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Molecular Genetics and Genomics

, Volume 293, Issue 4, pp 945–955 | Cite as

Ten alien chromosome additions of Gossypium hirsutumGossypium bickii developed by integrative uses of GISH and species-specific SSR markers

  • Dong Tang
  • Shouli Feng
  • Sai Li
  • Yu Chen
  • Baoliang Zhou
Original Article
  • 145 Downloads

Abstract

Gossypium bickii: (2n = 26, G1G1), a wild diploid cotton, carries many favourable traits. However, these favourable traits cannot be directly transferred into G. hirsutum (2n = 52, AADD) cultivars due to the differences in genomes. Monosomic alien addition lines (MAALs) are considered an invaluable tool for the introgression of genes of interest from wild relatives into cultivated crops. In this study, the G. hirsutumG. bickii amphidiploid (2n = 78, AADDG1G1) was backcrossed with G. hirsutum to develop alien additions containing individual G. bickii chromosomes in a G. hirsutum background. Genomic in situ hybridization was employed to detect the number of alien chromosomes added to the backcross progenies. A total of 183 G. bickii-specific DNA markers were developed to discriminate the identities of the G. bickii chromosomes added to G. hirsutum and assess the alien chromosome transmissibility. Chromosomes 4Gb and 13Gb showed the highest transmissibility, while chromosomes 1Gb, 7Gb and 11Gb showed the lowest. Ten of the 13 possible G. hirsutum-G. bickii MAALs were isolated and characterized, which will lay the foundation for transferring resistance genes of G. bickii into G. hirsutum, as well as for gene assignment, physical mapping, and selective isolation and mapping of cDNAs for particular G. bickii chromosomes. The strategies of how to use MAALs to develop varieties with the trait of interest from wild species (such as glanded plant-glandless seed) were proposed and discussed.

Keywords

Genomic in situ hybridization (GISH) Gossypium hirsutum Monosomic alien addition line (MAAL) Simple sequence repeat (SSR) Wild cotton 

Introduction

Gossypium bickii is a diploid wild cotton species (2n = 26, G1 genome) belonging to the tertiary germplasm pool. This species possesses many favourable traits, such as delayed pigment gland morphogenesis in the seed, which results in glandless seeds, whereas the plant remains glanded. Generally, hybridizing G. bickii with the cultivated tetraploid G. hirsutum is unsuccessful and it is extremely difficult to transfer genes of interest from G. bickii into G. hirsutum due to the lack of chromosome pairings and genetic recombination. Additionally, the F1 hybrids are completely sterile. To enhance the transfer of favourable genes from wild relatives into cultivated crops, monosomic alien chromosome addition lines (MAALs) are generally considered an invaluable tool.

MAALs are plants with an alien chromosome from one donor species added to the genome of a recipient species. As a bridge for the introgression of genes of interest from wild relatives into cultivated crops, MAALs have been used to introgress disease resistance (Kang et al. 2011; Lei et al. 2012; Tang et al. 2014) and improve tiller number and yield (Du et al. 2013) in common wheat. MAALs have also been widely used to introgress disease resistance into Oryza, Allium and sugar beets (Gao and Jung 2002; Luo et al. 2012; Vu et al. 2012). In cotton, G. hirsutumG. somalense MAALs have been used to improve fibre properties (Zhou et al. 2004).

Development of MAALs in cotton, however, has lagged behind other crops because of the high number and small size of chromosomes, making it difficult to discriminate alien chromosome additions. Previously, alien addition lines were determined based on classical cytogenetic analysis combined with a morphological survey (Hau 1981; Rooney et al. 1991; Mergeai 1992). This technique is time-consuming and unreliable, and no complete set of MAALs was reported in G. hirsutum until 2014 (Chen et al. 2014), despite many efforts. With the advent of molecular markers and molecular cytogenetic techniques, Zhou et al. (2004) identified two MAALs containing G. somalense chromosomes in a G. hirsutum background by RAPD and classical cytogenetic analysis. Ahoton et al. (2003) isolated six of the possible 13 MAALs carrying G. australe chromosomes in a G. hirsutum background. Sarr et al. (2011) isolated five MAALs of G. australe in G. hirsutum. Chen et al. (2014) obtained a complete set of alien chromosome addition lines of G. australe in G. hirsutum, and 11 MAALs of G. anomalum in G. hirsutum were developed by Wang et al. (2016).

To develop a set of MAALs for G. bickii in a G. hirsutum background, the G. hirsutumG. bickii amphidiploid (2n = 6x = 78, AADDG1G1) was employed as a maternal parent and consecutive backcrosses with G. hirsutum were performed to isolate the MAALs. The genomic in situ hybridization technique was employed to identify the number of additional chromosomes in the G. hirsutum background and a set of G. bickii-specific SSR markers were developed to discriminate the identity of the alien chromosomes. The results indicated that ten of a possible 13 MAALs were isolated, which will lay the foundation for transferring resistance genes of G. bickii into G. hirsutum, as well as for gene assignment, physical mapping, and selective isolation and mapping of cDNAs for particular G. bickii chromosomes. Finally, the strategies of how to use MAALs to develop varieties with the trait of interest from wild species were proposed and discussed.

Materials and methods

Plant materials

The G. hirsutum–G. bickii allohexaploid (2n = 6x = 78, AADDG1G1) was preserved in Nanjing Agricultural University (NAU) and crossed as the female parent with G. hirsutum ‘TM-1’ (2n = 4x = 52, AADD). A pentaploid (2n = 5x = 65, AADDG1) was obtained in 2013 and named C13-31-3. The pentaploid was used as the female parent in continuous backcrossing with TM-1 between 2014 and 2016. All seeds were germinated to allow roots to be excised for use in GISH analysis and then planted in plastic pots and grown in a phytotron under a 16-h/8-h light/dark and 28/24 °C day/night temperature regime. Young leaves were collected for SSR marker analysis at NAU. The progenies that contained alien chromosomes from G. bickii were transplanted into clay pots at Pailou Experimental Station (PES) of NAU. All plants were moved during the winter into the greenhouse at PES for preservation.

Chromosome preparation

To determine alien chromosome numbers in the backcross progenies, mitotic chromosomes were prepared as described by Hanson et al. (1995) and Wang (2001). Roots were pre-treated with 25 µg/ml cycloheximide for 2 h in an incubator at 28 °C where they grew to 3 cm in length. The root tips were collected and fixed in Carnoy’s solution (ethanol:acetic acid = 3:1, v/v) over 2 h. After fixation, the root tips were digested in an enzyme mixture containing 4% cellulase and 1% pectinase at 37 °C for 35 min and squashed with 45% acetic acid. All slides were stored at − 70 °C until use. After removing the coverslips, the slides were dehydrated through an ethanol series (70, 90, and 100%; 5 min each) prior to use in GISH.

Probe labelling and genomic in situ hybridization (GISH) analysis

Total genomic DNA was extracted and purified from young leaves according to the cetyltrimethyl ammonium bromide (CTAB) DNA extraction method (Paterson et al. 1993) with some modifications. Gossypium bickii genomic DNA was labelled with Bio-16-dUTP or digoxigenin-11-dUTP (Roche Diagnostics, Mannheim, Germany) by nick translation. GISH was performed following the procedure described by Wang et al. (2006) with some modifications. In this study, Cot-1 DNA was replaced by genomic DNA extracted from Upland cotton that was used as blocking DNA. The ratio of probe to block was 1:1. Photographs were taken using an Olympus BX51 fluorescence microscope (Media Cybernetics, Bethesda, MD, USA) with an Evolution VF CCD camera. Images were merged using Image-Pro Express software (Media Cybernetics, Bethesda, MD, USA).

Simple sequence repeat (SSR) marker analysis

Gossypium bickii-specific SSR markers were developed to discriminate the identities of alien chromosomes from the G. hirsutum–G. bickii MAALs as described by Chen et al. (2014). PCR amplification and PCR product separation and analysis were performed as described previously by Zhang et al. (2000). To employ a nomenclature consistent with that used for wheat chromosomes (Sears 1956) and our previous report (Chen et al. 2014), the 13 G. bickii chromosomes were designated 1Gb to 13Gb, based on the SSR marker distributions on chromosomes in the D-subgenome of the tetraploid map, where Gb indicates that G. bickii belongs to the G genome species in Gossypium and the superscript letter ‘b’ refers to the initial letter in the species name, bickii.

Morphological analysis

Morphological traits were evaluated in three biological replicates of all cotton plants during the flowering stage with the boll size measured 35-day post-anthesis and fibre examined after maturation.

Results

Development of a set of G. bickii-specific SSR markers

A total of 1506 D-subgenome SSR markers were screened for polymorphisms between G. bickii and G. hirsutum with 183 co-dominant markers identified to differentiate homoeologous G. hirsutum and G. bickii chromosomes and to determine alien chromosome additions for the G. hirsutum–G. bickii MAALs. The number of SSR markers on each chromosome ranged from 9 to 19 with an average of 14 (Fig. 1). Chromosome coverage ranged from 60.0 to 93.4% with a density of 5.95–10.14 cM (Table S1).

Fig. 1

Genetic linkage map of G. bickii chromosome-specific SSR markers based on the linkage map of tetraploid cotton reported by Guo et al. (2007)

Transmissibility of alien G. bickii chromosomes in the G. hirsutum background during backcrossing

The G. bickii chromosome-specific SSR markers were used to identify the pentaploid (BC1) chromosomes and all 13 chromosomes from G. bickii existed in the pentaploid. The pentaploid was used as the female parent in backcrosses with G. hirsutum ‘TM-1’, which generated 48 backcrossed seeds due to the lower fertility of the pentaploid (BC1). Twenty-seven (56.25%) seeds germinated and produced plants (BC2). GISH analysis indicated that these 27 plants carried between 1 and 8 alien G. bickii chromosomes (Figs. 2, S1). The largest number of plants (7/27) contained four alien chromosomes, whereas one plant contained only a single G. bickii chromosome, 4Gb (Table 1). A large number of alien chromosomes were lost in the backcross progenies (Fig. S2). However, SSR marker analysis demonstrated that the 27 plants covered all 13 G. bickii chromosomes. Chromosome 13Gb showed the highest transmissibility, present in up to 55.56% of the BC2 progeny (Table 1; Figs. 2, S2), whereas chromosomes 8Gb, 10Gb, 7Gb and 1Gb exhibited relatively lower transmissibility. BC2 plants with multiple alien chromosome additions were further backcrossed with G. hirsutum ‘TM-1’, which generated 378 seeds with 299 BC3 plants produced by germinating on Murashige and Skoog medium. For the BC3 generation, GISH analysis indicated that transmission of alien chromosomes significantly decreased to 0.42 per individual. Of the BC3 individuals, 199 had no alien chromosomes added, whereas 81 contained a single G. bickii chromosome and 19 contained two–four G. bickii chromosomes (Table 2). Further SSR marker analysis demonstrated that 12 of the 13 G. bickii G1 genome chromosomes (all except 7Gb) were present in the 100 BC3 plants that contained alien chromosomes. Chromosomes 4Gb and 13Gb showed the highest transmissibility, while chromosomes 1Gb, 7Gb, 8Gb, 9Gb, 10Gb and 11Gb showed the lowest (Table 3). In addition, the BC3 individual with two alien chromosomes (3Gb and 4Gb) was self-pollinated, and 31 seeds were produced. Of the 31 plants, 13 had no alien chromosomes, 14 had one alien chromosome, and four contained two alien chromosomes, showing a higher transmissibility in self-pollination than backcrossing.

Fig. 2

Genomic in situ hybridization revealed multiple G. hirsutumG. bickii alien chromosome additions. a G. hirsutum ‘TM-1’ (2n = 52); b G. bickii (2n = 26); ch indicates the multiple alien chromosome additions, 2n = 52 + 8, 2n = 52 + 6, 2n = 52 + 4, 2n = 52 + 3, 2n = 52 + 2, 2n = 52 + 1; i shows an example, where no alien chromosome was added. Green fluorescent signals indicate G. bickii alien chromosomes. Scale bar = 5 µm

Table 1

Transmission of alien chromosomes from BC1 to BC2 in G. hirsutum × G. bickii

Chromosome component

1Gb

2Gb

3Gb

4Gb

5Gb

6Gb

7Gb

8Gb

9Gb

10Gb

11Gb

12Gb

13Gb

No. plants

52

             

0

52 + 1

   

1

         

1

52 + 2

 

1

2

1

 

2

 

1

   

1

2

5

52 + 3

2

1

 

3

2

   

2

1

1

 

3

5

52 + 4

1

3

4

2

1

 

1

1

2

3

3

3

4

7

52 + 5

 

1

1

2

3

2

1

1

4

 

1

2

2

4

52 + 6

  

2

2

1

1

1

1

1

 

1

1

1

2

52 + 7

 

2

 

1

2

2

1

1

  

2

1

2

2

52 + 8

 

1

 

1

 

1

 

1

 

1

1

1

1

1

Sum

3

9

9

13

9

8

4

6

9

5

9

9

15

27

Transmissibility (%)

11.11

33.33

33.33

48.15

33.33

29.63

14.81

22.22

33.33

18.52

33.33

33.33

55.56

 
Table 2

Number of alien chromosomes in (G. hirsutum × G. bickii) BC3

Chromosome component

No. individuals

Percentage (%)

52

199

66.56

52 + 1

81

27.09

52 + 2

13

4.35

52 + 3

4

1.34

52 + 4

2

0.67

Table 3

Transmission of alien chromosomes from BC2 to BC3 in G. hirsutum × G. bickii

Chromosome component

1Gb

2Gb

3Gb

4Gb

5Gb

6Gb

7Gb

8Gb

9Gb

10Gb

11Gb

12Gb

13Gb

No. plants

52

             

199

52 + 1

 

11

10

13

3

10

 

3

1

2

 

13

15

81

52 + 2

 

4

4

8

3

1

   

1

 

2

3

13

52 + 3

1

 

1

1

2

   

1

2

1

 

3

4

52 + 4

 

1

2

 

2

  

1

   

2

 

2

Sum

1

16

17

22

10

11

0

4

2

5

1

17

21

299

Transmissibility (%)

0.33

5.35

5.69

7.36

3.34

3.68

0.00

1.34

0.67

1.67

0.33

5.69

7.02

 

Determination of the identities of monosomic alien addition lines (MAALs) by SSR markers

In the BC3 generation, GISH analysis indicated that 81 individuals each carried one alien chromosome; therefore, these were monosomic alien addition lines (MAALs). The G. bickii chromosome-specific SSR marker analysis showed that these 81 BC3 individuals carrying one alien chromosome (Fig. 3) belonged to ten different MAALs (Fig. 4). SSR marker analysis demonstrated that the MAALs carrying one G. bickii chromosome included chromosomes 2Gb, 3Gb, 4Gb, 5Gb, 6Gb, 8Gb, 9Gb, 10Gb, 12Gb, and 13Gb. Among them, chromosome 13Gb had the highest incidence, and chromosome 9Gb the lowest (Table 4; Fig. S3). No MAALs were isolated for chromosomes 1Gb, 7Gb and 11Gb. Chromosomes 1Gb and 11Gb remain in triple monosomic addition lines with chromosomes 3Gb and 4Gb and chromosomes 8Gb and 10Gb, respectively, while chromosome 7Gb was lost in the BC3 generation due to its low transmissibility.

Fig. 3

Genomic in situ hybridization revealed ten G. hirsutum–G. bickii MAALs. aj Represent MAALs 2Gb, 3Gb, 4Gb, 5Gb, 6Gb, 8Gb, 9Gb, 10Gb, 12Gb, and 13Gb, respectively; arrows indicate G. bickii alien chromosomes; red fluorescent signals represent the 52 chromosomes of G. hirsutum. Scale bar = 5 µm

Fig. 4

G. bickii-specific SSR marker analysis of MAALs. The G. bickii-specific amplicons were obtained using 13 individual chromosome-specific primer pairs for markers; a to m correspond to D1 to D13 in cultivated tetraploid cotton and are NAU7728, NAU4025, NAU174, NAU7800, NAU159, NAU1987, NAU2186, NAU394, NAU20, NAU6267, NAU4004, NAU7835, and NAU8214. P1 G. hirsutum; P2 G. bickii; A the hexaploid of G. hirsutum and G. bickii; 2 to 6 are MAALs 2Gb, 3Gb, 4Gb, 5Gb and 6Gb; 8 to 10 are MAALs 8Gb, 9Gb and 10Gb; 12 and 13 are MAALs 12Gb and 13Gb, respectively; M is a molecular size marker (50 bp ladder); arrows (red) indicate chromosome-specific markers for G. bickii

Table 4

Number of individuals for each MAAL in BC3 of G. hirsutum × G. bickii

MAAL

Individuals

Percentage (%)

MAAL-2Gb

11

13.75

MAAL-3Gb

10

12.50

MAAL-4Gb

12

15.00

MAAL-5Gb

3

3.75

MAAL-6Gb

10

12.50

MAAL-8Gb

3

3.75

MAAL-9Gb

1

1.25

MAAL-10Gb

2

2.50

MAAL-12Gb

13

16.25

MAAL-13Gb

15

18.75

Morphological observation of MAALs

Distinct morphological features were observed for the ten MAALs and they differed from the parents and amphidiploid (hexaploid) plant (Figs. 5, 6; Table 5). For example, the MAAL with 10Gb showed dwarfism with a plant height of only 57.0 cm and a high number of branches, whereas MAALs for 6Gb, 9Gb, and 12Gb were similar in plant height to TM-1 (Fig. 5a). MAAL for 5Gb showed a small leaf size similar to that of G. bickii, while MAALs for 2Gb and 12Gb showed large leaf sizes similar to that of TM-1 (Fig. 5b). MAALs for 4Gb and 10Gb had small flower buds and bracts similar to G. bickii (Fig. 6a). MAALs for 5Gb had light yellow petals and anthers and were different from all the others (Fig. 6b). This MAAL was easily discriminated by its very small bolls, which were similar in size to that of G. bickii (Fig. 6c). MAALs for 6Gb showed brown-coloured fibres (Fig. 6d).

Fig. 5

Morphological traits of MAALs of G. bickii individual chromosomes in G. hirsutum. a Represents plants; b represents leaves. P1 G. hirsutum; P2 G. bickii; A the amphidiploid of G. hirsutum and G. bickii; 2G to 6G are MAALs 2Gb, 3Gb, 4Gb, 5Gb and 6Gb; 8G to 10G are MAALs 8Gb, 9Gb and 10Gb; 12G and 13G are MAALs 12Gb, and 13Gb, respectively

Fig. 6

Flower, boll and fibre traits of MAALs of G. bickii individual chromosomes in G. hirsutum. P1 G. hirsutum; P2 G. bickii; A the amphidiploid of G. hirsutum and G. bickii; 2G to 6G are MAALs 2Gb, 3Gb, 4Gb, 5Gb and 6Gb; 8G to 10G are MAALs 8Gb, 9Gb and 10Gb; 12G and 13G are MAALs 12Gb, and 13Gb, respectively

Table 5

Morphological characteristics of ten MAALs

Trait

P1

P2

Monosomic alien addition line

 

TM-1

G. bickii

2Gb

3Gb

4Gb

5Gb

Anther number

125.80 ± 3.37

96.35 ± 4.37

95.23 ± 7.95

83.00 ± 2.65

95.75 ± 25.25

111.50 ± 12.02

Style length (cm)

3.26 ± 0.15

1.50 ± 0.21

1.97 ± 0.31

1.53 ± 0.15

1.60 ± 0.35

2.30 ± 0.14

Stigma length (cm)

1.18 ± 0.75

0.40 ± 0.08

0.85 ± 0.15

0.87 ± 0.76

0.68 ± 0.17

1.40 ± 0.28

Pedicel length (cm)

1.50 ± 0.31

1.01 ± 0.11

1.16 ± 0.56

1.10 ± 0.12

0.55 ± 0.13

0.85 ± 0.71

Locule number

4.00

4.00

4.00

4.00

4.00

4.00

Ovule number per locule

8.91 ± 0.28

4.00

7.43 ± 0.69

8.25 ± 0.50

7.31 ± 0.48

7.88 ± 0.83

Petal length (cm)

5.18 ± 0.34

3.96 ± 0.05

4.01 ± 0.31

3.52 ± 0.13

3.64 ± 0.31

4.27 ± 0.19

Petal width (cm)

4.73 ± 0.74

4.24 ± 0.05

3.97 ± 0.42

3.40 ± 0.71

3.89 ± 0.49

4.07 ± 0.13

Bract length (cm)

5.53 ± 0.29

0.90

4.33 ± 0.59

4.16 ± 0.56

3.72 ± 0.65

4.17 ± 0.15

Bract width (cm)

3.27 ± 0.58

0.10

2.47 ± 0.46

2.13 ± 0.17

2.13 ± 0.21

2.53 ± 0.31

Bract teeth number

12.90 ± 1.43

1.00

13.16 ± 2.25

8.33 ± 0.98

6.89 ± 1.36

8.00 ± 0.89

Sepal length (cm)

3.26 ± 0.18

1.40 ± 0.09

2.04 ± 0.69

2.97 ± 0.15

3.22 ± 0.39

3.05 ± 0.21

Sepal width (cm)

0.80 ± 0.62

1.00

1.44 ± 0.71

1.00 ± 0.80

0.86 ± 1.50

0.90 ± 0.70

Leaf width (cm)

13.90 ± 0.99

3.36 ± 0.15

14.14 ± 1.43

10.50 ± 1.41

13.85 ± 1.48

7.10 ± 0.42

Leaf length (cm)

11.50 ± 1.33

6.96 ± 0.4

11.95 ± 0.86

8.10 ± 0.63

11.00 ± 1.01

5.60 ± 0.64

Petiole length (cm)

8.68 ± 1.76

1.57 ± 0.40

8.90 ± 0.71

5.00 ± 0.43

7.98 ± 1.09

4.30 ± 2.19

Boll width (cm)

3.40 ± 0.15

1.06 ± 0.08

2.97 ± 0.37

3.41 ± 0.64

3.35 ± 0.16

1.55 ± 0.56

Boll length (cm)

4.50 ± 0.25

1.04 ± 0.04

4.17 ± 0.34

3.80 ± 0.36

3.43 ± 0.79

2.35 ± 0.39

Peduncle length (cm)

1.84 ± 0.44

1.13 ± 0.05

1.27 ± 0.14

1.37 ± 0.27

0.67 ± 0.14

0.96 ± 0.23

Plant height (cm)

146.17 ± 5.01

96.30 ± 3.21

107.20 ± 11.43

97.00 ± 15.6

104.30 ± 23.24

86.50 ± 3.53

Trait

Monosomic alien addition line

 

6Gb

8Gb

9Gb

10Gb

12Gb

13Gb

Anther number

108.33 ± 10

110.00 ± 15

90.67 ± 7.02

99.33 ± 10.12

102.00 ± 9.08

103.00 ± 9.08

Style length (cm)

2.61 ± 0.24

2.23 ± 0.58

2.27 ± 0.58

2.00 ± 0.26

2.23 ± 0.15

2.14 ± 0.48

Stigma length (cm)

0.96 ± 0.12

1.13 ± 0.15

1.20 ± 0.11

0.67 ± 0.21

0.70 ± 0.11

0.88 ± 0.23

Pedicel length (cm)

1.33 ± 0.87

0.78 ± 0.15

1.05 ± 0.09

0.57 ± 0.15

1.33 ± 0.15

1.60 ± 0.73

Locule number

4.66 ± 0.51

4.00

4.00

4.00

4.00

4.00

Ovule number per locule

7.16 ± 0.40

7.50 ± 0.53

7.38 ± 0.52

7.17 ± 0.72

8.66 ± 1.15

8.40 ± 0.54

Petal length (cm)

4.59 ± 0.44

4.14 ± 0.31

3.97 ± 0.39

4.03 ± 0.61

4.10 ± 0.24

4.52 ± 0.18

Petal width (cm)

4.48 ± 0.51

4.14 ± 0.31

3.78 ± 0.23

3.37 ± 0.38

4.09 ± 0.19

4.55 ± 0.43

Bract length (cm)

5.01 ± 0.68

4.16 ± 0.11

4.17 ± 0.17

3.61 ± 0.62

4.52 ± 0.35

4.74 ± 0.52

Bract width (cm)

2.94 ± 0.35

2.18 ± 0.18

2.33 ± 0.15

1.44 ± 0.28

2.71 ± 0.31

3.14 ± 0.42

Bract teeth number

11.61 ± 1.85

11.89 ± 1.89

8.15 ± 1.17

5.33 ± 1.66

15.44 ± 1.42

12.46 ± 1.84

Sepal length (cm)

2.80 ± 0.18

3.53 ± 0.31

2.80 ± 0.89

2.60 ± 0.10

2.36 ± 0.06

2.74 ± 0.29

Sepal width (cm)

1.15 ± 0.05

0.93 ± 0.15

0.85 ± 0.50

1.03 ± 0.12

1.26 ± 0.06

1.12 ± 0.04

Leaf width (cm)

8.70 ± 3.59

11.40 ± 2.55

9.60 ± 0.52

9.40 ± 0.57

10.90 ± 1.02

10.20 ± 0.67

Leaf length (cm)

6.90 ± 0.23

8.70 ± 2.93

6.90 ± 0.92

8.20 ± 0.14

7.50 ± 0.49

7.00 ± 0.81

Petiole length (cm)

9.72 ± 4.06

5.80 ± 1.41

4.60 ± 0.41

5.20 ± 0.28

8.40 ± 1.59

4.90 ± 0.73

Boll width (cm)

3.05 ± 0.23

2.97 ± 0.19

2.55 ± 0.87

2.31 ± 0.15

2.73 ± 0.63

3.08 ± 0.37

Boll length (cm)

4.24 ± 0.51

3.42 ± 0.46

3.61 ± 0.98

3.52 ± 0.28

4.47 ± 0.45

4.41 ± 0.46

Peduncle length (cm)

1.42 ± 0.46

1.33 ± 0.37

1.24 ± 0.31

1.49 ± 0.33

1.72 ± 0.31

1.50 ± 0.39

Plant height (cm)

124.00 ± 34.25

92.00 ± 2.83

117.00 ± 3.41

59.00 ± 2.83

103.6 ± 11.58

101.8 ± 17.23

Discussion

Cotton seeds are an important source of oil and protein with the seed containing 21% oil and 23% protein (Sunilkumar et al. 2006). However, gossypol and its derivatives exist in cotton, which limits the utilization of cotton seed as a food source for human beings and non-ruminant animals due to the toxicity of these compounds (Bottger et al. 1964). Fortunately, Australian diploid wild species (G. bickii, G. australe and G. sturtianum) have gossypol-free seeds, but high levels of gossypol in all the above-ground plant parts (Fryxell 1992). Numerous studies have focused on the transfer of this trait from G. bickii into G. hirsutum. However, so far, there have been very few reports on the successful development of cultivars with glanded plant-glandless seed phenotypes. Because the distant evolutionary relationship between G. bickii and Upland cotton has given rise to linkage drag, no sufficient genetic recombination has occurred, which makes it very difficult to select an ideal recombinant for breeding.

Early in 1960, two alleles, Gl2 and Gl3, controlling the glanded trait of Upland cotton were located on chromosomes 12 and 26 of the A and D genomes, respectively (McMichael 1960). Zhu and Ji (2001) and Zhu et al. (2004) suggested that the gene controlling the pigment gland trait of G. bickii (Gl2b) shows dominant epistasis to that of the A chromosome (Gl2) and recessive epistasis to that of the D chromosome (Gl3) in G. hirsutum. Therefore, Zhu et al. (2005) made the cross between the amphidiploid of (G. arboreum × G. bickii, genome composition AAGG, genotype Gl2Gl2Glb2Glb2) and an Upland cotton germplasm with the genotype Gl2Gl2g13g13 in 2005. From the above progenies, they obtained individuals with the glanded plant-glandless seed phenotype, whose genotype was suggested to be Gl2bGl2bgl3gl3. Since the genotypes G. hirsutum–G. bickii MAALs are Gl2bGl2Gl2Gl3Gl3 and the gene Gl3 shows dominant epistasis to Gl2b, it did not have the glanded plant-glandless seed trait.

Therefore, to transfer the Gl2b target gene into Upland cotton, the dominant gene Gl3 in Upland cotton has to be replaced or removed. The G. hirsutum–G. bickii MAALs developed in this study can be used as a bridge for the development of cotton with glanded plant-glandless seed traits. One strategy is to induce chromosome translocations between chromosomes 26 (D12) in G. hirsutum and 12Gb in G. bickii and to replace the Gl3 gene with the 12Gb gene. Another strategy is to use an aneuploid of cotton. To achieve this, a cross is first performed between monosome 12 (2n − 1) (available in Upland cotton, Endrizzi and Ramsay 1980) and MAAL-12Gb (2n + 1), where the former produces n and (n − 1) gametes, and the latter produces n and (n + 1) gametes. As the combination of the (n − 1) gamete (without chromosome 12) and the (n + 1) (with chromosome 12Gb) has occurred, the chromosome component of the hybrid F1 is (2n + 1–1). The F1 should then be self-pollinated to produce the F2 generation. In the F2 population, the chromosome substitution line of chromosome 12 will be produced and individuals with the genotype Gl2bGl2bGl3Gl3 will be selected. Next, the individuals with the genotype Gl2bGl2bGl3Gl3 should be crossed with Gl2Gl2g13g13 to produce F1 and F2 populations. From the F2 population, we would then be able to develop individuals with the genotype Gl2bGl2bgl3gl3 for breeding in the future.

In addition, G. bickii also possesses many other favourable traits, such as resistance to diseases (Verticillium wilt) and pest insects (aphids and mites) and tolerance to abiotic stresses (drought), which would be valuable in breeding. The development of G. hirsutum–G. bickii MAALs in this study will lay the foundation for further transferring genes of G. bickii into G. hirsutum, as well as for gene assignment, physical mapping, and selective isolation and mapping of cDNAs for particular G. bickii chromosomes.

Notes

Funding

This study was funded by the National Key Research and Development Program of China (2016YFD0100203), the National Key Technology Support Program of China during the Twelfth 5-year plan period (2013BAD01B03-04), the Priority Academic Program Development of Jiangsu Higher Education Institutions and Jiangsu Collaborative Innovation Center for Modern Crop Production.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest in the reported research.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

438_2018_1434_MOESM1_ESM.docx (20 kb)
Supplementary Table S1. G. hirsutum-G. bickii chromosome-specific SSR markers (DOCX 20 KB)
438_2018_1434_MOESM2_ESM.tif (860 kb)
Supplementary Fig. S1. Number of G. bickii chromosomes added in the BC2 generation (TIF 859 KB)
438_2018_1434_MOESM3_ESM.tif (1.7 mb)
Supplementary Fig. S2. Incidence of each alien chromosome from G. bickii in the BC2 generation (TIF 1741 KB)
438_2018_1434_MOESM4_ESM.tif (251 kb)
Supplementary Fig. S3. Number individuals of each MAAL (TIF 251 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Crop Genetics and Germplasm Enhancement, MOE Hybrid Cotton R&D Engineering Research CenterNanjing Agricultural UniversityNanjingPeople’s Republic of China
  2. 2.Key Laboratory of Cotton Breeding and Cultivation in Huang-Huai-Hai Plain, Ministry of AgricultureCotton Research Center of Shandong Academy of Agricultural SciencesJinanPeople’s Republic of China

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