Conversion of Fertility Restoration of the Sorghum IS1112C (A3) Male-Sterile Cytoplasm from Two Genes to One Gene

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1 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* ABSTRACT line IS1112C typically result in an F 1 with approximately The restoration of male fertility in sorghum [Sorghum bicolor 50% seed set (Worstell et al., 1984; our unpublished ob- (L.) Moench] lines carrying the IS1112C male-sterile cytoplasm is servations). characterized by limited seed set. Restoration requires complemensystem The elucidation of the biology and genetics of this tary gametophytic action of two restoring alleles, Rf3 and Rf4, for (Pring et al., 1999; Tang et al., 1998) revealed the individual gamete viability, and an F 1 heterozygous for the two restor- probable basis for this limited seed set. The IS1112C ing loci is expected to exhibit 25% viable pollen. The objective of male-sterile cytoplasm is a gametophytic fertility restothis study was to demonstrate the feasibility of converting restoration ration system requiring complementary action of two to single gene behavior. Single-seed descent B3Tx398/IS1112C F 5:6 genes for individual pollen viability. The restoring allines were used to generate homozygosity at the rf3 and rf4 loci, and leles have been designated Rf3 and Rf4.AnF 1 heterozycritical segregants were identified by progeny tests, assays for action gous for the two restoring alleles yields 25% viable polof the Rf3 allele, and genomic markers for the rf4 locus. Using these criteria, we constructed the genotypes Rf3Rf3rf4rf4 and rf3rf3rf4rf4 len (Tang et al., 1998) and exhibits reduced seed set, in normal, male-fertile cytoplasm lines, and in IS1112C male-sterile precluding utilization of the IS1112C male-sterile cyto- cytoplasm lines. Pollination of either male-sterile line with a matching plasm as an alternative source for hybrid production. male-fertile line resulted in about 25% pollen staining in the F 1, demis A chimeric mitochondrial open reading frame, orf107, onstrating complementation of the two restoring loci. Pollination of associated with the expression of CMS in the IS1112C either male-sterile line with IS1112C, Rf3Rf3Rf4Rf4, resulted in cytoplasm (Tang et al., 1996b, 1998, 1999). Aberrations about 50% pollen staining. These characteristics substantiate comple- in transcriptional characteristics of orf107 are associated mentary action of the restoring alleles, and are consistent with success- with fertility restoration; an enhanced transcript proful conversion to a single gene fertility restoration system. cessing activity (TPA) is detected in leaves or pollen of lines restored to fertility, resulting in cleavage of about T 70% of whole-length transcripts, within orf107, thus prehe production of hybrid sorghum seed is depen- cluding abundant transcripts for potential translation. dent on the utilization of cytoplasmic-nuclear male The enhanced TPA has tentatively been assigned to the sterility (CMS). The widely used current source of CMS restoring allele Rf3. In IS1112C, Rf3 is tightly linked to in sorghum is derived from milo (Schertz et al., 1989; the single dominant allele Mmt1, which conditions en- Stephans and Holland, 1954), and is assigned to the hanced TPA 5 to sorghum mitochondrial urf209 (Tang A1, or milo group of male sterility-inducing cytoplasms et al., 1996a, 1998; Pring et al., 1999). A possible mode (Pring et al., 1995; Schertz et al., 1989, 1997; Xu et al., of action of Rf4 has not been identified, but the locus 1995). Fertility restoration in lines carrying this cyto- has been assigned to linkage group E and PCR-based plasm is exacted through a mechanism requiring com- molecular markers for the locus have been developed plementary action of two genes; Schertz et al. (1989) (Wen et al., 2002). surveyed a number of sorghum lines and established The objective of this study was to exploit these rethat one or two major genes are required for full restora- sources in an approach to converting this two-gene restion. The first of these genes to be mapped has been toration system to one gene. We demonstrate the sucassigned the designation rf1, and the locus is located on cessful development of male-fertile and male-sterile linkage group H (Klein et al., 2001). lines carrying either of the two required alleles in a homozygous state, substantiate complementary gene ac- Many sources of CMS in sorghum have been charaction, and show successful single-gene fertility restoration terized in terms of differential fertility restoration patterns resulting in approximately 50% viable pollen. (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 MATERIALS AND METHODS cytoplasm (A3 group; Schertz et al., 1989) is problematic Genetic Lines in that male-sterile lines crossed with the parental, fertile IS1112C (Rf3Rf3Rf4Rf4), the male-sterile line A3Tx398 (IS1112C cytoplasm, rf3rf3rf4rf4) and the maintainer line B3Tx398 (rf3rf3rf4rf4) were originally obtained from K.F. USDA-ARS, Crop Genetics and Environment Unit, Department of Schertz, USDA-ARS, College Station, TX. The A3Tx398 line Plant Pathology, 1453 Fifield Hall, University of Florida, Gainesville, FL USA. Joint contribution of the USDA-ARS and the Univer- has been backcrossed with B3Tx398 for at least 12 generations. sity of Florida Agric. Exp. Stn. Published as Journal series No. R Plants were grown in greenhouses or field plots near Gaines- Received 15 Oct *Corresponding author (drpg@mail.ifas.ufl. ville, FL. edu). Crosses and Line Selections Published in Crop Sci. 43: (2003). Crop Science Society of America The line B3Tx398 was emasculated and pollinated with 677 S. Segoe Rd., Madison, WI USA IS1112C in 1996, and its progeny were driven to homozygosity 1747

2 1748 CROP SCIENCE, VOL. 43, SEPTEMBER OCTOBER 2003 by single seed descent (SSD). At the F 5, SSD single plants tary manner wherein both alleles must be present for were scored for presence of Mmt1 or mmt1 by transcript analy- individual gamete viability in plants carrying the IS1112C ses. The F 5:6, harvested F 6 seeds from selfed F 5 plants, were used cytoplasm (Tang et al., 1998; Pring et al., 1999). Viable as male parent sources for subsequent crosses with A3Tx398. gametes transmit both restoring alleles to subsequent gen- Linkage of Mmt1 and Rf3 allowed identification of lines carerations. The gametophytic nature of fertility restoration rying Mmt1 as candidates for an Rf3Rf3rf4rf4 line, which we designated (B3)FL3. Conversely, lines carrying mmt1 reprepossible to obtain independent segregation of the two thus renders them functionally linked. As such, it is not sented candidates for a rf3rf3rf4rf4 line, which we designated (B3)FL4. The genotypes of these candidates were verified in restoring alleles in plants carrying the IS1112C cytoplasm. a series of crosses in which progeny were scored for segregation of iodine pollen staining ability, seed set and the presence male-fertile cytoplasm, B3Tx398/IS1112C progeny were To facilitate segregation of Rf3 and Rf4 in a normal, or absence of the rf4-linked flanking markers LW8 and LW9, advanced to the F 5 by SSD. A total of 40 F 5 plants were or a combination of all of the above criteria. Each of these scored indirectly for Rf3 by scoring for action of the two developed lines became a maintainer for its corresponding tightly linked Mmt1. Direct scoring for Rf3 by transcript sterile line and a tester for the reciprocal line. For example, analyses is not possible because the normal cytoplasm (B3)FL3 was the maintainer for a male-sterile line carrying of BTx398 does not harbor orf107. Eleven of 40 plants Rf3Rf3rf4rf4, which we designated (A3)FL3, and a tester for the development of a male-sterile line carrying rf3rf3rf4rf4, studied were identified as carrying Mmt1 (Fig. 1), and, which we designated (A3)FL4. thus, carry Rf3, while 29 plants were mmt1mmt1 and Progeny of many of the crosses or lines tillered readily, assumed to be rf3rf3. allowing multiple pollinations of the same plants. Mature panicles were collected, and the plants were cut back and subse- Development of (B3)FL3 quently pollinated with other lines for additional test crosses. [genotype (N)Rf3Rf3rf4rf4] Transcript Analyses Ten of the 11 F 5:6 lines identified as Mmt1 were crossed The Rf3-associated enhanced transcript processing activity as male with A3Tx398 to identify lines that were (TPA) conferred within the IS1112C-specific mitochondrial Rf3Rf3rf4rf4, which would produce sterile F 1 progeny open reading frame orf107 was assessed by transcript analyses (Fig. 2). Four of the lines produced sterile progeny; the of progeny carrying the IS1112C cytoplasm, as previously de- remaining six lines included fertile plants among the scribed (Tang et al., 1996b). The single dominant allele Mmt1, progeny, indicating presence of Rf4, and were disrewhich confers enhanced TPA at a site 5 to sorghum mitochon- garded. Among the four candidate lines, the line drial orf209, is tightly linked to Rf3 in IS1112C (Tang et al., 4 was designated as (B3)FL3. The A3Tx398/ F ), and was assayed as described by Tang et al. (1996a). The was examined for pollen staining with iodine and progline B3Tx398 is mmt1mmt1, thus B3Tx398/IS1112C progeny eny were grown and selfed in the greenhouse and in exhibiting Mmt1-enhanced TPA were considered to carry Rf3. Probes and hybridization conditions for identifying the altered the field in two consecutive summers. Among a total of transcripts of orf107 and urf209 were as previously described 39 progeny from selfed panicles grown in the field (22 (Tang et al., 1996a, b). in 2000, 17 in 2001), two plants set seed with only one seed/panicle. Although most of the pollen grains were DNA Marker Analyses clearly not stainable with iodine, occasionally a few Two codominant markers (Wen et al., 2002) spanning the grains (at an estimated frequency of 10 4 or less) exhibrf4 locus were utilized to examine lines for homozygosity. The ited a trace of starch deposition. This observation may marker LW9, a sequence tagged site (STS) marker located explain the occurrence of extremely low number of seed 0.74 centimorgans (cm) from the rf4 locus, is displayed as a set in the selfed individuals. Two additional identified 366-base pair (bp) fragment in IS1112C and a 358-bp fragment lines were field-tested in two summers and two seed in B3Tx398. Marker LW8, a cleavable amplified polymorphic were obtained from 32 selfed panicles. Homozygosity sequence (CAPS) marker located 3.18 cm from the rf4 locus, at the rf3 locus was confirmed by pollination of the is 275 bp in IS1112C and 310 bp in B3Tx398. PCR conditions (A3)FL4 lines (see below). and agarose gel electrophoresis were as described (Wen et al., 2002). Development of (B3)FL4 RESULTS [genotype (N)rf3rf3Rf4Rf4] The two fertility restoration genes Rf3 and Rf4 exhibit a gametophytic mode of action and act in a complemen- The 29 mmt1mmt1 lines from the F 6 population described above carry the genotypes rf3rf3rf4rf4 or Fig. 1. Transcript analysis of segregants for Mmt1 among B3Tx398/IS1112C F 5 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.

3 TANG & PRING: SINGLE GENE FERTILITY RESTORATION OF IS1112C CYTOPLASM SORGHUM 1749 Fig. 2. Strategy for selecting lines (B3)FL3 (Rf3Rf3rf4rf4) and (B3)FL4 (rf3rf3rf4rf4). B3Tx398/IS1112C F 5:6 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 F 1 s(rf3rf3rf4rf4) identified rf4rf4 lines, while progeny that segregated 1 fertile:1 sterile identified the rf3rf3rf4rf4 lines, designated (B3)FL4. 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 F 6 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. F 6 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 IS1112Cspecific 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 greenhousegrown F 1 s 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 F 1 plants were exam- ined in the greenhouse. In total, these progeny segre- gated 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 geno- type (N)rf3rf3Rf4Rf4. In contrast, nine F 1 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 flank- ing markers LW8 and LW9, and the inferred genotype for S01-16 is (N)rf3rf3rf4rf4. 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 F 5:6 were used to polli- nate 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 pol- linated with (B3)FL4, which was expected to com- plement the genotype (A3)Rf3Rf3rf4rf4 such that all progeny should be fertile, with 25% pollen staining (Rf3rf3Rf4rf4). In contrast, Rf3rf3rf4rf4 progeny polli- nated 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) ifthe 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 F 5:6 //(B3)FL3 plants were each pollinated with (B3)FL4 and IS1112C, by exploiting til- Fig. 3. Identification of Rf4Rf4 lines among rf3rf3 F 5:6 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.

4 1750 CROP SCIENCE, VOL. 43, SEPTEMBER OCTOBER 2003 Fig. 4. Strategy for selecting (A3)FL3 (Rf3Rf3rf4rf4). A3Tx398 was pollinated with Mmt1 lines identified from the B3Tx398/IS1112C F 5:6 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. Development of (A3)FL4 [genotype (A3)rf3rf3Rf4Rf4] The (A3)rf3rf3Rf4Rf4 line (Fig. 6) was constructed by pollinating A3Tx398 with (B3)FL4, then backcross- ing with (B3)FL4, generating the Genotypes 1 (A3)rf3- rf3rf4rf4:1 (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 stain- ing in progeny. These lines should be associated with 25% pollen staining when pollinated with (B3)FL3 and 50% pollen staining when pollinated with IS1112C. 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. Progeny resulting from pollination with (B3)FL3 are expected to be heterozygous Rf3rf3Rf4rf4 and exhibit 25% pollen staining. We observed a mean of 23% stain- lering of this line. Mature panicles from a cross were removed, and tillers were subsequently pollinated with other male parents. Fourteen A3Tx398/Rf3Rf3 F 5:6 //(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 malesterile plants, and were disregarded. Progeny from one of the former, an (A3)FL3 candidate line, were exam- ined 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). 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 restora- tion 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-ster- ile (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. 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).

5 TANG & PRING: SINGLE GENE FERTILITY RESTORATION OF IS1112C CYTOPLASM SORGHUM 1751 Table 1. Pollen staining and field plot seed set of F 1 progeny Table 2. Pollen staining and field plot seed set among F 1 progeny of male-sterile line (A3)FL3 (Rf3Rf3rf4rf4) pollinated with of male-sterile (A3)FL4 [(A3)rf3rf3Rf4Rf4] candidate lines (B3)FL4 [(N)rf3rf3Rf4Rf4], IS1112C (Rf3Rf3Rf4Rf4) and pollinated with (B3)FL3 [(N)Rf3Rf3rf4rf4)] and IS1112C (B3)FL3 [(N)Rf3Rf3rf4rf4]. Seed set was estimated in 10% (Rf3Rf3Rf4Rf4). Candidate (A3)FL4 lines 2, 3, and 5 were increments. pollinated with (B3)FL3, seed was collected, and panicles on subsequent tillers were pollinated with IS1112C. Seed set was Percent iodine estimated in 10% increments. pollen staining Percent seed set Population Mean Range n Mean Range n Percent iodine pollen staining Percent seed set (A3)FL3/(B3)FL (A3)FL3/IS1112C Population Mean Range n Mean Range n (A3)FL3/(B3)FL (A3)FL4-2/(B3)FL (A3)FL4-3/(B3)FL (A3)FL4-5/(B3)FL Combined able pollen and 74% seed set (Table 2). These data are (A3)FL4-2/IS1112C (A3)FL4-3/IS1112C consistent with previous observations of heterozygotes (A3)FL4-5/IS1112C (Tang et al., 1998), with the exception of one plant Combined 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 homozy- gous at the rf4 locus, i.e., exhibiting single-gene restora- tion 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. DISCUSSION 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 ex- tensively utilized since 1956 without apparent complica- tions, and the A2 source is being utilized in some appli- cations (Schertz et al., 1997). The IS1112C source of CMS does not seem to be adaptable for utilization in hybrid production, on the 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 F 1 s were fertile with 25% iodine-stained pollen, identifying the line as (A3)FL4. basis of the approximate 50% seed set in the F 1 (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 BC 1 F 1 or BC 3 F 1 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 comple- mentation 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 F 5:6 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 twogene 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

6 1752 CROP SCIENCE, VOL. 43, SEPTEMBER OCTOBER 2003 germplasm examined to date is reflected in the large (Tang et al., 1996b). The distribution of Rf4 in these number of lines identified as maintainers (Pedersen and materials is unknown, but the high frequency of main- Toy, 1990; Tang et al., 1998; Schertz et al., 1989; Worstell tainer lines for the IS1112C cytoplasm clearly indicates et al., 1984). Restoration capability for A3Tx398 has that the presence of both restorers is rare. been observed in progeny resulting from bulked pollinations with eight sudan grass populations (Pedersen and ACKNOWLEDGMENTS Toy, 1990). We similarly have recovered restoration capability (unpublished data) in pollen bulks of NP28 This manuscript is dedicated in memory of Dr. K.F. Schertz, USDA-ARS, College Station, TX, who provided the impetus and NP35 sudan grass (Gorz et al., 1990a, 1990b), but for this work and developed critical data and germplasm for have not yet characterized the genetics of restoration. this project. We thank Drs. C.D. Chase, W.L. Rooney, and Since most currently examined sorghum lines are main- R.P. Wise for critical reviews, and acknowledge the technical tainers, they are suitable for development of male-sterile assistance of Yen Dao and Theresa Cao. Supported in part lines. Introgression of either Rf3Rf3 or Rf4Rf4 into these by the U.S. Department of Agriculture-National Research lines, and of Rf3Rf3Rf4Rf4 into pollinator lines, would Initiative Competitive Grants Program, This provide single-gene fertility restoration. was a cooperative investigation of the U.S. Department of There are current barriers in introgression of the re- Agriculture-Agricultural Research Service and the Institute storing loci into agronomically important lines. The of Food and Agricultural Sciences, University of Florida. IS1112C-derived LW8 CAPS marker is polymorphic in B3Tx398, B3Tx623, and IS3620C, but monomorphic in REFERENCES Tx7000 (Wen et al., 2002; unpublished data). The LW9 Gabay-Laughnan, S., G. Zabala, and J.R. Laughnan S-type marker is monomorphic in B3Tx623, B3Tx7000, and cytoplasmic male sterility in maize. p In C.S. Levings, III, IS3620C. Thus only B3Tx398 is polymorphic for both and I. K. Vasil (ed.) The molecular biology of plant mitochondria. rf4 flanking markers. Surveys of diverse germplasm for Kluwer Acad Pubs, Dordrecht, the Netherlands. markers linked to the rf4 locus have thus not been initi- Gorz, H.J., F.A. Haskins, and K.P. Vogel. 1990a. Registration of NP28 ated; identification of markers potentially polymorphic sudangrass germplasm, a composite of 90 low-dhurin self-pollinated in other lines, obtained by exon capture, is under devel- lines. Crop Sci. 30:758. Gorz, H.J., F.A. Haskins, and K.P. Vogel. 1990b. Registration of opment (Wen et al., 2002). Additionally B3Tx623, NP33, NP34, and NP35, three broadly based random-mating popu- B3Tx7000, IS3620C, and several other lines, are rf3rf3 lations of sudangrass. Crop Sci. 30: but carry Mmt1 (Tang et al., 1998; unpublished data), Ichikawa, N., N. Kishimoto, A. Inagaki, A. Nakamura, Y. Koshino, and thus urf209 TPA cannot be utilized to identify Rf3 Y. Yokozeki, M. Oka, S. Samoto, H. Akagi, K. Higo, C. Shinjyo, in these lines. Markers for the rf3 locus are under develof a rice line containing the Rf-1 gene which is involved in restora- T. Fujimura, and K. Shimada A rapid PCR-aided selection opment; two AFLP markers have been identified but have not been adequately characterized. Thus, B3Tx398 tion of the cytoplasmic male sterility. Mol. Breed. 3: Klein, R.R., P.E. Klein, A.K. Chhabra, J. Dong, S. Pammi, K.L. Childs, is the only currently known line that is mmt1mmt1, J.E. Mullet, W.L. Rooney, and K.F. Schertz Molecular mapwhich allows an assay for the apparent presence of the ping of the rf1 gene for pollen fertility restoration in sorghum tightly linked Rf3 in normal cytoplasm, male-fertile (Sorghum bicolor L.). Theor. Appl. Genet. 102: lines. Pedersen, J.F., and J.J. Toy Forage yield, quality, and fertility Single-gene gametophytic fertility restoration sys- of sorghum sudan grass hybrids in A1 and A3 cytoplasm. Crop tems have the disadvantage of requiring homozygosity Sci. 37: Pring, D.R., H.V. Tang, and K.F. Schertz Cytoplasmic male of the restoring locus in restorer lines to generate a sterility and organelle DNAs of sorghum. p In C.S. Levheterozygous F 1, which is associated with 50% pollen ings, III, and I.K. Vasil (ed.) The molecular biology of plant mitoviability. Such restoration systems have been used, such chondria. Kluwer Acad Pubs, Dordrecht, the Netherlands. as maize S male-sterile cytoplasm (Gabay-Laughnan Pring, D.R., H.V. Tang, W. Chen, W. Howad, and F. Kempken et al., 1995), or apparently are presently utilized, such A unique two-gene gametophytic male sterility system in sorghum as the Chinsurah boro II male-sterile cytoplasm of rice, involving a possible role of RNA editing in fertility restoration. J. Hered. 90: Oryza sativa L. (Ichikawa et al., 1997). Conversion of Schertz, K.F., A. Sotomayor-Rios, and S. Torres-Cardona Cytothe IS1112C CMS fertility restoration system to a single plasmic-nuclear male sterility opportunities in breeding and genetgene would require homozygosity at both the rf3 and ics. Proc. Grain Sorg. Res. Util. Conf. 16: rf4 loci in restoring lines. The selection of homozygous Schertz, K.F., S. Sivaramakrishnan, W.W. Hanna, J. Mullet, Y. Sun, fertile Rf3Rf3Rf4Rf4 lines can be easily accomplished U.R. Murty, D.R. Pring, K.N. Rai, and B.V.S. Reddy Alter- in lines carrying the IS1112C cytoplasm by selection of nate cytoplasms and apomixis of sorghum and pearl millet. p. 213 F2 individuals resulting from pollination of lines in A In Proceedings of the International Conference on Genetic Improvement of Sorghum and Pearl Millet. INTSORMIL/ICRIcytoplasm with the Rf3Rf3Rf4Rf4 IS1112C. We have SAT, Lubbock, TX. selected such lines in a BC 7 F 2 in the Tx398 and Tx7000 Stephans, J.C., and R.F. Holland Cytoplasmic male-sterility for backgrounds by identification of individuals with 100% hybrid sorghum seed production. Agron. J. 46: pollen staining (unpublished data). Tang, H.V., D.R. Pring, F.R. Muza, and B. Yan. 1996a. Sorghum The (A3)FL3 and (A3)FL4 lines can be used in sursterile cytoplasm. Curr. Genet. 29: mitochondrial orf25 and a related chimeric configuration of a maleveys to detect the complementary restoring alleles Tang, H.V., D.R. Pring, L.C. Shaw, F.A. Salazar, F.R. Muza, B. Yan, among important germplasm by pollination of these and K.F. Schertz. 1996b. Transcript processing internal to a mitomale-sterile lines and fertility observations of F 1 s. To chondrial open reading frame is correlated with fertility restoration date we have not detected Rf3 among a limited set of in male-sterile sorghum. Plant J. 10: sorghum lines, as detected by transcript analyses of F 1 s Tang, H.V., R. Chang, and D.R. Pring Cosegregation of single

7 TANG & PRING: SINGLE GENE FERTILITY RESTORATION OF IS1112C CYTOPLASM SORGHUM 1753 genes associated with fertility restoration and transcript processing Wise, R.P., C.R. Bronson, P.S. Schnable, and H.T. Horner The of sorghum mitochondrial orf107 and urf209. Genetics 150: genetics, pathology, and molecular biology of T-cytoplasm male Tang, H.V., W. Chen, and D.R. Pring Mitochondrial orf107 sterility in maize. Adv. Agron. 65: transcription, editing, and nucleolytic cleavage conferred by the Worstell, J.V., H.J. Kidd, and K.F. Schertz Relationships among gene Rf3 are expressed in sorghum pollen. Sex. Plant Reprod. male-sterility inducing cytoplasms of sorghum. Crop Sci. 24: : Wen, Y., H.V. Tang, W. Chen, R. Chang, D.R. Pring, P.E. Klein, K. Xu, G.-W., Y.-X. Cui, K.F. Schertz, and G.E. Hart Isolation Childs, and R.R. Klein Development and mapping of AFLP of mitochondrial DNA sequences that distinguish male-sterilitymarkers linked to the sorghum fertility restorer gene rf4. Theor. inducing cytoplasms in Sorghum bicolor (L.) Moench. Theor. Appl. Appl. Genet. 104: Genet. 90: