HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Zhang, J. Articles by Stewart, J. McD. Search for Related Content PubMed Articles by Zhang, J. Articles by Stewart, J. McD. Agricola Articles by Zhang, J. Articles by Stewart, J. McD. Related Collections Crop Genetics Cotton

CROP BREEDING, GENETICS & CYTOLOGY

Identification of Molecular Markers Linked to the Fertility Restorer Genes for CMS-D8 in Cotton

^a Dep. of Agronomy and Horticulture, New Mexico State Univ., Las Cruces, NM 88003
^b Dep. of Crop, Soil, and Environmental Sci., Univ. of Arkansas, Fayetteville, AR 72701

^* Corresponding author (jinzhang{at}nmsu.edu).

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The identification of molecular markers closely linked to restorer genes of the cytoplasmic male sterile (CMS) D8 system could facilitate the development of parental lines for hybrid cotton (Gossypium hirsutum L.). Our objective was to develop molecular markers closely linked to the two independent dominant restorer genes, Rf₁ from the D2 restorer line transferred from G. harknessii Brandegee (D₂ genome) and Rf₂ from the D8 restorer line. Bulked segregant analysis was used to identify random amplified polymorphic DNA (RAPD) markers that were linked to the restorer genes. Three testcrosses were used for mapping Rf₂ and two for Rf₁ relative to the markers. Two RAPD markers, UBC111₃₀₀₀ and UBC188₅₀₀, were associated with Rf₂ in coupling phase. UBC188₅₀₀ was closely linked to Rf₂ with an average genetic distance of 2.9 cM. A survey of cotton species and cultivars revealed that UBC188₅₀₀ was absent in normal cotton cultivars and most Gossypium spp. However, it was present in the D8 restorer line, G. raimondii Ulbr. (D₅), G. trilobum (DC.) Skovst. (D₈), and selected wild species from Australia, indicating that Rf₂ and the DNA fragment yielding the RAPD marker were both introgressed into the tetraploid cottons from the D₈ genome. A new RAPD marker, UBC169₇₀₀, and a previously identified RAPD marker, UBC659₁₅₀₀, cosegregated with Rf₁ in the two populations examined. The three RAPD markers, UBC188₅₀₀, UC169₇₀₀, and UBC659₁₅₀₀ were converted into reliable and genome-specific sequence tagged site (STS) markers on the basis of their sequence information. These markers are restorer-specific and should be useful in marker-based selection for developing restorer parental lines and constructing a high-resolution linkage map containing Rf₁ and Rf₂.

Abbreviations: AFLP, amplified fragment length polymorphism • CMS, cytoplasmic male sterile/sterility • EB, ethidium bromide • F, fertile • MAS, marker-assisted selection • PCR, polymerase chain reaction • RAPD, random amplified polymorphic DNA • RFLP, restriction fragment length polymorphism • S, sterile • SSR, simple sequence repeat • STS, sequence tagged site

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
CYTOPLASMIC MALE STERILITY in higher plants results from abnormal microsporogenesis or microgametogenesis leading to no pollen production or dysfunctional pollen. A CMS system provides an efficient means to produce commercial quantities of hybrid seed in plants (Kaul, 1988). It also offers a model system to study cytoplasm and nuclear genome interactions at the molecular, biochemical, and genetic levels. Cytoplasmic male sterility is maternally inherited, and in numerous species it is related to the unique arrangements of mitochondrial DNA and unusual open reading frames (Schnable and Wise, 1998). Fertility can be restored to cytoplasmically induced male sterility by introducing nuclear restorer genes. Restorer genes have been shown to suppress the expression of CMS-associated novel chimeric genes, but the mechanism of restoration appears to vary among different CMS and restorer systems (Schnable and Wise, 1998).

In cotton (G. hirsutum), several different sources of CMS have been developed (Meyer, 1975; Jia, 1990; Stewart, 1992; Meshram et al., 1994; Yuan et al., 1996), which makes this material potentially useful for hybrid cotton production and for studying the interactions between CMS and restorer genes (Rf). With its male sterility conferred by the exotic cytoplasm from G. harknessii (with genome designation D_2-2), CMS-D2 (Meyer, 1975) is the first system developed for cotton. Developed by Stewart (1992) from the cytoplasm of G. trilobum (with genome designation D₈), CMS-D8 is the second publicly accepted CMS system. Its male fertility can be independently restored by two restorer genes, namely sporophytic Rf₁ (designated D2 restorer gene hereafter) from the D_2-2 genome and gametophytic Rf₂ (designated D8 restorer gene hereafter) from the D₈ genome (Weaver and Weaver, 1977; Stewart and Zhang, 1996; Stewart et al., 1996; Zhang and Stewart, 2001a, 2001b). The two restorer gene loci reside on the same chromosome and are tightly linked with a genetic distance of 0.9 cM (Zhang and Stewart, 2001b). No morphological markers have been found to be linked to either of the Rf genes (Kohel et al., 1984; Zhang, 1999) except for the cracked root trait that is linked to Rf₁ with a recombination fraction of 14% (Weaver and Weaver, 1979). Since these morphological markers are limited in number and often have deleterious effects on plant growth, other genetic marker systems are needed to map the two Rf genes. In cotton, a detailed genetic map based on DNA restriction fragment length polymorphisms (RFLPs) was constructed and updated for an interspecific cross and was comprised of 705 RFLP loci assigned to 41 linkage groups with a total genetic distance of 4675 cM (Reinisch et al., 1994; Jiang et al., 2000). Several other linkage maps based on RFLP, simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP) were published recently (Ulloa et al., 2002; Zhang et al., 2002; Lacape et al., 2003).

In the past several years, interest in molecular mapping of restorer genes has increased in many plant species, including rice (Oryza sativa L.; Zhang et al., 1997a), wheat (Triticum aestivum L.; Ma and Sorrells, 1995), rye (Secale cereale L.; Borner et al., 1998), maize (Zea mays L.; Wise and Schnable, 1994), sorghum [Sorghum bicolor (L.) Moench; Pammi et al., 1994], rape (Brassica napus L.; Jean et al., 1997), sunflower (Helianthus annuus L.; Quillet et al., 1995), common bean (Phaseolus vulgaris L.; Jia et al., 1997), sugar beet (Beta vulgaris L.; Laporte et al., 1998), onion (Allium cepa L.; King et al., 1998), and petunia (Petunia x hybrida hort. ex E. Vilm.; Bentolila et al., 1998). Since a number of Rf genes in plants have been mapped along with increasing numbers of molecular markers, isolation of some of these Rf genes in petunia and radish (Raphanus sativus L.) via map-based cloning strategies has been accomplished recently (Bentolila et al., 2002; Brown et al., 2003; Koizuka et al., 2003). One of the restorer genes in maize, Rf₂, was also cloned and sequenced by transposon tagging (Cui et al., 1996). Several RAPD and SSR markers were reported to be linked with the D2 restorer gene (Rf₁) in cotton (Guo et al., 1998; Lan et al., 1999; Liu et al., 2003). However, the applicability of these markers is questionable since they were either not confirmed or coded with no primer sequence information available.

Initial D8 restorer lines of cotton were low yielding and late maturing, suggesting that the introduction of the D8 restorer gene, Rf₂ (Zhang and Stewart, 2001a), also resulted in introduction of other genes deleterious to the other phenotypes. Recently, improvement in the restorer lines has resulted in better fiber quality and yield potential. The success is possibly due to the elimination of additional D₈ genetic material. However, to estimate the length of the introgressed segment carrying the Rf₂ restorer gene and flanking DNA, genetic markers near the Rf₂ locus are needed. Furthermore, saturation of the Rf₂ segment with additional markers will permit the construction of a physical map of this chromosomal region and might allow cloning of the restorer gene via map-based cloning strategies (Zhang et al., 1997b).

Hybrid cotton has been used in India and China extensively and successfully with hybrid seeds produced by hand emasculation and a nuclear male sterility system (Sun et al., 1994; Basu, 1995). While the use of the CMS-D2 system has been hampered by the deleterious effect of the cytoplasm on cotton yield, the new CMS-D8 system offers cotton breeders new hope in economic use of hybrid cotton. The objectives of this study were to (i) identify RAPD markers linked to the CMS-D8 restorer genes by the bulked segregant analysis approach; (ii) convert these RAPD markers into STS markers and make them available to cotton research community; and (iii) study the existence of these markers in other cultivated and wild Gossypium spp. to provide molecular evidence on the origin and possible distribution of the restorer genes for CMS-D8.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Plant Materials
Five segregating populations were used (Table 1).

1. Population 1, a population of 79 plants from (D8R x T-586)F₁ x ARK8518 in which D8R containing D₈ cytoplasm was used as female to cross with T-586 and the resulting F₁ was testcrossed as female to ARK8518 (rf₂rf₂). For simplicity, D8R is a D8 restorer line homozygous for the fertility restoration (Rf₂Rf₂). T-586 is a multiple dominant marker line and contains the recessive restorer gene (rf₂rf₂) and nine dominant marker loci-red plant (R₁), okra leaf (L₂), tomentum (T₁), petal spot (R₂), yellow pollen (P₁), yellow petals (Y₁), brown lint (Lc₁), green lint (Lg), and naked seed (N₁) (Endrizzi et al., 1984). ARK8518 is a selection from a breeding line of upland cotton developed by F. M. Bourland. This population was used for bulked segregant analysis to identify Rf₂–associated markers.
   
2. Population 2, a BC₁F₁ population of 250 plants from (D8R x TM-1)F₁ x TM-1. TM-1 (rf₂rf₂) is the genetic standard for upland cotton, derived from repeated selfing from Deltapine 14 (Kohel et al., 1970).
   
3. Population 3, 137 plants derived from a cross between CMS-D8 and the F₁ from A₂D₈ x TM-1. A₂D₈, a synthetic tetraploid between G. arboreum L. (A₂) and G. trilobum (D₈) having A₂ cytoplasm and supposedly containing Rf₂Rf₂ from D₈, was used as female to cross with TM-1. The pollen grains from the F₁ were pollinated onto CMS-D8. This population was used to determine if more than one Rf gene was present in the D₈ genome. This population and Population 2 were also used to confirm the linkage results obtained from Population 1.
   
4. Population 4, 103 plants derived from (CMS-D8 x D2R) F₁ x ARK8518. The D2R lines, B418R or B416R (Cook and Namken, 1995), have the restorer gene, Rf₁Rf₁ (Zhang and Stewart, 2001b).
   
5. Population 5, 110 plants derived from (D2R x ARK8518) F₁ x ARK8518. These last two populations were used to identify RAPD markers linked to Rf₁.
   

DNA Extraction
Population 1 DNA was extracted by the macro-prep method described by Altaf et al. (1997), and DNA from Populations 2, 3, 4, and 5 was extracted by the rapid mini-prep method described by Zhang and Stewart (2000).

Bulked Segregant RAPD Analysis
Two bulking methods were used to conduct bulked segregant analysis (Michelmore et al., 1991). (i) Two DNA pools from 10 fertile (F) and 10 sterile (S) plants in the contrasting bulks from Populations 2, 3, and 4 were formed in which the DNA from the individual plants was represented in equal molar ratios. (ii) Young leaves from each S plant and each F plant in Population 1 were bulked to form the two respective F and S pools for DNA extraction. DNA from a series of plants was adjusted to 10 ng µL^–1. The pooled DNAs served as templates for polymerase chain reaction (PCR)-based RAPD analysis. Decamer oligonucleotide primers (UBC101 to UBC200) and UBC659 (5'-CGGTTTCGTA-3'), UBC686 (5'-CGTGACAGGA-3'), and OVP-15 (5'-GGAAGCCAAC-3') were obtained or synthesized from the University of British Columbia, Vancouver, Canada, and a single primer was used in each PCR reaction. The amplification reactions were in a total volume of 25 µL, consisting of 1 µL of template DNA (10 ng µL^–1), 2.5 µL of 10 x buffer II, 3.75 µL of 10 mM MgCl₂, 0.5 µL of 10 mM dNTPs, 0.33 µL of 50-µM primer, 0.1 µL of Taq polymerase (5 U µL^–1) and 16.82 µL of ddH₂O. The 10 x buffer II, 10 mM MgCl₂, and Taq polymerase were all purchased from Perkin-Elmer (Foster City, CA). Amplification was performed in a Hybaid Omnigene thermocycler (Hybaid Omn-E-02HL) for 45 cycles after initial denaturation at 94°C for 2 min. Each cycle consisted of 15 s at 94°C, 30 s at 40°C, and 90 s at 72°C. A 5-min final extension at 72°C followed the end of the cycling program. The amplification aliquots were resolved by gel electrophoresis in 1.4 or 1.0% agarose gels with 0.5 x 1,1,2,2-tetrabromoethane buffer, and were stained with 10 µg µL^–1 of ethidium bromide (EB). In most cases, EB was added directly to the gels during preparation. The bands of amplified DNA were visualized under UV light, and the sizes of the amplified DNA fragments were estimated based on a 100-bp DNA ladder (MBI, Amherst, NY). Once a putative polymorphic amplification was detected in the two bulks, genomic DNA from 5 to 10 individual F and S plants each were used to verify the results. Following confirmation of Rf-linkage, all plants in each population were surveyed for the markers to establish genetic distance.

Segregation and Linkage Analysis
Chi-square was used to test goodness-of-fit of segregation ratios for male fertility and RAPD markers, and for linkage between the restorer genes (Rf₁ or Rf₂) and RAPD markers. Linkage analysis was also performed with the Mapmaker program (Lander et al., 1987). The statistical thresholds were set at a minimum LOD of 3.0 and a maximum recombination fraction of 0.40. The Haldane mapping function (Haldane, 1919) was used to convert the recombination fraction into genetic mapping distance (centimorgans). Loci were named after the primer that revealed the polymorphism, with the estimated fragment size as subscript.

Sequence Tagged Site Marker Development
Polymorphic RAPD fragments between F and S plants were excised from agarose gels and extracted with a freeze-squeeze method. Briefly, the excised agarose gel slice containing the DNA fragment was placed in a 1.5-mL microcentrifuge tube and frozen at –70°C. Next, the tube was centrifuged for 15 min at room temperature at 14000 rpm to collect the aqueous DNA. The DNA solution was then subjected to a phenol extraction and ethanol precipitation. The purified DNA was either used directly for cloning or reamplified by the same RAPD primer and PCR conditions as described before. The PCR products without further purification were cloned directly into a pGEM-T vector (Promega, Madison, WI) following the manufacturer's instructions. About 10 transformed white bacterial colonies for each of the RAPD fragments were selected for plasmid DNA extraction by the alkaline lysis method (Sambrook et al., 1989). The extracted plasmid DNA was further purified with a Wizard kit (Promega) following the manufacturer's manual. The recombined plasmid DNA was sequenced with an ABI 377 DNA Sequencer (Applied Biosystems, Inc., Foster City, CA). The sequence information was used to design STS primers that usually included the original RAPD primer sequence plus extra bases for site specific amplification.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Segregation Analysis of Male Fertility
Because of the distinctive flower morphology of CMS-D8 and CMS-D2, the difference between CMS and restored plants was evident, and in all cases the phenotype could be scored without ambiguity. In the testcrosses, (D8R x T586) F₁ x ARK8518 and (D8R x TM-1) F₁ x TM-1, the segregation in male fertility gave a 1:1 fertile-to-sterile ratio (Table 1). Fertility restoration of CMS-D8 by the D8 restorer was confirmed to be controlled by a single dominant gene in the two populations segregating for the Rf₂ restorer gene. From the development of the CMS-D8 and the D8 restorer, and subsequent genetic studies, the D8 restorer gene, Rf₂, was demonstrated to be transferred into the tetraploid cotton from the D₈ genome (Stewart, 1992; Stewart et al., 1996; Zhang and Stewart, 2001a).

The cross CMS-D8 x (A₂D₈ x TM-1)F₁ also gave a 1:1 fertile-to-sterile ratio (Table 1), indicating the presence of only one Rf gene in the synthetic tetraploid (A₂D₈). This restorer gene is undoubtedly the Rf₂ gene from D₈ because the D8 restorer line (D8R) containing Rf₂ was developed by repeatedly backcrossing fertile plants from the original hexaploid (D₈AD₁) and its progenies as females to upland cotton (AD₁). Obviously, G. arboreum (A₂) does not provide any restoring factors. If more than one restorer gene were detected in this cross, it would indicate that either the original donor D₈ species contained more than one restorer gene or A₂ species contributed at least one. Since the male parent (A₂D₈ x TM-1) F₁ had A₂ cytoplasm, the normal segregation ratio for fertility implied that the A₂ cytoplasm, like the AD₁ cytoplasm (Stewart and Zhang, 1996; Zhang and Stewart, 2001a), has no effect on transmission of the recessive nonrestorer allele (rf₂) through pollen. The A₂ genome and A_h subgenome of tetraploid cotton differ by three reciprocal chromosome arm translocations (Endrizzi et al., 1984); consequently, the pairing between them will result in progenies with chromosome deficiencies and duplications, and abnormal segregation of genes located on the translocated chromosome arms would be expected. The normal segregation ratio indicated that the Rf₂ gene is not located on these translocated A₂ or A_h chromosomes, which is consistent with the linkage test between Rf₂ and seven morphological markers located on A_h subgenome chromosomes (Zhang, 1999). Homeologous chromosomal recombination involving A and D genomes or subgenomes was not detected. The chance that Rf₂ was on the A subgenome should be very low. The localization of Rf₁ on chromosome 4 (A subgenome) by Liu et al. (2003) is probably erroneous since Rf₁ was transferred into cotton from another D genome species (D_2-2) and is tightly linked to Rf₂. Because of substantial cryptic differences between the D₈ genome and D_h subgenome, synapsis between these homeologous chromosomes could give rise to gametic abnormalities. Such abnormalities could have negative effects on male fertility and even female fertility. The number of male-fertile plants was less than male-sterile plants; however, deviation from a 1:1 ratio was not significant, indicating that any effect of abnormal meiosis in the (A₂D₈ x TM-1) F₁ on segregation of the Rf₂ locus was negligible.

In each of the two testcrosses, that is, (CMS-D8 x D2R)F₁ x ARK8518 and (D2R x ARK8518)F₁ x ARK8518, where the D2 restorer was involved, a ratio of 1 fertile to 1 sterile plant was obtained (Table 1). The segregation confirmed that male fertility restoration to CMS-D8 by the D2 restorer is conditioned by one dominant restorer gene, Rf₁ (Zhang and Stewart, 2001b).

Inheritance of RAPD Markers
The contrasting S and F bulked DNA samples were used as templates to screen decamer primers for DNA polymorphisms. Altogether, about 400 loci and 350 loci from Population 1 and Population 4, respectively, were amplified with 100 primers. In Population 1, only about half of the primers produced PCR amplification products, with the number of bands ranging from 1 to 15. On average, there were 6.8 bands (loci) per primer amplified by the effective primers. In Population 4, about 70 primers (70%) gave PCR products with the number of bands ranging from 1 to 10. On average, 4.6 bands per primer were amplified by the effective primers in this population. Even though differences in the average number of bands per primer between the two populations did exist, the number of bands amplified by the primers was significantly correlated between the two populations (r = 0.314, P < 0.05, n = 54). The PCR conditions produced relatively consistent and reliable amplification from the primers with major RAPD bands conserved from the same primers.

Several random primers were found to amplify 14 polymorphic bands between S and F bulks in the two populations, and DNA from five to 10 individual plants from each of the bulks were used to verify the results. Even though positive association with the restorer genes was not confirmed in most cases, segregation of the polymorphic bands was evaluated. All 14 RAPD markers except for one exhibited dominant inheritance and had 1:1 segregation ratio, as expected. The results indicated the relative reliability of RAPD markers in genetic studies.

RAPD/STS Markers Linked to Rf₂
Among 100 10-mer primers screened, 11 produced polymorphisms between the S and F bulks from Population 1. Polymerase chain reaction analysis of individual plants with these primers identified two that amplified DNA fragments that distinguished the fertile from sterile segregants. One of the RAPD markers, UBC188₅₀₀, amplified by primer UBC188 (5'-GCTGGACATC-3') was tightly linked to Rf₂ in coupling phase (Fig. 1 and Table 2). The recombination fractions for the three mapping populations ranged from 0 to 4.8% with an average of 2.8%. The converted genetic distance was 2.9 cM based on the Haldane mapping function. When observations were combined across the three populations, the recombination fraction (genetic distance) was 2.7% (2.8 cM). Notably, no recombination was observed in Population 3 where the trispecific hybrid (A₂D₈ x TM-1) was used. The dissimilarity between the homeologous chromosomes from the D_h subgenome and the D₈ genome on which the Rf₂ gene is located might suppress genetic recombination. Fang et al. (1998) showed that genetic distance among markers varied among populations in Poncirus trifoliata (L.) Raf. Others (Paterson et al., 1990; Wise and Schnable, 1994; Williams et al., 1995) suggested that recombination can be suppressed in interspecific hybrids or by chromosome inversion.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Polymerase chain reaction product profile of DNA amplified with primer UBC188 in Population 2: (D8R x TM-1) F1 x TM1. Lanes labeled "F" and "S" represent individual fertile and sterile plants, respectively. Lane "M" is a 100-bp ladder. The arrow indicates the RAPD marker, UBC188500, which was present only in the fertile plants.

At present, whether the RAPD marker UBC111₃₀₀₀ was derived from the D₈ genome is not known. But, at least a 3-cM chromosome segment from the D₈ genome was retained during the development of the D8 restorer in upland cotton background (see below). If UBC111₃₀₀₀ should prove to be derived from the D₈ genome, most likely double crossover occurred during the backcrossing process because the genetic distance between Rf₂ and UBC111₃₀₀₀ is rather large.

The RAPD marker UBC188₅₀₀ was highly reliable and produced consistent results. Nevertheless, conversion of it into a STS marker (Paran and Michelmore, 1993) would allow its global and prudent use. Sequence tagged site primers, designed on the basis of genomic sequence information, are usually >18 bases in length, much longer than the RAPD primers. Their PCR amplification is genome site specific and robust, producing consistent results. Surprisingly, after the UBC188₅₀₀ fragment was cloned and sequenced, two sequences (386 and 540 bp) were identified that showed approximately 50% sequence identity with three major inserts on the 540-bp fragment. Among several pairs of STS primers designed from the sequences, one pair (UBC188₅₀₀STS-F: 5'-GCTGGACATC-plus 11 bp-3'; UBC188₅₀₀STS-R: 5'-GCTGGACATC-plus 10 bp-3') produced reliable and polymorphic results.

Screen for UBC188₅₀₀ in Gossypium Species and Cultivars
Fourteen cotton cultivars or breeding lines and 15 diploid Gossypium spp., including D₈, were tested for the presence of the UBC188₅₀₀ fragment (Table 3). Except for the D8R, which gave amplification of the RAPD fragment and restored male fertility to CMS-D8, all the normal upland cotton (G. hirsutum) and Pima cotton (G. barbadense) genotypes without the Rf₂ gene did not amplify UBC188₅₀₀ and produced all male-sterile F₁ plants when crossed to CMS-D8. Among the Gossypium spp. representing seven genomes, only G. trilobum (D₈) and G. raimondii Ulbrich (D₅) in the D-genome group and the species from Australia (G. sturtianum J.H. Willis, G. bickii Prokh., and G. pilosum Fryxell) showed amplification of this specific band. As shown before in Population 3, G. arboreum (A₂) had no restorer factor for CMS-D8 and also did not produce the UBC188₅₀₀ fragment. This result indicated that Rf₂ and the UBC188₅₀₀ fragment were from the D₈ genome since the D8 restorer was developed from the interspecific hybrid involving the D₈ genome (Stewart, 1992), and since the tetraploid cultivars without Rf₂ also yielded no UBC188₅₀₀ fragment. The UBC188₅₀₀ fragment could be used as a specific marker for CMS-D8 restorer development. However, at present, we do not know if the UBC188₅₀₀ in other species is the same as that in the D₈ genome and the D8 restorer, or, if these species have the restorer gene. To clarify the former, simple DNA cloning and sequencing would be needed; however, to clarify the latter, extensive interspecific crossing and backcrossing between these wild species and tetraploid cotton would be required, which is very difficult and is also beyond the scope of this report.

View larger version (50K): [in this window] [in a new window]   Fig. 2. Random amplified polymorphic DNA profile of DNA amplified with primer UBC169 in Population 5: (D2R x ARK8518)F1 x ARK8518. Lanes labeled "S" and "F" represent sterile and fertile plants, respectively. Lane M is 100-bp ladder. The arrow shows an extra band (UBC169700) in fertile plants.

The RAPD/STS markers are source (restorer gene) specific: UBC169₇₀₀ (UBC169₇₀₀STS) and UBC659₁₅₀₀ (UBC169₁₅₀₀STS) did not detect fertility-related polymorphism for Rf₂ in Populations 1, 2, and 3, while UBC188₅₀₀ (UBC188₅₀₀STS) did not detect polymorphism associated with Rf₁ in Populations 4 and 5. The Rf₂ and the DNA marker UBC188₅₀₀/UBC188₅₀₀STS are both from the D₈ genome since normal tetraploid cottons lack the Rf₂ gene and the RAPD marker. Similarly, the Rf₁ cosegregating markers, UBC169₇₀₀/UBC169₇₀₀STS and UBC659₁₅₀₀/UBC659₁₅₀₀STS, are both likely to be derived from the original Rf₁ donor (D_2-2). However, these two markers should be used to screen Gossypium spp. including D_2-2 for verification of origin. Since these two markers are very tightly linked to Rf₁, very few, if any, crossovers could occur between the markers and the restorer gene during D₂ restorer development. To obtain recombinants between Rf₁ and the two markers and estimate their genetic distances, a large segregating population is needed.

A Linkage Map Comprising Rf₁ and Rf₂
Random amplified polymorphic DNA markers are still widely used in plants, especially species for which development of other markers, such as SSR, AFLP, RFLP, or STS is limited (Tar'an et al., 2003). RAPD markers are generally not as reliable and specific as other PCR-based markers such as AFLP, SSR, or STS. Because of the short RAPD primers used in the PCR reactions, some nonspecific amplification from mispriming might occur. However, major RAPD bands with perfect primer annealing and strong amplifications are stable and conserved, as illustrated previously for the RAPD marker UBC188₅₀₀ and UBC659₁₅₀₀ and some other non-Rf gene-specific polymorphic RAPD markers that showed expected segregating ratios. However, UBC169₇₀₀ was not a major RAPD band that consistently amplified from the fertile plants containing Rf₁ allele. To ensure the global use of these Rf gene-associated markers, these RAPD markers were converted into reliable and genome-specific STS markers on the basis of their sequence information.

Since Rf₁ and Rf₂ are tightly linked with a recombination fraction of 0.9% (Zhang and Stewart, 2001b), the Rf₂ gene could be either located between Rf₁ (and markers UBC659₁₅₀₀/UBC659₁₅₀₀STS and UBC169₇₀₀/UBC169₇₀₀STS) and UBC188₅₀₀/UBC188₅₀₀STS or flanked by Rf₁ (and markers UBC659₁₅₀₀/UBC659₁₅₀₀STS, UBC169₇₀₀/UBC169₇₀₀STS, and UBC188₅₀₀/UBC188₅₀₀STS) and UBC111₃₀₀₀ (Fig. 3) . On the basis of fertility scoring, distinction between fertile plants with Rf₁ and with Rf₂ was not possible in test-crosses of (D8R x D2R)F₁ crossed as female to the normal-fertility nonrestoring lines (e.g., ARK8518 or TM-1). In this type of testcross, the three fertile genotypes, Rf₁rf₁Rf₂rf₂, Rf₁rf₁rf₂rf₂, and rf₁rf₁Rf₂rf₂, cannot be differentiated from each other by their phenotypes. Even though progeny tests can be made by selfing individual BC₁F₁ plants or backcrossing again, this would be very time consuming. Also, the progeny test by selfing cannot separate the double heterozygous genotype (Rf₁rf₁Rf₂rf₂) from rf₁rf₁Rf₂rf₂ since they all are fertile, while progeny test by backcrossing cannot separate Rf₁rf₁rf₂rf₂ from rf₁rf₁Rf₂rf₂ since they both produce a 1:1 fertile-to-sterile ratio. Therefore, as DNA markers very closely linked with Rf₁, UBC659₁₅₀₀/UBC659₁₅₀₀STS and UBC169₇₀₀/UBC169₇₀₀STS will be useful for constructing high-density linkages comprising Rf₁ and Rf₂. However, because UBC188₅₀₀/UBC188₅₀₀STS, UBC659₁₅₀₀/UBC659₁₅₀₀STS, and UBC169₇₀₀/UBC169₇₀₀STS are derived from D₈ and D₂ species, respectively, they are restorer-specific and dominant. It is difficult to map them simultaneously when the two restorers are used in different populations. An important advantage of STS markers is that they may be codominant (Barret et al., 1998; Lahague et al., 1998), which facilitates distinction of homozygous dominant from heterozygous dominant plants in a segregating population. However, the three converted STS markers for the two Rf genes are all dominant.

View larger version (16K): [in this window] [in a new window]   Fig. 3. A consensus map comprising Rf1, Rf2, and four DNA markers. Rf1 is either (a) flanked by Rf2 and UBC1113000, or (b) flanked by Rf2 and UBC188500.

The present study has developed two new RAPD/STS markers, UBC188₅₀₀/UBC188₅₀₀STS and UBC169₇₀₀/UBC169₇₀₀STS, and verified UBC659₁₅₀₀ and its STS marker for the Rf genes. The genetic distance between UBC188₅₀₀ and Rf₂ is less than 2.9 cM, whereas UBC169₇₀₀/UBC169₇₀₀STS and UBC659₁₅₀₀/UBC659₁₅₀₀STS cosegregate with Rf₁. As in rice and rape lines where MAS has been established for selection of Rf genes (Hansen et al., 1997; Ichikawa et al., 1997), these markers should be very good candidates for MAS for the two Rf genes in cotton.

To establish a high-resolution map surrounding the Rf genes, additional markers and marker systems should be explored. Since a molecular map based on RAPD, SSR, AFLP, and morphological markers in the trispecific hybrid (A₂D₈ x T586) has been established (Altaf et al., 1998), the Rf genes can be integrated in the map by use of a common set of the specific markers. The saturated map encompassing the two Rf genes is prerequisite to the isolation of the Rf genes by map-based cloning strategies.

Received for publication July 7, 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

• Altaf, M.K., R.G. Cantrell, J. Zhang, and M.K. Wajahatullah. and J.McD. Stewart. 1997. Molecular and morphological genetics of a trispecies F2 population of cotton. p. 448–452. In Proc. Beltwide Cotton Conf., New Orleans, LA. 7–10 Jan. 1997. National Cotton Council of America, Memphis, TN.
• Altaf, M.K., J. Zhang, J.McD. Stewart, and R.G. Cantrell. 1998. Integrated molecular map based on a trispecific F2 population of cotton. p. 491–492. In Proc. Beltwide Cotton Conf., San Diego, CA. 5–9 Jan. 1998. National Cotton Council of America, Memphis, TN.
• Barret, B., R. Delourme, N. Foisset, and M. Renard. 1998. Development of a scar marker for molecular tagging of dwarf BR E1ZH (Bzh) gene in Brassica napus L. Theor. Appl. Genet. 97:828–833.
• Basu, A.K. 1995. Hybrid cotton results and prospects. p. 335–341. In G.A. Constable and N.W. Forrester (ed.) Challenging the future. Proc. World Cotton Res. Conf., 1, Brisbane, Australia. 14–17 Feb. 1994. CSIRO, Melbourne, Australia.
• Bentolila, S., A.A. Alfonso, and M.R. Hanson. 2002. A pentatricopeptide repeat-containing gene restores fertility to cytoplasmic male-sterile plants. Proc. Natl. Acad. Sci. USA 99:10887–10892.[Abstract/Free Full Text]
• Bentolila, S., J. Zethot, T. Gerats, and M.R. Hanson. 1998. Locating the petunia Rf gene on a 650-kb DNA fragment. Theor. Appl. Genet. 96:980–988.
• Borner, A., V. Korzun, A. Polley, S. Malyshev, and G. Melz. 1998. Genetics and molecular mapping of a male fertility restorer locus (Rfg1) in rye (Secale cereale). Theor. Appl. Genet. 97:99–102.
• Brown, G.G., N. Formanova, J. Hua, R. Wargachuk, C. Dendy, P. Patil, M. Laforest, J. Zhang, W.Y. Cheung, and B.S. Landry. 2003. The radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats. Plant J. 35:262–272.[ISI][Medline]
• Cook, C.G., and L.N. Namken. 1995. Registration of B411, B416, and B418 parental lines of cotton. Crop Sci. 35:1518.[Free Full Text]
• Cui, X., R.P. Wise, and P.S. Schnable. 1996. The Rf2 nuclear restorer gene of male sterile T-cytoplasm maize. Science (Washington, DC) 272:1334–1336.[Abstract]
• Endrizzi, J.E., E.L. Turcotte, and R.J. Kohel. 1984. Qualitative genetics, cytology, and cytogenetics. p. 81–129. In R.J. Kohel and C.F. Lewis (ed.) Cotton. Agron. Monogr. 24. ASA, CSSA, and SSSA, Madison, WI.
• Fang, D.Q., G.T. Federici, and M.I. Roose. 1998. A high-resolution linkage map of the citrus tristeza virus resistance gene region in Poncirus trifilata (L.) Raf. Genetics 150:883–889.[Abstract/Free Full Text]
• Guo, W., T.Z. Zhang, J.J. Pan, and R.J. Kohel. 1998. Identification of RAPD marker linked with fertility-restoring gene of cytoplasmic male sterile lines in upland cotton. Chin. Sci. Bull. 43:52–54.
• Haldane, J.B.S. 1919. The combination of linkage values and the calculation of distances between the loci of linked factors. J. Genet. 8:299–309.[ISI]
• Hansen, M., C. Hallden, N.D. Nilsson, and T. Sall. 1997. Marker-assisted selection of restored male-fertile Brassica napus plants using a set of dominant RAPD markers. Mol. Breed. 3:449–456.
• Ichikawa, N., N. Kishimoto, A. Inakaki, A. Nakamura, Y. Koshino, Y. Yokozeki, M. Oka, S. Samoto, H. Akagi, K. Higo, C. Shinjyo, T. Fujimura, and H. Shimada. 1997. A rapid PCR-aided selection of a rice line containing the Rf-1 gene which is involved in restoration of the cytoplasmic male sterility. Mol. Breed. 3:195–202.
• Jean, M., G.G. Brown, and L.S. Landry. 1997. Genetic mapping of nuclear fertility restorer genes for the ‘Polima’ cytoplasmic male sterility in canola (Brassica napus L.) using DNA markers. Theor. Appl. Genet. 95:321–328.
• Jia, M.H., S. He, W. Vanhouten, and S. Mackenzie. 1997. Nuclear fertility restorer genes map to the same linkage group in cytoplasmic male sterile bean. Theor. Appl. Genet. 95:205–210.
• Jia, Z.C. 1990. The development of cytoplasmic male sterile line 104–7A and its three line. Chin. Cottons 17(6):11–12.
• Jiang, C.X., R.J. Wright, S.S. Woo, T.A. DelMonte, and A.H. Paterson. 2000. QTL analysis of leaf morphology in tetraploid Gossypium (cotton). Theor. Appl. Genet. 100:409–418.
• Kaul, M.L.H. 1988. Male sterility in higher plants. Springer-Verlag, Berlin.
• King, J.J., J.M. Bradeen, O. Bark, J.A. McCallum, and M.J. Havey. 1998. A low-density genetic map of onion reveals a role for tandem duplication in the evolution of an extremely large diploid genome. Theor. Appl. Genet. 96:52–62.
• Kohel, R.J., J.E. Quisenberry, and R.E. Dilbeck. 1984. Linkage analysis of the male-fertility restorer gene, Rf, in cotton. Crop Sci. 24:992–994.[Abstract/Free Full Text]
• Kohel, R.J., T.R. Richmond, and C.F. Lewis. 1970. Texas marker—1. Description of a genetic standard for Gossypium hirsutum L. Crop Sci. 10:670–671.[Abstract/Free Full Text]
• Koizuka, N., R. Imai, H. Fujimoto, T. Hayakawa, Y. Kimura, J. Kohno-Murase, T. Sakai, S. Kawasaki, and J. Imamura. 2003. Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish. Plant J. 34:407–415.[ISI][Medline]
• Lacape, J.M., T.B. Nguyen, S. Thibivilliers, B. Bojinov, B. Courtois, R.G. Cantrell, B. Bur, and B. Hau. 2003. A combined RFLP-SSR-AFLP map of tetraploid cotton based on a Gossypium hirsutum x Gossypium barbadense backcross population. Genome 46:612–626.[Medline]
• Lahague, F., P. This, and A. Bouquet. 1998. Identification of a codominant scar marker linked to the seedlessness character in grapevine. Theor. Appl. Genet. 97:950–959.[ISI]
• Lan, T.H., C.G. Cook, and A.H. Paterson. 1999. Identification of a RAPD marker linked to a male fertility restoration gene in cotton (Gossypium hirsutum L.). J. Agric. Genom. 4:1–5.
• Lander, E.S., P. Green, J. Abrahamson, A. Barlow, M.J. Daly, S.E. Lincoln, and L. Newburg. 1987. MAPMAKER: An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174–181.[Medline]
• Laporte, V., D. Merdinoglu, P. Saumitou-Laprade, G. Butterlin, P. Vernet, and J. Cuguen. 1998. Identification and mapping of RAPD and RFLP markers linked to a fertility restorer gene for a new source of cytoplasmic male sterility in Beta vulgaris ssp. maritima. Theor. Appl. Genet. 96:989–996.
• Liu, L., W. Guo, X. Zhu, and T. Zhang. 2003. Inheritance and fine mapping of fertility restoration for cytoplasmic male sterility in Gossypium hirsutum L. Theor. Appl. Genet. 106:461–469.[ISI][Medline]
• Ma, Z.Q., and M.E. Sorrells. 1995. Genetic analysis of fertility restoration in wheat using restriction fragment length polymorphisms. Crop Sci. 35:1137–1143.[Abstract/Free Full Text]
• Meshram, L.D., R.A. Ghongade, and M.W. Marawar. 1994. Development of male sterile systems from various sources in cotton (Gossypium spp.). P.K.V. Res. J. 18(1):83–86.
• Meyer, V.G. 1975. Male sterility from Gossypium harknessii. J. Hered. 66:23–27.[Free Full Text]
• Michelmore, R.W., I.P. Paran, and R.V. Kesseli. 1991. Identification of markers linked to disease resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating population. Proc. Natl. Acad. Sci. USA 88:9828–9832.[Abstract/Free Full Text]
• Pammi, S., J.E. Mullet, A.S. Reddy, and K.F. Schertz. 1994. RAPD markers linked to fertility restorer gene (Rf1) in Sorghum bicolor using bulked segregant analysis. Int. Sorghum Millet Newsl. 35:92.
• Paran, F., and R.N. Michelmore. 1993. Development of reliable PCR-based markers linked to downy mildew resistance gene in lettuce. Theor. Appl. Genet. 85:985–993.[ISI]
• Paterson, A.H., J.W. DeVerna, B. Lanini, and S.D. Tanksley. 1990. Fine mapping of quantitative trait loci using selected overlapping recombinant chromosomes in an interspecies cross of tomato. Genetics 124:735–742.[Abstract]
• Quillet, M.C., N. Madjidian, Y. Griveau, H. Serieys, M. Tersac, M. Lorieux, and A. Berville. 1995. Mapping genetic factors controlling pollen variability in an interspecific cross in Helianthus sect. Helianthus. Theor. Appl. Genet. 91:1195–1202.[ISI]
• Reinisch, A.J., J. Dong, C.L. Brubaker, D.M. Stelly, J.F. Wendel, and A.H. Paterson. 1994. A detailed RFLP map of cotton, Gossypium hirsutum x G. barbadense: Chromosome organization and evolution in a disomic polyploid genome. Genetics 138:829–847.[Abstract]
• Sambrook, J., E.F. Fritsch, and T. Maniatas. (ed.) 1989. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
• Schnable, P.S., and R.P. Wise. 1998. The molecular basis of cytoplasmic male sterility and fertility restoration. Trends Plant Sci. 3:175–180.
• Stewart, J. McD. 1992. A new cytoplasmic male sterile and restorer for cotton. p. 610. In Proc. Beltwide Cotton Conf., Nashville, TN. 6–10 Jan. 1992. National Cotton Council of America, Memphis, TN.
• Stewart, J.McD., C.E. Black-Brown, and J. Zhang. 1996. Sporophytic and gametophytic male dysfunction conditoned by the D-8 cytoplasm of cotton. p. 86. In 1996 Agronomy abstracts. ASA, Madison, WI.
• Stewart, J. McD., and J. Zhang. 1996. Cytoplasmic influence on the inheritance of the D8 restorer gene. p. 622–623. In Proc. Beltwide Cotton Conf., Nashville, TN. 9–12 Jan. 1996. National Cotton Council of America, Memphis, TN.
• Sun, J.Z., J.L. Liu, and J.F. Zhang. 1994. Heterosis in cotton: Advances and problems. (In Chinese with English abstract.) Acta Gossypii Sinica 6:5–10.
• Tar'an, B., T. Warkentin, D.J. Somers, D. Miranda, A. Vandenberg, S. Blade, S. Woods, D. Bing, A. Xue, D. DeKoeyer, and G. Penner. 2003. Quantitative trait loci for lodging resistance, plant height and partial resistance to mycosphaerella blight in field pea (Pisum sativum L.). Theor. Appl. Genet. 107:1482–1491.[ISI][Medline]
• Ulloa, M., W.R. Meredith, Jr., Z.W. Shappley, and A.L. Kahler. 2002. RFLP genetic linkage maps from four F(2.3) populations and a joinmap of Gossypium hirsutum L. Theor. Appl. Genet. 104:200–208.
• Weaver, D.B., and J.B. Weaver, Jr. 1977. Inheritance of pollen restoration in cytoplasmic male-sterile cotton. Crop Sci. 17:497–499.[Abstract/Free Full Text]
• Weaver, J.B., Jr., and D.B. Weaver. 1979. Cracked root mutant in cotton: Inheritance and linkage with fertility restoration. Crop Sci. 19:307–309.[Abstract/Free Full Text]
• Williams, C.G., M.M. Goodman, and C.W. Stuber. 1995. Comparative recombination distributions among Zea mays L. inbred lines, wide crosses and interspecific hybrids. Genetics 141:1573–1581.[Abstract]
• Wise, R.P., and P.S. Schnable. 1994. Mapping complementary genes in maize: Positioning the Rf1 and Rf2 nuclear-fertility restorer loci of Texas (T) cytoplasm relative to RFLP and visible markers. Theor. Appl. Genet. 88:785–795.
• Yuan, J., Z. Zhang, K.L. Liu, X.R. Hao, and H.F. Wang. 1996. The discovery and observation of sterility and nucleocytoplasmic interactions in cotton variety Jin-A. Acta Agric. Boreali Sinica 11(4):29–32.
• Zhang, G., T.S. Bharaj, Y. Lu, S.S. Virmani, and N. Huang. 1997a. Mapping of the Rf-3 nuclear fertility-restoring gene for WA cytoplasmic male sterility in rice using RAPD and RFLP markers. Theor. Appl. Genet. 94:27–33.
• Zhang, J. 1999. Mendelian and molecular genetics of cytoplasmic-nuclear interactions in cotton. Ph.D. diss. Univ. of Arkansas, Fayetteville.
• Zhang, J., W. Guo, and T. Zhang. 2002. Molecular linkage map of allotetraploid cotton (Gossypium hirsutum L. x Gossypium barbadense L.) with a haploid population. Theor. Appl. Genet. 105:1166–1174.[ISI][Medline]
• Zhang, J., and J.McD. Stewart. 2000. Economical and rapid method for extracting cotton genomic DNA. J. Cotton Sci. 4:193–201.
• Zhang, J., and J.McD. Stewart. 2001a. CMS-D8 restoration in cotton is conditioned by one dominant gene. Crop Sci. 41:283–288.[Abstract/Free Full Text]
• Zhang, J., and J.McD. Stewart. 2001b. Inheritance and genetic relationships of the D8 and D2-2 restorer genes for cotton cytoplasmic male sterility. Crop Sci. 41:289–294.[Abstract/Free Full Text]
• Zhang, J., Khan Wajahatullah, and J.McD. Stewart. 1997b. Identification of RAPD markers linked to the D8 CMS restorer gene in cotton. p. 152. In 1997 Agronomy abstracts. ASA, Madison, WI.

This article has been cited by other articles:

				J. Zhang, J. M. Stewart, and T. Wang Linkage Analysis between Gametophytic Restorer Rf2 Gene and Genetic Markers in Cotton Crop Sci., January 1, 2005; 45(1): 147 - 156. [Abstract] [Full Text] [PDF]

This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Zhang, J. Articles by Stewart, J. McD. Search for Related Content PubMed Articles by Zhang, J. Articles by Stewart, J. McD. Agricola Articles by Zhang, J. Articles by Stewart, J. McD. Related Collections Crop Genetics Cotton