Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 2013 Mar;193(3):727–737. doi: 10.1534/genetics.112.146092

Diversifying Sunflower Germplasm by Integration and Mapping of a Novel Male Fertility Restoration Gene

Zhao Liu *, Dexing Wang †, Jiuhuan Feng *, Gerald J Seiler *, Xiwen Cai *, Chao-Chien Jan ‡,1
PMCID: PMC3583994  PMID: 23307903

Abstract

The combination of a single cytoplasmic male-sterile (CMS) PET-1 and the corresponding fertility restoration (Rf) gene Rf1 is used for commercial hybrid sunflower (Helianthus annuus L., 2n = 34) seed production worldwide. A new CMS line 514A was recently developed with H. tuberosus cytoplasm. However, 33 maintainers and restorers for CMS PET-1 and 20 additional tester lines failed to restore the fertility of CMS 514A. Here, we report the discovery, characterization, and molecular mapping of a novel Rf gene for CMS 514A derived from an amphiploid (Amp H. angustifolius/P 21, 2n = 68). Progeny analysis of the male-fertile (MF) plants (2n = 35) suggested that this gene, designated Rf6, was located on a single alien chromosome. Genomic in situ hybridization (GISH) indicated that Rf6 was on a chromosome with a small segment translocation on the long arm in the MF progenies (2n = 34). Rf6 was mapped to linkage group (LG) 3 of the sunflower SSR map. Eight markers were identified to be linked to this gene, covering a distance of 10.8 cM. Two markers, ORS13 and ORS1114, were only 1.6 cM away from the gene. Severe segregation distortions were observed for both the fertility trait and the linked marker loci, suggesting the possibility of a low frequency of recombination or gamete selection in this region. This study discovered a new CMS/Rf gene system derived from wild species and provided significant insight into the genetic basis of this system. This will diversify the germplasm for sunflower breeding and facilitate understanding of the interaction between the cytoplasm and nuclear genes.

Keywords: cytoplasmic male sterility (CMS), fertility restoration gene (Rf), genomic in situ hybridization (GISH), molecular mapping

THE combination of cytoplasmic male-sterility (CMS) and corresponding fertility restoration (Rf) genes has been widely utilized for large-scale hybrid seed production of many crops, including cultivated sunflower (Helianthus annuus L., 2n = 34) (Serieys 1996; Horn et al. 2003). For over 40 years, the hybrid sunflower seed industry has largely relied on a single CMS, CMS PET-1, discovered from wild H. petiolaris subsp. petiolaris Nutt. and its corresponding fertility restoration gene Rf1 (Leclercq 1969; Dominguez-Gimenez and Fick 1975; Miller and Fick 1997; Horn et al. 2003; Jan and Vick 2007). Alternative CMS/Rf gene systems could expand the diversity of the sunflower crop and reduce the risks inherent with using a single CMS/Rf system. Also, identification and characterization of additional CMS/Rf gene systems will enrich knowledge of the interactions between cytoplasm and nuclear genes.

Seventy-two sunflower CMS sources have been identified (Serieys 2005), but only about a half of them have known corresponding Rf genes. Generally, one to four dominant Rf genes are needed for fertility restoration (Serieys 1996). However, only seven Rf genes have been mapped, i.e., Rf1, Msc1, Rf3-RHA 340, Rf3-RHA 280, and Rf5 for CMS PET-1, Rf4 for a new alloplasmic CMS GIG2, and Rf-PEF1 for CMS PEF1 (Gentzbittel et al. 1995; Jan et al. 1998; Horn et al. 2003; Abratti et al. 2008; Feng and Jan 2008; Schnabel et al. 2008; Yue et al. 2010; Liu et al. 2012; Qi et al. 2012). Rf1 was mapped to linkage group (LG) 6 on the RFLP map of Gentzbittel et al. (1995), and to LG 2 by Jan et al. (1998). This gene was also mapped to LG 13 of the SSR map, as well as a recently mapped Rf5 gene, which is from a restorer line Rf ANN-1742 (Yu et al. 2003; Horn et al. 2003; Kusterer et al. 2005; Yue et al. 2010; Qi et al. 2012). Msc1 was mapped to LG 12 of the RFLP map of Gentzbittel et al. (1999). Both Rf3-RHA 340 and Rf3-RHA 280 were mapped to LG 7 of the SSR map (Abratti et al. 2008; Liu et al. 2012), and Rf4 was reported to be on LG 3 of the SSR map (Feng and Jan 2008). The Rf-PEF1 gene was mapped to an AFLP linkage group that differed from LG 13 of the SSR map (Schnabel et al. 2008). Several Rf genes have been cloned from Arabidopsis, radish, rice, maize, and Petunia (Cui et al. 1996; Liu et al. 2001; Bentolila et al. 2002; Brown et al. 2003; Wang et al. 2006; Gillman et al. 2007). Extensive effort has been made to clone Rf1 in sunflower using a map-based cloning strategy (Horn et al. 2003).

Amphiploids are derived from interspecific or intergeneric crosses by chromosome doubling of the F1 hybrids with colchicine or by the spontaneous merging of two or more differentiated genomes. They have been used as an important “bridge” for transferring disease-resistance genes, abiotic stress-resistance/-tolerance genes, and other genes from wild relatives of wheat (Dvorák et al. 1988; Jiang et al. 1994; Colmer et al. 1995; Martín et al. 1999; Soliman et al. 2001), rye (Wojciechowska and Pudelska 2005; Islam et al. 2007; Kang et al. 2011; Malik et al. 2011), and triticale (Kwiatek et al. 2012). Amphiploids are also useful for studying the evolution and genetic diversity within a genus, such as Arabidopsis (Nasrallah et al. 2000), maize (Poggio et al. 2005; González et al. 2006), wheat (Kumar et al. 2010), and Brassica (Allender and King 2010; Bansal et al. 2012).

Several interspecific sunflower amphiploids have been produced via embryo rescue and colchicine treatment with successful gene transfer reported (Jan and Chandler 1989; Jan and Fernandez-Martinez 2002; Pérez-Vich et al. 2002; Feng and Jan 2008). Broomrape-resistance genes for race F in Spain were transferred from several wild Helianthus species into cultivated sunflower using interspecific amphiploids (Jan and Fernandez-Martinez 2002; Pérez-Vich et al. 2002). Amphiploids have shown resistance to Sclerotinia, a major fungal disease of sunflower (Jan et al. 2006; Feng et al. 2007). Also, Feng and Jan (2008) detected a new type of CMS, CMS GIG2, in backcrossed progenies of an amphiploid (Amp) of H. giganteus 1934/HA 89. They identified the Rf4 gene for this CMS in H. maximiliani 1631 utilizing an Amp NMS HA 89/H. maximiliani 1631.

Recently, a CMS line, CMS 514A, derived from the cross between H. tuberosus and an inbred line 7718B, was developed at the Liaoning Academy of Agricultural Sciences, Liaoning, China, but no Rf gene has been identified. Thirty-three maintainer and restorer lines from five countries, as well as 20 tester lines from the U.S. Department of Agriculture–Agricultural Research Service (USDA–ARS) Northern Crop Science Laboratory that are commonly used for Rf gene detection, failed to restore fertility in CMS 514A. This suggested the uniqueness of this CMS compared to other CMS systems, including CMS PET-1, CMS CMG1, CMS CMG2, and CMS CMG3 (Wang et al. 2007). The objectives of this study were to: (1) identify the Rf gene for CMS 514A from five interspecific amphiploids, and a hexaploid H. californicus (PI 664602); (2) introgress the Rf gene into a cultivated sunflower background using traditional crossing and backcrossing method and study the inheritance of the Rf gene; (3) conduct mitotic cytogenetic studies and genomic in situ hybridization (GISH) to characterize the alien chromosome or segments in the progenies; and (4) map the Rf gene using SSR and expressed sequence tag (EST)–SSR markers.

Materials and Methods

Plant materials

Five interspecific amphiploids (Amp H. atrorubens/HA 89, Amp H. mollis/P 21, Amp H. cusickii/P 21, Amp H. grosseserratus/P 21, and Amp H. angustifolius/P 21, 2n = 68), and the F1 progeny of hexaploid H. californicus/HA 89 (2n = 68), were crossed with CMS 514A in 2003. The male-fertile (MF) F1 plants from these crosses were backcrossed with HA 89 and HA 821 to transfer the Rf gene into a cultivated background. HA 89 and HA 821 are oilseed maintainer lines publicly released by USDA. HA 821 was used to increase the diversity of the background instead of using HA 89 only.

Mitotic chromosome counting and GISH

Root tips collected from seedlings were placed in distilled water at 2° for 18 hr and fixed in ethanol:acetic acid (V:V) = 3:1. Chromosome numbers in root tip cells were determined for the individual plants in each generation using the standard Feulgen staining method. The MF plants with 2n = 35 and 34 derived from the cross of CMS 514A × Amp H. angustifolius/P 21 (2n = 68) were used for cytogenetic analysis. Chromosome spreads were made following the method of Liu et al. (2007) with minor modifications. The root tips were digested at 37° for 2.5 hr in an enzyme mixture consisting of 2% cellulase (Sigma, St. Louis, MO) and 24% pectinase (Sigma) in 10 mM sodium citrate buffer (4 mM citric acid and 6 mM sodium citrate). The treated root tips were squashed in 45% acetic acid. Cover slips were removed after being frozen over liquid nitrogen for 5 min.

Genomic DNA of H. angustifolius (plant code G04/795) was used as a probe after being sheared in boiling water for 10 min and labeled with digoxigenin–11-dUTP using the nick translation method according to the manufacturer’s instructions (Roche Applied Science, Nutley, NJ). Genomic DNA of HA 89 was used as a blocking DNA after shearing in boiling water for 20 min and placed on ice for 5 min, with the ratio of blocking DNA to probe DNA of 30:1. Labeled probes were detected with anti-dig-rhodamine or anti-dig-fluorescein (Roche). Chromosomes were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Sigma) in Vectashield (Vector Laboratories, Burlingame, CT). Slides were analyzed using a fluorescence Axioplan2 imaging microscope (Zeiss, Germany). Images were captured by a charge-coupled device (CCD) camera (Zeiss AxioCam HRM) and processed using Axiovision 3.1 software and Adobe Photoshop 6.0.

Mapping population

Ten to 12 progenies derived from three plants, which were genetically similar to the F1 hybrids [G08/598, G08/613, and G08/621, pedigree CMS 514A/6/(CMS 514A//Amp H. angustifolius/P 21/3/2*HA 89/4/HA 821/5/HA 89 and Self) SIB], were used as test populations. An F2 population derived from G08/613 was used for the mapping of the Rf gene. The population was planted in the greenhouse in 2009 totaling 262 individuals. The MF F2 individuals were self-pollinated to obtain F3 seeds. Also, plants with poor pollen fertility were crossed with HA 89 to obtain adequate seed for progeny testing.

Pollen fertility analysis and F2 phenotype confirmation

The pollen fertility of the F2 progenies was determined visually and by pollen stainability for each MF plant. Pollen staining followed Alexander’s method (Alexander 1969) and was analyzed as previously reported by Liu et al. (2012). The F3 and testcross progenies of the F2 population were visually scored to confirm the fertility of each F2 plant, using 20–50 progenies from each F2 individual grown in the field in Fargo, North Dakota, in 2009.

DNA extraction and PCR analysis

Genomic DNA was extracted according to the protocol of the Qiagen DNAeasy 96 plant kit (Qiagen, Valencia, CA). The bulked segregant analysis (BSA) method was used for polymorphism screening (Michelmore et al. 1991), using equal quantities of DNA from 10 plants for each bulk. Four bulks were used, including the homozygous fertile (bulk F) and sterile (bulk S) bulks of F2 plants with 2n = 34, a fertile bulk with 2n = 35 (bulk 2n = 35) and a sterile bulk with 2n = 34 (bulk 2n = 34) in the BC4F2 progeny. The PCR amplification and genotyping followed Liu et al. (2012).

Molecular marker screening

In total, 370 pairs of SSR primers mapped to the 17 sunflower linkage groups from the Compositae database (http://compositdb.ucdavis.edu) were used for polymorphism screening among the four bulks. An additional 65 SSR markers and 28 EST–SSR markers from the candidate LG 3 of 23 maps in the Sunflower CMap Database (http://sunflower.uga.edu/cgi-bin/cmap/map_search) were used to screen for polymorphisms among the parents and the F1 plants. Polymorphic markers were used for genotyping the mapping population after confirmation.

Statistical analysis and linkage map construction

The deviation analyses of the fertility trait and marker loci were compared with the expected Mendelian ratios in the F2 generation using the chi-square test. The MAPMAKER/Exp v. 3.0b program (Whitehead Institute, Cambridge, MA) (Lander et al. 1987) was used for linkage analysis of the phenotypes and molecular genotypes following Liu et al. (2012).

Results

Identification of the Rf gene in Amp H. angustifolius/P 21

The chromosome number of the five amphiploids and the F1 hybrids of H. californicus/HA 89 (2n = 68) were stable and maintained during sib-crossing. All of the F1 plants from the crosses of CMS 514A with the F1s of H. californicus/HA 89 and four of the five amphiploids were male sterile (MS) (Table 1). However, the F1 plants derived from the crosses with Amp H. angustifolius/P 21 were all MF. Chromosome counts revealed that 12 of the 13 F1s derived from this cross had 51 chromosomes, and the remaining one had 47. Since P 21 does not restore the male fertility of CMS 514A (Wang et al. 2007), it suggests that the Rf gene came from H. angustifolius of the Amp H. angustifolius/P 21 and was designated Rf6.

Table 1. The F1 progeny fertility restoration of five amphiploids (Amp) and the F1 of H. californicus/HA 89 crossed with CMS 514A.

Cross No. of plant No. of fertile plant
CMS 514A//H. californicus/HA 89 4 0
CMS 514A//Amp H. atrorubens/HA 89 6 0
CMS 514A//Amp H. mollis/P 21 11 0
CMS 514A//Amp H. cusickii/P 21 6 0
CMS 514A//Amp H. grosseserratus/P 21 0 0
CMS 514A//Amp H. angustifolius/P 21 13 13
Total 50
Open in a new tab

Integration of Rf6 into cultivated sunflower

The F1 plants (2n = 51) derived from the cross of CMS 514A × Amp H. angustifolius/P 21 were crossed with HA 89. All F1 plants produced seeds, with an average seed set of 2.2% (593 seeds/26500 florets). The MF:MS ratio of 40 BC1F1 plants was 11:29, with the pollen stainability averaging 19.8% (range 0.7–79.1%), and a chromosome number of 2n = 37-46 for the MF plants (supporting information, Table S1). Four plants that produced very small amount of pollen (S+) were scored as MS. The MF progenies were backcrossed with HA 89 or crossed with HA 821 to further reduce their chromosome number to 2n = 34 (Figure 1). Three MF BC1F1 plants, with pollen stainability of 52.3, 79.1, and 19.3%, respectively, were crossed with HA 89. The chromosome numbers of BC2F1 progenies were reduced to 34–39, and 6 of 26 plants were MF with an average pollen stainability of 47.3% (range 12–87%). After crossing with HA 89, HA 821, or CMS BC2F1, 8 MF plants (2n = 35) were obtained among 36 progenies, with an average pollen stainability of 47.6% (range 9–96%).

Figure 1.

Figure 1

Open in a new tab

Scheme for introgression of the fertility restoration gene Rf6 for CMS 514A from an interspecific amphiploid (Amp) of H. angustifolius/P 21 (2n = 68) into cultivated sunflower.

After self-pollination and backcrossing with HA 89, seven of the eight MF plants with 2n = 35 resulted in 1 MF progeny with 2n = 36 (pollen stainability of 27.4%), 10 MF with 2n = 35 (pollen stainability of 56.5%, range 18.1–91.6%), and 2 MF with 2n = 34 (pollen stainabilities of 24.4 and 56.9%) from a total of 62 progenies (Table S2). Significant variation in pollen stainability among plants was observed. However, Rf6 restored the male fertility to >90% in some cases, suggesting the potential use of this material for sunflower breeding. The frequency of MF and MS plants with 2n = 36, 35, and 34 was 1.6 and 1.6%, 16.1 and 1.6%, and 3.2 and 75.8%, respectively. Since the majority of the MF plants (76.9%) had 2n = 35, it indicated that Rf6 was on the alien H. angustifolius chromosome in these 7 MF plants. Moreover, 79.0% of these 62 progenies had 2n = 34, with only 2 MF plants (4.0%), which suggested a low transmission rate of this alien chromosome in the progenies and a low frequency of recombination between the region containing Rf6 and the cultivated sunflower chromosome.

One MF plant, G07/517 (2n = 34), with pollen stainability of 56.9%, was crossed to an MS individual, G07/553 (2n = 34). The seed set of this cross was 100%, suggesting that all the female gametes were fertile. Of the 31 F1 hybrids derived from this cross, six plants (19.4%) were MF, and three were self-compatible with 100% seed set. These three plants, G07/610, G07/612, and G07/623, had improved pollen stainability of 74.7, 96.0, and 90.2%, respectively (Table S2), suggesting that MF plants with 2n = 34 and acceptable male and female fertility were found.

Genetic analysis of the alien chromosome carrying Rf6

Testcross progenies of CMS 514A pollinated with two MF plants, G07/596 and G07/598 (2n = 35, derived from the self-pollination of G07/513), resulted in 55 MS (2n = 34) and four MF plants (2n = 35). The MF plants were all 2n = 35, whereas the MS plants were all 2n = 34 (Table 2), which suggested that Rf6 was located on the alien chromosome in G07/596 and G07/598. The low number of the plants with 2n = 35 also indicated that this alien chromosome did not segregate randomly into the daughter cells in meiosis. The pollen stainability of G07/596 and G07/598 was 60.4 and 81.9%, respectively, while those for the four 2n = 35 MF progeny plants were improved further, averaging 95.0% (range 92.8–98.6%), including G08/672 and G08/683 (derived from CMS 514A × G07/596), and G08/638 and G08/651 (derived from CMS 514A × G07/598).

Table 2. Alien chromosome transmission in testcross and self-pollinated progenies of the male-fertile (MF) 2n = 35 plants.

Cross Total plants 2n = 34 MF:MS 2n = 35 MF:MS 2n = 35 (%)a 2n = 36 MF:MS 2n = 36 (%)b
CMS 514A × (G07/596 and G07/598) (MF, 2n = 35) 59 0:55 4:0 6.8
G09/2614 (MF, 2n = 35) × HA 89 59 0:39 20:0 33.9
G08/638 and G08/672 (MF, 2n = 35) selfed-total 116 1:75 25:10 30.2 4:1 4.3
G08/638 (MF, 2n = 35) selfed (1) 57 1:36 8:9 29.8 2:1 5.3
G08/672 (MF, 2n = 35) selfed (2) 59 0:39 17:1 30.5 2:0 3.4
Open in a new tab
a

Percentage = number of 2n = 35 plants/total plants × 100.

b

Percentage = number of 2n = 36 plants/total plants × 100.

Comparison of the chromosome constitutions of the MS 2n = 34 and MF 2n = 35 plants revealed a large chromosome in the 2n = 35 plants (Figure 2A), which was not present in the 2n = 34 plants (Figure 2B). One MF plant, G09/2614 (2n = 35), derived from the self-pollination of G08/672 was emasculated and pollinated with HA 89 to study the transmission of the alien chromosome in the progeny when it was used as the female parent. Among the 59 progenies of this cross, 20 were MF with 2n = 35, and the remaining 39 were MS with 2n = 34 (Table 2 and Table S3). A higher transmission rate of the alien chromosome carrying Rf6 was observed when the MF plants (2n = 35) were used as the female parent (33.9%) vs. as the male parent (6.8%). However, it was still lower than the expected percentage of 50% if this alien chromosome segregated randomly during meiosis. The above analysis indicated that the selection pressure on male gametes was stronger than that on female gametes.

Figure 2.

Figure 2

Open in a new tab

Chromosome spreads of male-fertile (MF) (2n = 35) and male-sterile (MS) (2n = 34) plants. (A) Chromosome spread of an MF plant with 2n = 35, G09/2617, derived from self-pollination of G08/672. (B) Chromosome spread of an MS plant with 2n = 34, G09/2567, derived from self-pollination of G08/638. The arrow in A shows the larger chromosome compared to other chromosomes, which is assumed to be the alien chromosome from H. angustifolius carrying Rf6. Bars, 5 μm.

G08/638 and G08/672 (2n = 35) were self-pollinated and produced 57 and 59 F2 plants, respectively. They were examined for chromosome number and male fertility (Table 2). Among the 116 plants, 35 had 2n = 35, 5 had 2n = 36, and the remaining 76 had 2n = 34. Again, the distorted segregation of the alien chromosomes was observed in this generation. Pollen fertility examination of the 57 F2 plants derived from G08/638 indicated that all except 1 with 2n = 34 were MS and that not all plants with 2n = 35 and 36 were MF. In the second group of plants derived from G08/672, 39 plants were 2n = 34 and MS, 2 plants were 2n = 36 and MF, and 17 plants were 2n = 35 and MF, except for 1 plant with 2n = 35, which was MS. The above genetic analyses indicated that the recombination rate between the alien chromosome and cultivated sunflower was very low; however, the expression of Rf6 was complicated in other cases, such as for the F2 plants derived from G08/638. It was likely that Rf6 had been recombined into the genome of the cultivated sunflower. Thus, in the progenies there were both MF plant with 2n = 34 and MS plants with 2n = 35 or 36; i.e., the large chromosomes in the gametes did not carry Rf6 gene in the latter cases.

Genetic analysis of the Rf6 gene

Three MF plants, G07/610, G07/612, and G07/623 (2n = 34), were checked for male fertility in their selfed progenies and testcrossed with CMS 514A to study the inheritance and restoration of Rf6. Of the 29 plants derived from the self-pollinated G07/610, 19 were MF (pollen stainability averaged 64.5%, but varied widely from 6.6 to 96.1%), and the remaining 10 were MS. Similarly, the MF:MS ratio of the 29 plants derived from G07/612 was 20:9. The pollen stainability also varied widely (range 27.3–99.4%), with a higher average of 78.9%. The ratio of MF:MS among the 30 plants derived from G07/623 was 20:10, with an even higher average pollen stainability of 91.5% (range 51.1–98.3%). The overall ratio of the MF to MS (59:29) were closer to 2:1 (χ2 =0.01, P = 0.94) than to 3:1 (χ2 =2.97, P = 0.085). The male fertility and pollen stainability of each individual is shown in Table S4.

The testcrosses of G07/610, G07/612, and G07/623 to CMS 514A showed different ratios of MF to MS plants. Among the 12 progenies derived from each cross combination, the ratio of MF to MS was 2:10, 4:8, and 8:4, respectively. The overall ratio of MF to MS in the three testcrosses was 14:22, fitting the expected ratio of 1:1 (χ2 =1.78, P = 0.18), indicating that these three male parents were heterozygous at the Rf6 gene locus. Pollen stainability varied from 88.2 to 99.5%, with an average of 96.8%. Three plants with 100% seed set, G08/598, G08/613, and G08/621, were selected for further study (Figure 1). Their pollen stainability was 96.9, 99.5, and 95.7%, respectively.

Mitotic GISH and cytogenetic analyses

The alien chromosome or segments from the H. angustifolius genome can be differentiated from the cultivated sunflower chromosomes by GISH. Male fertility data and chromosome counts for the testcross progenies of CMS 514A with G07/596 and G07/598, and the self-pollinated progeny of G08/672, as well as the progeny derived from G09/2614 × HA 89 (Table 2), suggested that Rf6 was located on the alien chromosome in the MF plants with 2n = 35 chromosomes. GISH results for both G08/638 and G08/672 (2n = 35) showed an obvious signal on one large chromosome compared to other cultivated sunflower chromosomes (Figure 3A for G08/638). The ratio of the short arm to the long arm of this alien chromosome was 0.6351 based on five cell observations. GISH results together with genetic analysis of the MF plants (2n = 35) suggested that this chromosome contains the Rf6 gene for CMS 514A.

Figure 3.

Figure 3

Open in a new tab

Genomic in situ hybridization (GISH) analyses of the alien H. angustifolius chromosome or segments in different progenies. The genomic DNA of H. angustifolius was labeled with digoxigenin–11-dUTP and detected by anti-dig-rhodamine (red), the chromosomes were counterstained by DAPI (blue). (A) a heterozygous MF plant, G08/638 (2n = 35), with an alien chromosome. (B) A heterozygous MF plant with 2n = 34, G08/2660, derived from self-pollination of G08/613, with one small translocation. (C) A homozygous MF plant with 2n = 34, G08/2663, derived from self-pollination of G08/613, with two small translocations. (D) A heterozygous MF plant with 2n = 34, G08/2649, derived from self-pollination of G08/621, with one whole short arm and one small segment translocations. (E) A homozygous MF plant with 2n = 34, G08/2670, derived from self-pollination of G08/598, with one whole short arm and two small translocations. (F) An MS plant with 2n = 34, G08/2657, derived from self-pollination of G08/621, with one whole short arm translocation. (G) An abnormal plant, G08/2651, derived from self-pollination of G08/621, with two whole short arm and one small segment translocations. The arrows show the alien chromosome or segments from H. angustifolius. Bars, 5 μm.

The F2 progenies derived from G08/598, G08/613, and G08/621 were tested for male fertility and the alien chromosome segments (Figure 3 and Table S5). A total of 27 plants were analyzed by GISH. Interestingly, three plants heterozygous for Rf6 derived from G08/613 had only one small translocation, and two plants homozygous for Rf6 contained two small translocations (Figure 3, B and C), whereas seven plants heterozygous for Rf6 from both G08/598 and G08/621 had two translocations, i.e., one with a whole short arm translocated and another with the same as G08/613 progenies (Figure 3D). For the two homozygous MF plants derived from G08/598, two small translocations together with one whole short arm translocation were detected in one plant, G08/2670 (Figure 3E), and only the two small segment translocations were detected in G08/2680. In addition, no alien chromosome segment was detected in three MS plants (G08/2656, G08/2675, and G08/2676), and only the whole short arm translocation was detected in two other MS plants, G08/2657 and G08/2658, derived from G08/621 (Figure 3F). Therefore, the GISH results for the F2 individuals indicated that Rf6 was located on the small H. angustifolius chromosomal segment involved in the translocation. The translocation point for the small segment was located at about the one-fourth distance from the end of the long arm of the chromosome.

Moreover, three translocations were detected using GISH analysis in two F2 plants with morphological abnormalities. One plant, G08/2651, derived from G08/621, had two whole arm translocations and one small segment translocation (Figure 3G) and was physically abnormal with a tiny capitulum and a short plant (about 40 cm). The second plant, G08/2681, derived from G08/598, was MF and had one whole arm translocation and two small segment translocations, but wilted during flowering. This wilting trait was also observed in two other MF F2 individuals (G08/2652 and G08/2672), and both had one whole arm and one small segment translocations. Therefore, genetic unbalanced gametes might be produced when Rf6 linked with undesirable genes or the alien chromosome or segments negatively interact with the cultivated sunflower background, causing morphological abnormalities.

Fertility segregation in the mapping population

Based on the fertility segregation, abnormal traits, and GISH analysis, a population derived from G08/613 was used to map Rf6. This population included 262 F2 individuals, with 166 MF and 89 MS plants, producing 221 usable F3 or testcross progeny families. Progeny test was not performed for 34 MF plants due to shortage of seed and 7 of 9 wilting plants died before flowering. Phenotypic analysis identified 19 homozygous MF, 113 heterozygous MF, and 89 MS plants in the F2 population. Chi-square test indicated the ratio of MS:MF or homozygous MF:heterozygous MF:homozygous MS phenotypes significantly deviated from the expected Mendelian ratio 1:3 or 1:2:1 (χ2 >13, P < 0.0005) (Table 3).

Table 3. Segregation of the Rf6 locus and marker loci in the F2 population derived from the cross CMS 514A/6/(CMS 514A//AMP H. angustifolius/P 21/3/2*HA 89/4/HA 821/5/HA 89 and Self) SIB.

Observed no.
Traits or markers No. of F2 plants A H B C Ratio expected χ2 P-value
Rf6a 255 89 166 1:3 13.33 2.6 × 10−4
Rf6b 221 89 113 19 1:2:1 44.46 2.2 × 10−10
ORS822 220 85 135 1:3 21.82 3.0 × 10–6
HT088 220 85 118 17 1:3 43.20 4.9 × 10−11
ORS433-a 220 82 138 1:3 17.67 2.6 × 10–5
ORS13 220 83 118 19 1:2:1 38.40 4.6 × 10−9
ORS1114 220 83 118 19 1:2:1 38.40 4.6 × 10−9
HT734 220 82 119 19 1:2:1 37.55 7.0 × 10−9
ORS488 220 82 119 19 1:2:1 37.55 7.0 × 10−9
ORS525 221 86 135 1:3 22.82 1.8 × 10−6
ORS433-bc 216 58 158 1:3 0.40 0.53
Open in a new tab

Symbols: A, homozygous MS (rfrf); H, heterozygous MF (Rfrf); B, homozygous MF (RfRf); C, RfRf or Rfrf.

a

Phenotyping data in the F2 generation.

b

Phenotyping data of the F2 individuals after progeny test.

c

ORS433-b is not linked to the Rf6 gene, thus not included in Figure 5B.

In this F2 mapping population, a large variation in fertility was observed (15–100%). About 82% of the plants had fertility >50%, and 73% >80%. The average pollen stainability for homozygous MF F2 plants was 93.9%. Lower pollen stainability was observed for the heterozygous MF F2 plants with 81.5%. For the MF F2s without progeny test, the pollen stainability was 87.2%. The overall average pollen stainability in this population was 83.5%.

Chromosomal location of Rf6

A total of 463 molecular markers were used for mapping. Only nine markers (1.9%) showed no products or weak bands. The 370 pairs of SSR primers from 17 sunflower linkage groups were used to screen polymorphisms using BSA, which averaged 21 primers per linkage group. Four polymorphic markers were identified for the two fertile (bulk F and bulk 2n = 35) and sterile bulks (bulk S and bulk 2n = 34), including ORS433, ORS488, ORS822, and ORS1114. Two polymorphic markers (ORS432 and ORS1021) were identified for bulk 2n = 35 and bulk 2n = 34, but not for bulk F and bulk S. The polymorphic markers were validated using the 10 individuals constituting each bulk. Validation of the markers is shown in Figure 4, using the ORS433 primer pair. The ORS432, ORS488, and ORS1114 markers were mapped to LG 3 of Tang et al. (2003) and RHA 280 × RHA 801_RIL (Sunflower CMap: http://www.sunflower.uga.edu/cgi-bin/cmap/viewer?data_source=pbio_cmap;ref_map_accs=RHA280xRHA801ril3), whereas ORS433, ORS822, and ORS1021 were multi-loci markers mapped to several LGs, including LG 3. The alien chromosome carrying Rf6 in the MF 2n = 35 plants was expected to be on LG 3 according to the results from the BSA. The four polymorphic primers between the fertile and sterile bulks were used to genotype the mapping population. The results showed a close linkage among the markers and Rf6, further confirming the chromosomal location of Rf6 on LG 3.

Figure 4.

Figure 4

Open in a new tab

Representative results of the markers amplified by the ORS433 primer pair among the four bulks and the individuals constituting each bulk, respectively, on a nondenaturing polyacrylamide gel. (1) Bulk S; (2) bulk F; (3) bulk 2n = 34; (4) bulk 2n = 35. The bulks 3 and 4 consist of the MS plants (2n = 34) and MF plants (2n = 35) from the selfed progenies of a BC4F1 MF plant (2n = 35), respectively; M indicates a 100-bp plus ladder Gelpilot (Qiagen); the arrows indicates the dominant markers. Marker ORS433-a (∼190 bp) is linked to Rf6, whereas marker ORS433-b (about 175 bp) is not.

In addition, 65 SSR and 28 EST-SSR markers on LG 3 from 23 maps in the Sunflower CMap Database were used to screen the polymorphism among the parents and the F1 plants. Thirteen polymorphic markers were identified, including seven SSR markers (ORS13, ORS134, ORS525, ORS683, ORS777, ORS1130, and ORS1144), and six EST-SSR markers (HT088, HT499, HT734, HT779, HT845, and HT1029). However, only ORS13, ORS525, HT088, and HT734 were linked to Rf6 after confirmation using the individuals of the four bulks. These four markers were also used for genotyping the mapping population. Segregations of Rf6 and markers using the whole F2 population are shown in Table 3.

Molecular mapping of Rf6

The chi-square test showed that eight markers were severely distorted from the expected Mendelian ratios. Using the eight markers mentioned above, Rf6 was located on a map constructed using 221 F2 individuals, covering a distance of 10.8 cM (Figure 5). Rf6 was located between the co-dominant HT088 and two co-segregated markers ORS13 and ORS1114. The closest markers were ORS13 and ORS1114, at a distance of 1.6 cM. Noticeably, the primer pair ORS433 produced two dominant markers, i.e., ORS433-a (about 190 bp) and ORS433-b (about 175 bp), with only ORS433-a linked to Rf6, whereas ORS433-b was not (Figure 4 and Figure 5). ORS433-b did not show the same segregation distortion in the mapping population as ORS433-a (Table 3). Therefore, the segregation distortion occurred only in the mapped chromosomal region harboring Rf6. Compared to the reference maps of Tang et al. (2003) (Figure 5A), and RHA 280 x RHA 801_RIL (in press) (Figure 5C) in the Sunflower CMap Database, the eight linked marker loci for Rf6 were grouped at the end of LG 3, although the order of the markers were reversed (Figure 5B). The markers ORS1021 and ORS432 were linked to the alien chromosome in the MF plants with 2n = 35, but not linked to Rf6 (region I on Figure 5, A and C). Their map positions suggested that the break point of the translocation carrying Rf6 might be located between the markers ORS432 and ORS13 (Figure 5A) or between ORS432 and ORS488 (Figure 5C).

Figure 5.

Figure 5

Open in a new tab

The position of the fertility restoration gene Rf6 on LG 3 of the sunflower map. (A) A partial map of LG 3 of Tang et al. (2003). (B) mapping result of Rf6 on LG 3, using 221 F2 plants. (C) a partial map of LG 3 of RHA 280 × RHA 801_RIL (Sunflower CMap). The distances are given in centimorgans (cM). The corresponding markers are noted by lines between the maps. Region I indicates the region is not linked to Rf6, and region II indicates the region where the possible break point of the translocation with Rf6 is located.

Discussion

CMS 514A/Rf6, a new CMS/Rf system

Fifty-nine different germplasm sources were used to identify the Rf gene for CMS 514A (Wang et al. 2007; this study). The Rf6 gene was determined to have originated from H. angustifolius. These results suggested that Rf6 is probably different from other reported Rf genes. The average pollen fertility of Rf1 is 98% for hybrid 894 (Seiler 2000) and 94% for Rf3 from RHA 280 (Liu et al. 2012), while the average pollen fertility of Rf6 was ∼80%, with a large variation in the F2 mapping population. Rf6 was mapped to LG 3 of the sunflower SSR public map (Tang et al. 2003), with eight linked markers in this study.

Seven Rf genes were previously mapped on the sunflower genetic maps, with only Rf4 reported on LG 3 (Feng and Jan 2008; reviewed by Liu et al. 2012). The closest marker linked to Rf4 is ORS1114 at a distance of 0.9 cM. Rf6 was also mapped to LG 3, with the closest markers ORS13 and ORS1114 at a distance of 1.6 cM. Considering the different origins, these two Rf genes are probably not the same. The H. angustifolius amphiploid was the only one of the five tested that restored male fertility for CMS 514A, while three amphiploids (H. atrorubens, H. grosseserratus, and H. angustifolius) restored male fertility for CMS GIG2. The H. mollis amphiploid failed to restore the male fertility for either CMS (Sunflower CMap). Therefore, these two CMSs are likely not the same. Currently, the allelic relationship analysis between Rf4 and Rf6 is under investigation to provide more information for their practical use in sunflower breeding. The combination of the markers closely linked to Rf6 will be useful for marker-assisted selection.

We introgressed Rf6 into cultivated sunflower using a traditional crossing and backcrossing scheme. The GISH analysis with different fertile or sterile F2 plants indicated that only the translocation located terminally on a chromosome, estimated to be about one-fourth of the long arm, is related to fertility restoration of CMS 514A. Molecular marker analysis suggested that the translocation break point on the alien chromosome for Rf6 in the MF plants (2n = 35) may be located between markers ORS432 and ORS13 or ORS488 on the reference maps (region II on Figure 5, A and C). In addition, the primer pair ORS433 produced two markers, with only ORS433-a closely linked to Rf6 in the F2 population. Considering ORS433 is a multilocus marker in the public sunflower maps and ORS433-b was not mapped onto LG 3 in this study, we are not sure whether the ORS433-a marker is the same ORS433 marker located on the region I of LG 3 or is a new marker in this study. Therefore, additional markers are needed to characterize this region to more precisely determine the translocation break point.

Rf6 and the linked marker loci showed segregation distortion

Segregation distortion is the deviation of the frequency of genotypes from the expected Mendelian ratio within a segregating population. Segregation distortion has been observed in fungi, plants, insects, and mammals (Lyttle 1991; Liu et al. 2010). In plants, segregation distortion has been encountered in maize (Mangelsdor and Jones 1926), rice (McCouch et al. 1988), wheat (Zhang and Dvorák 1990; Faris et al. 1998; Kumar et al. 2007), barley (Graner et al. 1991; Li et al. 2010), tobacco (Cameron and Moav 1957), tomato (Paterson et al. 1988), alfalfa (Echt et al. 1994), and coffee (Ky et al. 2000). Segregation distortion, also called “meiotic drive,” may be caused by genetic elements, including gametic selection (pollen tube competition, lethal pollen, and preferential fertilization), zygotic selection, interspecific sterility genes (S), and chromosome translocation (Lyttle 1991; Kumar et al. 2007; Gutiérrez et al. 2010; Liu et al. 2010). It has been suggested that meiotic drive elements are highly important for the evolution of recombination and sexual reproduction (Hurst and Werren 2001; Jaenike 2001; Li et al. 2010).

Segregation distortion has been reported for sunflower populations derived from the interspecific crosses involving wild species in the mapping of a downy mildew resistance gene on LG 1, PlARG, which originated from H. argophyllus Torrey and Gray (Dußle et al. 2004; Wieckhorst et al. 2010). Significant segregation distortion of the codominant markers closely linked to the PlARG gene was observed in the F2 population derived from the cross of CMS HA 342 × ARG1575-2, but not in the ones derived from the HA 342 × ARG1575-2 and NDBLOSsel × KWS04, indicating the influence of the CMS cytoplasm and chromosome segment(s) from the wild species on the fertility and segregation ratios in the population. In our study, a severe deviation was detected for Rf6 and the linked marker loci in a mapping population, which probably indicated suppressed recombination or gamete selection in this region. GISH and molecular marker results suggested that Rf6 was on the chromosome with the small segment translocation. Moreover, only one amphiploid was discovered to contain the Rf gene for CMS 514A after testing 59 different germplasm sources, including the maintainer of CMS 514A, which restores fertility to other CMS types (Wang et al. 2007; this study). Taken together that CMS 514A has an H. tuberosus cytoplasm and the Rf6 gene was from the wild species H. angustifolius, the segregation distortion may be caused by several factors, such as gametic selection, interspecific S gene, and chromosome translocation (Lyttle 1991; Kumar et al. 2007; Gutiérrez et al. 2010; Liu et al. 2010).

Due to the limited number of marker loci polymorphic between the parents, the linkage group covered a genetic distance of only 10.8 cM. Chromosomal inversion could be one of the reasons for the suppressed recombination. Detailed comparison of the marker orientations among the maps constructed here and the reference maps would help to explain this question. Therefore, more markers such as single nucleotide polymorphism (SNP) markers, SSR, or other types of markers are necessary to fine map Rf6, as well as the segregation distortion regions. Additionally, a low transmission rate of the alien chromosome or its segments into the cultivated sunflower was detected during backcrossing. Abnormal growth, such as reduced vigor, wilting before or near flowering stage, and sterile sections on the flowering capitulum of MF plants, was also noted in some backcrossing progenies.

In conclusion, this study identified an Rf gene, Rf6, that restores the male fertility of a recently identified CMS source, CMS 514A, originated from a wild species, H. angustifolius, via an interspecific amphiploid H. angustifolius/P 21 (2n = 68). This gene was introgressed into the cultivated sunflower background after several crosses and backcrosses. The alien chromosome or segments were characterized using GISH and molecular marker analyses. Rf6 was located on LG 3 of the sunflower public SSR map, with eight linked markers in a mapping population. Progenies with different translocations were developed during the crossing process. These could facilitate the development of a unique fertility restorer for CMS 514A and could be useful in studying the interactions between cytoplasm and nuclear genes.

Supplementary Material

Supporting Information
supp_193_3_727__index.html (2.1KB, html)

Acknowledgments

The authors thank Lisa A. Brown for technical assistance in this study and Ridhima Katyal, Jordan Hogness, Alexis Ganser, Yuni Chen, and Marjorie A. Olson for their help in conducting this study. We appreciate Drs. Chengsong Zhu (Kansas State University), Wentao Li (University of California-Davis), Zahirul Talukder, and Yunming Long (North Dakota State University) for valuable discussion during data analysis. We also thank Drs. Larry G. Campbell, Lili Qi, Prem P. Jauhar, Steven S. Xu, and Brady A. Vick for critical review of the manuscript.

Footnotes

Communicating editor: F. F. Pardo Manuel de Villena

Literature Cited

  1. Abratti G., Bazzalo M. E., León A., 2008.  Mapping a novel fertility restoration gene in sunflower, pp. 617–621 in 17th International Sunflower Conference, edited by I. Velasco. Córdoba, Spain.
  2. Alexander M. P., 1969.  Differential staining of aborted and nonaborted pollen. Stain Technol. 44: 117–122. [DOI] [PubMed] [Google Scholar]
  3. Allender C. J., King G. J., 2010.  Origins of the amphiploid species Brassica napus L. investigated by chloroplast and nuclear molecular markers. BMC Plant Biol. 10: 54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bansal P., Banga S., Banga S. S., 2012.  Heterosis as investigated in terms of polyploidy and genetic diversity using designed Brassica juncea amphiploid and its progenitor diploid species. PLoS ONE 7: e29607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bentolila S., Alfonso A. A., Hanson M. R., 2002.  A pentatricopeptide repeat-containing gene restores fertility to male-sterile plants. Proc. Natl. Acad. Sci. USA 99: 10887–10892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown G. G., Formanova N., Jin H., Wargachuk R., Dendy C., et al. , 2003.  The radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats. Plant J. 35: 262–272. [DOI] [PubMed] [Google Scholar]
  7. Cameron D. R., Moav R. M., 1957.  Inheritance in Nicotiana tabacum XXVII: pollen killer, an alien genetic locus inducing abortion of microspores not carrying it. Genetics 42: 326–335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Colmer T. D., Epstein E., Dvorák J., 1995.  Differential solute regulation in leaf blades of various ages in salt-sensitive wheat and a salt-tolerant wheat × Lophopyrum elongatum (Host) A. Love amphiploid. Plant Physiol. 108: 1715–1724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cui X., Wise R. P., Schnable P. S., 1996.  The rf2 nuclear restorer gene of male-sterile T-cytoplasm maize. Science 272: 1334–1336. [DOI] [PubMed] [Google Scholar]
  10. Dominguez-Gimenez J., Fick G. N., 1975.  Fertility restoration of male-sterile cytoplasm in wild sunflowers. Crop Sci. 15: 724–726. [Google Scholar]
  11. Dußle C. M., Hahn V., Knapp S. J., Bauer E., 2004.  PlArg from Helianthus argophyllus is unlinked to other known downy mildew resistance genes in sunflower. Theor. Appl. Genet. 109: 1083–1086. [DOI] [PubMed] [Google Scholar]
  12. Dvorák J., Edge M., Ross K., 1988.  On the evolution of the adaptation of Lophopyrum elongatum to growth in saline environments. Proc. Natl. Acad. Sci. USA 85: 3805–3809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Echt C. S., Kidwell K. K., Knapp S. J., Osborn T. C., McCoy T. J., 1994.  Linkage mapping in diploid alfalfa (Medicago sativa). Genome 37: 61–71. [DOI] [PubMed] [Google Scholar]
  14. Faris J. D., Laddomada B., Gill B. S., 1998.  Molecular mapping of segregation distortion loci in Aegilops tauschii. Genetics 149: 319–327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Feng J., Jan C. C., 2008.  Introgression and molecular tagging of Rf4, a new male fertility restoration gene from wild sunflower Helianthus maximiliani L. Theor. Appl. Genet. 117: 241–249. [DOI] [PubMed] [Google Scholar]
  16. Feng J., Seiler G. J., Gulya T. J., Li C., Jan C. C., 2007.  Sclerotinia stem and head rot resistant germplasm development utilizing interspecific amphiploids. 29th Sunflower Research Workshop, January 10–11, 2007, Fargo, ND. Available at: www.sunflowernsa.com/research/research-workshop/documents/Feng_etal_Amphiploids_2007.pdf
  17. Gentzbittel L., Vear F., Zhang Y.-X., Bervillé A., Nicolas P., 1995.  Development of a consensus linkage RFLP map of cultivated sunflower (Helianthus annuus L.). Theor. Appl. Genet. 90: 1079–1086. [DOI] [PubMed] [Google Scholar]
  18. Gentzbittel L., Mestries E., Mouzeyar S., Mazeyrat F., Badaoui S., et al. , 1999.  A composite map of expressed sequences and phenotypic traits of the sunflower (Helianthus annuus L.) genome. Theor. Appl. Genet. 99: 218–234. [Google Scholar]
  19. Gillman J. D., Bentolila S., Hanson M. R., 2007.  The petunia restorer of fertility protein is part of a large mitochondrial complex that interacts with transcripts of the CMS-associated locus. Plant J. 49: 217–227. [DOI] [PubMed] [Google Scholar]
  20. González G., Comas C., Confalonieri V., Naranjo C. A., Poggio L., 2006.  Genomic affinities between maize and Zea perennis using classical and molecular cytogenetic methods (GISH-FISH). Chromosome Res. 14: 629–635. [DOI] [PubMed] [Google Scholar]
  21. Graner A., Jahoor A., Schondelmaier J., Siedler H., Pillen K., et al. , 1991.  Construction of a RFLP map of barley. Theor. Appl. Genet. 83: 250–256. [DOI] [PubMed] [Google Scholar]
  22. Gutiérrez A. G., Carabalí S. J., Giraldo O. X., Martínez C. P., Correa F., et al. , 2010.  Identification of a rice stripe necrosis virus resistance locus and yield component QTLs using Oryza sativa × O. glaberrima introgression lines. BMC Plant Biol. 10: 6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Horn R., Kusterer B., Lazarescu E., Prüfe M., Friedt W., 2003.  Molecular mapping of the Rf1 gene restoring pollen fertility in PET1-based F1 hybrids in sunflower (Helianthus annuus L.). Theor. Appl. Genet. 106: 599–606. [DOI] [PubMed] [Google Scholar]
  24. Hurst G. D., Werren J. H., 2001.  The role of selfish genetic elements in eukaryotic evolution. Nat. Rev. Genet. 2: 597–606. [DOI] [PubMed] [Google Scholar]
  25. Islam S., Malik A. I., Islam A. K., Colmer T. D., 2007.  Salt tolerance in a Hordeum marinum-Triticum aestivum amphiploid, and its parents. J. Exp. Bot. 58: 1219–1229. [DOI] [PubMed] [Google Scholar]
  26. Jaenike J., 2001.  Sex chromosome meiotic drive. Annu. Rev. Ecol. Syst. 32: 25–49. [Google Scholar]
  27. Jan C. C., Chandler J. M., 1989.  Sunflower interspecific hybrids and amphiploids of Helianthus annuus × H. bolanderi. Crop Sci. 29: 643–646. [Google Scholar]
  28. Jan C. C., Fernandez-Martinez J. M., 2002.  Interspecific hybridization, gene transfer, and the development of resistance to the broomrape race F in Spain. Helia 25: 123–126. [Google Scholar]
  29. Jan C. C., Vick B. A., 2007.  Inheritance and allelic relationships of fertility restoration genes for seven new sources of male-sterile cytoplasm in sunflower. Plant Breed. 126: 213–217. [Google Scholar]
  30. Jan C. C., Vick B. A., Miller J. F., Kahler A. L., Butler E. T., III, 1998.  Construction of an RFLP linkage map for cultivated sunflower. Theor. Appl. Genet. 96: 15–22. [Google Scholar]
  31. Jan C. C., Feng J., Seiler G. J., Gulya T. J., 2006.  Amphiploids of perennial Helianthus species × cultivated sunflower possess valuable genes for resistance to Sclerotinia stem and head rot. 28th Sunflower Research Workshop, January 11–12, Fargo, ND. Available at: www.sunflowernsa.com /research/research-workshop/documents /Jan_Amphiploids_06.pdf
  32. Jiang J., Morris K. L., Gill B. S., 1994.  Introgression of Elymus trachycaulus chromatin into common wheat. Chromosome Res. 2: 3–13. [DOI] [PubMed] [Google Scholar]
  33. Kang H., Wang Y., Fedak G., Cao W., Zhang H., et al. , 2011.  Introgression of chromosome 3Ns from Psathyrostachys huashanica into wheat specifying resistance to stripe rust. PLoS ONE 6: e21802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kumar S., Gill B. S., Faris J. D., 2007.  Identification and characterization of segregation distortion loci along chromosome 5B in tetraploid wheat. Mol. Genet. Genomics 278: 187–196. [DOI] [PubMed] [Google Scholar]
  35. Kumar S., Friebe B., Gill B. S., 2010.  Fate of Aegilops speltoides-derived, repetitive DNA sequences in diploid Aegilops species, wheat-Aegilops amphiploids and derived chromosome addition lines. Cytogenet. Genome Res. 129: 47–54. [DOI] [PubMed] [Google Scholar]
  36. Kusterer B., Horn R., Friedt W., 2005.  Molecular mapping of the fertility restoration locus Rf1 in sunflower and development of diagnostic markers for the restorer gene. Euphytica 143: 35–42. [Google Scholar]
  37. Kwiatek M., Błaszczyk L., Wiśniewska H., Apolinarska B., 2012.  Aegilops-Secale amphiploids: chromosome categorization, pollen viability and identification of fungal disease resistance genes. J. Appl. Genet. 53: 37–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ky C. L., Barre P., Lorieux M., Trouslot P., Akaffou S., et al. , 2000.  Interspecific genetic linkage map, segregation distortion and genetic conversion in coffee (Coffea sp.). Theor. Appl. Genet. 101: 669–676. [Google Scholar]
  39. Lander E. S., Green P., Abrahamson J., Barlow A., Daly M. J., et al. , 1987.  MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174–181. [DOI] [PubMed] [Google Scholar]
  40. Leclercq P., 1969.  Une stérilité male cytoplasmic chez le tournesol. Ann. Amélior Plant 19: 99–106. [Google Scholar]
  41. Li H., Kilian A., Zhou M., Wenzl P., Huttner E., et al. , 2010.  Construction of a high-density composite map and comparative mapping of segregation distortion regions in barley. Mol. Genet. Genomics 284: 319–331. [DOI] [PubMed] [Google Scholar]
  42. Liu F., Cui X., Horner H. T., Weiner H., Schnable P. S., 2001.  Mitochondrial aldehyde dehydrogenase activity is required for male fertility in maize. Plant Cell 13: 1063–1078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Liu X., Guo L., You J., Liu X., He Y., et al. , 2010.  Progress of segregation distortion in genetic mapping of plants. Res. J. Agronomy 4: 78–83. [Google Scholar]
  44. Liu Z., Li D. Y., Zhang X. Y., 2007.  Genetic relationships among five basic genomes St, E, A, B and D in Triticeae revealed by genomic Southern and in situ hybridization. J. Integr. Plant Biol. 49: 1080–1086. [Google Scholar]
  45. Liu Z., Mulpuri S., Feng J., Vick B. A., Jan C. C., 2012.  Molecular mapping of the Rf3 fertility restoration gene to facilitate its utilization in breeding confection sunflower. Mol. Breed. 29: 275–284. [Google Scholar]
  46. Lyttle T. W., 1991.  Segregation distorters. Annu. Rev. Genet. 25: 511–557. [DOI] [PubMed] [Google Scholar]
  47. Malik A. I., Islam A. K., Colmer T. D., 2011.  Transfer of the barrier to radial oxygen loss in roots of Hordeum marinum to wheat (Triticum aestivum): evaluation of four H. marinum-wheat amphiploids. New Phytol. 190: 499–508. [DOI] [PubMed] [Google Scholar]
  48. Mangelsdor P. C., Jones D. F., 1926.  The expression of Mendelian factors in the gametophyte of maize. Genetics 11: 423–455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Martín A., Cabrera A., Esteban E., Hernández P., Ramírez M. C., et al. , 1999.  A fertile amphiploid between diploid wheat (Triticum tauschii) and crested wheat grass (Agropyron cristatum). Genome 42: 519–524. [PubMed] [Google Scholar]
  50. McCouch S. R., Kochert G., Yu Z., Wang Z., Khush G. S., et al. , 1988.  Molecular mapping of rice chromosomes. Theor. Appl. Genet. 76: 815–829. [DOI] [PubMed] [Google Scholar]
  51. Michelmore R. W., Paran I., Kesseli R. V., 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 populations. Proc. Natl. Acad. Sci. USA 88: 9828–9832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Miller J. F. Fick G. N., 1997.  Genetics of sunflower, pp. 441–495 in Sunflower Technology and Production, Agronomy Monograph 35, edited by A. A. Schneiter. ASA, CSSA, and SSSA, Madison, WI. [Google Scholar]
  53. Nasrallah M. E., Yogeeswaran K., Snyder S., Nasrallah J. B., 2000.  Arabidopsis species hybrids in the study of species differences and evolution of amphiploidy in plants. Plant Physiol. 124: 1605–1614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Pérez-Vich B., Akhtouch B., Munoz-Ruz J., Fernandez-Martinez J. M., Jan C. C., 2002.  Inheritance of resistance to a highly virulent race F of Orobanche cumana Wallr. in a sunflower line derived from interspecific amphiploids. Helia 25: 137–143. [Google Scholar]
  55. Paterson A. H., Lander E. S., Hewitt J. D., Peterson S., Lincoln S. E., et al. , 1988.  Resolution of quantitative traits into Mendelian factors by using a complete linkage map of restriction fragment length polymorphisms. Nature 335: 721–726. [DOI] [PubMed] [Google Scholar]
  56. Poggio L., Gonzalez G., Confalonieri V., Comas C., Naranjo C. A., 2005.  The genome organization and diversification of maize and its allied species revisited: evidences from classical and FISH-GISH cytogenetic analysis. Cytogenet. Genome Res. 109: 259–267. [DOI] [PubMed] [Google Scholar]
  57. Qi L. L., Seiler G. J., Vick B. A., Gulya T. J., 2012.  Genetics and mapping of the R11 gene conferring resistance to recently emerged rust races, tightly linked to male fertility restoration, in sunflower (Helianthus annuus L.). Theor. Appl. Genet. 125: 921–932. [DOI] [PubMed] [Google Scholar]
  58. Schnabel U., Engelmann U., Horn R., 2008.  Development of markers for the use of the PEF1 cytoplasm in sunflower hybrid breeding. Plant Breed. 127: 587–591. [Google Scholar]
  59. Seiler G. J., 2000.  Registration of ten interspecific germplasms derived from wild perennial sunflower. Crop Sci. 40: 587–588. [Google Scholar]
  60. Serieys H., 1996.  Identification, study and utilization in breeding programs of new CMS sources. FAO Progress Report. Helia 19: 144–158. [Google Scholar]
  61. Serieys H., 2005.  Identification, study and utilization in breeding programs of new CMS sources, pp. 47–53 in Proceedings of the 2005 Sunflower Subnetwork Progress Report. FAO Subnetwork, Rome.
  62. Soliman M. H., Rubiales D., Cabrera A., 2001.  A fertile amphiploid between durum wheat (Triticum turgidum) and the × Agroticum amphiploid (Agropyron cristatum × T. tauschii). Hereditas 135: 183–186. [DOI] [PubMed] [Google Scholar]
  63. Tang S., Kishore V. K., Knapp S. J., 2003.  PCR-multiplexes for a genome-wide framework of simple sequence repeat marker loci in cultivated sunflower. Theor. Appl. Genet. 107: 6–19. [DOI] [PubMed] [Google Scholar]
  64. Wang D.-X., Cui L.-J., Jan C. C., 2007.  Establishment of new cytoplasmic male sterility by introduction of cytoplasm from wild species in sunflower. Chinese J. Oil Sci. 29: 416–419. [Google Scholar]
  65. Wang Z., Zou Y., Li X., Zhang Q., Chen L., et al. , 2006.  Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing. Plant Cell 18: 676–687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wieckhorst S., Bachlava E., Dußle C. M., Tang S., Gao W., et al. , 2010.  Fine mapping of the sunflower resistance locus PlARG introduced from the wild species Helianthus argophyllus. Theor. Appl. Genet. 121: 1633–1644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Wojciechowska B., Pudelska H., 2005.  Production and characterization of amphiploids of Aegilops kotschyi and Ae. biuncialis with Secale cereale, and of backcross hybrids of Ae. biuncialis × S. cereale amphiploids with 2× and 4× S. cereale. J. Appl. Genet. 46: 157–161. [PubMed] [Google Scholar]
  68. Yu J. K., Tang S., Slabaugh M. B., Heesacker A., Cole G., et al. , 2003.  Towards a saturated molecular genetic linkage map for cultivated sunflower. Crop Sci. 43: 367–387. [Google Scholar]
  69. Yue B., Vick B. A., Cai X., Hu J., 2010.  Genetic mapping for the Rf1 (fertility restoration) gene in sunflower (Helianthus annuus L.) by SSR and TRAP markers. Plant Breed. 129: 24–28. [Google Scholar]
  70. Zhang H. B., Dvorák J., 1990.  Characterization and distribution of an interspersed repeated nucleotide sequence from Lophopyrum elongatum and mapping of a segregation-distortion factor with it. Genome 33: 927–936. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information
supp_193_3_727__index.html (2.1KB, html)
26a72330213ab672be6446e9bad5a50b_genetics.112.146092-1.pdf (220.8KB, pdf)
5f8aed73c19fa196684a465de09a349f_genetics.112.146092-6.pdf (58.9KB, pdf)
c7b366e6a24ad5c72608e56e65ad4520_genetics.112.146092-4.pdf (91.1KB, pdf)
fa6e58e349c6efe50d4fea6cfd282242_genetics.112.146092-3.pdf (70.7KB, pdf)
8fbaefde3b0cf1bc29382d603c62f99b_genetics.112.146092-2.pdf (58.8KB, pdf)
d7b1f060819e8e9a47baf9dca84b74ca_genetics.112.146092-5.xls (37.5KB, xls)

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES