Open Access

Characterization of eleven monosomic alien addition lines added from Gossypium anomalum to Gossypium hirsutum using improved GISH and SSR markers

BMC Plant BiologyBMC series – open, inclusive and trusted201616:218

https://doi.org/10.1186/s12870-016-0913-2

Received: 12 June 2016

Accepted: 29 September 2016

Published: 7 October 2016

Abstract

Background

Gossypium anomalum (BB genome) possesses the desirable characteristics of drought tolerance, resistance to diseases and insect pests, and the potential for high quality fibers. However, it is difficult to transfer the genes associated with these desirable traits into cultivated cotton (G. hirsutum, AADD genome). Monosomic alien addition lines (MAALs) can be used as a bridge to transfer desired genes from wild species into G. hirsutum. In cotton, however, the high number and smaller size of the chromosomes has resulted in difficulties in discriminating chromosomes from wild species in cultivated cotton background, the development of cotton MAALs has lagged far behind many other crops. To date, no set of G. hirsutum-G. anomalum MAALs was reported. Here the amphiploid (AADDBB genome) derived from G. hirsutum × G. anomalum was used to generate a set of G. hirsutum-G. anomalum MAALs through a combination of consecutive backcrossing, genomic in situ hybridization (GISH), morphological survey and microsatellite marker identification.

Results

We improved the GISH technique used in our previous research by using a mixture of two probes from G. anomalum and G. herbaceum (AA genome). The results indicate that a ratio of 4:3 (G. anomalum : G. herbaceum) is the most suitable for discrimination of chromosomes from G. anomalum and the At-subgenome of G. hirsutum. Using this improved GISH technique, 108 MAAL individuals were isolated. Next, 170 G. hirsutum- and G. anomalum-specific codominant markers were obtained and employed for characterization of these MAAL individuals. Finally, eleven out of 13 MAALs were identified. Unfortunately, we were unable to isolate Chrs. 1Ba and 5Ba due to their very low incidences in backcrossing generation, as these remained in a condition of multiple additions.

Conclusions

The characterized lines can be employed as bridges for the transfer of desired genes from G. anomalum into G. hirsutum, as well as for gene assignment, isolation of chromosome-specific probes, development of chromosome-specific “paints” for fluorochrome-labeled DNA fragments, physical mapping, and selective isolation and mapping of cDNAs/genes for a particular G. anomalum chromosome.

Keywords

Gossypium hirsutum Gossypium anomalum Chromosome Monosomic alien addition line Genomic in situ hybridization Microsatellite marker

Background

Cotton is the leading natural textile fiber crop in the world. Approximately 5 % of the world’s arable land is used for cotton planting, generating about $630.6 billion in 2011 [1]. Cotton belongs to the Gossypium genus of Malvaceae, which contains five tetraploid species (2n = 4× = 52, AADD genome) and approximately 45 diploid species (eight genomes from A to G and K, 2n = 2× = 26) [2]. Upland cotton (G. hirsutum) is the most widely cultivated species and its production accounts for over 95% of the world’s cotton production [3]. During the development of its cultivars, cotton has been subjected to long-term artificial selection, which narrowed its genetic base and gave rise to several difficulties in breeding. Cotton breeders face a scarcity of genetically diverse resources, therefore expanding the genetic base of cotton cultivars is imperative. Wild or untapped species have many excellent characteristics and contain abundant desirable genes, which have yet to be unlocked by pre-breeding. G. anomalum (2n = 2× = 26, BB genome) which is native to Africa, mainly Angola and Namibia [2], has the favorable characteristics of drought tolerance and resistance to diseases (cotton wilt, angular leaf spot) and insect pests (springtails, aphids): more importantly, it also possesses genes with the potential to produce high quality fibers (good fiber strength and fineness) [4] and cytoplasmic male sterility [57]. However, it is difficult to transfer these desirable genes into cultivated cotton through conventional breeding methods due to the isolation of wild species from cultivated species, which limits chromosome pairing and genetic recombination.

Monosomic alien addition lines (MAALs) contain only one alien chromosome in addition to the receptor background chromosomes. MAALs can be used as a bridge to transfer desired genes from wild species into G. hirsutum [8]. Over the past two decades, MAALs have been widely available for numerous crops [9], and these can be used for effectively identifying favorable genes in wild species, allowing for more accurate and faster transfer of such genes to create introgression lines, the effect of specific alien chromosomes to be examined, homeologies with chromosomes of cultivated species to be compared [10, 11], and physical maps of specific chromosomes to be constructed [12]. In cotton, however, the high number and smaller size of the chromosomes has resulted in difficulties in discriminating chromosomes from wild species in cultivated cotton background, therefore the development of cotton MAALs has lagged far behind many other crops. No set of cotton MAALs was reported until cotton molecular genetic maps were constructed and a genomic in situ hybridization (GISH) technique for cotton was developed. Previously, only one complete set of G. hirsutum-G. australe MAALs had been developed using simple sequence repeat (SSR) markers and GISH [9, 13, 14]. Two G. hirsutum-G. somalense MAALs and several G. hirsutum-G. sturtianum MAALs have also been obtained [11, 15].

In this study, the G. hirsutum-G. anomalum hexaploid was used as a maternal parent in the continuous backcrossing with upland cotton (recipient parent, G. hirsutum acc. TM-1), and eleven MAALs were isolated using GISH and SSR markers. These MAALs may be useful for mining and transferring favorable genes from G. anomalum into G. hirsutum on a genome-wide scale, mapping genes on chromosomes, analyzing genome structure and evolution, and micro-cloning for chromosome-specific library construction.

Results

Alien chromosomes from G. anomalum in G. hirsutum were examined by the improved GISH

The GISH technique used in our previous research was improved as follows. Genomic DNA extracted from G. anomalum and G. herbaceum was labeled with digoxigenin-11-dUTP and Bio-16-dUTP (Roche Diagnostics, Mannheim, Germany) by nick translation, respectively. The labeled DNA was mixed at a variety of ratios for GISH analysis using chromosomes from the mitotic metaphases as target templates. The results indicate that a ratio of 4:3 is the most suitable for discrimination of chromosomes from G. anomalum and the At-subgenome of G. hirsutum. At this ratio the chromosomes from G. anomalum only hybridized with the G. anomalum probe to produce a red signal, while chromosomes of the At-subgenome of G. hirsutum cross-hybridized with both the G. anomalum and G. herbaceum probes to produce a white signal and chromosomes of the Dt-subgenome of G. hirsutum were stained with 4’,6-diamidino-2-phenylindole (DAPI) (Roche Diagnostics), producing a blue color. Therefore, the GISH technique has been improved and can be further used to differentiate chromosomes from G. anomalum and the At-subgenome of G. hirsutum (Fig. 1).
Fig. 1

Genomic in situ hybridization of the putative alien chromosomes of G. anomalum in the G. hirsutum background using two G. herbaceum and G. anomalum probes. Genomic DNA from G. anomalum and G. herbaceum was labeled with digoxigenin-11-dUTP and Bio-16-dUTP by nick translation, respectively. Chromosomes of the At-subgenome of G. hirsutum were cross-hybridized with both the G. anomalum and G. herbaceum probes and produced white signals and chromosomes of the Dt-subgenome of G. hirsutum were stained with 4′,6-diamidino-2-phenylindole (DAPI) and produced blue signals. Chromosomes from G. anomalum were hybridized with G. anomalum probe and produced red signals. a mitotic chromosome spread of the 52 chromosomes of G. hirsutum. b mitotic chromosome spread of the 26 chromosomes of G. anomalum. cl mitotic chromosome spread showing the 52 G. hirsutum (white and blue) chromosomes and three (c), two (d), and one (e, f, g, h, i, j, k and l) individual chromosomes of G. anomalum (red), respectively. Scale bar = 5μm

Progenies of the pentaploid of (G. hirsutum × G. anomalum) × G. hirsutum backcrossed with G. hirsutum were subjected to GISH to determine the number of alien chromosomes transferred from G. anomalum to G. hirsutum using visible fluorescent hybridization signals. Thirty eight individuals of the BC1 population were examined by GISH analysis (Additional file 1: Table S1). The analysis demonstrated that 27 (71.05 %) carried 2 to 6 alien chromosomes, and 6 (15.79 %) carried 7 to 9 alien chromosomes. Only two (5.26 %) individuals carried one chromosome, 6Ba and 13Ba of G. anomalum, resepctively. One (2.63 %) plant had no alien chromosomes and the final two (5.26 %) plants had 13 alien chromosomes from G. anomalum (Fig. 1; Table 1).
Table 1

Incidence of alien chromosomes in the BC1 to BC2 G. hirsutum × G. anomalum generations

Chromosome number

1Ba

2Ba

3Ba

4Ba

5Ba

6Ba

7Ba

8Ba

9Ba

10Ba

11Ba

12Ba

13Ba

No. individuals

52

0

0

0

0

0

0

0

0

0

0

0

0

0

122

52 + 1

0

10

1

17

0

16

3

6

1

34

2

7

11

108

52 + 2

1

9

6

31

1

16

5

1

0

19

5

13

1

54

52 + 3

3

2

6

9

1

8

5

1

0

5

4

7

4

19

52 + 4

1

2

4

2

1

4

1

1

2

2

0

4

1

6

52 + 5

2

1

3

2

3

1

1

2

1

1

1

1

1

4

52 + 6

4

3

4

1

2

1

3

3

2

3

3

2

4

7

52 + 7

1

1

2

0

2

1

1

0

1

0

1

2

2

2

52 + 8

2

1

2

2

0

1

1

1

1

2

1

1

1

2

52 + 9

2

2

2

1

0

2

2

2

1

2

1

1

0

2

52 + 13

2

2

2

2

2

2

2

2

2

2

2

2

2

2

SUM

16

31

30

65

10

50

22

17

9

68

18

38

25

328

Incidence (%)

4.65

7.33

7.58

15.89

2.69

12.47

5.62

4.16

2.44

16.87

4.65

9.29

6.36

 

Monosomic addition (%)

0.00

9.26

0.93

15.74

0.00

14.81

2.78

5.56

0.93

31.48

1.85

6.48

10.19

 

A total of 290 individuals from the BC2 generation were further analyzed by GISH. The results indicated that 106 (36.55 %) individuals had one alien chromosome of G. anomalum and 121 (41.72 %) had no alien chromosomes in the G. hirsutum background. 50 (17.24 %) and 10 (3.45 %) individuals carried two and three alien chromosomes, respectively, and another 1 (0.34 %) carried four alien chromosomes. The results demonstrated that most of the BC2 individuals carried 0-1 alien chromosomes, and only a small number contained multiple alien chromosomes (Fig. 1; Table 1).

Screening of a set of putative G. anomalum chromosome-specific SSR primer pairs

During the evolution of Gossypium, chromosomal translocations occurred between genomes A1, A2, and B1, while genome D remained relatively stable [16]. Numerous recent reports also show that translocations occurred between chromosomes in the At-subgenome of the tetraploids [17], while no large structural variation was found in the Dt-subgenome. Therefore, we only selected SSR primers from the Dt -subgenome of the tetraploid cotton linkage map to screen putative G. anomalum chromosome-specific SSR primer pairs. Of the 1402 pairs of primers we selected, 1072 amplified distinct fragments in G. hirsutum and G. anomalum, including 272 dominant markers of G. hirsutum, 194 dominant markers of G. anomalum and 452 codominant markers, while 154 pairs produced no amplified polymorphic bands and another 330 pairs produced vague bands, which were excluded from further study. Then, based on the tetraploid cotton linkage map constructed by our institute [17], the above 452 codominant markers were located, and of these, 170 well-amplified and evenly distributed codominant markers within an interval of 10 cM were finally selected for use in genotyping the entire BC1F1 and BC2F1 population. The 170 codominant markers were distributed on the Dt-subgenome chromosomes, ranging from 10 to 18 markers per chromosome, with coverage of 80.9–100.0 % and a density of 6.7–15.0 cM of each chromosome (Table 2; Fig. 2). The G. anomalum-specific SSR markers could be used to track and identify the alien chromosomes from G. anomalum in G. hirsutum.
Table 2

SSR primers used for screening G. anomalum chromosomes in the alien addition lines

Chromosome

1Ba

2Ba

3Ba

4Ba

5Ba

6Ba

7Ba

8Ba

9Ba

10Ba

11Ba

12Ba

13Ba

 

NAU7675-120

NAU1847-200

NAU2836-230

NAU6966-200

NAU3095-260

NAU3677-160

NAU8250-220

NAU0104-230

NAU3100-170

NAU7772-160

NAU8254-160

NAU3084-250

NAU6582-550

 

NAU3347-250

NAU3733-200

NAU0093-130

NAU0210-200

NAU2503-250

NAU2679-150

NAU7974-150

NAU8183-160

NAU1886-150

NAU2543-190

NAU7698-160

NAU0206-100

NAU6426-370

 

NAU7914-160

NAU0645-130

NAU5675-180

NAU0012-230

NAU3183-230

NAU1454-200

NAU2556-250

NAU0738-230

NAU3888-220

NAU3917-180

NAU3731-300

NAU5397-160

NAU3011-220

 

NAU3714-190

NAU8013-220

NAU0354-180

NAU0569-160

NAU0144-250

NAU1987-160

NAU2974-150

NAU2876-200

NAU6701-200

NAU4071-220

NAU0133-120

NAU7007-150

NAU7727-250

 

NAU0072-180

NAU5490-280

NAU0200-410

NAU3508-200

NAU6205-160

NAU2397-270

NAU0300-120

NAU5130-320

NAU0148-170

NAU7900-150

NAU0646-140

NAU3905-150

NAU3948-250

 

NAU3337-320

NAU5421-210

NAU3875-210

NAU0146-180

NAU4055-170

NAU6347-170

NAU4017-220

NAU7616-150

NAU6848-150

NAU3531-210

NAU7140-150

NAU2715-200

NAU0039-110

 

NAU6624-220

NAU1778-100

NAU0088-140

NAU0033-150

NAU0378-180

NAU0783-180

NAU0121-200

NAU0583-300

NAU2753-250

NAU3665-220

NAU5418-160

NAU7838-150

NAU8306-130

 

NAU0107-110

NAU6474-300

NAU3700-180

NAU7579-140

NAU6406-200

NAU0356-170

NAU3594-110

NAU5335-150

NAU0075-130

NAU0922-200

NAU6999-420

NAU8230-170

NAU2443-140

 

NAU7670-150

NAU7809-200

NAU2908-200

NAU7290-230

NAU5486-200

NAU4682-150

NAU4956-280

NAU6389-270

NAU7815-250

NAU3137-300

NAU5212-200

NAU7719-200

NAU7738-160

 

NAU1495-170

NAU3598-200

NAU8203-230

NAU7946-150

NAU2944-180

NAU2714-170

NAU2820-200

NAU0435-180

NAU6984-200

NAU4881-240

NAU2361-250

NAU7824-190

NAU6738-130

 

NAU6095-170

NAU3820-110

NAU3292-270

NAU6993-150

NAU0123-120

 

NAU7686-180

NAU0069-160

NAU7743-130

NAU8079-200

NAU3373-220

NAU1274-210

NAU4871-150

  

NAU1702-180

NAU5111-230

 

NAU6830-150

 

NAU2597-180

NAU3904-190

NAU0799-210

NAU0142-500

NAU2602-270

NAU8006-160

NAU3447-110

   

NAU0805-190

 

NAU7747-160

 

NAU7692-150

NAU0298-130

NAU8120-320

NAU7983-170

NAU6809-160

  
     

NAU2655-170

   

NAU0245-110

 

NAU6315-180

  
     

NAU7015-150

   

NAU0864-240

 

NAU6267-180

  
     

NAU3826-420

   

NAU4477-250

 

NAU6520-200

  
     

NAU3609-250

        
     

NAU3656-210

        

Total

11

12

13

11

18

10

13

13

16

13

16

12

12

Position

8.20-120.01

0.00-107.44

3.23-126.25

0.00-113.51

10.90-189.98

13.32-121.59

2.85-121.07

0.00-149.89

0.00-148.34

15.49-111.16

12.11-156.15

0.00-108.09

7.24-106.43

GDC (cM/)a

111.81

107.44

123.02

113.51

179.08

108.27

118.22

149.89

148.34

95.67

144.04

108.09

99.19

Mean densityb

10.16

8.95

9.46

10.32

9.95

10.83

9.09

11.53

9.27

7.36

9.00

9.01

8.27

PCC (%)c

88.93

95.88

97.44

88.94

94.27

78.54

91.35

90.33

98.14

82.84

79.51

80.86

84.64

Note: aGDC genetic distance coverage (cM); bGenetic distance (cM) between two adjacent markers on a chromosome; cPercentage of chromosome covered by markers (%)

Fig. 2

Genetic linkage map of G. anomalum chromosome-specific SSR markers based on the linkage map of tetraploid cotton reported by Zhao et al. (2012)

Identity of alien chromosomes from G. anomalum as discriminated by SSR analysis

One hundred seventy G. hirsutum- and G. anomalum-specific codominant markers distributed on 13 Dt-subgenome chromosomes of the tetraploids were used to identify the alien chromosomes in 108 MAALs and multiple alien addition lines. The results demonstrated that 34 (31.48 %) MAAL individuals were MAAL-10Ba (the largest group), followed by 17 (15.74 %) MAAL-4Ba, 16 (14.81 %) MAAL-6Ba, 11 (10.19 %) MAAL-13Ba, 10 (9.26 %) MAAL-2Ba, 7 (6.48 %) MAAL-12Ba, 3 (2.78 %) MAAL-7Ba, 2 (1.85 %) MAAL-11Ba, 1 (0.93 %) MAAL-3Ba, and 1 (0.93 %) MAAL-9Ba (Figs. 3 and 4; Table 1). Two MAALs were not found, MAAL-1Ba and MAAL-5Ba; therefore Chrs. 1Ba and 5Ba were not isolated and remained as multiple addition lines.
Fig. 3

Genomic in situ hybridization of the putative monosomic alien chromosomes of G. anomalum in the G. hirsutum background using G. herbaceum and G. anomalum probes. a mitotic chromosome spread of the 52 chromosomes of G. hirsutum, showing 26 chromosomes each of the At- (white) and Dt- (blue) subgenomes. b-l mitotic chromosome spread showing the 52 G. hirsutum (white and blue) chromosomes and different individual chromosomes from G. anomalum (red), corresponding to 2Ba to 4Ga (b, c and d) and 6Ga to 13Ga (e, f, g, h, i, j, k and l), respectively. Scale bar = 5μm

Fig. 4

A set of G. anomalum-specific SSR markers were used to identify individual alien chromosomes of G. anomalum in G. hirsutum. a-k the G. anomalum-specific amplicons were obtained using 11 individual chromosome-specific primer pairs for markers; NAU5421, BNL2443, NAU7579, NAU3677, dPL0492, BNL2597, BNL3383, NAU4881, NAU9520, dPL0379, and dPL0864. The chromosomes correspond to D2 to D4 and D6 to D13 in cultivated tetraploid cotton. P1, G. hirsutum; P2, G. anomalum; F1, the hexaploid of G. hirsutum and G. anomalum; 1-11 show that each of these plants possesses a single different individual chromosome from G. anomalum, corresponding to 2Ba to 4Ba, and 6Ba to 13Ba. M, molecular size marker (50 bp ladder). Arrows (red) indicate chromosome-specific markers for G. anomalum

During the development of MAALs, Chr. 10Ba appeared most frequently, with an incidence of 16.87 %, followed by 15.89 % for 4Ba, 12.47 % for 6Ba, and 9.29 % for 12Ba. Chrs. 5Ba and 9Ba showed very low incidences of 2.69 % and 2.44 %(Table 1).

Morphological traits of MAALs

Morphological data were gathered during the cotton growing stage. The results shown in Tables 3, 4 and 5 indicate that the eleven MAALs differed from one another and also differed from their parents in terms of their morphological traits, such as plant type, leaf shape, size of flower and boll (Figs. 5 and 6; Tables 3, 4 and 5). Most of these MAALs grew slower than the recipient, TM-1. We found that MAAL-8Ba leaves had a very dark green color. We also observed that MAAL-7Ba, MAAL-12Ba and MAAL-13Ba had relatively bigger leaves, while MAAL-8Ba, MAAL-9Ba and MAAL-10Ba had relatively smaller leaves than the other lines (Fig. 5b). In addition, MAAL-6Ba, MAAL-10Ba, MAAL-11Ba and MAAL-12Ba had relatively larger flowers than the others. Only MAAL-7Ba showed petal spots and MAAL-6Ba had very light brown fibers, indicating that genes for petal spots and light brown fibers are located on chromosomes 7Ba and 6Ba (Figs. 5a and 6d), respectively. MAAL-2Ba and MAAL-12Ba had relatively longer bolls and MAAL-7Ba had the widest boll diameter, while MAAL-8Ba had the shortest bolls and MAAL-10Ba had the smallest boll diameter (Fig. 6c). MAAL-6Ba, MAAL-7Ba and MAAL-9Ba had a relatively larger boll weight, while MAAL-8Ba, MAAL-10Ba and MAAL-11Ba had a relatively smaller boll weight than the others (Table 4). We found that MAAL-7Ba had longer fibers than the others (Fig.6d)
Table 3

Morphological characteristics of the eleven MAALs

Characters

TM-1

G. anomalum

Hexaploid F1

2Ba

3Ba

4Ba

6Ba

7Ba

8Ba

9Ba

10Ba

11Ba

12Ba

13Ba

Petal color

Creamy

Mauve

Creamy

Creamy

Creamy

Creamy

Creamy

Creamy

Creamy

Creamy

Creamy

Creamy

Creamy

Creamy

Petal spot

Absent

Big dark red

Big dark red

Absent

Absent

Absent

Absent

light red

Absent

Absent

Absent

Absent

Absent

Absent

Petal length (cm)

4.04 ± 0.13

3.77 ± 0.49

4.75 ± 0.13

4.14 ± 0.32

4.19 ± 0.29

4.1 ± 0.32

4.37 ± 0.38

3.92 ± 0.31

3.57 ± 0.52

3.78 ± 0.51

4.49 ± 0.44

4.84 ± 0.41

4.53 ± 0.48

3.68 ± 0.21

Petal width (cm)

4.43 ± 0.20

4.37 ± 0.57

5.28 ± 0.28

4.32 ± 0.37

4.13 ± 0.22

4.01 ± 0.39

4.67 ± 0.52

4.24 ± 0.45

3.59 ± 0.66

3.76 ± 0.21

4.42 ± 0.44

5.39 ± 0.68

4.77 ± 0.58

3.53 ± 0.54

Another number

104 ± 4.97

69.33 ± 8.50

112.25 ± 10.69

96.36 ± 5.00

85.33 ± 8.08

92.50 ± 9.98

96.19 ± 12.58

68.44 ± 12.28

67.22 ± 9.39

97.40 ± 10.88

108.27 ± 9.21

109.83 ± 12.30

105.91 ± 12.24

92.09 ± 8.51

Style length (cm)

2.26 ± 0.05

1.70 ± 0.10

2.55 ± 0.17

2.19 ± 0.21

2.02 ± 0.06

1.76 ± 0.18

2.74 ± 0.24

1.78 ± 0.25

2.27 ± 0.20

2.25 ± 0.40

2.46 ± 0.32

2.60 ± 0.29

1.84 ± 0.17

2.10 ± 0.19

Stigma length (cm)

1.06 ± 0.09

0.43 ± 0.15

1.18 ± 0.15

1.09 ± 0.18

1.23 ± 0.20

0.81 ± 0.15

1.52 ± 0.26

0.83 ± 0.11

1.28 ± 0.20

1.05 ± 0.11

0.95 ± 0.38

1.51 ± 0.28

0.85 ± 0.12

1.11 ± 0.07

Pedicel length (cm)

1.05 ± 0.21

0.90 ± 0.10

1.88 ± 0.25

1.42 ± 0.40

1.22 ± 0.38

0.83 ± 0.15

2.52 ± 0.82

1.25 ± 0.34

0.78 ± 0.13

1.21 ± 0.26

1.01 ± 0.30

0.87 ± 0.27

2.97 ± 1.40

0.73 ± 0.12

sepal length (cm)

3.06 ± 0.05

1.95 ± 0.13

3.05 ± 0.17

3.17 ± 0.23

3.33 ± 0.26

2.99 ± 0.35

3.40 ± 0.29

2.98 ± 0.29

2.86 ± 0.10

2.88 ± 0.20

2.90 ± 0.29

3.19 ± 0.37

3.09 ± 0.38

2.90 ± 0.25

sepal width (cm)

1.10 ± 0.14

0.93 ± 0.10

1.00 ± 0.20

1.19 ± 0.39

1.27 ± 0.12

0.96 ± 0.14

1.12 ± 0.14

1.34 ± 0.29

0.83 ± 0.11

0.85 ± 0.12

0.87 ± 0.14

1.10 ± 0.14

1.19 ± 0.24

1.05 ± 0.22

Bracteole length (cm)

4.72 ± 0.50

1.52 ± 0.08

4.72 ± 0.32

5.28 ± 0.45

4.97 ± 0.28

4.19 ± 0.72

4.83 ± 0.63

4.84 ± 0.66

3.76 ± 0.37

4.41 ± 0.38

4.35 ± 0.58

5.20 ± 0.47

5.18 ± 0.61

3.47 ± 0.34

Bracteole width (cm)

2.85 ± 0.24

0.47 ± 0.07

2.98 ± 0.31

3.15 ± 0.35

2.57 ± 0.23

2.75 ± 0.48

3.23 ± 0.46

2.74 ± 0.45

2.47 ± 0.36

2.84 ± 0.37

2.56 ± 0.44

3.30 ± 0.27

3.16 ± 0.42

2.53 ± 0.24

Leaf color

Green

light Green

Green

Green

Green

Green

Green

Green

Dark green

Green

Green

Green

Green

Green

leaf length (cm)

12.03 ± 1.17

4.40 ± 0.36

6.57 ± 0.38

10.58 ± 2.28

9.75 ± 2.47

9.34 ± 2.25

9.10 ± 1.96

10.19 ± 1.03

7.66 ± 1.65

7.75 ± 0.21

7.70 ± 0.98

9.33 ± 3.75

10.17 ± 1.90

9.36 ± 1.74

leaf width (cm)

11.70 ± 0.20

2.53 ± 0.21

8.40 ± 0.56

11.68 ± 2.67

10.80 ± 2.69

11.46 ± 1.57

10.72 ± 2.20*

12.53 ± 1.72

10.52 ± 2.87

8.73 ± 0.11

8.40 ± 1.29

10.90 ± 3.72

11.27 ± 1.20

12.08 ± 2.10

Petiole length (cm)

6.7 ± 1.49

7.67 ± 0.47

8.57 ± 0.90

6.51 ± 2.00

4.60 ± 1.27

6.83 ± 0.85

5.71 ± 1.73

6.55 ± 1.14

6.65 ± 2.56

8.75 ± 0.503

5.60 ± 1.27

7.03 ± 3.48

7.52 ± 0.92

9.51 ± 1.69

boll length (mm)

43.08 ± 2.06

20.08 ± 1.01

33.18 ± 1.35

43.90 ± 2.94

38.75 ± 1.03

34.52 ± 1.62

34.03 ± 1.94

36.16 ± 1.41

30.58 ± 2.84

41.78 ± 0.10

34.02 ± 1.96

38.60 ± 12.00

48.88 ± 1.94

35.05 ± 2.037

boll width (mm)

39.31 ± 1.38

10.44 ± 0.61

22.34 ± 1.72

31.70 ± 3.22

41.75 ± 1.02

39.05 ± 2.19

39.82 ± 2.10

42.25 ± 2.16

31.25 ± 2.10

31.86 ± 1.82

25.52 ± 1.89

31.70 ± 2.26

33.38 ± 2.24

40.74 ± 2.54

boll tip length (mm)

3.89 ± 0.68

3.46 ± 0.59

5.06 ± 1.57

4.44 ± 0.95

4.07 ± 0.55

3.72 ± 0.85

4.94 ± 1.93

3.18 ± 0.84

3.15 ± 1.59

2.98 ± 1.71

4.08 ± 1.17

4.17 ± 1.27

5.48 ± 1.68

2.04 ± 1.10

Table 4

The yield-related traits of the eleven MAALs

MAAL

Boll size (g)

Seed index (g/100)

Lint percentage (%)

2Ba

3.15

13.05

30.27

3Ba

3.89

14.17

34.45

4Ba

4.19

12.87

36.86

6Ba

5.02

14.94

32.24

7Ba

5.01

13.74

35.95

8Ba

2.98

10.29

36.70

9Ba

5.49

13.13

34.14

10Ba

2.30

9.35

29.46

11Ba

2.41

9.38

30.35

12Ba

4.25

14.92

28.13

13Ba

4.44

14.91

35.30

TM-1 (CK)

5.64

14.91

28.16

Table 5

Summary of the unique traits of the monosomic alien addition lines

MAAL

Unique traits

2Ba

Long leaves and long calyx teeth of bract

3Ba

Short petiole and long Sepal

4Ba

Short column and stigma, high lint percent

6Ba

Long column and stigma, light brown fiber

7Ba

Purple petal spot, large leaves, long fiber

8Ba

Small bracts and flowers with few anthers, dark green leaves

9Ba

High boll weight

10Ba

Small leaves and bolls, many fruit branch and bolls

11Ba

Large flowers and the maximum anthers

12Ba

Long tips of cone-shape bolls and long pedicels

13Ba

Short peduncle and fruit branch, round and big bolls

Fig. 5

Flower and leaf traits for MAALs of G. anomalum individual chromosomes in G. hirsutum. Flower-related traits were photoed on the flowering day (0 day post anthesis, 0 DPA). a (petal), b (top third leaf) and (c) (bract); P1, G. hirsutum. P2, G. anomalum. F1, the hexaploid of G. hirsutum and G. anomalum. 2–4 and 6–13 are plants that carried a single different individual chromosome from G. anomalum, corresponding to 2Ba, 3Ba, 4Ba, 6Ba, 7Ba, 8Ba, 9Ba, 10Ba, 11Ba, 12Ba and 13Ba. Scale bar = 50 mm

Fig. 6

Flower, boll and fiber traits of MAALs of G. anomalum individual chromosomes in G. hirsutum. Squares, pistils and bolls were photoed at -1 DPA, 0 DPA and 35 DPA, respectively. a (square), b (pistil), c (boll) and d (fiber); P1, G. hirsutum. P2, G. anomalum. F1, the hexaploid of G. hirsutum and G. anomalum. 2–4 and 6–13 are plants that carried a single individual chromosome from G. anomalum, corresponding to 2Ba, 3Ba, 4Ba, 6Ba, 7Ba, 8Ba, 9Ba, 10Ba, 11Ba, 12Ba and 13Ba. Scale bar = 50 mm

Discussion

MAALs are powerful tools in crop breeding since they can be used to produce alien translocation and substitution lines, to study interspecific relationships, and to construct single chromosome libraries. They can also be used in gene mining, gene assignment, gene expression pattern analysis, gene function analysis, physical gene mapping, isolation of chromosome-specific probes, selective isolation and mapping of cDNA/gene of a particular chromosome. Numerous reports have shown that the development of MAALs has been successfully achieved in many crops such as wheat [1821], rice [22] tomato [23], potato [24], cucumber [25], tobacco [26], oat [12], sugar beet [27, 28], and rapeseed [29, 30]. MAALs have played and are playing important roles in numerous types of plant genomic research. The development of MAALs in Gossypium began as early as the 1980s but greatly lagged behind other crops due to the large number (2n = 52) and small size of chromosomes, which led to difficulty in accurately discriminating each chromosome, therefore, little progress has been made in cotton. So far only one set of MAALs has been completed [9], and this work benefited from advances in the development of GISH and molecular markers in cotton.

However, in this study, due to the very close relationship between chromosomes of the At-subgenome in G. hirsutum and those in G. anomalum often leading to cross-hybridization in GISH, we had to first improve the GISH technique by adjusting the ratio of the two different probes used. We tried five different combinations and found that the ratio of 4:3 was more suitable than any others for the discrimination of chromosomes from G. anomalum and the At-subgenome of G. hirsutum. Therefore, using a combination of the improved GISH methodology, G. anomalum chromosome-specific SSR molecular markers and conventional morphological survey, eleven MAALs were isolated and characterized, and two remain to be isolated from multiple addition states by further backcrossing.

Several previous reports showed that G. anomalum contains the favorable characteristics of drought tolerance and resistance to diseases (cotton Verticillium wilt, angular leaf spot) and insect pests (springtails, aphids); and more importantly, it also possesses genes with the potential to produce high quality fibers (good fiber strength and fineness) [4] and cytoplasmic male sterility [57]. Our previous reports also demonstrated that using G. anomalum as a donor parent and G. hirsutum as a recipient parent, a series of introgression lines with longer, stronger and finer fibers has been developed [31]. Shen et al. [32] mapped QTLs on Chr. 7 affecting fiber length in an F2 population derived from G. anomalum introgression line 7235 crossed with TM-1. However, in this study, we investigated some agronomic traits of MAALs and observed that most MAALs had poor performances in fiber quality or fiber yield components, implying that the added alien chromosomes had negative effects on most agronomic traits (Tables 4 and 6; Fig. 6). For example, the bolls of all MAALs were lighter than those of the recipient TM-1; and the fibers of all six MAALs were shorter than TM-1 (the fiber properties of the other five MAALs were not measured due to a lack of fiber samples). The resultant phenomena may be caused by linkage drag, which means that there were very close linkages between favorable and unfavorable genes on the same chromosome, even though the fibers of some MAALs were found to be stronger than those of TM-1. Therefore, to enhance the transfer of desirable genes and eliminate undesirable genes from G. anomalum, it is necessary to break the linkage drags to promote chromosome recombination between G. hirsutum and G. anomalum. The development of chromosome translocation lines or introgression lines may be an alternative choice based on the MAALs. We deeply believe that these MAALs of G. hirsutum-G. anomalum would be a powerful tool for systematically transferring desirable genes chromosome by chromosome from G. anomalum into G. hirsutum, as well as for gene mining, gene assignment, gene function analysis, gene physical mapping, isolation of chromosome-specific probes, selective isolation and mapping of cDNAs for a particular chromosome, and genomic research.
Table 6

Fiber quality traits from some MAALs measured by HVI

MAAL

Fiber length (mm)

Fiber uniformity (%)

Micornaire

Fiber strength (cN/tex)

Fiber elongation rate (%)

TM-1

29.08

86.20

4.35

31.95

7.00

MAAL-2Ba

27.99

83.80

4.66

29.60

6.70

MAAL-4Ba

26.02

83.60

4.52

28.32

6.50

MAAL-6Ba

25.84

82.20

5.43

30.67

6.80

MAAL-8Ba

26.99

83.40

4.04

32.44

6.60

MAAL-10Ba

25.94

83.10

3.35

35.67

6.70

MAAL-13Ba

27.17

84.70

4.78

28.91

6.50

Conclusions

From this study, we draw two conclusions. (1) The GISH technique used in our previous research has been improved by using a mixture of two probes at a ratio of 4:3 (G. anomalum and G. herbaceum) to avoid cross-hybridization caused by the very close relationship between chromosomes from G. anomalum and the At-subgenome of G. hirsutum, which can be suitable for recognizing alien chromosomes of G. anomalum in G. hirsutum background. (2) Eleven out of 13 potential MAALs were isolated, which would be used, at the chromosome level, for effectively identifying favorable genes in G. anomalum, allowing for more accurate and faster transfer of such genes to create introgression lines, the effect of specific alien chromosomes to be examined, homeologies with chromosomes of cultivated species to be compared, and physical maps of specific chromosomes to be constructed.

Methods

Plant materials

In 2012, the amphiploid (allohexaploid) (2n = 6× = 78, AADDBB genome) (previously obtained in our institue) derived from the doubled triploid hybrid of G. hirsutum (2n = 4× = 52, AADD genome) × G. anomalum (2n = 2× = 26, BB genome, obtained from Cotton Research Institute of Chinese Academy of Agricultural Sciences) was backcrossed as a maternal parent with G. hirsutum acc TM-1, the genetic standard line of upland cotton. In 2013, two pentaploid individuals were obtained at Pailou Experimental Station of Nanjing Agricultural University (PES/NJAU) and used as both paternal and maternal parents in the backcross with TM-1. The BC1 seeds obtained were planted in plastic cups with sterilized soil and incubated in the phytotron at Nanjing Agricultural University in 2014 spring at 25–28 °C and with 80% relative humidity. When they reached the fifth true leaf stage, the seedlings were transplanted into clay pots at PES/NJAU. Lastly, 38 BC1 individuals were identified using SSR markers and GISH and consecutively backcrossed with TM-1. The BC2 seeds obtained were planted in the same way in spring 2015. In the winter, all plants were moved into the greenhouse at PES for preservation.

Scheme for developing the monosomic alien addition lines

The interspecific hexaploid was backcrossed with Gossypium hirsutum acc TM-1 (obtained from the Southern Plains Agricultural Research Center, USDA-ARS) to produce the pentaploid (2n = 5× = 65, AADDB genome), then the pentaploid progenies were further consecutively backcrossed with TM-1 to generate backcross progenies (BC1 and BC2). GISH was used to characterize alien chromosomes in all backcross progenies from the BC1 generation. When more than one alien chromosome was added from G. anomalum, the progenies were further backcrossed with TM-1 to produce monosomic alien addition lines. If only one alien chromosome was added to the background of Upland cotton, the progenies were further examined using chromosome-specific SSR markers of G. anomalum to determine the identity of the added chromosome.

G. anomalum, TM-1, BC1, and BC2 chromosome preparation

Cotton seeds were cultivated in an incubator at 29 °C and their root tips were cut off when they grew to 3 cm long (seedling plant). The tips were immersed in 25 μg/ml cycloheximide at room temperature for 2 h to accumulate metaphase cells and then transferred to Carnoy I fixative containing 95% ethanol and acetic acid (3:1, v/v) for at least 2 h, digested in double enzymolysis liquid (4 % cellulose: 1 % pectinase = 1:2) at 37 °C for 45 min, and squashed in a drop of 45 % acetic acid. Finally, slides containing at least 20 well-spread somatic chromosomes at mitotic metaphase were prepared and stored at -70 °C overnight.

Genomic in situ hybridization (GISH)

Due to the very close relationships that exist between chromosomes of the B genome in G. anomalum and those of the At subgenome in G. hirsutum, two probes were employed here to avoid cross-hybridization between these chromosomes. Genomic DNA extracted from G. anomalum and G. herbaceum (2n = 2× = 26, AA genome) were labeled with digoxigenin-11-dUTP and Bio-16-dUTP (Roche Diagnostics, Mannheim, Germany) by nick translation, respectively. The probe fragment size was between 200-500 bp. Fluorescence in situ hybridization was carried out as described by [33] and [9] with some modifications. The mixing ratio of DNA probes from G. anomalum and G. herbaceum were adjusted to five different ratios, 2:1, 4:3, 1:1, 2:3, and 1:2, to determine the optimal ratio for discrimination of chromosomes from G. anomalum and the At-subgenome of G. hirsutum.

DNA extraction and G. anomalum-specific primer screening

Genomic DNA was extracted from young leaves of the two parents, G. anomalum and G. hirsutum acc. TM-1, the interspecific hexaploid, the pentaploid, and the BC1 and BC2 individuals using the method described by [34] with some modifications. A total of 2,168 pairs of SSR primers were selected from the high density genetic linkage map of Sea island and Upland cotton constructed in our institute [17] and employed to screen G. anomalum-specific primers. PCR reactions were performed and their amplified products were separated by PAGE, as described by [35, 36]. The G. anomalum-specific marker primers obtained were further used to characterize each chromosome from G. anomalum.

MAAL nomenclature

Thirteen G. hirsutum-G. anomalum MAALs were named MAAL-1Ba to MAAL-13Ba, according to the method described by [9], in which B represents the B genome of G. anomalum and ‘a’ refers to the initial letter of anomalum. The chromosome numbers 1 to 13 in the B genome of G. anomalum correspond to the homoeologous chromosomes in the Dt-subgenome of tetraploid cotton.

Investigation of agronomic traits of monosomic alien addition line

At the point of transition from the vegetative to the reproductive stage, the shape and size of fully expanded leaves from the same position in TM-1, G. anomalum, hexaploid and MAAL plants were investigated. Floral morphological traits from these MAALs were investigated in the flowering period. The size of cotton bolls at 35 days post-anthesis was also measured by vernier caliper. Finally, the hundred-seed weight, ginning outturn and single boll weight of the matured bolls were investigated. All the data were analyzed using the SPSS software version 18.0.

Abbreviations

GISH: 

Genomic in situ hybridization

MAAL: 

Monosomic alien addition line

SSR: 

Simple sequence repeat

Declarations

Acknowledgements

We acknowledge Dr Kunbo Wang, vice director of Cotton Research Institute of Chinese Academy of Agricultural Sciences, for providing seeds of Gossypium anomalum. We are also grateful to Dr RJ Kohel of the Southern Plains Agricultural Research Center, USDA-ARS, for providing seeds of Gossypium hirsutum acc TM-1.

Funding

The National Key Research and Development Program of China (2016YFD0100203), the National Key Technology R&D Program of China during the Twelfth Five-year Plan Period [grant number 2013BAD01B03-04] and Jiangsu Collaborative Innovation Center for Modern Crop Production. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Authors’ contributions

BLZ conceived and designed the experiments; XXW, YYW, CW, YC, YC, SLF and TZ performed the experiments; XXW, YYW and BLZ analyzed the data; YC, YC and TZ contributed reagents/materials/analysis tools; BLZ and XXW wrote the manuscript. All authors confirmed their contribution, read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
State Key Laboratory of Crop Genetics & Germplasm Enhancement, Nanjing Agricultural University
(2)
Key Laboratory of Cotton Breeding and Cultivation in Huang-Huai-Hai Plain, Ministry of Agriculture, Cotton Research Center of Shandong Academy of Agricultural Sciences

References

  1. Tang S, Teng Z, Zhai T, Fang X, Liu F, Liu D, Zhang J, Liu D, Wang S, Zhang K, Shao Q, Tan Z, Paterson AH, Zhang Z. Construction of genetic map and QTL analysis of fiber quality traits for Upland cotton (Gossypium hirsutum L.). Euphytica. 2015;201:195–213.View ArticleGoogle Scholar
  2. Fryxell PA. A revised taxonomic interpretation of Gossypium L.(Malvaceae). Rheedea. 1992;2:108–65.Google Scholar
  3. Chen ZJ, Scheffler BE, Dennis E, Triplett BA, Zhang TZ, Guo WZ, Chen XY, Stelly DM, Rabinowicz PD, Town CD, Arioli T, Brubaker C, Cantrell RG, Lacape JM, Ulloa M, Chee P, Gingle AR, Haigler CH, Percy R, Saha S, Wilkins T, Wright RJ, Deynze AV, Zhu YX, Yu SX, Abdurakhmonov I, Katageri I, Kumar PA, Rahman M, Zafar Y, Yu JZ, Kohel RJ, Wendel JF, Paterson AH. Toward sequencing cotton (Gossypium) genomes. Plant Physiol. 2007;145:1303–10.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Qian SY, Huang JQ, Peng YJ, Zhou BL, Ying MC, Shen DZ, Hu TX, Xu YJ, Gu LM, Ni WC, Cheng S. Studies on the hybridof Gossypium hirsutum L. and G. anomalum and application inbreeding. Sci Agri Sin. 1992;25:44–51.Google Scholar
  5. Ganesh SN, Vivek PC, Subhash SM, Ashok SJ. Interspecific hybridization in Gossypium L.: characterization of progenies with different ploidy-confirmed multigenomic backgrounds. Plant Breed. 2013;132:211–6.View ArticleGoogle Scholar
  6. Mehetre SS. Wild Gossypium anomalum: a unique source of fibre fineness and strength. Curr Sci. 2010;99:58–71.Google Scholar
  7. Narayanan SS, Singh J, Varma PK. Introgressive gene transfer in Gossypium. Goals, problems, strategies and achievements. Cot Fib Trop. 1984;39:123–35.Google Scholar
  8. Stewart, JM. Potential for crop improvement with exotic germplasm and genetic engineering. In: Constable GA, Forrester NW, editors. Challenging the Future: Proceedings of the World Cotton Research Conference-1, Brisbane Australia, February 14–17, 1995. Melbourne; p. 313–327Google Scholar
  9. Chen Y, Wang Y, Wang K, Zhu XF, Guo WZ, Zhou BL. Construction of a complete set of alien chromosome addition lines from Gossypium australe in Gossypium hirsutum: morphological, cytological, and genotypic characterization. Theor Appl Genet. 2014;127:1105–21.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Hau B. Ligne ′es d’addition sur Gossypium hirsutum L.I. Utilisation de l’hybridation interspe ′cifique et de lame ′thode des ligne ′es d’addition pour l’ame ′lioration ducotonnier. Cot Fib Trop. 1981;26:247–58.Google Scholar
  11. Rooney WL, Stelly DM, Altman DW. Identification of four Gossypium sturtianum monosomic alien addition derivatives from a backcrossing program with G. hirsutum. Crop Sci. 1991;31:337–41.View ArticleGoogle Scholar
  12. Kynast RG, Riera-Lizarazu O, Vales MI, Okagaki RJ, Maquieira S, Chen G, Ananiev EV, Odland WE, Russell CD, Stec AO, Livingston SM, Zaia HA, Rines HW, Phillips RL. A complete set of maize individual chromosome additions to the oat genome. Plant Physiol. 2001;125:1216–27.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Ahoton L, Lacape JM, Baudoin JP, Mergeai G. Introduction of Australian diploid cotton genetic variation into upland cotton. Crop Sci. 2003;43:1999–2005.View ArticleGoogle Scholar
  14. Sarr D, Lacape JM, Rodier-Goud M, Jacquemin JM, Benbouza H, Toussaint A, Palm R, Ahoton L, Baudoin JP, Mergeai G. Isolation of five new monosomic alien addition lines of Gossypium australe F. Muell in G. hirsutum L. by SSR and GISH analyses. Plant Breed. 2011;130:60–6.View ArticleGoogle Scholar
  15. Zhou ZH, Yu P, Liu GH, He JX, Chen JX, Zhang XX. Morphological and molecular characterization of two G. somalense monosomic alien addition lines (MAALs). Chin Sci Bull. 2004;49:910–4.Google Scholar
  16. Gerstel DU, Sarvella PA. Additional observations on chromosomal translocations in cotton hybrids. Evolution. 1956;10:408–14.View ArticleGoogle Scholar
  17. Zhao L, Lv Y, Cai C, Tong X, Chen X, Zhang W, Du H, Guo X, Guo W. Toward allotetraploid cotton genome assembly: integration of a high-density molecular genetic linkage map with DNA sequence information. BMC Genomics. 2012;13:539. http://www.biomedcentral.com/1471-2164/13/539.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Friebe B, Qi LL, Nasuda S, Zhang P, Tuleen NA, Gill BS. Development of a complete set of Triticum aestivum-Aegilops speltoides chromosome addition lines. Theor Appl Genet. 2000;101:51–8.View ArticleGoogle Scholar
  19. Kishii M, Yamada T, Sasakuma T, Tsujimoto H. Production of wheat–Leymus racemosus chromosome addition lines. Theor Appl Genet. 2004;109:255–60.View ArticlePubMedGoogle Scholar
  20. Wang XE, Chen PD, Liu DJ, Zhang P, Zhou B, Friebe B, Gill BS. Molecular cytogenetic characterization of Roegneria ciliaris chromosome additions in common wheat. Theor Appl Genet. 2001;102:651–7.View ArticleGoogle Scholar
  21. Kong F, Wang H, Cao A, Qin B, Ji J, Wang S, Wang X. Characterization of T. aestivum-H. californicum chromosome addition lines DA2H and MA5H. J Genet Genomics. 2008;35:673–8.View ArticlePubMedGoogle Scholar
  22. Multani DS, Khush GS, delos Reyes BG, Brar DS. Alien genes introgression and development of monosomic alien addition lines from Oryza latifolia Desv. to rice, Oryza sativa L. Theor Appl Genet. 2003;107:395–405.View ArticlePubMedGoogle Scholar
  23. Chetelat RT, Rick CM, Cisneros P, Alpert KB, DeVerna JW. Identification, transmission, and cytological behavior of Solanum lycopersicoides Dun. monosomic alien addition lines in tomato (Lycopersicon esculentum Mill.). Genome. 1998;41:40–50.View ArticleGoogle Scholar
  24. Ali SNH, Ramanna MS, Jacobsen E, Visser RGF. Establishment of a complete series of a monosomic tomato chromosome addition lines in the cultivated potato using RFLP and GISH analyses. Theor Appl Genet. 2001;103:687–95.View ArticleGoogle Scholar
  25. Chen JF, Luo XD, Qian CT, Jahn MM, Staub JE, Zhuang FY, Lou QF, Ren G. Cucumis monosomic alien addition lines: morphological, cytological, and genotypic analyses. Theor Appl Genet. 2004;108:1343–8.View ArticlePubMedGoogle Scholar
  26. Chen CC, Chen SK, Liu MC, Kao YY. Mapping of DNA markers to arms and sub-arm regions of Nicotiana sylvestris chromosomes using aberrant alien addition lines. Theor Appl Genet. 2002;105:8–15.View ArticlePubMedGoogle Scholar
  27. Reamon-Ramos SM, Wricke G. A full set of monosomic addition lines in Beta vulgaris from Beta webbiana: morphology and isozyme markers. Theor Appl Genet. 1992;84:411–8.PubMedGoogle Scholar
  28. Gao D, Guo D, Jung C. Monosomic addition lines of Beta corolliflora Zoss in sugar beet: cytological and molecular-marker analysis. Theor Appl Genet. 2001;103:240–7.View ArticleGoogle Scholar
  29. Srinivasan K, Malathi VG, Kirti PB, Prakash S, Chopra VL. Generation and characterisation of monosomic chromosome addition lines of Brassica campestris - B. oxyrrhina. Theor Appl Genet. 1998;97:976–81.View ArticleGoogle Scholar
  30. Budahn H, Schrader O, Peterka H. Development of a complete set of disomic rape-radish chromosome-addition lines. Euphytica. 2008;162:117–28.View ArticleGoogle Scholar
  31. Zhou BL, Song C, Shen XL, Zhang XG, Zhang ZL. Construction of gene pools with superior fiber properties in Upland cotton through interspecific hybridization between Gossypium hirsutum and Gossypium wild species. Acta Agron Sin. 2003;29:514–9.Google Scholar
  32. Shen X, Guo W, Zhu X, Yuan Y, Yu JZ, Kohel RJ, Zhang T. Molecular mapping of QTLs for fiber qualities in three diverse lines in Upland cotton using SSR markers. Mol Breed. 2005;15:169–81.View ArticleGoogle Scholar
  33. Wang K, Song XL, Han ZG, Guo WZ, Yu JZ, Sun J, Pan JJ, Kohel RJ, Zhang TZ. Complete assignment of the chromosomes of Gossypium hirsutum L by translocation and fluorescence in situ hybridization mapping. Theor Appl Genet. 2006;113:73–80.View ArticlePubMedGoogle Scholar
  34. Paterson AH, Brubaker CL, Wendel JF. A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis. Plant Mol Biol Rep. 1993;11:122–7.View ArticleGoogle Scholar
  35. Zhang J, Guo W, Zhang T. Molecular linkage map of allotetraploid cotton (Gossypium hirsutumL. × Gossypium barbadense L.) with a haploid population. Theor Appl Genet. 2002;105:1166–74.View ArticlePubMedGoogle Scholar
  36. Zhang J, Stewart J. Economical and rapid method for extracting cotton genomic DNA. J Cotton Sci. 2000;4:193–201.Google Scholar

Copyright

© The Author(s). 2016

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