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Development of a set of monosomic alien addition lines from Gossypium raimondii in Gossypium hirsutum toward breeding applications in cotton

BMC Plant Biology volume 25, Article number: 150 (2025) Cite this article

Abstract

Background

Gossypium raimondii Ulbr is a diploid wild cotton (2n = 26, D5D5) that originated in west-central Peru of South America and possesses desirable characteristics that are absent in the Upland cotton G. hirsutum. Many beneficial genes were lost from G. hirsutum in the process of domestication, leading to a narrowed genetic base and greater vulnerability to biotic and abiotic stresses. This genetic base can be expanded through distant hybridization using the superior genes of G. raimondii.

Results

In this study, putative hexaploid F1 plants of G. hirsutum - G. raimondii were generated by interspecific hybridization. Analysis of its mitotic metaphase plates revealed the presence of 78 chromosomes, with each of the six chromosome-specific fluorescence in situ hybridization (FISH) probes (3D5, 5D5, 6D5, 7D5, 9D5, and 10D5) of G. raimondii exhibiting bright and distinct signals on its respective pair of chromosomes. Then, the fertile hexaploid F1 plants were continuously backcrossed with G. hirsutum and a set of G. hirsutum - G. raimondii monosomic alien addition lines (MAALs) were developed using SSR markers in successive backcrosses and self-crossing from BC2F1 to BC4F2. These MAALs were confirmed by chromosome-specific anchored SSRs and FISH. This set of MAALs exhibited abundant variation in morphological traits, agronomic characteristics, yield, and fiber quality traits, as well as in drought and salt resistance at seedling stage. Notably, MAAL_9D5 and MAAL_10D5 demonstrated excellent fiber length (FL), fiber uniformity (FU), fiber strength (FS), micronaire value, and fiber elongation (FE); At seeding stage, MAAL_8D5, MAAL9D5, MAAL_10D5, MAAL_12D5, and MAAL_13D5 showed salt resistance potential; while MAAL_1D5, MAAL_3D5, MAAL_4D5, MAAL_7D5, MAAL_8D5, MAAL_12D5, and MAAL_13D5 exhibited drought resistance potential. These MAALs will provide important genetic bridge materials for gene transfer from G. raimondii as well as for the study of Gossypium species genomes and their evolution.

Conclusions

A set of Gossypium hirsutum - Gossypium raimondii MAALs were developed and they showed abundant variation in morphological, agronomic, yield, and fiber quality traits, as well as in drought and salt resistance at seedling stage.

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Background

The genus Gossypium comprises five tetraploid species (2n = 4x = 52, AD) and over 45 diploid species (2n = 2x = 26, classified eight genomic groups: A-G and K) [1, 2]. Among these, the two tetraploid species Gossypium hirsutum (AD)1 and G. barbadense (AD)2, along with the two diploid species G. herbaceum (A1) and G. arboreum (A2) are cultivated. The domesticated allotetraploid species G. hirsutum L. (2n = 4x = 52, (AD)1), commonly known as Upland cotton, accounts for approximately 90% of annual global cotton production because of its relatively high yield potential with moderate fiber quality and wide adaptability [3]. However, the diversity of modern cultivated upland cotton has been dramatically curtailed due to extensive selection within limited cotton resources, and this lack has become a major bottleneck for cotton breeding progress. The wild relatives of cotton represent an impressive range of genetic diversity with desired agronomic traits, which can be used to improve cultivated upland cotton via wide hybridization.

G. raimondii Ulbr. is a diploid wild cotton species (2n = 26, D5D5) native to Peru. Although it is not directly utilized for commercial production, it is an important reservoir of beneficial genes for elite fiber quality, resistance to verticillium wilt, insects resistance [4, 5], and tolerance to drought [6] and salt [7]. Additionally, it is also a source of fertility restorer for cytoplasmic male sterility [8]. Therefore, G. raimondii constitutes a rich resource that can be mined for useful variants lost during cotton domestication. Moreover, G. raimondii has garnered a significant attention in the fields of cotton speciation and genome research, as it is the donor of the Dt genome in allotetraploid cotton. Nevertheless, only a limited number of systematic efforts have been made to identify and transfer these novel traits from G. raimondii into the G. hirsutum background [9].

Monosomic alien addition lines (MAALs) consist of a single chromosome from one species plus the entire diploid complement of another species as the background. The development of MAALs is an effective strategy for transferring useful genes from distant wild relatives; such lines have been generated across a wide variety of crops, including wheat [10], Brassica oleracea [11], bunching onion [12], kale oilseed rape [13], sugar beet [14], rice [15], tomato [16], soybean [17], radish [18], Brassica alboglabra [19], tobacco [20], and oats [21]. In cotton, several sets of alien chromosome additions have been developed from the diploid wild species G. anomalum [22], G. bickii [23], and G. australe [24, 25]. However, no alien addition lines from G. raimondii into G. hirsutum have been produced to date. Notably, despite the D5 genome being closely related to Dt, the direct transfer of chromosomes from G. raimondii into G. hirsutum is challenging due to a high degree of genome incompatibility, which results in serious interspecific reproductive barriers.

The aims of the present study are to develop a first set of MAALs from G. raimondii under the G. hirsutum background using molecular marker and cytogenetic techniques, and to identify favorable agronomic traits for cotton breeding programs. These MAALs will facilitate the introduction of beneficial genes and traits for cotton improvement.

Results

Morphological, cytological, and molecular analyses of the putative synthesized hexaploid

The morphological characteristics and genotype of the putative hexaploid plants were found to be intermediate between G. hirsutum and G. raimondii (Fig. 1). Specifically, the shapes and sizes of leaves, bolls, and bracts were intermediate. Additionally, the proliferation of the putative hexaploid plants was sensitive to photoperiod with flowering and boll setting occurring during autumn and spring in Nanjing.

Fig. 1
figure 1

Morphological characteristics and amplification patterns of G. raimondii, G. hirsutum var. Deltapine 15, and the (G. hirsutum × G. raimondii)2 hexaploid. a G. raimondii; b G. hirsutum var. Deltapine 15; c (G. hirsutum × G. raimondii)2 hexaploid. d amplification patterns of G. raimondii, G. hirsutum var. Deltapine 15, and two plants of (G. hirsutum × G. raimondii)2 hexaploid. Red arrows indicate specific bands of G. raimondii chromosome. Scale bar = 5 cm

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Analysis of mitotic metaphase plates revealed the presence of 78 chromosomes in the (G. hirsutum × G. raimondii)2 hexaploid (Fig. 2), which aligns with the expected chromosome number for a hexaploid hybrid (2n = 6x = 78, A1A1D1D1D5D5) derived from G. hirsutum (2n = 4x = 52, A1A1D1D1) and G. raimondii (2n = 2x = 26, D5D5), thus confirming the doubled hybrid status of the obtained material. Furthermore, each of the six chromosome-specific fluorescence in situ hybridization (FISH) probes (3D5, 5D5, 6D5, 7D5, 9D5, and 10D5) exhibited bright and distinct signals on its respective pair of chromosomes (Fig. 2).

Fig. 2
figure 2

Cytological identification of metaphase mitotic cells in the (G. hirsutum × G. raimondii)2 hexaploid. Scale bar = 10 μM

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Genome-wide screening of a set of G. raimondii-specific SSR loci based on BC2F1 segregation data

Based on the SSR physical mapping information integrated from previous studies [26], a total of 1,117 SSR primer pairs (approximately 3–4 markers/5 cM) were selected. The selection included 276 pairs of CICR primer, 369 pairs of SWU primer, 138 pairs of NAU primer, 4 pairs of CGR primer, and 330 pairs of JAAS primer derived from the genome sequence of G. raimondii. These primers were used to analyze three parent plants (G. raimondii, G. hirsutum Deltapine 15, and G. hirsutum var. Su8289), five hexaploid F1 plants, and ten randomly selected BC2F1 plants. Finally, 235 evenly distributed, well amplified, informative microsatellite markers were screened and employed to genotype the entire BC2F1 population, which consisted of 140 individuals.

Segregation data from the 235 SSR loci were used to conduct linkage analysis using MapMaker/EXP 3.0b [27], resulting in the identification of 223 SSR loci across 13 linkage groups; 12 SSR loci were not linked to any group. As severe recombination suppression was observed, the 223 loci were placed at their approximate positions based on the combined marker map, rather than the BC2F1 segregation data. The 13 linkage groups were assumed to correspond to the 13 chromosomes of G. raimondii. The number of G. raimondii-specific loci on each chromosome ranged from 10 to 21, with an average of 17 markers per chromosome, and the average distance between adjacent markers on a chromosome varied from 8.57 to 13.22 cM. The coverage of G. raimondii-specific SSR marker loci on a chromosome ranged from 94.19% (1D5) to 99.67% (2D5), with an average coverage of 97.97% (Fig. 3 and Table 1). 

Fig. 3
figure 3

Map of G. raimondii-specific SSR loci based on the combined map information. The scales represent centiMorgans (cM)

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Table 1 Numbers and coverage of G. raimondii-derived SSR markers on 13 chromosomes
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Development of MAALs

Among the total of 140 BC2F1 individuals, 121 plants carried 53–63 chromosomes, covering all G. raimondii chromosomes. Many of these plants exhibited sterility, with some occasionally producing flowers that had incomplete stigmas or anthers, did not dehisce properly, or were sensitive to photoperiod. Only 42 plants produced viable hybrid seeds.

From the BC2F1 to the BC4F2 generation, plants that carried the target chromosomes alongside as few other G. raimondii chromosomes (including introgression segments) as possible were selected for successive backcrosses with Su8289 to generate the BC4F1 generation in 2021, which was in turn selfed to produce the BC4F2 generation in 2022. To efficiently monitor G. raimondii chromosome content and reduce labor cost, 107 G. raimondii-specific markers were used to discriminate the identity of the alien chromosomes and the background genotype in each generation (marker names listed in Supplemental Table 1). These markers were selected on nearness to the recombination breakpoints in the BC2F1 generation and represented genomic regions delimited by specific recombination events. Finally, a set of 12 monosomic alien addition lines from G. raimondii under the G. hirsutum background was developed (Supplemental Fig. 1). Of which, the plants of MAAL-5D5 showed weak viability and fertility, and failed to advance to the BC4F1 generation.

The transmission rates of alien chromosomes in the BC3F1, BC4F1, and BC4F2 generations are summarized in Table 2. Transmission rates of the whole alien chromosomes ranged from 9.76% (13D5) to 34.48% (2D5) in BC3F1, from 12.12% (3D5) to 36.26% (2D5) in BC4F1, and from 9.09% (4D5) to 68.66% (1D5) in the BC4F2 generation. Generally, the rates were higher during selfing compared to backcrossing, with the exception of 4D5 and 12D5. The incidence rates of introgression were significantly greater for chromosomes 2D5, 4D5, 6D5, 8D5, and 11D5 by selfing than by backcrossing. Conversely, the introgression of 12D5 remained consistently low (8.25–14.49%) regardless of whether selfing or backcrossing was employed.

Table 2 Identification of alien addition lines in different generations
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Identification by FISH

Based on the SSR marker analysis results, plants containing one G. raimondii chromosome (specifically 3D5, 6D5, 7D5, 9D5, and 10D5) were selected for further cytological identification using root tip cells. As illustrated in Fig. 4, some of the chromosomes in the mid mitotic stage were stained blue by DAPI, and a total of 53 chromosomes were counted, which is one more than the 52 chromosomes present in the G. hirsutum genome, indicating that we successfully obtained G. hirsutum - G. raimondii MAALs that respectively harbor chromosomes 3D5, 6D5, 7D5, 9D5, and 10D5.

Fig. 4
figure 4

Cytological identification of metaphase mitotic cells in MAAL-3D5, MAAL-6D5, MAAL-7D5, MAAL-9D5, and MAAL-10D5. Scale bar = 10 μM

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Phenotypic characteristics of the MAALs

The MAALs showed abundant variation in morphological traits, viability, agronomic characteristics, yield-related, and fiber quality traits. Notably, MAAL-3D5 was characterized by dwarfing and low viability; MAAL-10D5 demonstrated slower growth and later flowering compared to the recurrent parent, Su8289; and MAAL-6D5was sensitive to short photoperiods.

Concerning each line in detail:

  • MAAL-1D5 plants were quite similar to the recurrent parent, Su8289 (Figs. 1 and 5). However, their boll weight (BW) and lint percent (LP) were significantly lower than those of Su8289 (P < 0.05, P < 0.01), with respective values recorded in 2022 and 2023 of 3.54 ± 0.36 g, 3.75 ± 0.38 g and 34.25% ± 0.89, 31.86% ± 2.28 (Table 3). Additionally, in 2023, MAAL-1D5 exhibited a higher mean index of MIC value (5.33 ± 0.06) and a lower FE (6.47% ± 0.06) compared to Su8289 (P < 0.05) (Table 4).

    MAAL-2D5 plants were characterized by light green leaves and shorter fruit stems (Figs. 1 and 5). They also exhibited the lowest LP (29.65% ± 3.26 and 23.73% ± 2.14 in 2022 and 2023, respectively) and a lower BW (3.87 ± 0.38 g in 2022) (Table 3). No significant differences were observed in fiber-related traits between MAAL-2D5 and Su8289 (Table 4).

  • MAAL-3D5 plants demonstrated smaller leaves, petals, and boll size compared to the recurrent parent (Figs. 1 and 5). The mean BW and LP were significantly lower than those of Su8289 (P < 0.05, P < 0.01), with respective values recorded in 2022 and 2023 of 2.40 ± 0.24 g, 2.10 g and 29.93% ± 2.84, 27.90% ± 1.29 (Table 3). This line also had the lowest fiber uniformity (FU) (81.60% ± 0.45 in 2022) and MIC value (4.00 ± 0.20 in 2022) (Table 4).

  • MAAL-4D5 plants had smaller leaves and bolls (Figs. 1 and 5). The average BW and LP were significantly lower than those of Su8289 (P < 0.01), with BW recorded at 3.56 ± 0.65 g in 2022 and LP at 32.77% ± 2.02 and 34.90% ± 2.54 for 2022 and 2023, respectively (Table 3). In contrast, the average values for fiber strength (FS), MIC, and FE in 2023 were significantly higher than those of Su8289 (P < 0.05, P < 0.01), at 31.12 ± 2.17 cNˑtex−1, 5.48 ± 0.13, and 6.85% ± 0.16, respectively (Table 4).

  • MAAL-5D5 plants showed low viability and fertility, characterized by smaller leaves, and the smallest boll size (Figs. 1 and 5).

  • MAAL-6D5 plants demonstrated vigorous growth and were sensitive to day-length, responding to a short photoperiod. They featured larger petals and bracts, as well as longer stamens and fruit stems compared to the recurrent parent (Figs. 1 and 5). In 2023, the mean LP (32.60% ± 5.75) was significantly lower than that of Su8289 (P < 0.01) (Table 3), while the average FS (29.80 ± 1.11 cNˑtex−1), MIC (5.66 ± 0.38), and FE (6.80% ± 0.16) were all significantly higher (P < 0.05, P < 0.01) (Table 4).

  • MAAL-7D5 plants had pink stamen filaments similar to G. raimondii, along with bigger bracts (Figs. 1 and 5). They also featured three leaf lobes, where both Su8289 and the hexaploid F1 had five leaf lobes (Figs. 1 and 5). In both 2022 and 2023, the mean BW (3.57 ± 0.35 g and 3.93 ± 0.45 g) and LP (35.49% ± 1.70 and 33.24% ± 3.33) were significantly lower than in Su8289 (P < 0.05, P < 0.01) (Table 3). However, in 2023, the average fiber length (FL) (28.10 ± 1.13 mm), FU (84.93% ± 0.63), FS (30.63 ± 1.59 cNˑtex−1), MIC (5.50 ± 0.18), and FE (6.70% ± 0.08) were all significantly higher (P < 0.05, P < 0.01) (Table 4).

  • MAAL-8D5 plants exhibited dark green leaves and elongated pistils (Fig. 5). In 2022, the average BW (3.77 ± 0.30 g) and MIC (4.87 ± 0.15) were significantly and markedly lower than those of Su8289 (P < 0.05, P < 0.01) (Tables 3 and 4). In contrast, in 2023, the mean FS (30.37 ± 0.84 cNˑtex−1) was significantly higher than that of Su8289 (P < 0.01) (Table 4).

  • MAAL-9D5 plants were characterized by light green leaves and smaller bracts (Figs. 1 and 5). The average LP in 2022 and 2023 (34.08% ± 1.22 and 29.94% ± 3.85), as well as the average BW in 2023 (3.50 ± 0.16 g), were significantly lower (P < 0.01) (Table 3). It is worth noting that MAAL-9D5showed excellent fiber quality with significantly longer FL in 2022 and 2023 (28.16 ± 0.56 and 27.94 ± 0.57 mm), high FU (85.06% in 2023), stronger FS (31.53 ± 0.96 cNˑtex−1), lower MIC value (4.33 ± 0.56), and higher FE in 2023 (6.76% ± 0.05) than those of Su8289 (P < 0.05, P < 0.01) (Table 4).

  • MAAL-10D5 plants exhibited slower growth and delayed flowering compared to the recurrent parent Su8289. Additionally, they had smaller bracts (Figs. 1 and 5). The yield-related traits, including BW (3.90 ± 0.46 and 3.55 ± 0.44 g) and LP (33.12% ± 1.33 and 34.03% ± 2.42) in two years, were substantially lower than those of Su8289 (P < 0.01). Several fiber-related traits were significantly and markedly higher than those of Su8289 (P < 0.05, P < 0.01), including FL (28.27 ± 0.49 and 28.64 ± 1.11 mm in 2022 and 2023, respectively), FU (85.48% ± 0.65 in 2023), FS (34.13 ± 1.40 and 33.08 ± 1.47 cNˑtex−1 in 2022 and 2023, respectively), and FE (6.74% ± 0.11 in 2023) (Table 4). Conversely, the MIC value in 2022 was substantially lower at 4.53 ± 0.15 (P < 0.01) (Table 4).

  • MAAL-11D5 plants exhibited small, light green leaves along, and bigger petals (Figs. 1 and 5). Yield-related traits in this line, including BW in 2022 (2.87 ± 0.13 g) and LP in 2023 (31.42% ± 3.07), were significantly lower than those of Su8289 (P < 0.01) (Table 3). However, in 2023, the average FL and FS were longer (27.00 ± 0.57 mm) and stronger (30.86 ± 0.63 cNˑtex−1) compared to Su8289 (P < 0.05, P < 0.01) (Table 4).

  • MAAL-12D5 plants had small stamens and bolls (Figs. 1 and 5). The average FL in 2022 (24.73 ± 0.67 mm), FS in 2023 (26.48 ± 1.07 cNˑtex−1), and in both years BW (3.64 ± 0.33 g and 3.50 ± 0.26 g) and LP (30.86% ± 3.36 and 34.44% ± 1.92) were all significantly lower than those obtained for Su8289 (P < 0.05, P < 0.01) (Tables 3 and 4). Additionally, MIC values (6.07 ± 0.15 and 5.45 ± 0.31) were significantly higher than those of Su8289 (P < 0.05) (Table 4).

  • MAAL-13D5 plants featured longer petioles and larger petals (Figs. 1 and 5). The mean values of LP in 2023 (35.56% ± 2.12) and BW in 2022 and 2023 (3.32 ± 0.26 and 3.64 ± 0.36 g) were significantly lower than those of Su8289 (P < 0.01), while the average MIC value in 2022 (5.73 ± 0.31) was significantly higher (P < 0.05) (Tables 3 and 4).

Fig. 5
figure 5

Morphological characteristics of 13 MAALs. A-E Leaf, petal, bract leaf, stamen, and boll phenotype of MAALs. Scale bar = 5 cm

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Table 3 Difference analysis of yield-related traits between the recurrent parent Su8289 and MAALs
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Table 4 Difference analysis of fiber quality traits between the recurrent parent Su8289 and MAALs
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Salt and drought tolerance characteristics of the MAALs at seedling stage

The initial evaluation of salt tolerance was performed on the recurrent parent and ten MAALs from the BC4F2 generation. All plants exhibited normal growth under water treatment, while salt stress adversely affected their growth (Fig. 6a). Seven days after exposure to 400 mmolˑL−1 NaCl treatment, the cotyledons of Su8289, MAAL-1D5, MAAL-2D5, MAAL-3D5, MAAL-4D5, and MAAL-7D5 were shed, while MAAL-8D5, MAAL-9D5, MAAL-10D5, MAAL-12D5, and MAAL-13D5 only showed slight curling and yellowing (Fig. 6a). Three phenotypic traits related to salt tolerance were analyzed relative to controls. As shown in Fig. 6b, significantly varying degrees of salt tolerance were observed among the MAALs. Seven lines exhibited greater relative plant height than Su8289: MAAL-4D5, MAAL-7D5, MAAL-8D5, MAAL-9D5, MAAL-10D5, MAAL-12D5, and MAAL-13D5. Additionally, six lines demonstrated a greater relative fresh mass of aerial tissue: MAAL-4D5, MAAL-8D5, MAAL-9D5, MAAL-10D5, MAAL-12D5, and MAAL-13D5. Seven lines showed a greater relative dry mass of aerial tissue: MAAL-3D5, MAAL-4D5, MAAL-8D5, MAAL-9D5, MAAL-10D5, MAAL-12D5, and MAAL-13D5. Overall, MAAL-2D5 exhibited lower salt tolerance compared to Su8289, while MAAL-8D5, MAAL-9D5, MAAL-10D5, MAAL-12D5, and MAAL-13D exceeded Su8289 in phenotype under salt stress and across all three phenotypic traits. Notably, the line MAAL-8D5 achieved the highest average in all three evaluation indicators, indicating it to be the most salt-tolerant among the ten MAAL materials.

Fig. 6
figure 6

Phenotype and difference analysis of salt tolerance traits between the recurrent parent Su8289 and MAALs. *: P < 0.05; **: P < 0.01. Scale bar = 10 cm

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The initial evaluation of drought tolerance was conducted on the recurrent parent and nine MAALs of the BC4F2 generation. As illustrated in Fig. 7, after 13 days without watering, the recurrent parent Su8289 showed wilting symptoms, while seven MAALs (MAAL-1D5, MAAL-3D5, MAAL-4D5, MAAL-7D5, MAAL-8D5, MAAL-12D5, and MAAL-13D5) showed only mild symptoms of drought stress. In contrast, MAAL-9D5 and MAAL-10D5 exhibited severe drought symptoms. When plants were re-watered, none of the recurrent parent plants survived, nor did MAAL-9D5 and MAAL-10D5. However, the seven mildly stressed MAALs all successfully recovered, and their survival rate ranged from 93.33% to 98.33%.

Fig. 7
figure 7

Phenotype and survival rate analysis of the recurrent parent Su8289 and MAALs before and after drought treatment. **: P < 0.01. Scale bar = 10 cm

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Discussion

In distant hybridization, the hexaploid pathway is an efficient and commonly used strategy to restore the fertility of hybrids. Typically, hexaploids have regular chromosome pairing and genetic stability. However, intergenomic exchanges may occur at the hexaploid or pentaploid stages at a rate dependent on the pairing affinities of the two genomes [24, 28]. In this study, we used 1,117 pairs of SSR primer to screen polymorphic markers among the three parents and five hexaploid F1 plants. During this process, we found that three of the five hexaploid plants lacked a segment of chromosome 5D5 between NAU2942 and NAU3418, indicating heterogenetic recombination in the G. hirsutum × G. raimondii hexaploid. In addition, a relatively high frequency of intergenome recombination events was observed, particularly, in the selfing generation (Table 2); this rate was significantly higher than that previously reported between G. hirsutum and G. anomalum [26]. This elevated recombination rate further supports the notion that the Dt subgenome of G. hirsutum is closely related to G. raimondii (D5 genome).

Wild cotton species constitute a great pool that stores favorable genes with which cultivated cotton might be improved. Transferring favorable genes from this genetic pool through crosses is a common method used by breeders to improve cultivated cotton. The creation of MAALs is one way of mining these genetic resources and several MAALs of cotton species have been developed. In 2004, Zhou et al. [29] identified two G. hirsutum-G. somalense MAALs by cytology and random amplification polymorphic DNA (RAPD) analysis. In 2011, Sarr et al. [24] isolated five G. hirsutum-G. australe MAALs using SSR markers and genomic in situ hybridization. In 2014, Chen et al. [25] used morphology, molecular markers (SSR), and genomic in situ hybridization (GISH) techniques to create the first complete set of alien addition lines in cotton from the progeny of backcrosses between G. hirsutum and G. australe, comprising 11 MAALs and two multiple chromosome alien lines (MACALs). In 2016, Wang et al. [30] created 11 G. hirsutum-G. anomalum MAALs and two MACALs using SSR molecular markers and improved GISH technology. In 2018, Tang et al. [23] used GISH and SSR molecular markers to identify ten MAALs and three MACALs from G. bickii in G. hirsutum (2018). Finally, in 2020, Meng et al. [22] used SSR molecular markers and whole genome resequencing to create a complete set of 13 MAALs from G. anomalum in G. hirsutum. However, there has been no reports on the development of MAALs from G. raimondii so far. In this study, we isolated a complete set of 13 MAALs from G. raimondii in G. hirsutum via SSR molecular marker technology. We observed that the MAALs to exhibit abundant variation in morphological traits, viability, agronomic performance, fiber quality, and biotic and abiotic stress tolerance traits. Previous reports have indicated that G. raimondii may contain favorable characteristics related to fiber quality, drought and salt tolerance, as well as resistance to diseases and insect pests. Here, we confirmed that the genes controlling fiber quality inherited from G. raimondii to be located on chromosomes 9D5 and 10D5; genes controlling salt stress on chromosomes 8D5, 9D5, 10D5, 12D5, and 13D5; and genes controlling drought tolerance on chromosomes 1D5, 3D5, 4D5, 7D5, 8D5, 12D5, and 13D5. In addition, MAAL-6D5 did not flower during July and August in Nanjing, China, suggesting that the genes controlling photoperiod sensitivity might be located on chromosome 6D5. These MAALs are valuable genetic materials for alien gene transfer as well as for the study of Gossypium species genomes and their evolution.

Conclusions

In summary, putative hexaploid F1 plant of G. hirsutum - G. raimondii, possessing 78 chromosomes was obtained by interspecific hybridization. Each of its six chromosome-specific fluorescence in situ hybridization (FISH) probes of G. raimondii exhibited bright and distinct signals on its respective pair of chromosomes. This fertile hexaploid F1 plants were continuously backcrossed with G. hirsutum and a set of G. hirsutum-G. raimondii MAALs were developed using SSR markers across successive backcrosses and self-crossing from BC2F1 to BC4F2. This set of MAALs showed abundant variation in morphological, agronomic, yield, and fiber quality traits, as well as in drought and salt resistance at seedling stage.

Methods

Plant growth

In a previous study, we obtained triploid hybrids with the genome composition A1D1D5 by crossing G. hirsutum (A1A1D1D1) var. Deltapine 15 with G. raimondii (D5D5) [31]. Hybrid seedling plants were treated with 0.15% colchicine, resulting in the acquisition of a fertile putative hexaploid (A1A1D1D1D5D5), which was obtained and preserved at the Jiangsu Academy of Agricultural Sciences (JAAS). In 2016 and 2017, these putative hexaploid plants were backcrossed as females to G. hirsutum var. Su8289, yielding the BC1F1 (pentaploid) generation in 2018. The BC1F1 plants were subsequently backcrossed to Su8289 to produce the BC2F1 generation in 2019. From the BC2F1 to the BC4F2 generation, SSR marker-assisted selection (MAS) was employed to detect target alien chromosomes in each generation. Plants harboring additional chromosomes from G. raimondii were selected to make successive backcrosses with Su8289 to produce the BC4F1 generation in 2021, which was then selfed to produce the BC4F2 generation in 2022. Finally, a complete set of MAALs carrying all 13 G. raimondii chromosomes was obtained. Each MAAL was named according to the chromosome number of the extra G. raimondii-derived chromosome it carried. The Experiment Station for Plant Science, JAAS, Nanjing, China (N31°36ʹ E119°10ʹ). Figure 8 describes the procedure for production of MAALs.

Fig. 8
figure 8

Flow chart of development of MAALs of G. hirsutum - G. raimondii

Full size image

MAALs nomenclature

The diploid wild species G. raimondii (2n = 2x = 26, D5D5) contains 13 different chromosomes, collectively known as the D5 genome. This species is recognized as one of the ancestral donors of the Dt subgenome of G. hirsutum (2n = 4x = 52, A1A1D1D1). For naming lines, the convention adopted begins with ‘MAAL’, followed by numbers ranging from 1D5 to 13D5, corresponding to the alien chromosomes of G. raimondii [32]. Thus, the designations are MAAL-1D5, MAAL-2D5, MAAL-3D5, MAAL-4D5, MAAL-5D5, MAAL-6D5, MAAL-7D5, MAAL-8D5, MAAL-9D5, MAAL-10D5, MAAL-11D5, MAAL-12D5, and MAAL-13D5.

SSR marker screening and analysis

SSR markers were selected based on a combined SSR physical map, approximately 3–4 marker/5 cM. The sequences of SSR marker primers are available from the Cotton Marker Database (CMD) website (http://www.cottonmarker.org). To identify a set of informative G. raimondii-specific SSR markers evenly distributed across all 13 chromosomes, another 330 pairs of SSR primer were developed based on the genome sequence of G. raimondii. The mining program MISA (http://pgrc.ipk-gatersleben.de/misa/) was used to search SSR information in the genome of G. raimondii, and the resulting SSR flanking sequences were used to design PCR-specific primers for the development of SSR molecular markers. The G. raimondii-derived SSR markers generated in this study were designated as “JAAS18001 to JAAS18286 and JAAS19001 to JAAS19164”, with “JAAS” representing Jiangsu Academy of Agricultural Sciences. Screening of primers was performed in G. raimondii, G. hirsutum var. Deltapine 15, G. hirsutum var. Su8289, hexaploid F1, and ten individual plants randomly selected from the BC1F1 population that yielded primers with clear amplification products and demonstrated a uniform distribution across the G. raimondii genome.

Cotton genomic DNA was extracted from young, fresh cotton leaves using Paterson’s modified cetyltrimethylammonium bromide (CTAB) method [33]. PCR reactions were performed using a PCR instrument in a 10 μL reaction system, which contained 5 μL of PCR Master Mix (2 × TSINGKE Master Mix), 0.1 μL of each primer, and 2 μL of DNA template (20–50 ng). The PCR products were electrophoresed using the polyacrylamide gel electrophoresis (PAGE) analysis. The gels were photographed and recorded. Then, the images were cropped to display the target bands more clearly.

Chromosome preparation for FISH

FISH analyses were performed in the wild diploid cotton species G. raimondii (D5, 2n = 26), six MAALs of G. raimondii in the G. hirsutum background (specifically MAAL-3D5, MAAL-5D5, MAAL-6D5, MAAL-7D5, MAAL-9D5, and MAAL-10D5), and synthesized hexaploid plants (AhAhDhDhD5D5) derived from an interspecific hybrid between G. hirsutum var. Deltapine 15 and G. raimondii. All plants were cultivated under standard conditions in an artificial climate chamber at Jiangsu Academy of Agricultural Sciences, China. Mitotic metaphase chromosome spreads were prepared following the protocol established by Wang et al. [34] with minor modifications. Root tips harvested from mature plants were pretreated with 2 mM 8-hydroxyquinoline at approximately room temperature for 4 h. Subsequently, the root tips were fixed in freshly prepared methanolacetic acid solution (3:1) and preserved at -20°C prior to further processing. For root tip digestion, an enzymatic solution containing 2% cellulase (Yakult Pharmaceutical, Tokyo, Japan) and 1% pectolyase (Sigma Chemical, St. Louis, MO, United States) was applied and incubated at 37°C for 2 h. The root tips were then gently squashed with coverslips. After removing coverslips, the slides were dried at room temperature and stored at -20°C before conducting FISH analysis.

Design and labeling of the oligo probes

Oligos were designed with G. raimondii as the reference assembled genome [32]. Chromosome-specific oligo probes were designed for chromosomes 3D5, 5D5, 6D5, 7D5, 9D5, and 10D5 using Chorus2 (https://github.com/zhangtaolab/Chorus2) as described previously [35], with several modifications. In summary, the genomic sequences were first filtered to remove repetitive sequences in the G. raimondii genome using RepeatMasker2 (http://www.repeatmasker.org/). Subsequently, the remaining sequences were dissected into 59 nt oligos with a step size of 5 nt. Next, we mapped the oligo sequences to the genome, excluded those mapping to two or more loci with 75% homology, and calculated the melting temperature (Tm) and hairpin Tm for each oligo, retaining only those with a dTm greater than 10 degrees Celsius (where dTm = Tm of the melting temperature minus Tm of the hairpin). These constituted an oligo pool as a single probe. Oligos for each chromosome were synthesized by MYcroarray (Ann Arbor, Michigan, United States) and were directly labeled with digoxin.

FISH analysis of MAALs

FISH was conducted following published protocols with several modifications [34]. For chromosome denaturation, 100 μL of 70% formamide in 2 × saline sodium citrate (SSC) was applied to each slide, and denaturation was performed at 70°C for 65 s. The slides were then dehydrated in an ethanol series (75% and 100%; 5 min each) and dried at room temperature. For molecular hybridization, the hybridization mixture (15 μL per slide) was comprised of 50% formamide, 10% dextran sulfate, 1.5 μL of 20 × SSC, and 50 ng labeled probe. This mixture was applied to the denatured chromosomes and incubated for 12 h at 37°C. Subsequently, the slides were washed sequentially with 2 × SSC, 50% formamide in 2 × SSC, and 2 × SSC at 42°C for 5 min each. Chromosomes were then counterstained with DAPI in an antifade solution (Vector Laboratories, United States) under coverslips. Chromosomes and FISH signals were observed under a fluorescence microscope (Olympus BX63), and images were captured and merged using CellSens Dimension software V1.9 software with a CCD camera. Final image adjustments were performed with Adobe Photoshop 2021.

Morphological character

Phenotypic evaluation of the MAALs was carried out in the BC4F1 and BC4F2 generations. Boll weight, lint percentage, and fiber quality (fiber length, fiber strength, micronaire, neatness and elongation) were investigated in the field at the Experiment Station for Plant Science, Jiangsu Academy of Agricultural Sciences, Nanjing, China. Photographs were taken of leaves, petals, stamens, bracts, and bells. BW was measured in g, and LP was calculated as lint weight/seed weight × 100%. A total 15 g lint sample of each MAAL was sent to the Supervision, Inspection and Test Center of Cotton Quality, Ministry of Agriculture and Rural Affairs, Anyang, China, for testing of the following fiber quality traits: FL (mm), FS, (cNˑtex−1), MIC, FU, and FE.

Salt and drought treatment at seedling stage

The seeds of ten different MAALs and the recurrent parent Su8289 were allowed to germinate on wet filter paper at 28°C for 1.5 days with over 90% activation observed. Subsequently, the germinating seeds were planted in paper cups containing equal amounts of mixed substrate (70 g) and grown in a light growth incubator with 16 h light and 8 h dark at 28 ± 2°C. After developing two true leaves and one top bud, the well-grown plants were watered with 100 mL of either water (control) or salt (400 mmolˑL−1 NaCl solution). Each treatment group comprised 20 healthy and uniformly growing plants. After seven days of treatment, the plant height, fresh mass of aerial tissue, and dry mass of aerial tissue were measured and the relative values of each were determined as the salt-treated group mean / control group mean. Significance analysis was performed using SPSS Statistics software.

The seeds of nine different MAALs and the recurrent parent Su8289 were germinated on wet filter paper at 28 °C for 1.5 days. Following germinating, the seeds were planted in paper cups with equal amounts of mixed substrate and grown in a light growth incubator with 16 h light and 8 h dark at 28 ± 2°C under normal watering conditions. When seedlings had grown two true leaves, the plants were subjected to natural drought (water was withheld) for 13 days. After this drought period, the plants were re-watered, and recovery was checked after 24 h. Each group contained at least 20 plants.

Data availability

The data and materials used in this study are available from the corresponding author on reasonable request.

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Acknowledgements

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Funding

We would like to acknowledge support from the National Key Research and Development Plan of China (2022YFD1200300), Jiangsu Key R&D Program (BE2022364), and Jiangsu Collaborative Innovation Center for Modern Crop Production.

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Author notes

  1. Zhenzhen Xu and Xiaoxi Lu contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Cotton and Rapeseed (Nanjing), Ministry of Agriculture and Rural Affairs, The Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, P. R. China

    Zhenzhen Xu, Xiaoxi Lu, Wenjie Ding, Wei Ji, Wuzhimu Ali, Heyang Wang, Shan Meng, Qi Guo, Peng Xu, Xianglong Chen, Liang Zhao, Huan Yu & Xinlian Shen

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Contributions

XS, ZX, and XL conceived and designed the study. ZX, XL, WD, WJ, WA, HW, SM, QG, and XC conducted the fieldwork and lab experiments. XL, WJ, SM, PX, LZ, and HY conducted the data analyses. XS, ZX, and XL led the writing of the manuscript. All authors contributed substantially to the final writing. All authors read and approved the final manuscript.

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Correspondence to Xinlian Shen.

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Xu, Z., Lu, X., Ding, W. et al. Development of a set of monosomic alien addition lines from Gossypium raimondii in Gossypium hirsutum toward breeding applications in cotton. BMC Plant Biol 25, 150 (2025). https://doi.org/10.1186/s12870-025-06084-x

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