Research Report

Mapping of Nuclear Male-Sterile Gene ms14 Using SSR Markers in Cotton  

Peizheng Wang , Lei Bian , Cong Cao
Hainan Tropical Ocean University, Sanya, Hainan, 572022, China
Author    Correspondence author
Molecular Plant Breeding, 2016, Vol. 7, No. 35   doi: 10.5376/mpb.2016.07.0035
Received: 25 Oct., 2016    Accepted: 14 Dec., 2016    Published: 23 Dec., 2016
© 2016 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Wang P.Z., Bian L., and Cao C., 2016, Mapping of nuclear male-sterile gene ms14 using SSR markers in cotton, Molecular Plant Breeding, 7(35): 1-5 (doi: 10.5376/mpb.2016.07.0035)

Abstract

Nuclear male sterility (NMS) is a very important character for cotton breeding and genetics programs. Identification of linked molecular markers with NMS will greatly facilitate breeding for this trait. Among all of the Nuclear male-sterile genes, only Dong-A (ms14) was utilized successfully in hybrid production of cotton. Dong-A (ms14), a recessive NMS line developed from spontaneous mutation in upland cotton, has been applied most widely and played an increasing role in hybrid cultivar development in China. An interspecific F2 population comprised of 180 individual plants was developed by crossing a cultivar of Kang A (Dong-A derived Lines, Gossypium hirsutum L.) to a cultivar of 601588 (G. barbadense L.). Basing on this population the ms14 gene was mapped on chromosome 2 and closely linkaged with BNL3971 marker within a genetic distance of 16.7 cM. The BNL3971 marker could be used for the marker assisted selection in breeding a new cultivar line, and provide the information for closely location and further gene isolation by map based cloning.

Keywords
Cotton; Molecular mapping; Nuclear male-sterile genes; ms14

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 (ms1, 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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