Introduction

Male-sterility is a useful characteristic in hybrid production, which has made a tremendous contribution to food security in world. Male-sterility is a condition in plants in which the male gametophytic function is prevented, but the potential for female reproduction remains. Male sterility may be divided into nuclear male sterility (NMS) and cytoplasmic male sterility (CMS) according to its inheritance or origin CMS. CMS remains the best system for hybrid seed production for its maternal inheritance which ensures 100% sterility in the female parent (Wang et al., 2014). However NMS, which result the segregation of fertile plants from the sib-maintenance of the female parent has not been widely used in seed production due to uneconomical in many cases. NMS and CMS types of male sterility have been both found in cotton. CMS lines have not been applied successfully in hybrid production as some detrimental effects to F₁ hybrid yield or maintainer and restorer lines could not guarantee a completely sterile female parent or a fully fertile hybrid (Weaver, 1986) and at present no adequate CMS/restorer system is available. However, NMS is an effective mean of producing cotton hybrid seed in China (Zhang et al., 1992) and India (Grover and Pental, 2003) for its obvious advantages, such as stable and complete sterility performance, extensive distribution of restorers, and no negative cytoplasmic effects. But the most severe shortcoming of GMS comes from the fact that offspring of GMS plants pollinated by heterozygous pollinators always segregate in a ratio of 1:1 which creates a need to eradicate the male fertile plants from the offspring which is uneconomical in many cases (Miao et al., 2003).

Allelic relationships among NMS genes in cotton have been reported and 19 NMS genes (ms_1, ms₂, ms₃, ms₅ms₆, ms₈ms₉, ms₁₃, ms₁₄, ms₁₅, ms₁₆, Ms₄ms₄, Ms₇ms₇, Ms₁₀ms₁₀, Ms₁₁ms₁₁, Ms₁₂ms₁₂, Ms₁₇ms₁₇, Ms₁₈ ms₁₈ and Ms₁₉ms₁₉) in cotton have been reported (Richmond and Kohel, 1961; Justus et al., 1963; Allison and Fisher, 1964; Weaver, 1968; Bowman and Weaver, 1979; Turcotte and Feaster, 1985; Percy and Turcotte, 1999; Zhang et al., 1992). Among these GMS lines, only ms₁₄ and ms₅ms₆ lines have been utilized successfully in developing hybrid cotton in China and India (Weaver, 1968; Basu, 1996). Dong A (ms₁₄) was first identified as a spontaneous mutation in Sichuan province, China (Huang and Shi, 1988). Several cultivars based ms₁₄ gene have been successfully bred and spread in hybrid seed production in China. The area of hybrid cotton produced by ms₁₄ gene is grown on about 4×10⁴ hectares every year from 1984 in China (Zhang and Pan, 1999).

The improvement of hybrid performance requires a continuous development of new lines by the introduction of sterile gene into the cultivated cotton lines. However, the development of new male-sterile lines that can only be identified and selected after flowering at next generation is time and cost-intensive. With the availability of molecular markers and genetic linkage maps in cotton, marker-assisted selection can finally be applied to this important crop. Therefore, the availability of closely linked PCR-based markers for the sterile gene (s) would be a considerable advantage for cotton breeding. In several crops, NMS genes have been tagged with various types of molecular markers and specific genes have been identified or cloned, examples include the rice (Subudhi et al., 1997; Wang et al., 2003), Chinese cabbage (Miao et al., 2003), rapeseed (Yi et al., 2006; Huang et al., 2007). To date, NMS genes (ms₁₅, ms₅, ms₆, ms₈ and ms₉) have been anchored on the genetic linkage map in tetraploid cotton using molecular marker techniques (Chen et al., 2009).However, there have been no molecular marker studies on ms₁₄. In this study Kang A is an inbred line carrying the single gene ms₁₄ controlling NMS, and 601588 are an inbred lines with the NMS gene ms₁₄. One F₂ mapping populations developed from crosses between the KA and 601588 lines were scored for fertility/sterility. The objectives were to identify molecular markers linked with ms₁₄ NMS genes, and place these genes on the genetic map of cotton.

1 Results and Analysis

1.1 Segregation of the ms₁₄ gene in the F₂ population

The male-sterile plants of Kang A (ms₁₄ms₁₄) were crossed with these plants of 601588 (Ms₁₄Ms₁₄) to produce F₁ which were selfed to produce the F₂. The F₂ population was constructed with 180 plants which were identified to be consisted of 145 male-fertile plants (Ms_) and 35 male-sterile plants (ms₁₄ms₁₄) showed in Table 1. The segregation ratio of fertile to sterile plants in this F₂ population fitted a 3:1 ratio (X² =0.435, p < 0.05). The segregation of fertility (69 sterile and 64 fertile) in the population of sib-mating between MS and MF in Kang A fitted a 1 fertile :1 sterile ratio (X² =0.006, p < 0.05) (Table 1). So the results confirmed that the sterility of in Kang A was controlled by a single recessive gene.

Table 1 The X2 test of segregation ratio of fertility in NMS lines and F2 population Note: *significant at the 0.05 probability level (c2(0.05, df=1) = 3.84)

1.2 Mapping the ms₁₄ gene by SSR markers

Between parents, 2000 SSR markers were screened, and 112 of them showing clear and stable polymorphic PCR products in the F₂ population were selected to construct the genetic linkage map for mapping the ms₁₄ gene. Linkage map analysis showed that the ms₁₄ gene and three SSR markers (BNL3971, NAU663 and BNL1897) were mapped to the same linkage group together (Figure 1). The three SSR markers were placed on one side of ms₁₄ gene, and among them BNL3971 was most closely linked with ms₁₄ gene at a distance of 16.7 cM. These three markers were all corresponded on Chr2 of existing linkage maps reported by Guo et al.. Therefore, it is reasonable to conclude that the ms₁₄ gene is located on Chr2.

2 Discussion

2.1 The first NMS interspecific hybrid released for commercial cultivation in China

Heterotic hybrid varieties in cotton can show more than a 10%-20% yield advantage over the best conventional ones under the same cultivation conditions (Li, 2005). The commercial use of cotton heterosis in India and China is available to make emasculations and crosses by hand (Huang and Shi, 1988). In China, the area of hybrid cotton since 2000 has been around 20% of the total cotton growing area (Li, 2005). As facing lack and high cost of labor, cost of hybrid cotton seed is increasing. To solve this problem, NMS lines have been developed and extensively used in cotton production for eliminating tedious hand emasculation which systems can reduce cost of hybrid seed by almost 50% (Zhou et al., 2006). The interspecific hybrids could combine the yield of G. hirsutum and fiber quality of G. barbadense (Basu, 1996). In this research the hybrid cultivar (XinLuzhong 31) crossing Kang A to a cultivar of 601588, which has the almost same fiber quality of G. barbadense L. and have better lint percent and yield than that of G. barbadense L., was the first released interspecific in northwester of China.

2.2 Mapping the ms₁₄ gene with linked to SSR markers

The major use of NMS in cotton breeding is development of new inbred line and the production of F₁ hybrid seed. SSR markers linked to the ms locus provide a useful approach for early identification of lines carrying the male-sterile allele without need for progeny testing. In this study, we mapped the NMS gene (ms₁₄) on Chr2, which was flanked by three SSR markers (BNL3971, NAU663 and BNL1897). BNL3971 marker was most closely linked with the ms₁₄ gene at a distance of 16.7 cM. During previous studies, ms₅, ms₈ and ms₁₅ were located on Chr12 (Chen et al., 2009) and ms₆ and ms₉ were located on Chr 26 (Rhyne, 1991), thus the results supported that these genes should not be allelic with ms₁₄. This marker will be potentially useful in marker-assisted selection programs aimed at introgressing ms alleles into elite cotton cultivars, furthermore, will also help to isolate NMS genes by map-based cloning in the future.

3 Materials and Methods

3.1 Plant materials

The male sterility of Kang A (G. hirsutum L.) which derived from Dong-A Lines (ms₁₄) is controlled by single recessive genes (designated our previous study). The NMS lines have been maintained by sib-mating between male fertile (MF) individuals (ms₁₄ms₁₄) and sterile (MS) individuals (Ms₁₄ms₁₄). The male-sterile plants of Kang A were crossed with a cultivar of 601588 (G. barbadense L.) and the subsequent F₁ progenies were allowed to self-pollinate for F₂ seeds. The F₂ populations were planted at the Cotton Experiment Station of Xinjiang Condy Agriculture Science & Technology Development Co. for evaluation of pollen fertility. Plants were examined at flowering for male sterility/fertility through visual examination. Plants with full anther extrusion and pollen production were classified as male fertility (MF), and plants lacking anther extrusion and pollen grains were classified as male sterility (MS) (Figure 1). Genetic maps were constructed by genotyping 180 F₂ progeny of this population.

Figure 1 Mapping the gene of ms14 on Chr2

3.2 DNA extraction and PCR reaction

Cotton DNA was extracted and SSR PCR amplifications were performed following Paterson et al. (1993).

3.3 Data analysis

We used 2000 SSR primers which were available from CMD (http://www.cottonssr.org). Markers were first screened from the parents and their F₁ progeny to detect polymorphisms, and subsequently used to genotype the F₂ plants. A chi-square analysis was used to analyze marker data to test for goodness-of-fit to an expected segregation ratio. Linkage maps were constructed by MAPMAKER/Exp Version 3.0b Software (Barlow et al., 1987) and linkage groups were identified by pairwise comparisons. Marker groupings were determined using the ″Group″ command at a maximum recombination fraction of 50 cM and a minimum LOD score greater than 4.0. Marker order was confirmed with the ″ripple″ command. Recombination frequencies were converted into map distances (cM) using the Kosambi mapping function. The SSR loci chromosome assignments were used as bridge loci with existing linkage maps (Wang et al., 2006; Guo et al., 2007) by checking the molecular sizes of the parental.

Authors' contributions

Wang Peizheng carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. Bian Lei carried out the statistical analysis. Cao Cong participated in the design of the study and performed. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by Provincial College Students Innovation and Entrepreneurship Training Program (20150125, 20150109).

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