Identification of QTL controlling post-flowering period in soybean

The length of the reproductive period affects the grain yield of soybean (Glycine max [L.] Merr), and genetic control of the period might contribute to yield improvement. To detect genetic factor(s) controlling the reproductive period, a population of recombinant inbred lines (RILs) was developed from a cross between Japanese landrace ‘Ippon-Sangoh’ and, Japanese cultivar ‘Fukuyutaka’ which differ in their duration from flowering to maturation (DFM) relative to the difference in the duration from sowing to flowering (DSF). In the RIL population, the DFM correlated poorly (r = −0.16 to 0.34) with the DSF in all field trials over 3 years. Two stable QTLs for the DFM on chromosomes (Chr-) 10 and 11 as well as two stable QTLs for the DSF on Chr-10 and -16 were identified. The QTL on Chr-11 for the reproductive period (designated as qDfm1; quantitative trait locus for duration from flowering to maturation 1) affected all three trials, and the difference in the DFM between the Fukuyutaka and Ippon-Sangoh was mainly accounted for qDfm1, in which the Fukuyutaka allele promoted a longer period. qDfm1 affected predominantly the reproductive period, and thus it might be possible to alter the period with little influence on the vegetative period.     

Mapping of QTLs controlling epicotyl length in adzuki bean (Vigna angularis)

Epicotyl length (ECL) of adzuki bean (Vigna angularis) affects the efficiency of mechanized weeding and harvest. The present study investigated the genetic factors controlling ECL. An F₂ population derived from a cross between the breeding line ‘Tokei1121’ (T1121, long epicotyls) and the cultivar ‘Erimo167’ (common epicotyls) was phenotyped for ECL and genotyped using simple sequence repeats (SSRs) and single-nucleotide polymorphism (SNP) markers. A molecular linkage map was generated and fifty-two segregating markers, including 27 SSRs and 25 SNPs, were located on seven linkage groups (LGs) at a LOD threshold value of 3.0. Four quantitative trait loci (QTLs) for ECL, with LOD scores of 4.0, 3.4, 4.8 and 6.4, were identified on LGs 2, 4, 7 and 10, respectively; together, these four QTLs accounted for 49.3% of the phenotypic variance. The segregation patterns observed in F₅ residual heterozygous lines at qECL10 revealed that a single recessive gene derived from T1121 contributed to the longer ECL phenotype. Using five insertion and deletion markers, this gene was fine mapped to a ~255 kb region near the end of LG10. These findings will facilitate marker-assisted selection for breeding in the adzuki bean and contribute to an understanding of the mechanisms associated with epicotyl elongation.     

An SSR-based genetic map of pepper (Capsicum annuum L.) serves as an anchor for the alignment of major pepper maps

Of the Capsicum peppers (Capsicum spp.), cultivated C. annuum is the most commercially important, but has lacked an intraspecific linkage map based on sequence-specific PCR markers in accord with haploid chromosome numbers. We constructed a linkage map of pepper using a doubled haploid (DH) population derived from a cross between two C. annuum genotypes, a bell-type cultivar ‘California Wonder’ and a Malaysian small-fruited cultivar ‘LS2341 ({"type":"entrez-nucleotide","attrs":{"text":"JP187992","term_id":"347495878"}}JP187992)’, which is used as a source of resistance to bacterial wilt (Ralstonia solanacearum). A set of 253 markers (151 SSRs, 90 AFLPs, 10 CAPSs and 2 sequence-tagged sites) was on the map which we constructed, spanning 1,336 cM. This is the first SSR-based map to consist of 12 linkage groups, corresponding to the haploid chromosome number in an intraspecific cross of C. annuum. As this map has a lot of PCR-based anchor markers, it is easy to compare it to other pepper genetic maps. Therefore, this map and the newly developed markers will be useful for cultivated C. annuum breeding.     

Genetic linkage map of Chinese native variety faba bean (Vicia faba L.) based on simple sequence repeat markers

Simple sequence repeat (SSR) marker is a powerful tool for construction of genetic linkage map which can be applied for quantitative trait loci (QTL) and marker‐assisted selection (MAS). In this study, a genetic map of faba bean was constructed with SSR markers using a 129 F₂ individuals population derived from the cross of Chinese native variety 91825 (large seed) and K1563 (small seed). By screening 11 551 SSR primers between two parents, 149 primer pairs were detected polymorphic and used for F₂ population analysis. This SSR‐based genetic linkage map consisted of 15 linkage groups with 128 SSR. The map encompassed 1587 cM with an average genetic distance of 12.4 cM. The genetic map generated in this study will be beneficial for genetic studies of faba bean for identification of marker‐locus‐trait associations as well as comparative mapping among faba bean, pea and grasspea.     

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.     

Density enhancement of a faba bean genetic linkage map (Vicia faba) based on simple sequence repeats markers

Genetic mapping for faba bean lags far behind other major crops. Density enhancement of the faba bean genetic linkage map was carried out by screening 5,325 genomic SSR primers and 2033 expressed sequence tag (EST)‐SSR primers on the parental cultivars '91825' and 'K1563'. Two hundred and fifteen genomic SSR and 133 EST‐SSR primer pairs that detected polymorphisms in the parents were used to screen 129 F₂ individuals. This study added 337 more SSR markers and extended the previous linkage map by 2928.45 cM to a total of 4516.75 cM. The number of SSR markers in the linkage groups varied from 12 to 136 while the length of each linkage group ranged from 129.35 to 1180.21 cM. The average distance between adjacent loci in the enhanced genetic linkage map was 9.71 cM, which is 2.79 cM shorter than the first linkage map of faba bean. The density‐enhanced genetic map of faba bean will be useful for marker‐assisted selection and breeding in this important legume crop.     

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.     

Fine mapping of the gene for susceptibility to black spot disease in Japanese pear (Pyrus pyrifolia Nakai)

Black spot disease, which is caused by the Japanese pear pathotype of the filamentous fungus Alternaria alternata (Fries) Keissler, is one of the most harmful diseases in Japanese pear cultivation. We mapped a gene for susceptibility to black spot disease in the Japanese pear (Pyrus pyrifolia Nakai) cultivar ‘Kinchaku’ (Aki gene) at the top of linkage group 11, similar to the positions of the susceptibility genes Ani in ‘Osa Nijisseiki’ and Ana in ‘Nansui’. Using synteny-based marker enrichment, we developed novel apple SSR markers in the target region. We constructed a fine map of linkage group 11 of ‘Kinchaku’ and localized the Aki locus within a 1.5-cM genome region between SSR markers Mdo.chr11.28 and Mdo.chr11.34. Marker Mdo.chr11.30 co-segregated with Aki in all 621 F₁ plantlets of a ‘Housui’ × ‘Kinchaku’ cross. The physical size of the Aki region, which includes three markers (Mdo.chr11.28, Mdo.chr11.30, and Mdo.chr11.34), was estimated to be 250 Kb in the ‘Golden Delicious’ apple genome and 107 Kb in the ‘Dangshansuli’ Chinese pear genome. Our results will help to identify the candidate gene for susceptibility to black spot disease in Japanese pear.     

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.     

Phenotyping of faba beans (<Emphasis Type="Italic">Vicia faba</Emphasis> L.) under cold and heat stresses using chlorophyll fluorescence

Temperature stress including low and high temperature adversely affect the growth, development and productivity of crops. Faba bean (Vicia faba L.) is an important crop as both human food source and animal feed, which contains a range of varieties that are sensitive to cold and heat stresses. In this study, 127 faba bean genotypes were collected from gene banks based on differences in geographical origin. The 127 genotypes were treated by single cold stress (2/2 °C day/night temperature (DT/NT)) and 42 genotypes were treated by either single episode of cold or heat (38/30 °C DT/NT) stress, or a combination of both at photosynthetic photon flux density of 250 µmol m^?2 s^?1. Chlorophyll fluorescence was used to detect the tolerance of faba beans to low and high temperatures. The maximum quantum efficiency of photosystem II (PSII), F_v/F_m, revealed pronounced differences in cold tolerance among the faba bean genotypes. The 42 genotypes were clustered into four groups according to cold and heat stresses, respectively, and the susceptibilities of faba beans under temperature stress could be distinguished. The combination of cold and heat stresses could aggravate the damage on reproductive organs, but not on the leaves, as indicated by the F_v/F_m. These results confirm that the use of F_v/F_m is a useful approach for detecting low and high temperature damage to photosystem II and to identify tolerant faba bean genotypes, however the results also indicate that the geographical origin of the genotypes could not directly be used to predict climate resilience. These sources of cold- and heat-tolerance could improve the temperature tolerance of faba bean in breeding programs.     

Genetic variability of faba bean genotypes for chocolate spot (<Emphasis Type="Italic">Botrytis fabae</Emphasis>) resistance and yield

Faba bean (Vicia faba L.) has high utility as a food and soil fertility improving crop. One of the major fungal pathogens of faba bean is Botrytis fabae, the causative agent of chocolate spot. The disease affects significantly the leaf, stem, pod and seed of faba bean compromise its productivity in the smallholder farming sector. Nonetheless, there are limited resistant/tolerant faba bean varieties available and disease control technology options. Therefore, it was prudent to evaluate faba bean landraces for chocolate spot resistance. Fifty landraces together with ten improved varieties were evaluated both in the field and in the greenhouse under natural and artificial inoculation with previously selected aggressive Botrytis fabae isolate (Iso-016) from West Gojjam, in Ethiopia. There were highly significant differences (p?<?0.001) among the landraces for reaction to the disease and agronomic traits. Significant positive correlation was recorded between reaction of genotypes in the field and greenhouse disease data. The overall mean disease epidemics varied from 92.5 to 697.5 for the area under disease progress curve (AUDPC). The highest level of resistance was found in the ICARDA lines, ILB-4726, ILB-938 and BPL-710. Of all 18 landrace collections displayed significantly lower disease reaction than the susceptible check. However the resistance was moderate. The selected eighteen landraces will be recommended for use in breeding for chocolate resistance. Overall, resistance was highly heritable, suggesting that phenotypic selection can be exploited to improve chocolate spot resistance in faba bean varieties.     

DNA markers linked to Pga1, an adzuki bean gene that confers resistance to Cadophora gregata race 1

Brown stem rot (BSR) caused by Cadophora gregata f. sp. adzukicola (syn. Phialophora gregata) is a serious soilborne disease of adzuki bean (Vigna angularis) in Japan. Cultivation of resistant cultivars is the most effective disease control method, therefore the selection of resistant lines is a priority for breeders. BSR-resistant adzuki bean lines have been screened in pathogen-infected fields. However, field selection using the pathogen and artificial inoculation methods is time-consuming and labor-intensive. In the present study, we used 105 F₃ lines derived from a cross between a BSR-resistant cultivar ‘Syumari’ and a susceptible cultivar ‘Buchishoryukei-1’ for BSR inoculation tests. Amplified fragment-length polymorphism (AFLP) analyses with 1024 primer sets revealed that six fragments were polymorphic between resistance and susceptible bulked groups. Five DNA markers (Pg77, Pg118, Pg138, Pg139 and Pg126) were developed from the nucleotide sequences of polymorphic AFLP markers and their flanking regions. Pg118, which was derived from E-ACT/M-ACT-118, was tightly linked to the resistance gene Pga1 and was converted into a codominant marker for its easier use in marker-assisted selection for adzuki bean BSR resistance. Finally, the applicability of the developed markers for BSR resistance was tested on 32 adzuki bean accessions or cultivars.     

Construction of integrated linkage map of a recombinant inbred line population of white lupin (Lupinus albus L.)

We report the development of a Diversity Arrays Technology (DArT) marker panel and its utilisation in the development of an integrated genetic linkage map of white lupin (Lupinus albus L.) using an F₈ recombinant inbred line population derived from Kiev Mutant/{"type":"entrez-protein","attrs":{"text":"P27174","term_id":"14195583","term_text":"P27174"}}P27174. One hundred and thirty-six DArT markers were merged into the first genetic linkage map composed of 220 amplified fragment length polymorphisms (AFLPs) and 105 genic markers. The integrated map consists of 38 linkage groups of 441 markers and spans a total length of 2,169 cM, with an average interval size of 4.6 cM. The DArT markers exhibited good genome coverage and were associated with previously identified genic and AFLP markers linked with quantitative trait loci for anthracnose resistance, flowering time and alkaloid content. The improved genetic linkage map of white lupin will aid in the identification of markers for traits of interest and future syntenic studies.     

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.     

甘蔗SSR和AFLP分子遗传连锁图谱构建

采用甘蔗商业品种Co419与野生种割手密Y75/1/2杂交,获得269个单株,组成F1群体,用F102/356与商业品种ROC25回交获得266个单株,组成BC1群体。利用筛选的多态性条带丰富的36对SSR引物和12对AFLP引物,对两个群体进行PCR扩增和分子遗传连锁分析,构建甘蔗分子遗传连锁图谱。用F1群体获得630个分离标记,经χ2检测,298个标记为单双剂量标记,占总标记数的47%;用BC1群体获得571个分离标记,有264个标记为单双剂量标记,占总标记数的46%;4个亲本获得单双剂量标记的数量依次为Co41902/356Y75/1/2ROC25。在LOD≥5.0,相邻标记遗传距离≤40cM的条件下,F1群体有134个单双剂量标记被纳入55个连锁群,其中39个连锁群归属8个同源组,16个未列入,总遗传距离为1458.3cM,标记间平均图距为10.9cM;BC1群体有133个单双剂量标记被纳入47个连锁群,其中34个连锁群归属于8个同源组,13个连锁群未列入,总遗传距离为1059.6cM,标记间平均图距为8.0cM。从4个亲本单双剂量标记进入的连锁群数来看,Co419最多,归入34个连锁群,其次为Y75/1/2,归入20个连锁群,第3为02/356和ROC25,归入19个连锁群。研究结果表明,从单双剂量标记比例、形成连锁群数量、总遗传距离来看,F1群体构图质量要优于BC1群体。     

Sat-BSA: an NGS-based method using local de novo assembly of long reads for rapid identification of genomic structural variations associated with agronomic traits

Advances in next generation sequencing (NGS)-based methodologies have accelerated the identifications of simple genetic variants such as point mutations and small insertions/deletions (InDels). Structural variants (SVs) including large InDels and rearrangements provide vital sources of genetic diversity for plant breeding. However, their analysis remains a challenge due to their complex nature. Consequently, novel NGS-based approaches are needed to rapidly and accurately identify SVs. Here, we present an NGS-based bulked-segregant analysis (BSA) technique called Sat-BSA (SVs associated with traits) for identifying SVs controlling traits of interest in crops. Sat-BSA targets allele frequencies at all SNP positions to first identify candidate genomic regions associated with a trait, which is then reconstructed by long reads-based local de novo assembly. Finally, the association between SVs, RNA-seq-based gene expression patterns and trait is evaluated for multiple cultivars to narrow down the candidate genes. We applied Sat-BSA to segregating F₂ progeny obtained from crosses between turnip cultivars with different tuber colors and successfully isolated two genes harboring SVs that are responsible for tuber phenotypes. The current study demonstrates the utility of Sat-BSA for the identification of SVs associated with traits of interest in species with large and heterozygous genomes.     

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.     

QTL mapping of fruit nutritional and flavor components in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) using genome-wide SSR markers and recombinant inbred lines (RILs) from an intra-specific cross

Fruit nutritional and flavor components are important targets for breeding new cultivars of tomato (Solanum lycopersicum L.). We developed 108 recombinant inbred lines (the K39 RILs) in the F₆ generation from a cross between two phenotypically different breeding lines, K03 and K09. A linkage map was constructed using 172 genome-wide simple sequence repeat markers, 3 single-nucleotide polymorphism markers, and 2 phenotypic markers. The K39 RIL map consists of 12 linkage groups (LGs) and covers a genetic distance of 1089 cM. We measured the fruit soluble solids content (SSC), titratable acidity (TA), glutamic acid content (GLU), and lycopene content (LYC) of each line in four generations (F₆, F₈, F₁₀, F₁₁), β-carotene content (CAR) in two generations, and pH in one generation. By composite interval mapping that considered yearly variations in components as non-genetic effects, we detected three quantitative trait loci (QTLs) for SSC, four for TA, two for CAR, and one each for GLU, LYC, and pH. Among them, we found two QTLs for TA in LGs 6 and 11, those for GLU and LYC were candidates for novel QTLs. QTLs detected in this study were clustered in five LGs, but we observed no apparent trade-off relationships among the QTLs in each LG. Being derived from an intra-specific cross of tomato breeding materials, these QTLs can be used in practical breeding for improving fruit quality with low risk of linkage drag.