Development of an SSR marker set for efficient selection for resistance to black spot disease in pear breeding

Black spot disease, which is caused by Alternaria alternata (Fries) Keissler Japanese pear pathotype, is one of the most harmful diseases in Japanese pear cultivation. Because of the potential harm of fungicides to consumers and the environment, resistant cultivars are desired. In this study, to enable efficient marker-assisted selection in pear breeding, we conducted comprehensive inoculation tests and genotyping with 207 pear cultivars. We identified a marker set (Mdo.chr11.27 and Mdo.chr11.34) suitable for selection for black spot resistance. In most susceptible cultivars, Mdo.chr11.27 amplified a 220-bp band and Mdo.chr11.34 amplified a 259-bp band. The genotype of Mdo.chr11.34 corresponds perfectly to the estimated genotype of Japanese pears susceptible to black spot disease. Using linkage analysis, we identified the positions of the gene for susceptibility to black spot disease in Chinese pear. Mdo.chr11.27 and Mdo.chr11.34 were tightly linked to susceptibility in Chinese pear, and the susceptibility gene was mapped at the top of linkage group 11, similar to that in Japanese pear. This marker set and the accumulation of phenotypic data will enable efficient marker-assisted breeding for black spot resistance in pear breeding.     

Development of cultivar-specific DNA markers based on retrotransposon-based insertional polymorphism in Japanese pear

We developed retrotransposon-based insertional polymorphism (RBIP) markers based on the long terminal repeat (LTR) sequences of copia-like retrotransposon Ppcrt4 and flanking genome sequences, which were derived from 454 sequencing data from Japanese pear (Pyrus pyrifolia) ‘Hosui’. Out of 40 sequences including both LTR and flanking genome regions, we developed 22 RBIP markers and used them for DNA profiling of 80 pear cultivars: 64 Japanese, 10 Chinese (Pyrus ussuriensis) and 6 European (Pyrus communis). Three RBIP markers were enough to differentiate ‘Hosui’ from the other Japanese pear cultivars. The 22 RBIP markers could also distinguish 61 of the 64 Japanese pear cultivars. European pears showed almost no amplification of the 22 RBIP markers, which might suggest that retrotransposons had transposed during Asian pear evolution or reflect the genetic relationship between Asian and European pears. Sixteen of the RBIP markers could be positioned on a genetic linkage map of ‘Hosui’. The RBIP loci were distributed in 10 linkage groups, and some loci were very closely located within the same linkage group. The information obtained will be applicable to developing cultivar-specific RBIP marker sets in plants.     

Transfer of the Rsv3 locus from ‘Harosoy’ for resistance to soybean mosaic virus strains C and D in Japan

Resistance to soybean mosaic virus (SMV) is imperative for soybean (Glycine max (L.) Merr.) production in the Tohoku region. Molecular markers for SMV resistance were previously reported for U.S. SMV strains, but they cannot be applied because of the differences in strain classification between Japan and the U.S. A U.S. variety ‘Harosoy’ has been used mainly as a donor of resistance to SMV strains C and D in a Japanese breeding program, resulting in resistant varieties such as ‘Fukuibuki.’ Because ‘Harosoy’ harbors the Rsv3 gene conferring resistance to the virulent SMV strain groups, G5 through G7, it appears that the Rsv3 gene confers resistance to strains C and D. In this study, we introduced resistance to the two strains from ‘Fukuibuki’ into a leading variety ‘Ohsuzu’ by recurrent backcrossing with marker-assisted selection. All lines selected with markers near Rsv3 showed resistance to the strains, suggesting that the Rsv3 locus is responsible for the resistance. Three years of trials showed that one of the breeding lines, ‘Tohoku 169,’ was equivalent to ‘Ohsuzu’ with respect to agricultural characteristics such as seed size, maturity date, and seed yield, except for the SMV resistance.     

Confirming a major QTL and finding additional loci responsible for field resistance to brown spot (Bipolaris oryzae) in rice

Brown spot is a devastating rice disease. Quantitative resistance has been observed in local varieties (e.g., ‘Tadukan’), but no economically useful resistant variety has been bred. Using quantitative trait locus (QTL) analysis of recombinant inbred lines (RILs) from ‘Tadukan’ (resistant) × ‘Hinohikari’ (susceptible), we previously found three QTLs (qBS2, qBS9, and qBS11) that conferred resistance in seedlings in a greenhouse. To confirm their effect, the parents and later generations of RILs were transplanted into paddy fields where brown spot severely occurred. Three new resistance QTLs (qBSfR1, qBSfR4, and qBSfR11) were detected on chromosomes 1, 4, and 11, respectively. The ‘Tadukan’ alleles at qBSfR1 and qBSfR11 and the ‘Hinohikari’ allele at qBSfR4 increased resistance. The major QTL qBSfR11 coincided with qBS11 from the previous study, whereas qBSfR1 and qBSfR4 were new but neither qBS2 nor qBS9 were detected. To verify the qBSfR1 and qBSfR11 ‘Tadukan’ resistance alleles, near-isogenic lines (NILs) with one or both QTLs in a susceptible background (‘Koshihikari’) were evaluated under field conditions. NILs with qBSfR11 acquired significant field resistance; those with qBSfR1 did not. This confirms the effectiveness of qBSfR11. Genetic markers flanking qBSfR11 will be powerful tools for marker-assisted selection to improve brown spot resistance.     

DNA profiling of pineapple cultivars in Japan discriminated by SSR markers

We developed 18 polymorphic simple sequence repeat (SSR) markers in pineapple (Ananas comosus) by using genomic libraries enriched for GA and CA motifs. The markers were used to genotype 31 pineapple accessions, including seven cultivars and 11 breeding lines from Okinawa Prefecture, 12 foreign accessions and one from a related species. These SSR loci were highly polymorphic: the 31 accessions contained three to seven alleles per locus, with an average of 4.1. The values of expected heterozygosity ranged from 0.09 to 0.76, with an average of 0.52. All 31 accessions could be successfully differentiated by the 18 SSR markers, with the exception of ‘N67-10’ and ‘Hawaiian Smooth Cayenne’. A single combination of three markers TsuAC004, TsuAC010 and TsuAC041, was enough to distinguish all accessions with one exception. A phenogram based on the SSR genotypes did not show any distinct groups, but it suggested that pineapples bred in Japan are genetically diversed. We reconfirmed the parentage of 14 pineapple accessions by comparing the SSR alleles at 17 SSR loci in each accession and its reported parents. The obtained information will contribute substantially to protecting plant breeders’ rights.     

Identificaiton of a clubroot resistance locus conferring resistance to a Plasmodiophora brassicae classified into pathotype group 3 in Chinese cabbage (Brassica rapa L.)

In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) genes Crr1 and Crr2 are effective against the mild Plasmodiophora brassicae isolate Ano-01 and the more virulent isolate Wakayama-01, but not against isolate No. 14, classified into pathotype group 3. ‘Akiriso’, a clubroot-resistant F₁ cultivar, showed resistance to isolate No. 14. To increase the durability of resistance, we attempted to identify the CR locus in ‘Akiriso’. CR in ‘Akiriso’ segregated as a single dominant gene and was linked to several molecular markers that were also linked to CRb, a CR locus from cultivar ‘CR Shinki’. We developed additional markers around CRb and constructed partial genetic maps of this region in ‘Akiriso’ and ‘CR Shinki’. The positions and order of markers in the genetic maps of the two cultivars were very similar. The segregation ratios for resistance to isolate No. 14 in F₂ populations derived from each of the two cultivars were also very similar. These results suggest that the CR locus in ‘Akiriso’ is CRb or a tightly linked locus. The newly developed markers in this study were more closely linked to CRb than previously reported markers and will be useful for marker-assisted selection of CRb in Chinese cabbage breeding.     

Genome-wide indel markers shared by diverse Asian rice cultivars compared to Japanese rice cultivar ‘Koshihikari’

Insertion-deletion (indel) polymorphisms, such as simple sequence repeats, have been widely used as DNA markers to identify QTLs and genes and to facilitate rice breeding. Recently, next-generation sequencing has produced deep sequences that allow genome-wide detection of indels. These polymorphisms can potentially be used to develop high-accuracy polymerase chain reaction (PCR)-based markers. Here, re-sequencing of 5 indica, 2 aus, and 3 tropical japonica cultivars and Japanese elite cultivar ‘Koshihikari’ was performed to extract regions containing large indels (10–51 bp) shared by diverse cultivars. To design indel markers for the discrimination of genomic regions between ‘Koshihikari’ and other diverse cultivars, we subtracted the indel regions detected in ‘Koshihikari’ from those shared in other cultivars. Two sets of indel markers, KNJ8-indel (shared in eight or more cultivars, including ‘Khao Nam Jen’ as a representative tropical japonica cultivar) and C5-indel (shared in five to eight cultivars), were established, with 915 and 9,899 indel regions, respectively. Validation of the two marker sets by using 23 diverse cultivars showed a high PCR success rate (≥95%) for 83.3% of the KNJ8-indel markers and 73.9% of the C5-indel markers. The marker sets will therefore be useful for the effective breeding of Japanese rice cultivars.     

Development of genomic and EST-SSR markers in radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) belongs to Brassicaceae family and is a close relative of Brassica. This species shows a wide morphological diversity, and is an important vegetable especially in Asia. However, molecular research of radish is behind compared to that of Brassica. For example, reports on SSR (simple sequence repeat) markers are limited. Here, we designed 417 radish SSR markers from SSR-enriched genomic libraries and the cDNA data. Of the 256 SSR markers succeeded in PCR, 130 showed clear polymorphisms between two radish lines; a rat-tail radish and a Japanese cultivar, ‘Harufuku’. As a test case for evaluation of the present SSRs, we conducted two studies. First, we selected 16 SSRs to calculate polymorphism information contents (PICs) using 16 radish cultivars and four other Brassicaceae species. These markers detected 3–15 alleles (average = 9.6). PIC values ranged from 0.54 to 0.92 (average = 0.78). Second, part of the present SSRs were tested for mapping using our previously-examined mapping population. The map spanned 672.7 cM with nine linkage groups (LGs). The 21 radish SSR markers were distributed throughout the LGs. The SSR markers developed here would be informative and useful for genetic analysis in radish and its related species.     

Molecular and genealogical analysis of grain dormancy in Japanese wheat varieties,with specific focus on MOTHER OF FT AND TFL1 on chromosome 3A

In the wheat (Triticum aestivum L.) cultivar ‘Zenkoujikomugi’, a single nucleotide polymorphism (SNP) in the promoter of MOTHER OF FT AND TFL1 on chromosome 3A (MFT-3A) causes an increase in the level of gene expression, resulting in strong grain dormancy. We used a DNA marker to detect the ‘Zenkoujikomugi’-type (Zen-type) SNP and examined the genotype of MFT-3A in Japanese wheat varieties, and we found that 169 of 324 varieties carry the Zen-type SNP. In Japanese commercial varieties, the frequency of the Zen-type SNP was remarkably high in the southern part of Japan, but low in the northern part. To examine the relationship between MFT-3A genotype and grain dormancy, we performed a germination assay in three wheat-growing seasons. On average, the varieties carrying the Zen-type SNP showed stronger grain dormancy than the varieties carrying the non-Zen-type SNP. Among commercial cultivars, ‘Iwainodaichi’ (Kyushu), ‘Junreikomugi’ (Kinki-Chugoku-Shikoku), ‘Kinuhime’ (Kanto-Tokai), ‘Nebarigoshi’ (Tohoku-Hokuriku), and ‘Kitamoe’ (Hokkaido) showed the strongest grain dormancy in each geographical group, and all these varieties, except for ‘Kitamoe’, were found to carry the Zen-type SNP. In recent years, the number of varieties carrying the Zen-type SNP has increased in the Tohoku-Hokuriku region, but not in the Hokkaido region.     

Development and validation of DNA markers linked to Sdvy-1, a common bean gene conferring resistance to the yellowing strain of Soybean dwarf virus

The yellowing strain of Soybean dwarf virus (SbDV-YS) causes yellowing and yield loss in common bean (Phaseolus vulgaris). The most effective control is achieved through breeding for resistance. An indeterminate climbing cultivar with a white seed coat, ‘Oofuku’, is resistant to SbDV-YS in inoculation tests. We crossed ‘Oofuku’ with an elite cultivar, ‘Taisho-Kintoki’, which is SbDV-YS-susceptible, determinate dwarf with a red-purple seed coat, and performed amplified-fragment-length polymorphism analysis of F₃ lines. From nucleotide sequences of the resistant-specific fragments and their flanking regions, we developed five DNA markers, of which DV86, DV386, and DV398 were closely linked to Sdvy-1, a resistance gene. Using the markers, we developed ‘Toiku-B79’ and ‘Toiku-B80’, the near-isogenic lines (NILs) incorporating Sdvy-1 in the background of ‘Taisho-Kintoki’. The NILs had similar growth habit, maturity date and seed shape to those of ‘Taisho-Kintoki’. The quality of boiled beans was also similar, except that the NILs had more seed coat cracking than ‘Taisho-Kintoki’. The NILs showed no SbDV-YS infection in inoculation tests. We suggest that Sdvy-1 is a useful source of SbDV-YS resistance in common bean.     

Major QTLs associated with green stem disorder insensitivity of soybean (Glycine max (L.) Merr.)

Green stem disorder (GSD) is one of the most serious syndromes affecting soybean (Glycine max) cultivation in Japan. In GSD, stems remain green even when pods mature. When soybean plants develop GSD, seed surfaces are soiled by tissue fluid and seed quality is deteriorated during machine harvesting. We performed quantitative trait locus (QTL) analyses for GSD insensitivity using recombinant inbred lines (RILs; n = 154) derived from a cross between an insensitive line (‘Touhoku 129’) and a sensitive leading cultivar (‘Tachinagaha’) during a 6-year evaluation. Three effective QTLs were detected. The influences of these QTLs were in the following order: qGSD1 (LG_H) > qGSD2 (LG_F) > qGSD3 (LG_L). At these three QTLs, ‘Touhoku 129’ genotypes exhibited more GSD insensitivity than ‘Tachinagaha’ genotypes. The lower incidence of GSD for ‘Touhoku129’ was attributable primarily to these three QTLs because RILs harboring a ‘Touhoku 129’ genotype at the three QTLs exhibited a GSD incidence similar to that of ‘Touhoku 129.’ Although a limitation of this study is that only one mapping population was evaluated, this QTL information and the flanking markers of these QTLs would be effective tools for resolving GSD in soybean breeding programs.     

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.)

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a ‘piping-leaf-type’ cultivar, ‘Yugafu’, and a ‘spiny-tip-leaf-type’ variety, ‘Yonekura’. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the ‘spiny-leaf type’ as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding.     

Genetic dissection of black grain rice by the development of a near isogenic line

Rice (Oryza sativa L.) can produce black grains as well as white. In black rice, the pericarp of the grain accumulates anthocyanin, which has antioxidant activity and is beneficial to human health. We developed a black rice introgression line in the genetic background of Oryza sativa L. ‘Koshihikari’, which is a leading variety in Japan. We used Oryza sativa L. ‘Hong Xie Nuo’ as the donor parent and backcrossed with ‘Koshihikari’ four times, resulting in a near isogenic line (NIL) for black grains. A whole genome survey of the introgression line using DNA markers suggested that three regions, on chromosomes 1, 3 and 4 are associated with black pigmentation. The locus on chromosome 3 has not been identified previously. A mapping analysis with 546 F₂ plants derived from a cross between the black rice NIL and ‘Koshihikari’ was evaluated. The results indicated that all three loci are essential for black pigmentation. We named these loci Kala1, Kala3 and Kala4. The black rice NIL was evaluated for eating quality and general agronomic traits. The eating quality was greatly superior to that of ‘Okunomurasaki’, an existing black rice variety. The isogenicity of the black rice NIL to ‘Koshihikari’ was very high.     

Fine mapping of the major Soybean dwarf virus resistance gene Rsdv1 of the soybean cultivar ‘Wilis’

Soybean dwarf virus (SbDV), a Luteoviridae family member, causes dwarfing, yellowing and sterility of soybean (Glycine max), leading to one of the most serious problems in soybean production in northern Japan. Previous studies revealed that the Indonesian soybean cultivar ‘Wilis’ is resistant to SbDV and that the resistance can be introduced into Japanese cultivars. A major QTL for SbDV resistance has been reported between SSR markers Sat_217 and Satt211 on chromosome 5. In this study, we named this QTL Rsdv1 (resistance to SbDV) and developed near-isogenic lines incorporating Rsdv1 (Rsdv1-NILs) using Sat_217 and Satt211 markers. The Rsdv1-NILs were resistant to SbDV in greenhouse inoculation and field tests, indicating that Rsdv1 alone is sufficient for the resistance phenotype. We fine-mapped Rsdv1 within the 44-kb region between Sat_11 and Sct_13. None of the six genes predicted in this region was closely related to known virus resistance genes in plants. Thus, Rsdv1 may confer resistance by a previously unknown mechanism. We suggest that Rsdv1 may be a useful source for the Japanese soybean breeding program to introduce SbDV resistance.     

Close linkage of a blast resistance gene,Pias(t), with a bacterial leaf blight resistance gene,Xa1-as(t), in a rice cultivar ‘Asominori’

It has long been known that a bacterial leaf blight-resistant line in rice obtained from a crossing using ‘Asominori’ as a resistant parent also has resistance to blast, but a blast resistance gene in ‘Asominori’ has not been investigated in detail. In the present study, a blast resistance gene in ‘Asominori’, tentatively named Pias(t), was revealed to be located within 162-kb region between DNA markers YX4-3 and NX4-1 on chromosome 4 and to be linked with an ‘Asominori’ allele of the bacterial leaf blight resistance gene Xa1, tentatively named Xa1-as(t). An ‘Asominori’ allele of Pias(t) was found to be dominant and difference of disease severity between lines having the ‘Asominori’ allele of Pias(t) and those without it was 1.2 in disease index from 0 to 10. Pias(t) was also closely linked with the Ph gene controlling phenol reaction, suggesting the possibility of successful selection of blast resistance using the phenol reaction. Since blast-resistant commercial cultivars have been developed using ‘Asominori’ as a parent, Pias(t) is considered to be a useful gene in rice breeding for blast resistance.     

Identification of QTLs for fruit quality traits in Japanese apples: QTLs for early ripening are tightly related to preharvest fruit drop

Many important apple (Malus × domestica Borkh.) fruit quality traits are regulated by multiple genes, and more information about quantitative trait loci (QTLs) for these traits is required for marker-assisted selection. In this study, we constructed genetic linkage maps of the Japanese apple cultivars ‘Orin’ and ‘Akane’ using F₁ seedlings derived from a cross between these cultivars. The ‘Orin’ map consisted of 251 loci covering 17 linkage groups (LGs; total length 1095.3 cM), and the ‘Akane’ map consisted of 291 loci covering 18 LGs (total length 1098.2 cM). We performed QTL analysis for 16 important traits, and found that four QTLs related to harvest time explained about 70% of genetic variation, and these will be useful for marker-assisted selection. The QTL for early harvest time in LG15 was located very close to the QTL for preharvest fruit drop. The QTL for skin color depth was located around the position of MYB1 in LG9, which suggested that alleles harbored by ‘Akane’ are regulating red color depth with different degrees of effect. We also analyzed soluble solids and sugar component contents, and found that a QTL for soluble solids content in LG16 could be explained by the amount of sorbitol and fructose.     

Identification of a novel gene (Apq1) from the indica rice cultivar ‘Habataki’ that improves the quality of grains produced under high temperature stress

The appearance of brown rice grown under high temperature conditions is an important characteristic for improvement in Japanese rice breeding programs. We performed a QTL analysis of the appearance quality of brown rice using chromosome segment substitution lines of the indica cultivar ‘Habataki’ in the ‘Koshihikari’ genetic background. A line carrying a ‘Habataki’ segment on chromosome 7 showed a high percentage of perfect grains produced under high temperature conditions during the ripening period. To verify the role of this segment, and to narrow down the region containing the useful allele, substitution mapping was performed using multiple paired lines. As a result, the chromosomal location of a gene that we named Appearance quality of brown rice 1 (Apq1) was delimited to a 48-kb region. In addition, we developed an Apq1-near isogenic line (NIL) to evaluate the effect of Apq1 on various agronomic traits. Under high temperature conditions during the ripening period, the Apq1-NIL produced significantly higher percentages of perfect grains than ‘Koshihikari’. Other agronomic traits, including yield and palatability, were similar between the Apq1-NIL and ‘Koshihikari’. Therefore, the ‘Habataki’ allele of Apq1 will be useful in breeding programs aimed at improving the quality of grains ripened under high temperature conditions.     

Comparative QTL mapping for male sterility of cultivated strawberry (Fragaria × ananassa Duch.) using different reference genome sequences

Male sterility is one of the reproductive isolation systems in plants and quite useful for F₁ seed production. We previously identified three independent quantitative trait loci (QTLs) for male sterility of cultivated strawberry, Here, we identified the specific subgenomes in which these QTLs are located by QTL-seq approach. QTLs qMS4.1, qMS4.2, and qMS4.3 were mapped separately in subgenomes Fvb4-4, Fvb4-3, and Fvb4-1, respectively, in ‘Camarosa’ genome assembly v. 1.0.a1. Candidate regions of qMS4.1 and qMS4.3 were clearly detected around 12–26 Mb in Fvb4-4 and 12–14 Mb in Fvb4-1, respectively; those of qMS4.2 were fragmented in Fvb4-3, which suggests that some scaffolds were incorrectly assembled in Fvb4-3. qMS4.3 was mapped to chr4X1 of ‘Reikou’ genome assembly r2.3, and qMS4.1 and qMS4.2 were both mapped to chr4Av, which indicates that differentiation of the subgenomes in which both QTLs are located was insufficient in ‘Reikou’ r2.3. Although ‘Camarosa’ genome assembly v. 1.0.a1 is an unphased map, which merges homologous chromosomes into one sequence, ‘Reikou’ genome assembly r2.3 is a phased map, which separates homologous chromosomes. QTL mapping to different reference genomes clearly showed the specific features of each reference genome, and that using different kinds of reference map could accelerate fine mapping and map-based cloning of certain genes of cultivated strawberry.     

Reverted glutathione S-transferase-like genes that influence flower color intensity of carnation (Dianthus caryophyllus L.) originated from excision of a transposable element

A glutathione S-transferase-like gene, DcGSTF2, is responsible for carnation (Dianthus caryophyllus L.) flower color intensity. Two defective genes, DcGSTF2mu with a nonsense mutation and DcGSTF2-dTac1 containing a transposable element dTac1, have been characterized in detail in this report. dTac1 is an active element that produces reverted functional genes by excision of the element. A pale-pink cultivar ‘Daisy’ carries both defective genes, whereas a spontaneous deep-colored mutant ‘Daisy-VPR’ lost the element from DcGSTF2-dTac1. This finding confirmed that dTac1 is active and that the resulting reverted gene, DcGSTF2rev1, missing the element is responsible for this color change. Crosses between the pale-colored cultivar ‘06-LA’ and a deep-colored cultivar ‘Spectrum’ produced segregating progeny. Only the deep-colored progeny had DcGSTF2rev2 derived from the ‘Spectrum’ parent, whereas progeny with pale-colored flowers had defective forms from both parents, DcGSTF2mu and DcGSTF2-dTac1. Thus, DcGSTF2rev2 had functional activity and likely originated from excision of dTac1 since there was a footprint sequence at the vacated site of the dTac1 insertion. Characterizing the DcGSTF2 genes in several cultivars revealed that the two functional genes, DcGSTF2rev1 and DcGSTF2rev2, have been used for some time in carnation breeding with the latter in use for more than half a century.     

Construction of a high-density reference linkage map of tea (Camellia sinensis)

A few linkage maps of tea have been constructed using pseudo-testcross theory based on dominant marker systems. However, dominant markers are not suitable as landmark markers across a wide range of materials. Therefore, we developed co-dominant SSR markers from genomic DNA and ESTs and constructed a reference map using these co-dominant markers as landmarks. A population of 54 F₁ clones derived from reciprocal crosses between ‘Sayamakaori’ and ‘Kana-Ck17’ was used for the linkage analysis. Maps of both parents were constructed from the F₁ population that was taken for BC₁ population. The order of most of the dominant markers in the parental maps was consistent. We constructed a core map by merging the linkage data for markers that detected polymorphisms in both parents. The core map contains 15 linkage groups, which corresponds to the basic chromosome number of tea. The total length of the core map is 1218 cM. Here, we present the reference map as a central core map sandwiched between the parental maps for each linkage group; the combined maps contain 441 SSRs, 7 CAPS, 2 STS and 674 RAPDs. This newly constructed linkage map can be used as a basic reference linkage map of tea.