On the cover

Since anaerobic germination (AG) is a key trait for the improvement of direct seeding in rice breeding, a simple and reliable method of evaluating AG is required. We germinated seeds in deoxygenated water and measured the coleoptile length several days later (main photo). We then compared the results with a conventional method using the survival rate in flooded soil (upper left panel). The anoxic water evaluation method saves space and time in a stable environment compared with flooded soil evaluation, indicating it is more applicable to the genetic analysis and breeding of AG in rice (This issue, p. 633–639).

(N. Kuya: NARO, Institute of Crop Science)

Climate change triggers increases in temperature, drought, and/or salinity that threaten potato production, because they necessitate specific amounts and quality of water, meanwhile lower temperatures generally support stable crop yields. Various cultivation techniques have been developed to reduce the negative effects of drought, heat and/or salinity stresses on potato. Developing innovative varieties with relevant tolerance to abiotic stress is absolutely necessary to guarantee competitive production under sub-optimal environments. Commercial varieties are sensitive to abiotic stresses, and substantial changes to their higher tolerance levels are not easily achieved because their genetic base is narrow. Nonetheless, there are several other possibilities for genetic enhancement using landraces and wild relatives. The complexity of polysomic genetics and heterozygosity in potato hamper the phenotype evaluation over abiotic stresses and consequent conventional introgression of tolerance traits, which are more challenging than previous successes shown over diseases and insects resistances. Today, potatoes face more challenges with severe abiotic stresses. Potato wild relatives can be explored further using innovative genomic, transcriptomic, proteomic, and metabolomic approaches. At the field level, appropriate cultivation techniques must be applied along with precision farming technology and tolerant varieties developed from various breeding techniques, in order to realize high yield under multiple stresses.

Isoflavones in soybean seeds are responsible for plant–microbe interactions and defend against pathogens, and are also beneficial to human health. We used two biparental populations and mini core collection of soybean germplasm to identify and validate QTLs underlying the content of isoflavone components. We identified a major QTL, qMGly_11, which regulates the content of malonylglycitin, on chromosome Gm11, in populations bred from parents with high, low, and null glycitein contents. qMGly_11 explained 44.5% of phenotypic variance in a population derived from a cross between ‘Aokimame’ (high) and ‘Fukuyutaka’ (low) and 79.9% of that in a population between ‘Kumaji-1’ (null) and ‘Fukuyutaka’ (low). The effect was observed only in the hypocotyl. We further confirmed the effect of qMGly_11 in a mini-core collection, where it explained 57.1% of the genetic diversity of glycitin production and 56.5% of malonylglycitin production. qMGly_11 increased the contents of glycitin and malonylglycitin at the expense of daidzin and malonyldaidzin in all analyzed populations. We discuss the gene responsible for this QTL and the availability of the null allele for metabolic engineering of soybean seed isoflavones.

The green rice leafhopper (GRH, Nephotettix cincticeps Uhler) is a serious insect pest of rice (Oryza sativa L.) in temperate regions of Asia. Wild Oryza species are the main source of resistance to insects. The W1413 accession of African wild rice (O. longistaminata A. Chev. & Roehrich) is resistant to GRH. To analyze its resistance, we developed 28 BC₃F₃ introgression lines carrying W1413 segments in the genetic background of Nipponbare, a susceptible rice cultivar, and evaluated their GRH resistance. Five BC₃F₃ populations were used for quantitative trait locus (QTL) analysis and seven BC₃F₄ populations for QTL validation. Four significant QTLs on the long arm of chromosome 2 (qGRH2), short arm of chromosome 4 (qGRH4), short arm of chromosome 5 (qGRH5), and long arm of chromosome 11 (qGRH11) were identified. The contribution of the W1413 allele at qGRH11 was the largest among the four QTLs; the other QTLs also contributed to GRH resistance. Chromosomal locations suggested that qGRH11 corresponds to the previously reported GRH resistance gene Grh2, qGRH4 to Grh6, and qGRH5 to Grh1. qGRH2 is a novel QTL for resistance to GRH. Thus, resistance of O. longistaminata to GRH can be explained by at least four QTLs.

Genetic mapping is a basic tool for eukaryotic genomic research. It allows the localization of genes or quantitative trait loci (QTLs) and map-based cloning. In this study, we constructed a linkage map based on DNA samples from a commercial strain L808, including two parental monokaryons and 93 single spore isolates considered with segregating to 1:1:1:1 at four mating types (A₁B₁, A₁B₂, A₂B₁ and A₂B₂). Using Simple Sequence Repeats (SSR), Sequence Related Amplified Polymorphism (SRAP), Target Region Amplified Polymorphism (TRAP) molecular markers, 182 molecular markers and two mating factors were located on 11 linkage groups (LGs). The total length of the map was 948.083 centimorgan (cM), with an average marker interval distance of 4.817 cM. Only two gaps spanning more than 20 cM was observed. The probability of 20 cM, 10 cM, 5 cM genetic distance cover one marker was 99.68%, 94.36%, 76.43% in our genetic linkage map, respectively. This is the first linkage map of Lentinula edodes using SSR markers, which provides essential information for quantitative trait analyses and improvement of genome assembly.

Ralstonia solanacearum causes bacterial wilt, a soil-borne disease and one of the most important maladies of potato and other Solanaceae crops. We analyzed the resistance of a potato clone to bacterial wilt by quantitative trait locus (QTL) analysis. A resistant diploid potato clone 10-03-30 was crossed with a susceptible diploid clone F₁-1 to generate a diploid, two-way pseudo-testcross F₁ population comprised of 94 genotypes. Dense linkage maps, containing 4,139 single nucleotide polymorphism markers with an average distance of 0.6 and 0.3 cM between markers, were constructed for both parents. The resistance level was evaluated by in vitro inoculation test with R. solanacearum (phylotype I/biovar 4/race 1). Five QTLs (qBWR-1 to -5) were identified on potato chromosomes 1, 3, 7, 10, and 11, and they explained 9.3–18.4% of the phenotypic variance. The resistant parent had resistant alleles in qBWR-2, qBWR-3, and qBWR-4 and susceptible alleles in qBWR-1 and qBWR-5. Accumulation of the resistant alleles in all five QTLs increased the level of resistance compared with that of the resistant parent. This is the first study to identify novel QTLs for bacterial wilt resistance in potato by using genome-wide markers.

This study examined contents of nine plant hormones in developing seeds of field-grown wheat varieties (Triticum aestivum L.) with different seed dormancy using liquid chromatography—mass spectrometry. The varieties showed marked diversity in germination indices at 15°C and 20°C. Contents of the respective hormones in seeds showed a characteristic pattern during seed maturation from 30-day post anthesis to 60-day post anthesis. Principal component analysis and hierarchical clustering analysis revealed that plant hormone profiles were not correlated with dormancy levels, indicating that hormone contents were not associated with preharvest sprouting (PHS) susceptibility. Indole acetic acid (IAA) contents of mature seeds showed positive correlation with the germination index, but no other hormone. Response of embryo-half seeds to exogenous abscisic acid (ABA) indicates that ABA sensitivity is correlated with whole-seed germinability, which can be explained in part by genotypes of MOTHER OF FT AND TFL (MFT) allele modulating ABA signaling of wheat seeds. These results demonstrate that variation in wheat seed dormancy is attributable to ABA sensitivity of mature seeds, but not to ABA contents in developing seeds.

Seed-cotton yield (SY) and lint yield (LY) are the most important yield traits of cotton. Thus, it is critical to dissect their genetic architecture. Upland cotton (Gossypium hirsutum) is widely grown worldwide. In this study, a genome-wide association mapping was performed based on the CottonSNP80K array to dissect the genetic architecture of SY and LY in Upland cotton. Twenty-three significant associations were detected within four environments, including 11 associated with SY and 12 associated with LY. Seven single nucleotide polymorphisms (SNPs), TM234, TM237, TM247, TM255, TM256, TM263, and TM264, were co-associated with the two traits, which may indicate pleiotropy or intergenic tight linkages. Five SNPs, TM13332, TM39771, TM57119, TM81653, and TM81660, were coincided with those of previous reports and could be used in marker-assisted selection. Combining functional annotations with expression analyses of the genes identified within 400 kb of the significantly associated SNPs, we hypothesize that the three genes, Gh_D05G1077 and Gh_D13G1571 for SY, and Gh_A11G0775 for LY, may have the potential to increase cotton yield. The results would provide useful information for understanding the genetic basis of yield traits in Upland cotton and for facilitating its high-yield breeding through molecular design.

Drought significantly influences maize morphology and yield potential. The elucidation of the genetic mechanisms of yield components and morphological traits, and tightly linked molecular markers under drought stress are thus of great importance in marker assisted selection (MAS) breeding. Here, we identified 32 QTLs for grain weight per ear, kernel ratio, and ear height-to-plant height ratio across two F_2:3 populations under both drought and non-drought conditions by single-environment mapping with composite interval mapping (CIM), of which 21 QTLs were mapped under water-stressed conditions. We identified 29 QTLs by joint analysis of all environments with mixed-linear-model-based composite interval mapping (MCIM), 14 QTLs involved in QTL-by-environment interactions, and 11 epistatic interactions. Further analysis simultaneously identified 20 stable QTLs (sQTLs) by CIM and MCIM could be useful for genetic improvement of these traits via QTL pyramiding. Remarkably, bin 1.07-1.10/6.05/8.03/8.06 exhibited four pleiotropic sQTLs that were consistent with phenotypic correlations among traits, supporting the pleiotropy of QTLs and playing important roles in conferring growth and yield advantages under contrasting watering conditions. These findings provide information on the genetic mechanisms responsible for yield components and morphological traits that are affected by different watering conditions. Furthermore, these alleles provide useful targets for MAS.

Direct seeding saves time and labour in the cultivation of rice. However, seedling establishment is often unstable, and yields are lower than in transplanting. Anaerobic germination (AG) is a key trait for improvement of direct seeding of rice. We established a simple and reliable method of evaluating AG in rice breeding. We germinated seeds in distilled water or deoxygenated water and measured coleoptile length several days later; compared the results of each method with survival rate in flooded soil; and used the anoxic water method for QTL analysis and for testing cultivars. Coleoptile elongation in anoxic water and survival rate in flooded soil were significantly correlated (r = 0.879, P < 0.01). A significant QTL, likely to be a major gene (AG1), was found in chromosome segment substitution lines and in a backcrossed F₂ population derived from tolerant and sensitive lines. Diverse rice genetic resources were classified into tolerant or sensitive accession groups reflecting their ecotypes. Our study revealed that anoxic water evaluation method saves space and time in a stable environment compared with flooded soil evaluation. It is applicable to QTL analysis and isolation of genes underlying anaerobic germination.

Leafy amaranths, which are consumed as traditional food in Asia and Africa, are now considered among the most promising vegetables. In Vietnam, leafy amaranths, particularly Amaranthus tricolor L., are important summer vegetables due to their excellent nutritional values and high tolerance to biotic and abiotic stresses. However, this species has not been subjected to systematic breeding. Here we describe species identification and evaluation of the genetic diversity of Vietnamese amaranth collection by using matK and simple sequence repeats (SSR) markers. Our phylogenetic analysis based on the matK marker classified the species of 68% of the accessions, of which 120 belonged to A. tricolor. We developed 21 SSR markers, which amplified a total of 153 alleles in 294 A. tricolor accessions originating from Vietnam and overseas, with a mean allelic richness of 7.29 per marker, observed heterozygosity of 0.14, expected heterozygosity of 0.38, and polymorphic information content of 0.35. The STRUCTURE and F_ST analysis indicated a positive relationship between geographic distance and genetic differentiation among most of the overseas groups and the Vietnamese collection, but not among geographic groups within the Vietnamese collection. Vietnamese amaranths could be divided into two major types, one common in East Asia and the other one unique to Vietnam.

Seedling vigor is of vital importance for the initial plant establishment of direct-seeded rice. Here, 166 recombinant inbred lines were employed to assess eight seedling vigor traits over the first 25 days of germination. Significant correlations were found between most traits, and statistical analysis has revealed, for the first time, a linear relationship that uses seedling height and fresh weight to accurately predict the accumulation of dry biomass. Further, a subset of 20 lines, spanning the phenotypic range of the larger population, were genotyped by using 16 simple sequence repeats (SSR) markers known to be associated with seedling vigor traits. Markers RM317 and RM348 linked with significantly different phenotypes, including seedling height and fresh weight. One combination, haplotype II-1 (RM317 (I/II) and RM348 (1/2)), consistently produced superior values for seedling height, root length, and leaf length. The new prediction tool for seedling dry weight, namely the haplotype of RM317 and RM348, will have a wide range of applications in breeding strategies by using marker-assisted selection to produce elite lines that optimize genetic composition contributing to seedling vigor.

Flowering cherry is an extremely renowned ornamental tree, consisting of a variety of species and cultivars. Because cherry species have no strict barriers for interspecific hybridization before fertilization, identification of the gene underlying post-zygotic hybrid inviability will help breeders identify specimens for breeding and help us understand speciation mechanisms. In this study, we mapped the genetic linkages and physical genome sequences for a presumed hybrid inviability locus (HIs-1) that we observed in the seedlings crossed between Cerasus × yedoensis ‘Somei-yoshino’ and its wild relative C. spachiana. By the surveying linkage maps of ‘Somei-yoshino’ and C. spachiana, we identified correlation with seedling inviability only in linkage group 4 (LG4) of the ‘Somei-yoshino’ map. When we produced a finer-scaled map of HIs-1 in LG4, we found that HIs-1 is located between two microsatellite (SSR) markers with a 3.8 cM span. Using eight SSR markers based on peach genome sequences, we further refined the candidate region for HIs-1. This region was located between Pp04C001 and Pp04C007 markers, spanning 240 Kb of the peach genome, in which 45 transcribed genes had been estimated. From these candidate genes, it will be feasible to identify molecular mechanisms involved in cherry hybrid inviability.

Seed coat cracking in soybeans [Glycine max (L). Merr.] leads to commercial and agronomic losses. The Japanese elite soybean cultivar ‘Fukuyutaka’ is often used as a parent for breeding, but its high rate of seed coat cracking is an obstacle to its further use in breeding programs. To establish a DNA marker-assisted selection system for seed coat cracking, genetic factors related to high rates of seed coat cracking were surveyed, and a quantitative trait locus (QTL) with a stable effect on seed coat cracking in both years of a two-year replication experiment was detected on chromosome 20. Comparison of a set of near-isogenic lines (NILs) around this locus verified that the presence of the ‘Fukuyutaka’ allele significantly increased seed coat cracking in the kernel. The locus is located in a genomic region spanning 3.2 Mb. Marker-assisted selection for the locus will improve the selection efficiency of ‘Fukuyutaka’-derived breeding populations.

A total of 47 rice accessions collected from Kenya were investigated the genetic variations and classified into two cluster groups, A and B, by polymorphism data of 65 simple sequence repeat (SSR) markers. Clusters A and B corresponded to Japonica and Indica Groups, respectively. The number of Japonica Group accessions was limited in comparison with those of the Indica Group. Based on their patterns of reaction to standard differential blast isolates (SDBIs), these accessions and 57 control cultivars including differential varieties and several accessions harboring partial resistance genes were classified again into three cluster groups: Ia (high resistance), Ib (intermediate resistance) and II (susceptible). The rice accessions from Kenya were classified only into groups Ia and Ib. The accessions from Kenya were finally classified into three categories, A-Ia, B-Ia and B-Ib, based on the two classifications of polymorphism of SSR markers and resistance. The Indica Group accessions had wider genetic variation for blast resistance than did the Japonica Group accessions. The three leading cultivars (Basmati 217, Basmati 370 and ITA 310) categorized into Cluster group Ia were susceptible to some SDBIs from Kenya. The genetic variation for blast resistance in Kenya was demonstrated as the first report using SDBIs.

Wide hybridization, which is a powerful tool to broaden genetic variation, has been used in breeding of many crops. However, in ornamental gentian few wide hybridizations have been reported. Interspecific hybridizations between two gentian cultivated species (Gentiana scabra and G. triflora) and 11 wild species, which were classified in five sections, were carried out using ovule culture. When G. scabra was used as a female parent, normal seedlings and hybrid plants were obtained from eight and five interspecific combinations, respectively. The yield of seedling produced from ovule culture depended on interspecific combinations, ranging from 0.3 to 427.7 normal seedling per flower. In the hybridization of G. triflora with five wild species, normal seedlings and plants were produced in five and four interspecific combinations, respectively. The yield of normal seedling ranging from 0.4 to 228.3 was different between not only interspecific combinations but also reciprocal crosses. Two cultivated species are classified in sect. Pneumonanthe, and successful production of hybrids was obtained from the hybridization with species classified in sections Pneumonanthe or Cruciata. The hybrid nature of the produced plants was confirmed by molecular marker and morphology. The production of interspecific hybrids opens a novel prospect in ornamental gentian breeding.

Carrot (Daucus carota) is cultivated in temperate regions for its taproot. Eastern and Western types have been differentiated. In Japan, the former type is categorized into Kintoki, Takinogawa oonaga, and Toso, with a few local cultivars. However, their genetic relationships are unclear because of the paucity of reports. We classified the Japanese Eastern and selected Western types based on simple sequence repeat (SSR) markers. Field traits, including root weight, length, diameter, and skin color, were also examined. Our field tests showed clear differences between the Kintoki and Western-type cultivars, confirming their differentiation. A phylogram based on nine SSRs classified 24 cultivars into groups I and II. Group I included all Eastern-type carrots examined (Kintoki and Toso groups, plus two local and two foreign cultivars), with the exception of an Indian cultivar (‘Pusa rudhira red’). Among them, red carrots including Kintoki were clustered into two subgroups. Western-type, Eastern-Western hybrid, and ‘Pusa rudhira red’ were included in group II. A population structure analysis revealed the split between the Eastern and the other types. This study elucidates the genetic characteristics of the Eastern type of carrot, which will be valuable information for carrot breeding, especially when using the Eastern type as a source.

To utilize a transposon-tagged mutant as a breeding material in rice, an endogenous DNA transposon, nDart1-0, was introduced into Koshihikari by successive backcrossing together with aDart1-27, an active autonomous element. The founder line for nDart1-tagged lines of Koshihikari carried nDart1-0 on chromosome 9 and transposed nDart1-12s on chromosomes 1 and 8 and nDart1-3 on chromosome 11. In nDart1-tagged lines, there were the most abnormal phenotypic mutants and many aberrant chlorophyll mutants at seedling stage. At mature stage, many semi-sterile mutants were observed. Dwarf, reduced culm number and lesion mimic mutants were also found. In total, 43.2% of the lines segregated some phenotypic mutants. Thus, the nDart1-tagged lines of Koshihikari are expected to be potentially useful for screening stress-tolerant mutants under abiotic or biotic stress conditions.

Low amylose content (AC) is a desirable trait for rice (Oryza sativa L.) cooking quality and is selected in soft rice breeding. The Wx^hp allele was derived from a Yunnan rice landrace in China, Haopi, with low AC. To efficiently and rapidly utilize the low amylose content-associated gene Wx^hp in rice molecular breeding programs, we developed a tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method, according to the single-nucleotide variation of the Wx^hp. Four Wx^hp-specific primers were used to perform PCR assays using genomic DNA extracted from several rice varieties. Based on the band pattern of the amplified products after electrophoresis, this method can accurately distinguish three Wx^hp-related genotypes (i.e., Wx^hp homozygotes, Wx^hp heterozygotes, and wild-type), and the genotypes completely correspond to the appearance of mature endosperm. This method represents a novel approach that is both inexpensive and highly efficient and can be widely used for genotyping Wx^hp alleles in rice germplasm collections and may aid breeding programs with marker-assisted selection (MAS).