PstI-genomic and cDNA clones revealing mapped restriction fragment-length polymorphisms (RFLP) in cucumber (Cucumis sativus L.) were sequenced in order to ensure that these clones remain available and to determine if any clones showing genetic linkage in cucumber are physically linked in Arabidopsis thaliana. Sequence comparisons using translated searches revealed that 80% of the cucumber cDNA clones showed significant (≤e-20) similarities to Arabidopsis expressed sequence tags (ESTs) or genomic sequences, as opposed to relatively few (32%) of the cucumber genomic clones. Two clones revealing RFLPs linked at 2 cM in cucumber showed significant (≤e-20) similarities to sequences separated by 347,616 basepairs on chromosome 4 of Arabidopsis.
Michael J. Havey
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.
Michael J. Havey
Restriction fragment length polymorphisms (RFLPs) in the chloroplast and nuclear genome are useful for estimation of phylogenetic relationships. Fifteen mutations at restriction enzyme sites in the chloroplast DNA were discovered. The wild species A. oschaninii and A. vavilovii were identical to A. cepa for all mutations. These species represent sources of wild germplasm closely related to the bulb onion. Nuclear RFLPs are now being used to estimate the genetic distances between accessions of A. oschaninii A. vavilovii, and open-pollinated populations of the cultivated bulb onion.
Michael J. Havey
Although always among the top five vegetables in value, little genetic information has been published on the bulb onion. Genetic and molecular analyses are hampered by the plant's biennial nature, severe inbreeding depression, and huge genome. Research is underway to construct a low-density genetic map of onion based on RFLPs, AFLPs, and RAPDs. Among open-pollinated populations (OPPs), levels on DNA polymorphisms were in agreement with those of other outcrossing diploid species. However, we identified little putative-allelic diversity among the OPPs (1.9 polymorphic bands per polymorphic probe–enzyme combination) supporting a bottleneck during the domestication of onion. Our segregating family is from the cross of two diverse inbreds and will be used to map quantitative trait loci conditioning phenotypically correlated production (maturity, storability, and firmness), consumer-preference (pungency, flavor, and bulb shape), and health-enhancing (anti-platelet aggregation) attributes of onion. We are also attempting to tag chromosome regions controlling relatively simply inherited traits that are difficult or expensive to characterize classically.
Yutaka Sato and Michael J. Havey
The production of hybrid-onion seed depends on cytoplasmic male sterility (CMS) systems. The male-sterile line is seed propagated using a normal (N) cytoplasmic maintainer line homozygous recessive at the nuclear male-fertility restoration locus (MS). Because of onion's biennial generation time, 4 to 8 years are required to establish the genotype at the MS locus. The development of maintainer lines would benefit greatly from a genetic marker linked to the MS locus. Such a marker would allow breeders to establish the nuclear genotype in seedlings and flower only those plants that are maintainers (N msms) or plants that can be used to develop maintainers (N MSms), reducing the number of plants to be testcrossed or backcrossed to a sterile line. We evaluated restriction fragment length polymorphisms (RFLPs), random amplification of polymorphic DNA (RAPDs), and amplified fragment length polymorphisms (AFLPs) to tag the chromosome region carrying the MS locus. No RAPDs or RFLPs cosegregated with MS. AFLP markers were identified that phenotypically correlated with restoration of male fertility.
Michael J. Havey and Borut Bohanec
The U.S. Department of Agriculture, Agricultural Research Service, announces the release of onion inbred ‘B8667 A&B’ and synthetic population ‘Sapporo-Ki (SKI) -1 A&B’. Both of these releases represent a cytoplasmic male-sterile (A) line with its maintainer (B) for seed propagation of male-sterile plants. ‘B8667 A&B’ is intended for production of red, long-day, well-storing hybrids. ‘SKI-1’ combines the earliness of the Japanese population ‘Sapporo-Ki’ with maintenance of cytoplasmic male sterility. The long-day synthetic population ‘Onion Haploid-1’ (‘OH-1’) is a joint release of the U.S. Department of Agriculture, Agricultural Research Service, and the University of Ljubljana, Slovenia. ‘OH-1’ is intended to serve as a responsive control for extraction of gynogenic haploids of onion.
Michael J. Havey and Farhad Ghavami
Single nucleotide polymorphisms (SNPs) were genotyped using a high-density array and DNAs from individual plants of important onion (Allium cepa L.) populations from major production regions and from the likely progenitor of onion, Allium vavilovii Popov et Vved. Genotypes at 1226 SNPs were used to estimate genetic relationships among these populations and revealed close associations among onions grown in Europe and those in North America, South America, and eastern Asia, supporting paths of introduction from Europe to the Americas and Asia. ‘Nasik Red’ is a population grown on the Indian subcontinent and was divergent from onions of European origin. Frequencies of SNPs among and within populations were used as a measure of informativeness, and 199 commonly polymorphic SNPs were identified distributed across the eight chromosomes of onion. These SNPs will be useful for estimations of relatedness among broader collections of onion populations, mapping of important phenotypes, fingerprinting of inbred lines and hybrids, and quality control of seed lots.
Sergio Melgar and Michael J. Havey
The most commonly used source of cytoplasmic male sterility in onion (Allium cepa) is controlled by the interaction of the cytoplasm [male-sterile (S) or normal male-fertile (N)] and one nuclear male-fertility-restoration locus (Ms). Scoring of genotypes at Ms is generally done by testcrossing male-fertile to male-sterile (S msms) plants, followed by scoring of testcross progenies for male-fertility restoration. We identified two N-cytoplasmic families, one that was homozygous dominant and the other segregating at Ms. Plants from each of these two families were individually testcrossed to male-sterile onion. Nuclear restoration of male fertility in testcross progenies was evaluated in the field over 4 years. For male plants homozygous dominant at Ms, we expected testcross families to show 100% male-fertility restoration, but observed mean values between 46% and 100%. For plants segregating at Ms, we again observed lower than expected frequencies of male-fertility restoration. These results demonstrate that the dominant Ms allele shows reduced penetrance, requiring that male-fertility restoration be scored over years to more confidently assign genotypes at Ms.
Steven J. Damon and Michael J. Havey
Natural variation exists in onion (Allium cepa L.) for amounts and types of epicuticular waxes on leaves. Wild-type waxy onion possesses copious amounts of these waxes, whereas the foliage of semiglossy and glossy phenotypes accumulates significantly less wax. Reduced amounts of epicuticular waxes have been associated with resistance to onion thrips (Thrips tabaci Lindeman), an important insect pest of onion. A segregating family from the cross of waxy and semiglossy onions was used to map single nucleotide polymorphisms (SNPs) and identify chromosome regions affecting amounts and types of epicuticular waxes as measured by gas chromatography–mass spectrometry. The amount of the primary epicuticular wax on onion leaves, hentriacontanone-16, was controlled by one region on chromosome 5. One region on chromosome 2 affected concentrations of several primary fatty alcohols. Results indicate that the region on chromosome 2 may be associated with the acyl reduction pathway, and the region on chromosome 5 may affect the decarbonylation pathway of epicuticular wax biosynthesis. Because lower amounts of epicuticular waxes are recessively inherited, SNPs tagging regions on chromosomes 2 and 5 will be useful for marker-assisted breeding to vary amounts and types of epicuticular waxes on onion foliage with the goal to develop cultivars resistant to onion thrips.
Joseph J. King and Michael J. Havey
The bulb onion (Allium cepa L.) is a diploid with an very large nuclear genome of 15300 Mbp/1C (107× arabidopsis, 16× tomato, 6× maize). We developed a low-density genetic map with morphological, RAPD, and RFLP markers to examine genome organization and to study QTL controlling phenotypically correlated bulb quality traits. A mapping population of 58 F3 families was derived from a cross of the inbreds Brigham Yellow Globe 15-23 (BYG) × Alisa Craig 43 (AC). These inbreds are distinct in solids, storability, pungency, and bulb shape. Analysis of 580 RAPD primers detected 53 (9%) polymorphisms between BYG and AC, but only 12 (2%) segregated at expected ratios among F3 families. Using probes from onion cDNA libraries and four restriction enzymes, 214 RFLPs were identified between mapping parents. A 112-point map includes 96 RFLPs, 13 RAPDs, a locus controlling complementary red bulb color, and two loci hybridizing with a clone of the enzyme alliinase (EC 184.108.40.206), which produces the flavors characteristic of Allium species. Duplicated loci were detected by ≈25% of RFLP probes and were unlinked, loosely linked (2 to 30 cM), or tightly linked (<2 cM). This frequency of duplication was comparable to species with polyploid ancestors (paleopolyploids) and was higher than that found in most true diploids. However, the distribution of duplicated loci suggests that, in contrast to whole genome duplications typical of paleopolyploids, the contemporary size and structure of the onion genome may be a product of intrachromosomal duplications (cryptopolyploidy) and subsequent structural rearrangements.