Akio Kojima

Read2Marker: a data processing tool for microsatellite marker development from a large data set.

Microsatellites, or simple sequence repeats (SSRs), are widely considered to be one of the best sources of DNA polymorphism that can be utilized for developing genetic markers (1). Recently, several methods for making microsatellite-enriched genomic libraries were reported (2,3) and, as a consequence, the random sequencing of a microsatellite-enriched genomic library becomes a possible strategy for the mass development of microsatellite markers. Despite the progress in microsatellite isolation, it remains timeconsuming and laborious to handle the large amounts of sequence data. A set of script programs, read2Marker, was developed as a solution to this problem (Figure 1). In cooperation with phred/ phrap (4), BLAST (5), Primer3 (6), and T-Coffee (7), read2Marker can process an unlimited number of raw output data files from an automatic sequencer. It can run on a MacOS X or Linux platform and generates a report on many aspects of the nonredundant microsatellite-containing clones such as whole sequence, microsatellite core sequences, and PCR primers. A set of trace data obtained by sequencing from both ends of a clone is base-called by phred and assembled by phrap. Based on quality values (QVs), the high QV part of the assembled sequence is used for further processing. When data in plain text format without QVs are to be processed, all of the nucleotides are automatically assigned a QV of 20 to disable the quality threshold aspect of the read2Marker program. Subsequently, a microsatellite core is screened in the clone sequence by a newly developed tool, srchssr2. Initially, srchssr2 searches for repeated sequences of four dinucleotide motifs (GA, GT, AT, GC) and 10 trinucleotide motifs (AAC, AAG, ACC, ACG, ACT, AGC, AGG, ATC, TAT, CGC) independently in the clone sequence, and the core with the highest repeat number is selected. Next, a core structure neighboring the selected longest core is rescreened. If the gap between these cores is shorter than a maximum gap length set by the user in advance, then the two cores and gap sequence are connected to make a new core sequence. These procedures are repeated, and finally the composite microsatellite core in the sequence is identified. Finally, a PCR primer set for amplifying the microsatellite core is designed using the Primer3 program. The algorithm for finding microsatellites with srchssr2 is quite simple, but it is efficient enough to find cores. It can find composite repeats composed of different motifs and extracts 5′and 3′-flanking sequences that will be used for a redundancy check in the next step. Several SSR-finding programs with more sophisticated algorithms such as Msatfinder (M.I. Thurston and D. Field, www.bioinf.ceh.ac.uk/msatfinder), TROLL (8), Sputnik (C. Abajian, espressosoftware.com/pages/sputnik. jsp), and Tandem repeats finder (9) are available, and some of them could be substituted for srchssr2 with some modification, especially in cases where screening for motifs of more than three nucleotides is required. In order to remove redundant sequences, a homology check is performed using the BLAST algorithm. Two outer sequences of the clone flanking the microsatellite core are independently set as query sequences and BLASTed to a database comprising unique clones. If at least one of the two core-flanking sequences does not provide a hit from the database, the clone is judged as unique and added to

Construction of SSR-based chromosome map in bunching onion ( Allium fistulosum )

We have constructed a linkage map of bunching onion (Allium fistulosum L., 2nxa0=xa016) using an F2 population of 225 plants. The map consists of 17 linkage groups with 212 bunching onion SSR markers and 42 bulb onion (A. cepa L.) SSR, InDel, CAPS or dCAPS markers, covering 2,069xa0cM. This is the first report of a linkage map mainly based on SSR markers in the genus Allium. With the 103 anchor markers [81 bunching onion SSRs, 11 bulb onion SSRs and 11 bulb onion non-SSRs (1 InDel, 9 CAPSs and 1 dCAPS)] whose chromosome assignments were identified in A. cepa and/or A. fistulosum, via the use of several kinds of Allium alien addition lines, 16 of the 17 linkage groups were connected to the 8 basic chromosomes of A. cepa.

Genetic mapping of AFLP markers in Japanese bunching onion (Allium fistulosum)

SummaryThe first genetic linkage map of Japanese bunching onion (Allium fistulosum) based primarily on AFLP markers was constructed using reciprocally backcrossed progenies. They were 120 plants each of (P1)BC1 and (P2)BC1 populations derived from a cross between single plants of two inbred lines: D1s-15s-22 (P1) and J1s-14s-20 (P2). Based on the (P2)BC1 population, a linkage map of P1 was constructed. It comprises 164 markers – 149 amplified fragment length polymorphisms (AFLPs), 2 cleaved amplified polymorphic sequences (CAPSs), and 12 simple sequence repeats (SSRs) from Japanese bunching onion, and 1 SSR from bulb onion (A. cepa) – on 15 linkage groups covering 947 centiMorgans (cM). The linkage map of P2 was constructed with the (P1)BC1 population and composed of 120 loci – 105 AFLPs, 1 CAPS, and 13 SSRs developed from Japanese bunching onion and 1 SSR from bulb onion – on 14 linkage groups covering 775 cM. Both maps were not saturated but were considered to cover the majority of the genome. Nine linkage groups in P2 map were connected with their counterparts in P1 map using co-dominant anchor markers, 13 SSRs and 1 CAPS.

Isolation of 1,796 SSR clones from SSR-enriched DNA libraries of bunching onion (Allium fistulosum)

Bunching onion (Allium fistulosum L.) is one of the most important vegetables in Japan. To establish a genetic basis for molecular breeding of bunching onion, we isolated 1,796 simple sequence repeat (SSR) clones by large-scale sequencing of SSR-enriched genomic DNA libraries. Of these, 1,331 (74.1%) contained (GT)n repeats (nxa0>xa05), while 314 (17.5%) were (GA)n-containing clones. The average number of SSR repeats was 10.5 and 10.4 in the (GT)n- and (GA)n-containing clones, respectively. In a sample of five bunching onion inbred lines, an average of 3.2 alleles were detected in the 100 SSR loci investigated, with the polymorphic information content averaging 0.55. These results indicate that bunching onion SSRs are very rich sources of highly informative genetic markers.

Direct determination of the chromosomal location of bunching onion and bulb onion markers using bunching onion–shallot monosomic additions and allotriploid-bunching onion single alien deletions

To determine the chromosomal location of bunching onion (Allium fistulosum L.) simple sequence repeats (SSRs) and bulb onion (A. cepa L.) expressed sequence tags (ESTs), we used a complete set of bunching onion–shallot monosomic addition lines and allotriploid bunching onion single alien deletion lines as testers. Of a total of 2,159 markers (1,198 bunching onion SSRs, 324 bulb onion EST–SSRs and 637 bulb onion EST-derived non-SSRs), chromosomal locations were identified for 406 markers in A. fistulosum and/or A. cepa. Most of the bunching onion SSRs with identified chromosomal locations showed polymorphism in bunching onion (89.5%) as well as bulb onion lines (66.1%). Using these markers, we constructed a bunching onion linkage map (1,261xa0cM), which consisted of 16 linkage groups with 228 markers, 106 of which were newly located. All linkage groups of this map were assigned to the eight basal Allium chromosomes. In this study, we assigned 513 markers to the eight chromosomes of A. fistulosum and A. cepa. Together with 254 markers previously located on a separate bunching onion map, we have identified chromosomal locations for 766 markers in total. These chromosome-specific markers will be useful for the intensive mapping of desirable genes or QTLs for agricultural traits, and to obtain DNA markers linked to these.

Inheritance mode of male sterility in bunching onion (Allium fistulosum L.) accessions

Cytoplasmic male sterility (CMS) is an indispensable trait for F1 hybrid seed production in bunching onion (Allium fistulosum L.). Expansion of the cytoplasmic diversity of F1 hybrid cultivars by introduction of various CMS resources has great potential to eliminate vulnerability to cytoplasm type-specific diseases. This study aimed to evaluate appearance frequency of male sterile plants in several bunching onion accessions and to identify CMS resources. In eight (‘Nogiwa Aigara’, ‘Bansei Hanegi’, ‘Amarume’, ‘Kimnung’, ‘Zhangqiu’, ‘INT/CHN/1990/GOTOU’, ‘Natsunegi’ and ‘Guangzhou’) of 135 accessions collected from Japan, China, Mongolia, Korea and Taiwan, male sterile plants appeared with varied frequencies from 1.7% (‘Nogiwa Aigara’ and ‘Bansei Hanegi’) to 24.5% (‘Zhangqiu’). The inheritance mode of Zhangqiu- and Guangzhou-derived male sterility was confirmed to be CMS by sib-crossings and interbreed crossings. Microscopic examination of microsporogenesis in the CMS plants revealed that microspore protoplasm rapidly degenerated without mitotic division after the release of microspores from tetrads. The CMS germplasm described here would be useful for the development of “A” lines to be used in F1 hybrid seed production of bunching onion. Male fertility in ‘Nogiwa Aigara’, ‘Bansei Hanegi’, ‘Kimnung’, ‘INT/CHN/1990/GOTOU’ and ‘Natsunegi’ was verified to be controlled by a single fertility restoration locus.

SSR-tagged breeding scheme for allogamous crops: a trial in bunching onion ( Allium fistulosum )

In several autogamous and vegetatively propagated crops, DNA markers have been used for cultivar identification. However, allogamous crops such as bunching onion (Allium fistulosum L.) are recalcitrant to marker-aided cultivar identification, as well as hybrid seed purity tests, due to the high degree of genetic heterogeneity within each cultivar. To aid cultivar identification and ensure its accuracy in bunching onion, we proposed the “SSR-tagged breeding” scheme in our previous study. The feasibility of this scheme was investigated here using a landrace of bunching onion with two populations tagged with two or four selected SSR markers. Compared with a control population, no significant differences were detected in the agronomic traits of the SSR-tagged populations. The targeted SSR loci were genetically uniform within each population whereas other loci maintained high heterogeneity. These results demonstrate that the SSR-tagged breeding scheme, even with a very small number of markers, is efficient for the identification of newly bred cultivars, and consequently for F1 purity tests, in allogamous crops in which inbreeding depression is as severe as in bunching onion.

QTL analysis for pseudostem pungency in bunching onion ( Allium fistulosum )

Low pungency is one of the most important agronomic traits in bunching onion (Allium fistulosum L.). Although the degree of pungency can be evaluated indirectly using a colorimetric test for pyruvic acid, DNA markers linked to low-pungency quantitative trait loci (QTLs) are still desired. In this study, we evaluated pungency in the bunching onion pseudostem through six trials conducted over 3xa0years using an F2:3 population. QTL analysis based on the genetic linkage map revealed that the major pungency QTL was located within a 24.2-cM interval on Chr. 2a. The low-pungency parent-derived allele at AFAT04B03, a simple sequence repeat locus linked to the pungency QTL, was rare among commercial bunching onion cultivars. In addition, individuals homozygous for the low-pungency parent-derived allele at AFAT04B03 were significantly less pungent than those that were homozygous or heterozygous. Thus, these findings suggest that AFAT04B03 is an effective selection marker for low pungency in bunching onion breeding.

Modes of inheritance of two apomixis components, diplospory and parthenogenesis, in Chinese chive (Allium ramosum) revealed by analysis of the segregating population generated by back-crossing between amphimictic and apomictic diploids

To investigate the mode of inheritance of apomixis in Chinese chive, the degrees of diplospory and parthenogenesis were evaluated in F1 and BC1 progenies derived from crosses between amphimictic and apomictic diploids (2n = 16, 2x). The F1 population was generated by crossing three amphimictic diploids 94Mo13, 94Mo49 and 94Mo50 with an apomictic diploid KaD2 and comprised 110 diploids and 773 triploids. All the diploid F1 plants examined were completely or highly eusporous and completely syngamic. All the triploid F1 plants examined were highly diplosporous and highly parthenogenetic. KaD2 could not transmit its high level of apomixis via monoploid pollen grains. The BC1 population, generated by crossing 94Mo49 with apomictic triploids found in the F1 offspring, exhibited heteroploidy; it comprised haploid, diploid, triploid, tetraploid and various aneuploid individuals. In this generation, clear segregation was observed between diplospory and parthenogenesis. Analysis of the BC1 population suggests that diplospory and parthenogenesis are each controlled by single dominant genes, D and P, respectively. However, all the BC1 plants characterized as parthenogenetic were diplosporous. The absence of phenotypically eusporous parthenogenetic plants can be explained by assuming that the presence of diplospory gene is a prerequisite for the parthenogenesis gene expression in Chinese chive.

Screening and incorporation of rust resistance from Allium cepa into bunching onion (Allium fistulosum) via alien chromosome addition

Bunching onion (Allium fistulosum L.; 2n = 16), bulb onion (Allium cepa L. Common onion group), and shallot (Allium cepa L. Aggregatum group) cultivars were inoculated with rust fungus, Puccinia allii, isolated from bunching onion. Bulb onions and shallots are highly resistant to rust, suggesting they would serve as useful resources for breeding rust resistant bunching onions. To identify the A. cepa chromosome(s) related to rust resistance, a complete set of eight A. fistulosum - shallot monosomic alien addition lines (MAALs) were inoculated with P. allii. At the seedling stage, FF+1A showed a high level of resistance in controlled-environment experiments, suggesting that the genes related to rust resistance could be located on shallot chromosome 1A. While MAAL, multi-chromosome addition line, and hypoallotriploid adult plants did not exhibit strong resistance to rust. In contrast to the high resistance of shallot, the addition line FF+1A+5A showed reproducibly high levels of rust resistance.