Published in Crop Sci. 43:1747-1753 (2003).
© 2003 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
CROP BREEDING, GENETICS & CYTOLOGY
Conversion of Fertility Restoration of the Sorghum IS1112C (A3) Male-Sterile Cytoplasm from Two Genes to One Gene
H. V. Tang and
D. R. Pring*
USDA-ARS, Crop Genetics and Environment Unit, Department of Plant Pathology, 1453 Fifield Hall, University of Florida, Gainesville, FL 32611 USA
* Corresponding author (drpg{at}mail.ifas.ufl.edu).
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ABSTRACT
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The restoration of male fertility in sorghum [Sorghum bicolor (L.) Moench] lines carrying the IS1112C male-sterile cytoplasm is characterized by limited seed set. Restoration requires complementary gametophytic action of two restoring alleles, Rf3 and Rf4, for individual gamete viability, and an F1 heterozygous for the two restoring loci is expected to exhibit 25% viable pollen. The objective of this study was to demonstrate the feasibility of converting restoration to single gene behavior. Single-seed descent B3Tx398/IS1112C F5:6 lines were used to generate homozygosity at the rf3 and rf4 loci, and critical segregants were identified by progeny tests, assays for action of the Rf3 allele, and genomic markers for the rf4 locus. Using these criteria, we constructed the genotypes Rf3Rf3rf4rf4 and rf3rf3Rf4Rf4 in normal, male-fertile cytoplasm lines, and in IS1112C male-sterile cytoplasm lines. Pollination of either male-sterile line with a matching male-fertile line resulted in about 25% pollen staining in the F1, demonstrating complementation of the two restoring loci. Pollination of either male-sterile line with IS1112C, Rf3Rf3Rf4Rf4, resulted in about 50% pollen staining. These characteristics substantiate complementary action of the restoring alleles, and are consistent with successful conversion to a single gene fertility restoration system.
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INTRODUCTION
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THE PRODUCTION OF hybrid sorghum seed is dependent on the utilization of cytoplasmic-nuclear male sterility (CMS). The widely used current source of CMS in sorghum is derived from milo (Schertz et al., 1989; Stephans and Holland, 1954), and is assigned to the A1, or milo group of male sterility-inducing cytoplasms (Pring et al., 1995; Schertz et al., 1989, 1997; Xu et al., 1995). Fertility restoration in lines carrying this cytoplasm is exacted through a mechanism requiring complementary action of two genes; Schertz et al. (1989) surveyed a number of sorghum lines and established that one or two major genes are required for full restoration. The first of these genes to be mapped has been assigned the designation rf1, and the locus is located on linkage group H (Klein et al., 2001).
Many sources of CMS in sorghum have been characterized in terms of differential fertility restoration (Schertz et al., 1989, 1997), and categorized into seven groups (Pring et al., 1995; Xu et al., 1995). Fertility restoration of lines carrying the IS1112C male-sterile cytoplasm (A3 group; Schertz et al., 1989) is problematic in that male-sterile lines crossed with the parental, fertile line IS1112C typically result in an F1 with approximately 50% seed set (Worstell et al., 1984; our unpublished observations).
The elucidation of the biology and genetics of this system (Pring et al., 1999; Tang et al., 1998) revealed the probable basis for this limited seed set. The IS1112C male-sterile cytoplasm is a gametophytic fertility restoration system requiring complementary action of two genes for individual pollen viability. The restoring alleles have been designated Rf3 and Rf4. An F1 heterozygous for the two restoring alleles yields 25% viable pollen (Tang et al., 1998) and exhibits reduced seed set, precluding utilization of the IS1112C male-sterile cytoplasm as an alternative source for hybrid production.
A chimeric mitochondrial open reading frame, orf107, is associated with the expression of CMS in the IS1112C cytoplasm (Tang et al., 1996b, 1998, 1999). Aberrations in transcriptional characteristics of orf107 are associated with fertility restoration; an enhanced transcript processing activity (TPA) is detected in leaves or pollen of lines restored to fertility, resulting in cleavage of about 70% of whole-length transcripts, within orf107, thus precluding abundant transcripts for potential translation. The enhanced TPA has tentatively been assigned to the restoring allele Rf3. In IS1112C, Rf3 is tightly linked to the single dominant allele Mmt1, which conditions enhanced TPA 5' to sorghum mitochondrial urf209 (Tang et al., 1996a, 1998; Pring et al., 1999). A possible mode of action of Rf4 has not been identified, but the locus has been assigned to linkage group E and PCR-based molecular markers for the locus have been developed (Wen et al., 2002).
The objective of this study was to exploit these resources in an approach to converting this two-gene restoration system to one gene. We demonstrate the successful development of male-fertile and male-sterile lines carrying either of the two required alleles in a homozygous state, substantiate complementary gene action, and show successful single-gene fertility restoration patterns resulting in approximately 50% viable pollen.
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MATERIALS AND METHODS
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Genetic Lines
IS1112C (Rf3Rf3Rf4Rf4), the male-sterile line A3Tx398 (IS1112C cytoplasm, rf3rf3rf4rf4) and the maintainer line B3Tx398 (rf3rf3rf4rf4) were originally obtained from K.F. Schertz, USDA-ARS, College Station, TX. The A3Tx398 line has been backcrossed with B3Tx398 for at least 12 generations. Plants were grown in greenhouses or field plots near Gainesville, FL.
Crosses and Line Selections
The line B3Tx398 was emasculated and pollinated with IS1112C in 1996, and its progeny were driven to homozygosity by single seed descent (SSD). At the F5, SSD single plants were scored for presence of Mmt1 or mmt1 by transcript analyses. The F5:6, harvested F6 seeds from selfed F5 plants, were used as male parent sources for subsequent crosses with A3Tx398. Linkage of Mmt1 and Rf3 allowed identification of lines carrying Mmt1 as candidates for an Rf3Rf3rf4rf4 line, which we designated (B3)FL3. Conversely, lines carrying mmt1 represented candidates for a rf3rf3Rf4Rf4 line, which we designated (B3)FL4. The genotypes of these candidates were verified in a series of crosses in which progeny were scored for segregation of iodine pollen staining ability, seed set and the presence or absence of the rf4-linked flanking markers LW8 and LW9, or a combination of all of the above criteria. Each of these two developed lines became a maintainer for its corresponding sterile line and a tester for the reciprocal line. For example, (B3)FL3 was the maintainer for a male-sterile line carrying Rf3Rf3rf4rf4, which we designated (A3)FL3, and a tester for the development of a male-sterile line carrying rf3rf3Rf4Rf4, which we designated (A3)FL4.
Progeny of many of the crosses or lines tillered readily, allowing multiple pollinations of the same plants. Mature panicles were collected, and the plants were cut back and subsequently pollinated with other lines for additional test crosses.
Transcript Analyses
The Rf3-associated enhanced transcript processing activity (TPA) conferred within the IS1112C-specific mitochondrial open reading frame orf107 was assessed by transcript analyses of progeny carrying the IS1112C cytoplasm, as previously described (Tang et al., 1996b). The single dominant allele Mmt1, which confers enhanced TPA at a site 5' to sorghum mitochondrial orf209, is tightly linked to Rf3 in IS1112C (Tang et al., 1998), and was assayed as described by Tang et al. (1996a). The line B3Tx398 is mmt1mmt1, thus B3Tx398/IS1112C progeny exhibiting Mmt1-enhanced TPA were considered to carry Rf3. Probes and hybridization conditions for identifying the altered transcripts of orf107 and urf209 were as previously described (Tang et al., 1996a, b).
DNA Marker Analyses
Two codominant markers (Wen et al., 2002) spanning the rf4 locus were utilized to examine lines for homozygosity. The marker LW9, a sequence tagged site (STS) marker located 0.74 centimorgans (cM) from the rf4 locus, is displayed as a 366-base pair (bp) fragment in IS1112C and a 358-bp fragment in B3Tx398. Marker LW8, a cleavable amplified polymorphic sequence (CAPS) marker located 3.18 cM from the rf4 locus, is 275 bp in IS1112C and 310 bp in B3Tx398. PCR conditions and agarose gel electrophoresis were as described (Wen et al., 2002).
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RESULTS
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The two fertility restoration genes Rf3 and Rf4 exhibit a gametophytic mode of action and act in a complementary manner wherein both alleles must be present for individual gamete viability in plants carrying the IS1112C cytoplasm (Tang et al., 1998; Pring et al., 1999). Viable gametes transmit both restoring alleles to subsequent generations. The gametophytic nature of fertility restoration thus renders them functionally linked. As such, it is not possible to obtain independent segregation of the two restoring alleles in plants carrying the IS1112C cytoplasm.
To facilitate segregation of Rf3 and Rf4 in a normal, male-fertile cytoplasm, B3Tx398/IS1112C progeny were advanced to the F5 by SSD. A total of 40 F5 plants were scored indirectly for Rf3 by scoring for action of the tightly linked Mmt1. Direct scoring for Rf3 by transcript analyses is not possible because the normal cytoplasm of BTx398 does not harbor orf107. Eleven of 40 plants studied were identified as carrying Mmt1 (Fig. 1), and, thus, carry Rf3, while 29 plants were mmt1mmt1 and assumed to be rf3rf3.
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Fig. 1. Transcript analysis of segregants for Mmt1 among B3Tx398/IS1112C F5 progeny. Action of Mmt1 results in enhanced transcript processing activity 5' to urf209, generating a 832 nt transcript from the progenitor 1044 nt transcript. Segregants B, C, E, F, and I represent genotypes Mmt1Mmt1 or Mmt1mmt1, while A, D, G, H, J, K, and L are mmt1mmt1.
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Development of (B3)FL3 [genotype (N)Rf3Rf3rf4rf4]
Ten of the 11 F5:6 lines identified as Mmt1 were crossed as male with A3Tx398 to identify lines that were Rf3Rf3rf4rf4, which would produce sterile F1 progeny (Fig. 2). Four of the lines produced sterile progeny; the remaining six lines included fertile plants among the progeny, indicating presence of Rf4, and were disregarded. Among the four candidate lines, the line 99-60-4 was designated as (B3)FL3. The A3Tx398/99-60-4 F1 was examined for pollen staining with iodine and progeny were grown and selfed in the greenhouse and in the field in two consecutive summers. Among a total of 39 progeny from selfed panicles grown in the field (22 in 2000, 17 in 2001), two plants set seed with only one seed/panicle. Although most of the pollen grains were clearly not stainable with iodine, occasionally a few grains (at an estimated frequency of 10-4 or less) exhibited a trace of starch deposition. This observation may explain the occurrence of extremely low number of seed set in the selfed individuals. Two additional identified lines were field-tested in two summers and two seed were obtained from 32 selfed panicles. Homozygosity at the rf3 locus was confirmed by pollination of the (A3)FL4 lines (see below).
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Fig. 2. Strategy for selecting lines (B3)FL3 (Rf3Rf3rf4rf4) and (B3)FL4 (rf3rf3Rf4Rf4). B3Tx398/IS1112C F5:6 individuals were scored for Mmt1, tightly linked to Rf3. Rf3rf3 or Rf3Rf3 individuals were used to pollinate A3Tx398 and resulting sterile plants identified the Rf3Rf3rf4rf4 lines, designated (B3)FL3. Segregants that were mmt1mmt1, and thus rf3rf3, were used to pollinate A3Tx398 to verify the genotype. The line A3Tx398/(B3)FL3 (Rf3rf3rf4rf4) was pollinated with the candidate lines; progeny that yielded only sterile F1s (Rf3rf3rf4rf4) identified rf4rf4 lines, while progeny that segregated 1 fertile:1 sterile identified the rf3rf3Rf4Rf4 lines, designated (B3)FL4.
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Development of (B3)FL4 [genotype (N)rf3rf3Rf4Rf4]
The 29 mmt1mmt1 lines from the F6 population described above carry the genotypes rf3rf3Rf4Rf4 or rf3rf3rf4rf4; we sought to identify lines carrying the former genotype. Two phases of progeny testing were conducted to identify the rf3rf3Rf4Rf4 lines (Fig. 2). We first verified the presumed genotypes by pollinating A3Tx398 and examining the progeny, which were expected to carry nonstaining pollen and exhibit male sterility. In the second test, the candidate lines were crossed as male with A3Tx398/(B3)FL3 (Rf3rf3rf4rf4), which is sterile. This test cross served two objectives: (i) to identify F6 lines with the Rf4Rf4 genotype and (ii) to demonstrate complementation of the restoring alleles through reconstitution from independently derived lines in a normal, male-fertile cytoplasm. F6 lines of genotype (N)rf3rf3rf4rf4 were expected to produce only sterile progeny. Lines which are rf3rf3Rf4Rf4, however, were expected to produce both restored and sterile progeny in a 1:1 ratio.
Nine candidate individuals were utilized for crossing and progeny testing and were genotyped with the LW8 and LW9 markers, which flank the rf4 locus (Wen et al., 2002). Eight of the nine plants carried the IS1112C-specific codominant LW9 (Fig. 3) and LW8 (not shown) markers, indicating homozygosity at the rf4 locus. The remaining plant, designated as S01-16 carried only the B3Tx398-specific alleles. Pollen staining of greenhouse-grown F1s resulting from pollination of A3Tx398 with the nine plants indicated that they were all sterile. The eight rf3rf3Rf4Rf4 candidate plants were crossed as male to A3Tx398/(B3)FL3 and the F1 plants were examined in the greenhouse. In total, these progeny segregated 28 fertile:19 sterile. The fertile plants exhibited approximately 25% iodine-stained pollen, consistent with the presumed Rf3rf3Rf4rf4 genotype. We concluded that these eight candidate plants had the genotype (N)rf3rf3Rf4Rf4. In contrast, nine F1 progeny were derived from the candidate plant S01-16 and all were sterile. Thus, this plant did not have the functional Rf4 allele, consistent with data obtained by scoring for flanking markers LW8 and LW9, and the inferred genotype for S01-16 is (N)rf3rf3rf4rf4.
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Fig. 3. Identification of Rf4Rf4 lines among rf3rf3 F5:6 lines crossed as male with A3Tx398/(B3)FL3. The codominant STS marker LW9 is 366 bp in IS1112C (A) and five segregants (C-G), while B3Tx398 (B) exhibits a 358-bp fragment.
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One of the eight lines, designated S00-12, was used to pollinate A3Tx398 and progeny were tested in the field in two years; no seed was observed among 44 selfed plants. Thus, in the absence of the Rf3 allele, the Rf4Rf4 lines produced completely sterile progeny.
Development of (A3)FL3 [genotype (A3)Rf3Rf3rf4rf4]
To develop an (A3)Rf3Rf3rf4rf4 line (Fig. 4), plants identified as Mmt1 from the F5:6 were used to pollinate A3Tx398. Sterile progeny (Rf3rf3rf4rf4) were pollinated with (B3)FL3, resulting in sterile plants with an expected 1:1 ratio of Rf3Rf3rf4rf4:Rf3rf3rf4rf4. These progeny were evaluated in two test crosses to identify the desired Rf3Rf3rf4rf4 genotype. Progeny were pollinated with (B3)FL4, which was expected to complement the genotype (A3)Rf3Rf3rf4rf4 such that all progeny should be fertile, with 25% pollen staining (Rf3rf3Rf4rf4). In contrast, Rf3rf3rf4rf4 progeny pollinated with (B3)FL4 should segregate 1 fertile:1 sterile. Similarly, IS1112C as a male parent was expected to result in progeny with 50% pollen staining (Rf3Rf3Rf4rf4) if the female was Rf3Rf3rf4rf4, while pollination of Rf3rf3rf4rf4 lines would result in 1:1 segregation exhibiting 50% (Rf3Rf3Rf4rf4) and 25% (Rf3rf3Rf4rf4) stained pollen. Fourteen A3Tx398/Rf3Rf3 F5:6//(B3)FL3 plants were each pollinated with (B3)FL4 and IS1112C, by exploiting tillering of this line. Mature panicles from a cross were removed, and tillers were subsequently pollinated with other male parents.
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Fig. 4. Strategy for selecting (A3)FL3 (Rf3Rf3rf4rf4). A3Tx398 was pollinated with Mmt1 lines identified from the B3Tx398/IS1112C F5:6 population. Fertile plants were disregarded. Sterile lines were pollinated with (B3)FL3, backcrossed with (B3)FL3, then pollinated with (B3)FL4. Progeny which were all fertile identified the female (A3)FL3.
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Fourteen A3Tx398/Rf3Rf3 F5:6//(B3)FL3/3/(B3)FL4 families were evaluated for fertility in a greenhouse. Ten families exhibited only fertile plants, with 123 plants scored, while four families segregated 30 fertile:17 male-sterile plants, and were disregarded. Progeny from one of the former, an (A3)FL3 candidate line, were examined for pollen stainability and seed set in field plots. We observed a mean of 21% stained pollen and 75% seed set (Fig. 5, Table 1). These data are consistent with complementation of the Rf3 and Rf4 restoring alleles in a Rf3rf3Rf4rf4 heterozygote, with pollen stainability as previously observed (Tang et al., 1998; Pring et al., 1999).
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Fig. 5. Iodine staining of pollen from line (A3)Rf3Rf3rf4r4 pollinated with A) (B3)FL4 (rf3rf3Rf4Rf4) and B) IS1112C (Rf3Rf3Rf4Rf4), resulting in approximately 25 and 50% staining, respectively. Note partially filled grains in each line, as previously described (Tang et al., 1998).
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Table 1. Pollen staining and field plot seed set of F1 progeny of male-sterile line (A3)FL3 (Rf3Rf3rf4rf4) pollinated with (B3)FL4 [(N)rf3rf3Rf4Rf4], IS1112C (Rf3Rf3Rf4Rf4) and (B3)FL3 [(N)Rf3Rf3rf4rf4]. Seed set was estimated in 10% increments.
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Progeny from the same candidate line pollinated with IS1112C were similarly examined for pollen stainability and percentage seed set. We observed a mean of 37% stained pollen and 96% seed set (Fig. 5, Table 1), consistent with conversion to a one-gene fertility restoration system.
The identified (A3)FL3 line was also pollinated with (B3)FL3 and B3Tx398 to substantiate the presumed genotypes. Six (A3)FL3/(B3)FL3 plants were male-sterile (Table 1), and 11 (A3)FL3/B3Tx398 plants were examined for action of Rf3; each exhibited enhanced TPA (not shown). These observations are consistent with the assumed genotype of (A3)FL3.
Development of (A3)FL4 [genotype (A3)rf3rf3Rf4Rf4]
The (A3)rf3rf3Rf4Rf4 line (Fig. 6) was constructed by pollinating A3Tx398 with (B3)FL4, then backcrossing with (B3)FL4, generating the Genotypes 1 (A3)rf3rf3Rf4Rf4:1 (A3)rf3rf3Rf4rf4. The desirable genotype (A3)rf3rf3Rf4Rf4 was identified by presence of only the IS1112C-specific STS marker LW9, as shown for the selection of (B3)FL4 (Fig. 3), and LW8 (not shown). The authenticity of the lines was verified by pollination with (B3)FL3 and IS1112C, and examination of pollen staining in progeny. These lines should be associated with 25% pollen staining when pollinated with (B3)FL3 and 50% pollen staining when pollinated with IS1112C.
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Fig. 6. Strategy for selecting (A3)FL4 (rf3rf3Rf4Rf4). A3Tx398 was pollinated with (B3)FL4, backcrossed with (B3)FL4, and progeny were examined for presence of the rf4-linked codominant markers LW8 and LW9. Selected females were pollinated with (B3)FL3; the F1s were fertile with 25% iodine-stained pollen, identifying the line as (A3)FL4.
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Among 20 backcross plants screened for the presence of the LW8 and LW9 markers, 14 were identified as homozygous for Rf4, five were heterozygous, and one was heterozygous for LW8 but homozygous for LW9. Fourteen homozygous plants were subjected to test crosses to verify the assumed genotype, and three were randomly selected for examination of pollen stainability and percentage seed set in field plots (Table 2). The three candidate rf3rf3Rf4Rf4 individuals were pollinated with (B3)FL3 and IS1112C.
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Table 2. Pollen staining and field plot seed set among F1 progeny of male-sterile (A3)FL4 [(A3)rf3rf3Rf4Rf4] candidate lines pollinated with (B3)FL3 [(N)Rf3Rf3rf4rf4)] and IS1112C (Rf3Rf3Rf4Rf4). Candidate (A3)FL4 lines 2, 3, and 5 were pollinated with (B3)FL3, seed was collected, and panicles on subsequent tillers were pollinated with IS1112C. Seed set was estimated in 10% increments.
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Progeny resulting from pollination with (B3)FL3 are expected to be heterozygous Rf3rf3Rf4rf4 and exhibit 25% pollen staining. We observed a mean of 23% stainable pollen and 74% seed set (Table 2). These data are consistent with previous observations of heterozygotes (Tang et al., 1998), with the exception of one plant that set no seed. The staining and seed data are also consistent with the assumption that (B3)FL3 is indeed Rf3Rf3. If this line was heterozygous, 50% of progeny would be sterile. Pollination with IS1112C should result in progeny heterozygous at the rf3 locus and homozygous at the rf4 locus, i.e., exhibiting single-gene restoration characteristics. We observed a mean of 44% stained pollen and 94% seed set with three (A3)FL4 candidates (Table 2). These data are consistent with single gene restoration behavior.
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DISCUSSION
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The development of alternative sources of CMS for sorghum seed production is desirable as part of a strategic goal of expanding cytoplasmic diversity in hybrids. Minimizing cytoplasmic uniformity is potentially important in consideration of the demonstrated disease toxin and insecticide sensitivity of hybrid maize produced in the Texas male-sterile cytoplasm (reviewed by Wise et al., 1999). Among the major seven groups of sorghum male-sterile cytoplasms, the milo, or A1, source of CMS in sorghum (Stephans and Holland, 1954) has been extensively utilized since 1956 without apparent complications, and the A2 source is being utilized in some applications (Schertz et al., 1997).
The IS1112C source of CMS does not seem to be adaptable for utilization in hybrid production, on the basis of the approximate 50% seed set in the F1 (Worstell et al., 1984; our observations). The description of fertility restoration of the IS1112C male-sterile cytoplasm as a two-gene gametophytic system was based on analyses of segregants wherein BC1F1 or BC3F1 populations were emasculated and pollinated with the maintainer line (Tang et al., 1998; Pring et al., 1999). In these analyses, presence of the Rf3 allele was assessed by transcript analysis and resultant fertility examinations. Lines which were Rf3 and sterile were assumed to rf4rf4, and lines which were rf3 and sterile were assumed to be Rf4rf4 or rf4rf4, by deduction.
We have developed male-sterile lines and normal cytoplasm, male-fertile lines carrying the genotypes Rf3Rf3rf4rf4 and rf3rf3Rf4Rf4, demonstrated complementation of the restoring alleles, and provide evidence of single-gene fertility restoration of either male-sterile line by the homozygous Rf3Rf3Rf4Rf4 line IS1112C. Individuals homozygous at the rf3 and rf4 loci were successfully recovered from the F5:6 SSD lines and utilized to generate specific genotypes, utilizing transcript analyses for the Rf3 and Mmt1-associated TPA (Tang et al., 1996a, 1996b, 1998) and the rf4-linked STS/CAPS markers LW8 and LW9 (Wen et al., 2002). The unique biology of this system, wherein turgid inviable pollen grains are retained in anthers at exsertion in male-sterile plants, and in plants carrying 25 or 50% stained viable pollen, allowed direct inspection of meiotic products to confirm pollen viability predictions.
Construction of the (A3)FL3(Rf3Rf3rf4rf4) and (A3)FL4 (rf3rf3Rf4Rf4) lines and analogous (N)FL3 (Rf3Rf3rf4rf4) and (N)FL4 (rf3rf3Rf4Rf4) lines permitted a reciprocal complementation test. The restoration of pollen staining capability and of seed set in crosses made in either direction, (A3)FL4/(N)FL3 or (A3)FL3/(N)FL4, is consistent with pollen staining and seed set of heterozygous Rf3rf3Rf4rf4 plants, and with the two-gene gametophytic model (Pring et al., 1999; Tang et al., 1998). Pollination of either sterile line with IS1112C results in approximately 50% pollen staining, predicted by the two-gene model.
The infrequent occurrence of fertility restoration of the IS1112C male-sterile cytoplasm among sorghum germplasm examined to date is reflected in the large number of lines identified as maintainers (Pedersen and Toy, 1990; Tang et al., 1998; Schertz et al., 1989; Worstell et al., 1984). Restoration capability for A3Tx398 has been observed in progeny resulting from bulked pollinations with eight sudan grass populations (Pedersen and Toy, 1990). We similarly have recovered restoration capability (unpublished data) in pollen bulks of NP28 and NP35 sudan grass (Gorz et al., 1990a, 1990b), but have not yet characterized the genetics of restoration. Since most currently examined sorghum lines are maintainers, they are suitable for development of male-sterile lines. Introgression of either Rf3Rf3 or Rf4Rf4 into these lines, and of Rf3Rf3Rf4Rf4 into pollinator lines, would provide single-gene fertility restoration.
There are current barriers in introgression of the restoring loci into agronomically important lines. The IS1112C-derived LW8 CAPS marker is polymorphic in B3Tx398, B3Tx623, and IS3620C, but monomorphic in Tx7000 (Wen et al., 2002; unpublished data). The LW9 marker is monomorphic in B3Tx623, B3Tx7000, and IS3620C. Thus only B3Tx398 is polymorphic for both rf4 flanking markers. Surveys of diverse germplasm for markers linked to the rf4 locus have thus not been initiated; identification of markers potentially polymorphic in other lines, obtained by exon capture, is under development (Wen et al., 2002). Additionally B3Tx623, B3Tx7000, IS3620C, and several other lines, are rf3rf3 but carry Mmt1 (Tang et al., 1998; unpublished data), and thus urf209 TPA cannot be utilized to identify Rf3 in these lines. Markers for the rf3 locus are under development; two AFLP markers have been identified but have not been adequately characterized. Thus, B3Tx398 is the only currently known line that is mmt1mmt1, which allows an assay for the apparent presence of the tightly linked Rf3 in normal cytoplasm, male-fertile lines.
Single-gene gametophytic fertility restoration systems have the disadvantage of requiring homozygosity of the restoring locus in restorer lines to generate a heterozygous F1, which is associated with 50% pollen viability. Such restoration systems have been used, such as maize S male-sterile cytoplasm (Gabay-Laughnan et al., 1995), or apparently are presently utilized, such as the Chinsurah boro II male-sterile cytoplasm of rice, Oryza sativa L. (Ichikawa et al., 1997). Conversion of the IS1112C CMS fertility restoration system to a single gene would require homozygosity at both the rf3 and rf4 loci in restoring lines. The selection of homozygous fertile Rf3Rf3Rf4Rf4 lines can be easily accomplished in lines carrying the IS1112C cytoplasm by selection of F2 individuals resulting from pollination of lines in A3 cytoplasm with the Rf3Rf3Rf4Rf4 IS1112C. We have selected such lines in a BC7F2 in the Tx398 and Tx7000 backgrounds by identification of individuals with 100% pollen staining (unpublished data).
The (A3)FL3 and (A3)FL4 lines can be used in surveys to detect the complementary restoring alleles among important germplasm by pollination of these male-sterile lines and fertility observations of F1s. To date we have not detected Rf3 among a limited set of sorghum lines, as detected by transcript analyses of F1s (Tang et al., 1996b). The distribution of Rf4 in these materials is unknown, but the high frequency of maintainer lines for the IS1112C cytoplasm clearly indicates that the presence of both restorers is rare.
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ACKNOWLEDGMENTS
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This manuscript is dedicated in memory of Dr. K.F. Schertz, USDA-ARS, College Station, TX, who provided the impetus for this work and developed critical data and germplasm for this project. We thank Drs. C.D. Chase, W.L. Rooney, and R.P. Wise for critical reviews, and acknowledge the technical assistance of Yen Dao and Theresa Cao. Supported in part by the U.S. Department of Agriculture-National Research Initiative Competitive Grants Program, 99-35300-7725. This was a cooperative investigation of the U.S. Department of Agriculture-Agricultural Research Service and the Institute of Food and Agricultural Sciences, University of Florida.
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NOTES
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Joint contribution of the USDA-ARS and the University of Florida Agric. Exp. Stn. Published as Journal series No. R-09082.
Received for publication October 15, 2002.
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