Abstract
Maintainer lines are used to seed propagate male-sterile lines for the development of hybrid onion (Allium cepa L.) cultivars. The identification of maintainer lines would be more efficient with molecular markers distinguishing genotypes at the nuclear male-fertility restoration (Ms) locus. Ms has been mapped to chromosome 2 of onion and linked genetic markers identified. However, linkages between these markers and Ms were detected using F2 or BC1 families at maximum linkage disequilibrium and, for many markers, their efficacy to predict genotypes at Ms in onion populations at or near linkage equilibrium remains unknown. In this research, near isogenic lines homozygous-dominant and -recessive at Ms were developed and screened for 930 single nucleotide polymorphisms (SNPs). Three SNPs tightly linked on chromosome 2 remained in linkage disequilibrium with genotypes at Ms among randomly selected plants from three open-pollinated populations of onion as well as among a collection of inbred lines. These SNPs should be useful for selection of the recessive ms allele to aid in the development of maintainer lines for hybrid onion development.
Hybrid onion seed is produced using cytoplasmic-genic male sterility (CMS) systems. The most commonly used source of CMS in onion is conditioned by male-sterile (S) cytoplasm (Havey, 1993). For plants possessing S cytoplasm, a single nuclear locus (Ms) controls male-fertility restoration; a dominant allele at Ms conditions male fertility, whereas plants homozygous-recessive at Ms are male-sterile (Jones and Clarke, 1943). Plants possessing normal (N) cytoplasm are always male-fertile regardless of their genotype at Ms. Male-sterile inbred lines are seed-propagated by crossing male-sterile plants (S msms) with maintainer plants that possess N cytoplasm and the homozygous-recessive genotype at Ms (Jones and Davis, 1944). Therefore, a main goal of hybrid onion breeding programs is to identify superior maintainer (N msms) lines to seed-propagate male-sterile lines for hybrid development. The identification of maintainer lines can be expensive and time-consuming because of the biennial generation time of onion and the relatively high cost of using insects for crossing (Pike, 1986). To identify maintainers, male-sterile (S msms) plants are crossed with male-fertile (S Ms– or N–) plants. The male fertility of progenies from the male-sterile parent is then established. If all testcross progenies are male-fertile, the male parent was homozygous-dominant at Ms. If the progenies are all male-sterile, the male parent must be N msms. Finally, if the testcross progenies are segregating for male fertility, the male parent must be heterozygous at Ms. Plants that are identified as N msms or N Msms can be then used to develop maintainer lines for backcrossing to sources of S cytoplasm.
Numerous molecular markers distinguishing N and S cytoplasms of onion have been developed (Engelke et al., 2003; Havey, 1993, 1995; Kim et al., 2009; Sato, 1998). Identification of molecular markers tightly associated with the nuclear Ms locus would greatly aid the development of maintainer lines. These markers would allow breeders to identify plants carrying the recessive ms allele early during the first year and carry forward for testcrossing only those plants with a higher probability of possessing recessive allele(s) at Ms. We previously reported that the genomic region carrying a restriction fragment length polymorphism (RFLP) revealed by cDNA AOB272 is tightly linked (0.9 cM) to Ms and converted this marker to a polymerase chain reaction (PCR)-based polymorphism (Gökçe et al., 2002). Others have either converted our previously described markers to PCR-detectable polymorphisms (Bang et al., 2011) or developed new markers linked to Ms (Huo et al., 2012; Yang et al., 2012). Although these markers are useful for selection of maintainer lines after crossing (i.e., at maximum linkage disequilibrium), they may not be in linkage disequilibrium with Ms among plants from open-pollinated (OP) populations (Gökçe and Havey, 2002). Yang et al. (2012) identified two markers linked to the Ms locus that were consistent across a sample of inbreds and hybrids; however, they proposed that their markers will be useful to genotype plants from OP populations without actually testing their use. In this research, I identified SNPs that are in linkage disequilibrium with alleles at the Ms locus across OP and inbred populations of onion.
Materials and Methods
Near-isogenic lines (NILs) differing for genotypes at Ms were developed by self-pollinating individual plants from the OP population ‘Sapporo-Ki’ (SK) and at the same time testcrossing to the male-sterile line ‘MSU611-1AxMSU611B’. Pollinations were completed using flies (Jones and Emsweller, 1934). At least 20 progenies from each testcross family were scored in the field for male-fertility restoration over 2 to 4 years. S1 progenies that were heterozygous at Ms (i.e., testcross family segregated for male-fertility restoration) were again self-pollinated and testcrossed to MSU611-1AxMSU611B. This process was repeated through the S3 generation, selecting in each generation single heterozygous plants for self-pollination and testcrossing. Two full-sib S3 progenies were identified as heterozygous at Ms and were self-pollinated to produce the S4 generation. S4 progenies were then identified as homozygous-dominant or -recessive at the Ms locus and were separately intercrossed to produce S4 NILs that are N MSMS vs. N msms. DNA was isolated from these two NILs as described by Bark and Havey (1995). A total of 930 SNPs were scored between the two NIL populations using the KASPar assay (LGC Genomics, Beverly, MA) as previously reported by Duangjit et al. (2013).
Individual plants from OP populations ‘Brigham Yellow Globe’ (BYG), ‘Mountain Danvers’ (MD), and SK were randomly selected and paired with male-sterile lines. Pollinations were completed using flies and seed was harvested separately from the male-fertile (S1 families) and male-sterile (testcross families) plants. At least 20 progenies from each testcross family were evaluated over at least 2 years and scored as male-fertile (male parent was MsMs), segregating for male fertility (male parent Msms) or male-sterile (male parent msms) as previously reported by Gökçe and Havey (2002). DNA was isolated as described by Bark and Havey (1995) from at least 25 plants from each S1 family and a collection of inbred lines developed and released by U.S. public-sector researchers (Table 1) and genotyped for SNPs polymorphic between the two NILs using KASPar (Duangjit et al., 2013).
Genotypes at the nuclear male-fertility restoration locus (Ms) and single nucleotide polymorphisms (SNPs) among public-sector onion inbreds.z
Results and Discussion
Testcrosses of the two NILs from SK to male-sterile MSU611-1AxMSU611B revealed 100% male-fertile and male-sterile testcross progenies, confirming that the NILs are homozygous-dominant and -recessive at the Ms locus, respectively. Of 930 SNPs, 29 remained polymorphic between the NILs (Table 2) and mapped to chromosomes 1, 2, 4, 5, and 8 (Duangjit et al., 2013). SNPs on chromosomes 1, 4, and 8 were located at the ends of linkage groups (Duangjit et al., 2013) and may have remained heterozygous between the two NILs as a result of reduced crossing over. The NILs were heterozygous for numerous SNPs on chromosomes 2 and 5 (Table 2).
Percent recombination detected between the recessive ms allele and single nucleotide polymorphisms (SNPs) among random plants from three open-pollinated populations of onion.
The Ms locus has been mapped to chromosome 2 and showed tight linkage to an RFLP revealed by cDNA clone AOB272 (Gökçe et al., 2002). SNPs isotig34671_610, isotig30856_1351, isotig29186_1830, isotig17237_4883, isotig28524_1455, isotig39878_664, and isotig29609_387 mapped to the same region on chromosome 2 as AOB272 in either or both the BYG15-23 × AC43 or OH1 × 5225 families (Duangjit et al., 2013). To estimate the degree of linkage disequilibrium with Ms, DNAs from 148 plants from OP populations BYG (31), MD (54), and SK (63) were genotyped for the 29 SNPs. These plants had been previously genotyped at Ms (Gökçe and Havey, 2002) and the estimated allelic frequencies combined across all three populations for Ms and ms were 0.59 and 0.41, respectively. Because these three onion populations have been maintained by open pollination, only markers very near Ms should remain in linkage disequilibrium. As expected, all SNPs on chromosomes 1, 4, 5, and 8 and many of those on chromosome 2 were over 30% recombinant between genotypes at Ms (Table 2). Importantly three SNPs on chromosome 2 [isotig34671_610, isotig30856_1351, and isotig29186_1830 (Table 3)] remained in disequilibrium with Ms across the three OP onion populations, with 11.0%, 13.0%, and 15.7% recombinant gametes from this random sample of plants (Table 2). No recombination among these SNPs was observed in segregating families from BYG15-23 × AC43 and OH1 × 5225 (Duangjit et al., 2013). These three SNPs were also consistent for genotypes at Ms across a collection of pubic-sector inbred lines of onion, although there was recombination detected in B1828B, MSU8155B, and W52 for SNP isotig34671_610 (Table 1). Putative annotations of and sequences flanking the three cDNAs in linkage disequilibrium with Ms are listed in Table 3. Although isotig30856_1351 shows significant similarity to pentatricopeptide repeat-containing proteins, a class of nuclear genes often conditioning male-fertility restoration (Schmitz-Linneweber and Small, 2008), recombination between the SNP in this cDNA and Ms was detected (Table 2).
Annotations and complementary (c) DNA sequences flanking single nucleotide polymorphisms (SNPs) tightly associated with the male-fertility restoration (Ms) locus of onion.z
The SNPs listed in Table 3 must be tightly associated with the Ms locus to remain in linkage disequilibrium in OP populations and should be useful for selecting plants carrying the recessive ms allele for development of maintainer lines. However, because recombination between these SNPs and the Ms locus was detected among the OP and inbred populations, the success of these SNPs to predict genotypes at Ms will vary among onion populations. I recommend that an onion population be crossed with S-cytoplasmic, male-sterile line(s) and testcross seed evaluated for male-fertility restoration. Once it is established that the recessive ms allele is present in the population, individual plants should be genotyped for isotig34671_610, isotig30856_1351, and isotig29186_1830 to determine if these SNPs are polymorphic, and to select individual plants carrying the SNP allele(s) putatively in coupling phase with the recessive ms allele (Table 2). If these markers remained in linkage disequilibrium with the Ms locus in the population, discarding plants with SNP alleles in coupling phase with the dominant Ms allele should allow for efficient development of maintainer lines from selected plants or increase the frequency of the recessive ms allele in progenies from intercrossing among the selected plants.
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