure 1. (contd)

450


D8(Chr.24) 0.0 7.6 11.9 15.3 15.7 16.8 17.1 18.0 21.7 25.8 28.3 31.8 31.9 33.3 36.9 39.9 42.4 43.3 43.6 46.1 46.4 51.3 52.7 55.4 56.6 57.1 58.9 60.2 62.1 64.1 65.7 66.9 67.9 69.6 70.2 70.4 71.1 71.6 73.2 74.0 74.8 76.1 77.9 82.8 92.2

NAU3192-230 NAU3793-630 NAU2602-270 NAU3668-250 HAU1846-160 NAU3158-350 NAU3904-230 NAU3324-210 NAU7105-420 NAU1350-270 HAU2738-1000 cgr5202-180 NAU7105-200 NAU4099-170 BNL2961-200 HAU2015-250 CIR041-200 NAU6310-230 HAU3247-450 BNL1521-200 JESPR305-270 HAU0356-500 NAU2292-460 NAU1587-160 NAU7133-300 HAU0722-100 NAU3667-230 TMP14-200 HAU2086-350 NAU3952-350 NAU6166-1800 HAU2522-190 NAU1207-220 NAU1531-230 NAU2240-210 NAU3988-200 JESPR33-170 NAU3632-350 CIR119-350 Gh573-200 BNL252-160 NAU1355-220 NAU3769-260 cgr5433-400 NAU6485-230

Genetic mapping of a cross between G. hirsutum and G. tomentosum A9(Chr.9) 0.0 11.1 18.9 22.0 29.5 35.1 37.5 39.4 43.4 46.3 47.2 47.7 48.3 51.2 55.2 55.8 57.0 57.7 58.6 61.4 65.3 67.1 71.3 74.4 0.0 4.8 5.2 9.6 10.5 17.4 17.9 18.3 21.3 22.8 23.7 25.4 26.1 28.6 32.1 33.1 35.5 36.2 40.3 43.0 43.5 44.7 46.1 49.1 49.9 50.2 52.5 56.8 59.1 61.2 66.7

HAU2496-550 NAU5461-500 HAU2496-270 NAU3888-200 Gh486-100 NAU6177-160 NAU2832-700 Gh486-130 TMF18-230 NAU859-200 NAU2200-130 NAU3101-200 NAU3159-310 MUSS432-800 JESPR296-120 TMP01-290 CIR019-250 Gh112-140 cgr5833-130 Gh584-120 NAU5318-480 NAU2964-200 NAU2575-175 Gh111-170 NAU6101-510 BNL3582-300 HAU3027-180 cgr5218-140 cgr6572-900 NAU3967-235 NAU7019-170 cgr6377-230 HAU2852-220 NAU3358-250 NAU2354-130 cgr6572-270 NAU1009-235 NAU1282-160 NAU2723-350 NAU5069-380 cgr5474-200 cgr5867-120 BNL1317-180 Gh247-140 cgr5707-160 NAU799-350 NAU3738-220 JESPR208-60 JESPR290-130 NAU2829-300 NAU3365-300 BNL1414-130 NAU1493-240 NAU5494-200 cgr5474-130 cgr5474-180

D9(Chr.23)

A10(Chr.10)

69.7 73.9 78.7 80.6 81.6 85.1 86.3 87.6 88.6 91.5 95.4 97.4 102.1 114.9 117.0 120.5 123.3

NAU2771-460 HAU2496-240 NAU3888-230 TMF17-230 HAU2067-300 NAU2771-170 CIR019-280 NAU4021-280 TMN07-200 NAU6564-300 NAU6508-350 NAU6700-150 JESPR296-280 NAU6581-200 NAU2575-180 NAU3986-150 NAU2753-150 NAU2322-250 NAU5472-150 cgr5867-400 NAU3966-230 BNL3140-120 NAU3967-350 JESPR290-145 BNL1414-160 Gh247-420 NAU2829-310 NAU5525-210 NAU5454-320 BNL1030-246 JESPR208-120 HAU1572-300 BNL1030-370 NAU6976-260 MUSS151-200 Gh416-190 BNL597-180 NAU7019-150

146.0

Gh416-130

0.0 9.5 11.9 14.2 17.5 18.5 26.6 30.6 32.3 33.1 34.1 35.4 38.3 39.2 43.2 48.4 50.6 55.7 59.8 66.4

0.0 10.3 11.8 13.4 16.3 18.4 21.1 22.7 24.4 30.8 32.4 35.3 37.4 38.6 41.6 44.5 46.8 49.9 53.1 56.7 60.2 62.5 67.0 70.9 71.9 74.3 77.9 79.1 89.1 97.3

NAU2538-190 NAU4071-400 NAU5359-350 NAU1290-260 HAU0611-1100 HAU2681-260 NAU440-270 cgr5406-180 NAU904-450 NAU1368-500 HAU2873-350 HAU2200-230 JESPR56-110 HAU1423-330 dPL0108-230 NAU5362-160 NAU456-200 NAU3574-160 Gh564-200 CIR037-460 NAU3619-360 TMO05-250 BNL1669-135 NAU5438-100 TMA18-180 NAU1408-250 CIR082-400 NAU6476-300 NAU3262-250 TMF09-200

D10(Chr.20) 0.0 4.8 8.7 14.1 14.8 17.5 19.4 22.1 24.3 26.0 27.4 29.5 31.0 33.1 35.0 35.1 37.6 38.5 39.5 40.8 42.3 43.7 44.5 46.7 48.2 48.7 49.5 50.6 52.8 54.2 55.5 56.2 57.8 58.6 60.5 61.6 61.8 62.8 63.7 64.0 65.4 66.7 67.3 67.5 69.3 69.5 70.8 71.9 73.5 74.1 75.7 78.1 79.0 79.5 83.7 88.1 89.4 94.6 97.6 99.2 99.3 109.5 109.6 115.2 119.2

CIR094-80 NAU1066-450 NAU6755-230 NAU5304-180 NAU5307-170 NAU3917-170 NAU2540-170 NAU2698-200 Gh629-200 NAU456-190 NAU3137-150 NAU904-180 BNL2570-230 NAU6693-250 NAU4973-200 HAU2200-500 MUCS576-400 HAU2825-320 NAU3531-230 NAU3434-235 NAU5013-200 cgr6439-160 HAU3104-300 NAU6240-120 NAU5546-160 NAU3618-150 BNL3948-100 TMJ18-320 dPL0108-220 Gh424-170 NAU3574-150 BNL3993-200 HAU2178-235 NAU6365-400 Gh564-160 JESPR171-180 HAU1491-300 HAU2508-340 NAU6269-240 NAU1044-420 NAU1210-400 NAU3665-190 cgr6701-230 NAU3013-250 TMF09-230 NAU6667-400 NAU6515-300 Gh48-100 BNL2882-160 NAU3665-250 NAU3404-180 Gh564-180 BNL3646-150 NAU6495-250 cgr6701-170 NAU1297-420 dc40044-270 Gh428-170 JESPR190-300 NAU6305-160 NAU6463-260 NAU1297-300 NAU2991-210 NAU2933-2150 NAU6659-200

Figure 1. (contd)

4450 cM cotton genome (Rong et al. 2004), and the average distance of 2.98 cM between the adjacent markers. By performing comparative analysis between the HT map and high-density molecular HB map (constructed by Dr Zhang’s group of Nanjing Agricultural University, Guo et al. 2007), we identified 420 common loci. Of these, 186 are

located on At and 234 are located on Dt subgenome (table 2). Comparisons among all common loci revealed that the distribution of loci is largely collinear on homologous chromosomes, accompanied by some small inversions or small rearrangements. Most of the orders of the 420 common loci along the chromosomes of HT and HB are quite similar,


451

Meiying Hou et al. A11(Chr.11) 0.0 10.3 14.9 15.7 17.7 18.7 22.0 22.7 24.4 26.1 26.8 28.1 29.7 31.6 39.4 42.6 46.4 50.2 53.9 55.8 57.8 58.3 58.7 60.1 63.5 67.2 67.5 67.8 68.2 70.3 72.9 75.9 76.7 77.3 77.6 80.0 81.0 86.0 93.9 101.5 104.4 108.2 109.1 110.5 121.8 123.2 128.6 131.5 133.0

NAU429-250 NAU2086-200 NAU6697-230 cgr5428-140 NAU3480-160 NAU2016-800 BNL1231-210 NAU3390-500 BNL1066-130 NAU3770-180 NAU429-225 TMP20-250 NAU5505-190 BNL836-330 NAU4962-200 Gh433-130 Gh316-180 NAU3703-200 BNL3592-190 TMP20-220 Gh316-150 TMC12-200 NAU1014-180 NAU491-200 NAU1014-160 BNL2632-250 NAU6999-100 NAU1063-230 TMN16-170 NAU1049-490 NAU3478-380 BNL4094-190 NAU4962-360 HAU2837-400 NAU3367-250 Gh433-160 NAU6334-250 NAU2852-300 NAU6673-210 NAU1162-190 Gh329-170 NAU5217-350 NAU1103-170 NAU4901-230 BNL1151-170 NAU7051-650 NAU7649-120 BNL2589-230 NAU1064-420

D11(Chr.21) 0.0 8.1 10.3 13.5 25.1 29.1 29.7 32.9 33.3 35.8 37.2 39.3 40.5 43.1 47.3 47.8 51.0 53.5 54.4 56.2 56.4 58.1 58.5 59.3 59.4 60.6 61.8 62.5 63.7 65.3 65.5 65.7 66.8 67.7 69.2 69.7 73.3 75.1 77.1 78.8 78.9 79.8 81.2 82.5 83.7 84.0 85.3 85.8 86.1 87.4 89.1 90.5 91.9 92.0 95.6 96.5 99.5 100.3 102.4 104.6 106.0 107.4 108.8 111.3 112.2 113.9 119.2 127.8 139.7

NAU6146-150 BNL1408-180 CIR414-200 TMG06-200 NAU4026-230 NAU4086-320 NAU3704-480 cgr5148-160 HAU1809-590 NAU6524-290 NAU6128-300 NAU6224-240 HAU2559-250 MUSB0849-230 cgr5015-200 HAU1311-280 cgr5747-360 NAU6594-350 cgr5412-150 NAU3354-200 NAU429-235 JESPR245-130 NAU987-150 NAU1270-150 Gh434-200 NAU5468-270 NAU2950-170 NAU7100-400 HAU1805-330 CIR077-240 NAU3240-410 cgr5233-150 NAU1342-300 NAU1057-300 NAU6658-150 NAU6598-240 cgr5543-160 NAU6697-190 NAU4865-190 NAU6593-300 cgr5602-160 NAU7100-350 NAU7140-120 NAU6530-310 NAU3377-170 cgr5543-280 JESPR154-140 NAU5064-250 NAU6222-250 NAU7139-200 NAU2257-350 NAU980-460 NAU3748-600 NAU6267-350 NAU3334-650 BNL1705-160 NAU1233-260 NAU3657-170 NAU3481-200 NAU3341-450 NAU5468-400 NAU2998-180 NAU3621-160 NAU1038-300 BNL3449-280 NAU3341-250 NAU3731-170 cgr5810-150 BNL1034-230 NAU3341-520 HAU2004-430

D12(Chr.26)

A12(Chr.12) 0.0 6.0 10.1 12.6 13.7 19.6 20.5 21.2 25.1 28.9 34.8 43.5 43.6

HAU2663-270 NAU2116-400 NAU8660-150 NAU2902-120 HAU1081-170 dPL0240-180 JESPR270-130 NAU3401-470 NAU3401-410 JESPR270-80 dPL0243-200 cgr6439-200 dPL0243-190

55.3 59.5

cgr6439-180 BNL3261-240

66.3

JESPR295-100

75.6 77.3 0.0 7.7 15.0 20.8 25.9 30.1 34.2 37.6 41.6 45.1 45.6 45.9 48.6 51.8 52.0 55.0 55.4 58.0 59.4 61.2 63.5 64.7 66.5

dPL0243-320 cgr6439-240 NAU1341-200 NAU3519-220 CIR348-155 CIR362-200 NAU3305-140 w1073-150 STV023-170 BNL1679-170 NAU5047-220 cgr5151-140 NAU5419-350 NAU1151-160 NAU3291-400 HAU1828-180 HAU0717-380 NAU5047-250 NAU852-500 dPL0380-170 NAU2868-400 NAU5419-230 Gh188-340 NAU4020-490 NAU971-200 NAU1021-200 BNL1673-175 dPL0380-180 NAU3611-1300 NAU3236-220

66.6 69.3 72.7 80.6

0.0 4.5 6.9 9.6 10.5 10.6 15.3 16.9 17.5 20.4 21.4 23.8 26.1 27.0 27.1 30.9 31.3 32.9 34.4 37.7 38.4 39.9 42.1 42.2 44.1 44.3 45.8 46.2 47.6 48.7 49.1 49.3 51.3 52.7 54.6 58.3 62.7 65.7 68.5 75.6 86.9

NAU3897-160 BNL3816-170 NAU1331-200 NAU5397-750 NAU3519-250 NAU3006-550 NAU6659-350 NAU4925-150 ne2 NAU1301-240 NAU1581-250 NAU2116-680 NAU3647-320 NAU3920-280 Gh603-150 NAU1301-260 MUCS590-450 HAU1292-310 HAU1452-240 HAU0908-280 NAU786-200 NAU3897-170 NAU877-200 NAU2010-280 NAU2715-290 JESPR295-14 BNL3368-300 NAU1565-380 Gh568-120 NAU3519-210 BNL2495-200 NAU1024-220 w1075-100 NAU1274-250 HAU1459-400 NAU4912-150 NAU5650-200 NAU1558-185 BNL3261-210 NAU1231-230 Gh629-170 NAU1231-290

Figure 1. (contd)

although several orders of loci exhibit differences due to reversals of neighbouring markers, which could be explained by occasional missing data or scoring errors in this small population (of 93 individuals). 452

In the present map, 55 duplicated loci were identified which combined with the 420 common loci, bridge 13 expected homeologous At/Dt chromosomes in tetraploid cotton. There are three duplicate loci on A1 and D1


Genetic mapping of a cross between G. hirsutum and G. tomentosum A13(Chr.13) 0.0 5.5 11.3 14.8 18.8 19.9 22.0 23.7 24.7 26.6 27.3 29.1 30.9 34.2 36.5 37.6 38.8 39.1 39.6 40.5 41.9 43.3 44.4 45.6 46.4 46.5 47.7 48.4 48.8 49.7 51.5 52.4 54.9 56.1 57.7 60.5 62.5 63.4 67.5 69.3 75.3 80.4 85.7 91.3 92.6

D13(Chr.18)

NAU3017-600 NAU4875-270 cgr5554-270 NAU3017-230 cgr5331-260 cgr5554-140 NAU3970-480 MUCS145-240 HAU1182-400 NAU748-300 NAU3398-210 HAU0539-235 CIR201-150 NAU1530-420 NAU4103-170 NAU4102-250 BNL2652-170 dPL0083-160 Gh592-130 Gh34-130 dPL0308-130 CIR144-470 HAU3061-410 NAU6746-200 NAU952-200 TME17-225 NAU1201-170 NAU3307-300 NAU817-200 NAU3138-180 cgr6359-200 NAU3989-300 JESPR153-135 MUCS440-480 HAU3061-230 NAU3570-230 cgr5242-135 TMP01-200 BNL1707-170 HAU2631-320 HAU2901-400 NAU6699-390 NAU6582-260 NAU856-220 cgr6812-150

0.0 18.6 24.2 28.0 31.9 33.3 34.0 35.4 37.4 37.9 40.4 45.9 47.4 48.2 50.7 57.6 59.9 60.6 62.4 64.7 66.1 69.0 71.5 73.2 74.7 75.7 78.1 78.8 78.9 79.9 80.3 85.1 85.3 85.4 87.6 88.0 89.0 89.4 89.7 91.4 92.7 94.2 96.6 97.9 98.2 101.0 102.8 103.9 106.1 106.8 109.4 111.9 114.2 115.4 117.7 121.5 127.9

NAU2886-300 NAU2980-260 NAU3991-250 NAU3827-230 NAU2780-250 HAU2977-210 HAU2276-1000 NAU2765-400 NAU2866-350 NAU2886-500 NAU3843-250 NAU3861-250 cgr5564-150 HAU2276-450 NAU3130-200 JESPR178-230 CIR221-200 NAU6724-350 NAU6738-350 MUCS128-190 NAU3011-200 NAU5275-240 cgr5446-210 BNL3479-450 CIR099-80 NAU3184-200 Gh501-200 JESPR56-220 NAU6305-170 HAU1036-360 HAU1036-240 NAU3638-210 NAU2443-140 HAU1909-300 dPL0308-140 NAU2443-130 NAU5392-240 NAU2697-180 TMB04-220 cgr5242-130 NAU6426-400 dPL0161-200 NAU1023-230 HAU083-250 dPL0308-135 NAU3816-250 BNL1721-205 Gh691-130 NAU2697-160 cgr5390-150 NAU6682-250 NAU3848-480 Gh433-70 BNL3558-220 NAU3534-250 NAU6672-270 cgr5856-480

Figure 1. (contd)

homeologous chromosomes, one each on A2/D2, A5/D5, A12/D12; two each on A6/D6, A11/D11, A13/D13; four each on A3/D3, A7/D7, A8/D8; five each on A4/D4; seven each on A10/D10; 13 each on A9/D9. Two postpolyploidization reciprocal translocations of A2/A3 and A4/A5 in the At subgenome were also further confirmed by several homologous loci, such as BNL3590 and NAU805 on A2 and D3, NAU5035 on A3 and D2 and Gh166, JESPR184 and NAU2376 on A5 and D4 (tables 1 and 2).

The density of marker varies between chromosomes, ranging from 1.83 cM (D10) to 5.28 cM (A7), which largely benefits from the greater number of loci on the Dt subgenome. There are 89 markers on chromosome A5, whose polymorphic loci are the greatest among the 26 chromosomes of cotton. The chromosome with the fewest markers is A7, with 25 polymorphic loci. Chromosome A5 has the greatest length, covering 219.3 cM, compared with the shortest genetic distance of 86.9 cM of chromosome D12. The largest gap between two adjacent loci is 36.3 cM on chromosome D3. The number of intervals remaining in the tetraploid map > 10 cM is 43, which are almost evenly distributed on the At and Dt subgenomes (21 vs 22). There are 554 and 650 markers anchored on the At subgenomes and Dt subgenomes, respectively. A total of 334 eSSR loci were localized to the At subgenome and 411 eSSR loci to the Dt subgenome, with a ratio of At : Dt = 1 : 1.2. In addition, 218 gSSR loci were localized to the At subgenome and 239 gSSR loci to the Dt subgenome, also with a ratio of At : Dt = 1 : 1.1. Moreover, more gSSR loci are distributed on the Dt subgenome than on the At subgenome, which suggests that the Dt subgenome has a higher DNA sequence divergence rate between tetraploid cotton species than the At subgenome (Small and Wendel 2000, 2002). Analysis of segregation distortion and the factors that contribute to this process

Of the 1800 discrete loci, 33.7% (607/1,800) loci showed segregation distortion (P < 0.05). Of these, 58.8% (357/607) were mapped on cotton chromosomes, with 265 loci segregating towards the maternal parent G. hirsutum allele (89 loci appear in At subgenome and 176 loci appear in Dt), 42 loci towards the paternal parent G. tomentosum allele (13 loci appear in the At subgenome and 29 loci in Dt) and 50 loci showed heterozygous allele (19 loci appear in the At subgenome, with 31 loci in Dt). In summary, these segregation distorted loci are unevenly distributed on 26 chromosomes, with 1–33 loci on each chromosome (table 3). More distorted loci are located on the Dt subgenome than on the At subgenome (236 vs 121), and approximately three-fourths are skewed towards the female parent. Segregation distortion loci are distributed on all chromosomes, with most on A5 (33 loci) but the highest percentage (>50%) of segregation distorted loci on A10, D2, D3, D8 and D5 in HT. The results also show that there are 35 segregation distortion regions (SDRs) on 26 cotton chromosomes, indicating that the distorted loci identified in this study are distributed unevenly on the cotton genome. SDRs are four times higher in the Dt subgenome than in the At subgenome. In the Dt subgenome, except for D1 and D9 chromosomes without SDRs, four SDRs were found on D5, D8 and D12, three on D2, D3 and D11, two on D4 and D7 and one on D6, D10 and D13. In the At subgenome, only four chromosomes have SDRs; two SDRs appears on A5, A10 and A12 and one appears on A8. The distribution of SDRs is very uneven


453

Meiying Hou et al. Table 1. Loci composition and recombination distances of chromosomes based on SSRs in the F2 population.

Chromosome

Total loci

Actual map length (cM)

Estimated map length (cM) with gaps

Actual average distance between loci (cM)

No. of gaps > 10 cM (largest)

A-subgenome A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 At-total

38 30 32 38 89 45 25 31 56 30 49 46 45 554

157.0 106.0 124.0 90.1 219.3 136.8 102.0 93.9 141.2 97.3 133.0 157.9 92.6 1650.9

187.0 136.0 124.0 90.1 219.3 166.8 132.0 93.9 171.2 97.3 133.0 187.9 92.6 1830.9

4.92 4.53 3.87 2.37 2.46 3.71 5.28 3.03 3.06 3.24 2.71 4.08 2.06 3.30

4 (32.37) 2 (23.7) 3 (24.9) 0 (9.8) 0 (10.0) 2 (18.8) 3 (12.5) 2 (10.6) 1 (11.1) 1 (10.3) 2 (11.3) 1 (11.7) 0 (6.0) 21 (32.37)

D-subgenome D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 Dt-total

43 46 43 37 53 53 56 45 39 65 71 42 57 650

135.9 125.0 162.5 91.7 120.7 162.7 159.6 92.2 146.0 119.2 139.7 86.9 127.9 1669.9

165.9 125.0 192.5 91.7 120.7 192.7 159.6 92.2 146.0 119.2 139.7 86.9 127.9 1759.9

3.86 2.72 4.48 2.48 2.28 3.64 2.85 2.05 3.74 1.83 1.97 2.07 2.24 2.71

3 (14.3) 2 (22.8) 4 (36.3) 0 (8.9) 1 (13.7) 4 (16.24) 1 (18.8) 0 (9.3) 2 (22.7) 1 (10.2) 2 (12.0) 1 (11.3) 1 (18.6) 22 (36.3)

1204

3320.8

3590.8

2.98

43 (36.3)

Total

between the At and Dt subgenomes; the phenomenon of uneven distribution of SDR was also reported by Zhang et al. (2008). To determine which factors contribute to distorted segregation, the allele frequency (p = q) and the distribution of different genotype frequencies (p2 :2pq:q2 ) in the F2 population were analysed for all 226 codominant markers (table 4). The results indicate that more than half of distorted segregation loci were influenced only by zygotic selection, while one-third were influenced by both gametic and zygotic selections and only a few were influenced only by gametic selection. Comparison of gSSR and eSSR distorted segregation loci showed that the number of eSSR distorted segregation loci is 1.5–2 times that of gSSR, which suggests that eSSR loci (functional loci) may tend to be distorted. Mapping of genes controlling hairiness and nectarilessness

Heavy/dense trichome distributions on leaves and stems of G. tomentosum, which are morphological traits that increase 454

resistance to sucking pests, have been transferred into G. hirsutum (Lee 1985). The gene controlling the trichome trait was termed T1 by Lee (1985). In the current study, in the F2 population of G. hirsutum × G. tomentosum, 67 individuals exhibited heavy, dense trichomes like the parent G. tomentosum and 26 exhibited normal or sparse trichomes like the maternal parent G. hirsutum L. acc 08N2162. The segregation ratio for heavy, dense trichomes : normal or sparse trichomes fits a ratio of 3 : 1 (χ 2c = 0.2903 < χ 20.05,1 = 3.841). T1 is located in the central region of chromosome A6, 0.6 cM from the nearest locus, NAU5759-300 and 1.5 cM from HAU0940-590. Nectarilessness, another morphological trait that confers resistance to pests, is controlled by two duplicated recessive genes in G. tomentosum, i.e., ne1 and ne2 , which are located on the A and D genomes, respectively (Rhyne 1965). Since Meyer and Meyer (1961) first transferred the nectariless traits from G. tomentosum to G. hirsutum (Holder et al. 1968), some upland cotton lines have both ne1 and ne2 genes and others may have only one of them or not. In this study, in the F2 population of G. hirsutum × G. tomentosum,


Genetic mapping of a cross between G. hirsutum and G. tomentosum Table 2. Common marker loci distributed on chromosomes between the two maps. Homeologous Chr. Pair

At HB/HT

Dt HB/HT

At/Dt HT

Duplicated loci NAU4073-170/NAU4073-180; BNL1355-600/BNL1355-170; NAU6951-270/NAU6951-300 cgr5876-160/cgr5876-170 NAU3884-190/NAU3884-240; NAU3309-190/NAU3309-170; NAU5443-180/NAU5443-160; NAU889-350/NAU889-190 NAU1577-230/NAU1577-210; NAU2477-230/NAU2477-250; NAU6992-240/NAU6992-220; NAU1325-170/NAU1325-150; NAU2235-220/NAU2235-150 NAU1221-235/NAU1221-310 NAU3715-310/NAU3715-370; CIR280-250/CIR280-260 NAU4030-170/NAU4030-180; NAU1043-240/NAU1043-250; NAU474-340/NAU474-260; NAU3280-350/NAU3280-400 HAU1846-510; HAU1846-140/HAU1846-160; HAU2086-250/HAU2086-350; NAU1531-140/NAU1531-230; NAU3904-180/NAU3904-230 HAU2496-550; HAU2496-270/HAU2496-240; NAU3888-200/NAU3888-230; JESPR296-120/JESPR296-280; CIR019-250/CIR019-280; NAU2575-175/NAU2575-180; NAU3967-235/NAU3967-350; NAU7019-170/NAU7019-150; cgr5867-120/cgr5867-400; Gh247-140/Gh247-420; JESPR208-60/JESPR208-120; JESPR290-130/JESPR290-145; NAU2829-300/NAU2829-310; BNL1414-130/BNL1414-160 NAU904-450/NAU904-180; HAU2200-230/HAU2200-500; dPL0108-230/dPL0108-220; NAU456-200/NAU456-190; NAU3574-160/NAU3574-150; Gh564-200/Gh564-160; Gh564-180; TMF09-200/TMF09-230 NAU429-250; NAU429-225/NAU429-235; NAU6697-230/NAU6697-190 NAU3519-220/NAU3519-250; NAU3519-210 dPL0308-130/dPL0308-135; cgr5242-135/cgr5242-130 BNL3590-180/BNL3590-185; NAU805-500/NAU805-200 NAU5035-240/NAU5035-250 NAU2376-380/NAU2376-1200; NAU2376-310; JESPR184-170/JESPR184-190; GH166-190/GH166-200

1

17

9

3

2 3

7 15

21 23

1 4

4

9

14

5

5 6 7

34 12 4

20 14 19

1 2 4

8

7

16

4

9

23

13

13

10

10

23

7

11 12 13

25 8 15

27 14 21

2 1 2 2(A2/D3) 1(D2/A3) 3(D4/A5)

186

234

55

Total

71 individuals had nectaries, while 22 were nectariless. The segregation ratio for nectary : nectariless also fit a ratio of 3:1 (χ 2c = 0.0323 < χ 20.05,1 = 3.841). This trait is located near the top region of chromosome 26, i.e. D12, 0.6 cM from the nearest locus NAU4925-150 and 2.9 cM from NAU1301-240 (figure 1). According to Meyer and Meyer (1961), Holder et al. (1968), Rhyne (1965) (by interspecific hybrids involving diploids and amphidiploids) and Endrizzi et al. (1984) (based on analysis of monosomic lines), the gene controlling nectarilessness on the D genome is ne2 , as the dominant Ne2 confers full-sized leaf nectarines. Therefore, in the current study, we determined that the gene controlling nectarilessness is ne2 and not ne1 (as reported by Waghmare et al. (2005)) as Ne1 confers smaller leaf nectaries (Holder et al. 1968). The results also indicate that G. hirsutum L. acc 08N2162 is genotyped as ne1 ne1 Ne2 Ne2 , since the genotype of G. tomentosum is known to be ne1 ne1 ne2 ne2 (Meyer and Meyer 1961). Our result further confirmed the previous studies that Ne2 gene was assigned to chromosome 26 or D12 by both linkage mapping and monosomic test (Holder et al. 1968; Endrizzi et al. 1984). The possible reason of this

incongruence with Waghmare et al. (2005) may be the use of different upland cotton parent crossed with G. tomentosum. With the different background, there may be different phenotypes (nectary size) of nectaries gene.

Discussion Comparison of chromosome structural changes

The polymorphism rates of SSR markers and genetic diversity influence the density of genetic maps that are constructed; the higher the polymorphism rate, the higher the map density. High polymorphism rates between parents indicate that the parents are remotely related. Normally, among G. hirsutum, 4.13–7.9% polymorphism rates are observed, while there is 18.2–47.9% polymorphism between G. hirsutum and G. barbadense (Park et al. 2005; Shen et al. 2005; Han et al. 2006; Guo et al. 2007; He et al. 2007; Qin et al. 2008; Lin et al. 2009; Zhang et al. 2009; Yu et al. 2011). In this study, a total of 8848 SSR (including gSSR and eSSR) primer pairs were used to screen polymorphisms


455

Meiying Hou et al. Table 3. Chromosome tagging information of deviated segregation loci. Chromosome

Distorted to G. hirsutum

Distorted to G. tomentosum

Distorted to F1

Total distorted loci

Frequency (%)

SDRs

A-subgenome A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 At-total

8 4 1 7 23 3 1 2 8 16 5 11 0 89

1 1 0 1 2 0 0 2 0 0 3 2 1 13

2 0 1 1 8 0 1 0 0 1 1 4 0 19

11 5 2 9 33 3 2 4 8 17 9 17 1 121

28.95 16.67 6.25 23.68 37.08 6.67 8.00 12.90 14.29 56.67 18.37 36.96 2.22 21.84

0 0 0 0 2 0 0 1 0 2 0 2 0 7

D-subgenome D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 Dt-total

3 24 21 14 25 14 10 18 3 10 17 14 3 176

3 0 2 3 3 2 3 1 1 1 2 2 6 29

1 3 0 1 0 0 2 5 4 2 9 1 3 31

7 27 23 18 28 16 15 24 8 13 28 17 12 236

16.28 58.70 53.49 48.65 52.83 30.19 26.79 53.33 20.51 20.00 39.44 40.48 21.05 36.31

0 3 3 2 4 1 2 4 0 1 3 4 1 28

265

42

50

357

29.65

35

Total

between G. hirsutum and G. tomentosum. Approximately 16% of the SSR primer pairs showed polymorphism. The polymorphism rate of HT was lower than that of interspecies of HB and higher than that of intraspecies of HH (among G. hirsutum). These results support previous studies suggesting that G. hirsutum is more closely related to G. tomentosum than to G. barbadense (Saha and Zipf 1997), which is

consistent with the results of Wendel and Cronn (2003). The construction of a higher density genetic map of HT necessitates the development of more new SSR markers or new types of markers. Comparisons among all common loci revealed that the distribution of loci is largely collinear on homologous chromosomes, and the orders of most of the loci along the

Table 4. Analysis of allele frequency and the distribution of different genotype frequencies to infer genetic distortion. Gametic selection eSSR Total (%)a (%)b

Gametic and zygotic selection simultaneously gSSR eSSR Total (%)d (%)d (%)b

gSSR (%)a

5% 1% 0.1%

8 (34.8) 4 (57.1) 0 (0.0)

15 (65.2) 3 (42.9) 0 (0.0)

23 (20.9) 7 (11.7) 0 (0.0)

23 (41.1) 13 (36.1) 10 (31.3)

33 (58.9) 23 (63.9) 22 (68.7)

56 (50.9) 36 (60.0) 32 (57.1)

8 (25.8) 6 (35.3) 12 (50.0)

23 (74.2) 11 (64.7) 12 (50.0)

31 (28.2) 17 (28.3) 24 (42.9)

12 (40.0)

18 (60.0)

30 (13.3)

46 (37.1)

78 (62.9)

124 (54.9)

26 (36.1)

46 (63.9)

72 (31.9)

Total (average)e

gSSR (%)c

Zygotic selection eSSR Total (%)c (%)b

Significance level

a Percentage of segregation distortion loci influenced by only gametic selection. b Percentage of segregation distortion loci all codominant. c Percentage of segregation distortion loci influenced by only zygotic selection. d Percentage of segregation distortion loci influenced by both gametic and zygotic e Average percentage of segregation distortion loci at all significance levels.

456

selections.


Genetic mapping of a cross between G. hirsutum and G. tomentosum chromosomes of HT and HB are very similar, although the orders of several loci are different due to the reversed orders of neighbouring markers, which could be explained by the occasional absence of data or by scoring errors in this small population (93 individuals). These results support the results of Waghmare et al. (2005), who determined that chromosome translocations did not play a role in the divergence of G. hirsutum, G. barbadense and G. tomentosum. Comparisons among all 420 common markers also revealed that no significant chromosomal rearrangements exist between G. tomentosum and G. barbadense, but at least three small, distinct inversions occurred on A3, D1 and D7. On the terminus of the A3 chromosome, the order of five loci, NAU5444, NAU3995, NAU1167, NAU5233 and NAU3016 in the HB map, is reversed in HT, but an occasional error occurred due to the fact that NAU3995 is very close to NAU1167. This supports the results of Waghmare et al. (2005). Two parallel cases were also discovered, including the internal region on D1, involving four loci, i.e. BNL2646, JESPR298, JESPR205 and MUCS322 in HT and at the terminus on D7 involving three loci including NAU3053, NAU4030 and NAU3459. The presence of these small, distinct inversions implies that tetraploid cotton genome divergence occurred following polyploidy formation due to tetraploid cotton monophyletic origin. Segregation distortion and comparisons with the HB molecular map

Segregation distortion phenomena have been popular subjects of genetic research since Mangelsdorf and Jones (1926) first reported them in maize. These phenomena are also commonly observed in both interspecific and infraspecific crosses of cotton (Lacape et al. 2003; Shen et al. 2005; Frelichowski et al. 2006; Han et al. 2006; Zhang et al. 2009) and other plants (Tanksley et al. 1992). These events are skewed mainly towards the maternal parent (TM-1). This study also confirms these phenomena. Moreover, higher percentage of segregation distortion loci were observed in HT than in HB. While segregation distortion loci in HB are distributed on only a few chromosomes such as A7 and its homeologous chromosome D7, segregation distortion loci are distributed on many chromosomes in HT. There were a total of 35 SDRs, which clustered on three chromosomes in At and eight in Dt. One possible reason for the severe skewed segregation, which involves functional genes, is that unlike the cultivated species G. barbadense, the wild species G. tomentosum has not been domesticated to fit artificial conditions, leading to the lower transmission rate of some lower viable or unviable gametes/zygotes containing the unfavourable dominant alleles from G. tomentosum, even though this species is closely allied with G. hirsutum. Kumar et al. (2007) also confirmed that gametophytic competition among gametes for preferential fertilization resulted in severe skewed segregation in

wheat. Another possible reason for the severe skewed segregation is the small size of the F2 population (93 individuals), which prompted us to enlarge the population for mapping. Most of the segregation distortion loci (265/357) skewed towards the maternal parent, G. hirsutum. Therefore, when G. tomentosum is used as a donor parent to develop chromosome segment substitution lines (CSSLs) in the upland cotton background, some chromosome segments with segregation distortion loci may not be easy to transfer into upland cotton. An enlarged backcross population might be needed to overcome this problem. Some segregation distortion loci (50/357) skewed towards the F1 hybrid, implying that the plants possessing these loci tend to be heterozygous in F1 , which is consistent with the results of Zhang et al. (2011).

Applicability of SSR-based genetic map to breeding

High-density genetic maps are important tools for mapping QTLs/genes conferring traits of interest, allowing mapbased gene cloning and marker-assisted selection. In particular, PCR-based SSR marker genetic maps are important tools for developing CSSLs for genetic dissection of important traits. In addition, such maps are useful for designing CSSLs for cotton breeding, as SSR markers are portable, codominant, simple and informative and SSR mapping is a costeffective and rapid. Reinisch et al. (1994) first reported a genetic linkage map for cotton and Rong et al. (2004) produced a saturated tetraploid cotton map based on the results of Reinisch et al. (1994); this map comprises 2584 loci at intervals of 1.72 cM and covers 4447.9 cM. Yu et al. (2011) recently produced a high density map based on the results of Zhang et al. (2008); this map includes 2316 loci on the 26 cotton chromosomes and covers 4418.9 cM, with an average of 1.91 cM between adjacent markers. Yu et al. (2012) constructed a HB genetic map comprising 2072 loci (1825 SSRs and 247 SNPs) and covering 3380 cM of the cotton genome (AD), with an average marker interval of 1.63 cM. Zhao et al. (2012) published an updated map based on Guo et al. (2007), consisting of 3414 loci in 26 linkage groups covering 3667.62 cM, with an average interlocus distance of 1.08 cM. To date, however, even though several genetic maps of cotton genomes have been constructed using diverse DNA molecular markers and mapping populations (Ulloa et al. 2002; Zhang et al. 2002, 2009, 2012; Lacape et al. 2003, 2009; Mei et al. 2004; Rong et al. 2004; Frelichowski et al. 2006; Lin et al. 2009; Yu et al. 2011; Lopez-Lavalle et al. 2012), only one map produced from a cross between G. hirsutum and G. tomentosum has been reported using RFLP markers (Waghmare et al. 2005), but this map is not suitable for high throughput applications for cotton breeding, as it is based on hybridization-based RFLP markers rather than PCR-based markers. In this study, a detailed genetic linkage map of a cross between G. hirsutum and G. tomentosum was produced entirely from SSR-based markers (PCR-based) and covering


457

Meiying Hou et al. a large region of the cotton genome, with 1204 loci (including two phenotypic traits, 745 eSSR loci and 457 gSSR loci) mapped onto 26 chromosomes of the cotton genome at LOD ≥ 4. This is the first entirely SSR-based map produced from a cross of G. hirsutum and G. tomentosum, with more than 1000 loci on the map and at least 25 loci on each chromosome. This map is sufficient for markerassisted selection, which will lay the foundation for developing chromosome segment substitution lines from G. tomentosum in an upland cotton background by marker-assisted high-throughput selection. Acknowledgements We thank Dr Yuanming Zhang and Shangqian Xie (State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University) for their constructive comments and suggestions. We are also grateful to Dr Kunbo Wang, vice director of Cotton Research Institute of Chinese Academy of Agricultural Sciences, for providing us pollens of G. tomentosum at Hainan wild cotton growing garden. This work was supported by The Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Received 8 April 2013, in revised form 12 May 2013; accepted 19 June 2013 Published on the Web: 27 November 2013


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