On the cover

The appearance quality (APQ) of grains is one of the most important traits in rice (Oryza sativa L.). To find useful lines with increased APQ for rice breeding, we cultivated chromosome segment substitution lines containing chromosome segments from indica, aus, and tropical japonica cultivars in the genetic ‘Koshihikari’ genetic background at the Kannondai field in Tsukuba, Japan, and conducted trait analysis. As a result, we found SL3215, SL2037, and SL2033 with reduced chalkiness but unaltered heading date and yield traits (This issue, p. 79–84).

(D. Ogawa: Institute of Crop Science, National Agricultural and Food Research Organization (NARO))

Grain chalkiness decreases the appearance quality (APQ) of rice (Oryza sativa L.) grains and reduces consumer satisfaction. Improving APQ is a crucial issue for both marketing and breeding. Here, we screened chromosome segment substitution lines (CSSLs) with higher APQ to find promising genetic resources. These CSSLs harbor chromosome segments derived from multiple donors in the genetic background of ‘Koshihikari’, a leading japonica rice cultivar in Japan. Three CSSLs had an increased percentage of perfect grains without panicle weight loss under field conditions across 3 years in Tsukuba city, Ibaraki prefecture, Japan. The positions of reduced chalkiness in grains differed among CSSLs, suggesting the different contribution of the harbored chromosome segments to APQ improvement. There were no significant differences in days to heading, culm length, panicle length, or panicle number in all three CSSLs, but 1000-grain weight was reduced in one. These results identify two promising genetic resources for further improvement of APQ in current japonica cultivars with reduced chalkiness but unaltered heading date and yield traits.

Cucumber is one of the most important vegetables in the Japanese market. To facilitate genomics-based breeding, there is a demand for reference genome of Japanese cucumber. However, although cucumber genome is relatively small, its assembly is a challenging issue because of tandem repeats comprising ~30% (~100 Mbp) of the genome. To overcome, we deployed the Oxford Nanopore sequencing that produces long reads with N50 length of >30 kbp. With this technology we achieved a chromosome-level assembly of cv. ‘Tokiwa’, a founder line of Japanese cucumber represented with the elongated fruit shape and high-crisp texture. Compared to the existing cucumber genomes, the Tokiwa genome is 20% longer and annotated with 10% more genes. The assembly with nanopore long reads also resolved tandem repeats spanning >100 kbp, demonstrating its strength in overcoming repetitive sequences.

Strong yellow color, caused by carotenoid accumulation, in semolina flour made from durum wheat (Triticum turgidum L. subsp. durum (Desf.)) is one of the most important traits for pasta production. The first step in the carotenoid biosynthesis pathway, which is catalyzed by phytoene synthase (PSY), is a bottleneck, and allelic variation of Psy-A1 in durum wheat produces different yellow pigment contents (YPC) in seeds. Durum wheat carrying leaf rust resistance gene Lr19, which was translocated from wheat relative Thinopyrum ponticum chromosome 7E to durum wheat chromosome 7A, is known to produce high YPC, and the causal gene is presumed to be Psy-E1, which is tightly linked to Lr19. In this study, Psy-E1 produced higher YPC than Psy-A1 alleles, such as Psy-A1k, Psy-A1l and Psy-A1o, in durum wheat. Segregation analysis demonstrated that Psy-E1 is located at the Psy-A1 locus on chromosome 7A. In a 2-year field test of near-isogenic materials, Psy-E1 was accompanied by yield loss with decreases in grain number per spike, test weight and thousand-kernel weight under moisture conditions typical of wheat-growing areas of Japan. Thus, Psy-E1 has the potential to contribute high YPC in durum wheat breeding programs, although the applicable cultivation environments are limited.

Polyphenol oxidase (PPO) is a key enzyme contributing to the time-dependent discoloration of wheat products. Developing cultivars with low PPO activity is one way to solve this problem. In this study, we focused on the Ppo-D1 gene, which has the second highest effect on grain PPO activity after the Ppo-A1 gene. Utilizing resequencing data, we found that the Ppo-D1 gene in the common wheat line ‘Fukuhonoka-NIL’, which exhibits low PPO activity, has an approximately 3 kb deletion in the 3′UTR and a 73 bp deletion in the third exon. The deletion in the third exon indicated that this allele was the ppo-D1d allele, previously identified in the wheat D genome progenitor, Aegilops tauschii Coss. Additionally, the ppo-D1d allele in ‘Fukuhonoka-NIL’ had very low expression, suggesting that this allele is non-functional. We developed a new co-dominant DNA marker for distinguishing the Ppo-D1a, Ppo-D1b and ppo-D1d alleles and demonstrated that F₂ plants homozygous for the ppo-D1d allele exhibited significantly lower grain PPO activity. Additionally, we determined that the ppo-D1d allele likely originated from Ae. tauschii ssp. tauschii (lineage 1) accessions. The ppo-D1d allele has not previously been found in common wheat (Triticum aestivum L., AABBDD genome), and thus the DNA marker developed in this study will be helpful for introducing this allele in common wheat breeding programs.

Cassava is an important staple crop in tropical and subtropical regions. Cassava mosaic disease (CMD) is one of the most dangerous diseases affecting cassava production in Africa. Since the first reported in Southeast Asia in 2015, the CMD prevalence has become a concern in Southeast Asia. To combat it, CMD resistance has been introduced from African cassava into Asian elite cultivars. However, efficient DNA markers for the selection of CMD resistance are not available. The CMD2 locus confers resistance to African cassava mosaic virus via non-synonymous substitutions in the DNA polymerase δ subunit 1 gene (MePOLD1). Here, we developed DNA markers to identify the mutations providing the resistance. We examined the association between the resistance score in CMD-infected fields and the genotypes of hybrids of CMD-resistant and ‑susceptible Asian lines. Our study provides powerful tools to the global cassava breeding community for selecting CMD resistant cassava.

Japanese red or white common bean (Phaseolus vulgaris L.) cultivars, used to make sweetened boiled beans, are called “kintoki” beans. Kintoki beans are planted to precede winter wheat for crop rotation in Hokkaido, northern Japan. Therefore, early maturity is an important trait for them. The aim of this study was to map the genomic region associated with days to maturity in kintoki beans by genome-wide association study (GWAS). Significant single nucleotide polymorphisms associated with days to maturity were detected on chromosome 1 (Pv01) by GWAS in 3 years, and the candidate region for early maturity was mapped to a 473-kb region. Sequencing analysis indicated that Phvul.001G221100, a phytochrome A3 gene, is likely to be responsible for early maturity in kintoki cultivars: the insertion of a cytosine in exon 1 at position 47 644 850 on Pv01 causes a frameshift that creates an early stop codon. Our findings suggest that the loss-of-function mutation of Phvul.001G221100 is derived from a leading cultivar, ‘Taisho-Kintoki’, and is originated from a spontaneous mutation in the oldest kintoki cultivar, ‘Hon-Kintoki’. The DNA markers targeting the functional insertion of phytochrome A3 will be useful for marker-assisted selection in kintoki bean breeding.

In barley (Hordeum vulgare L.), many DNA markers have been developed for the selection of traits related to various end-use purposes of breeding. To perform rapid marker-assisted selection of many lines, we developed Kompetitive Allele-Specific PCR (KASP) markers, which can be used for effective automatic genotyping of single nucleotide polymorphisms (SNPs). The KASP primers were designed for 17 SNPs in 14 genes related to important traits. The target allele of all primers tried was identified on the basis of high FAM fluorescence in comparison with that of HEX. To evaluate the suitability of the developed markers in breeding programs, we used them to genotype 62 representative cultivars and lines. Then, using six of the developed markers, we comprehensively analyzed a total of 2,941 lines collected from eight breeding sites with a genotyping success rate of 95.1%–99.8% (mean, 98.6%). All six markers showed differences in allele percentages among breeding programs, and specific allele combinations were observed in all four barley types. Our data will be useful for predicting phenotype segregation and designing cross combinations. The developed KASP marker set can be used for high-throughput genotyping and should make breeding more efficient when combined with an accelerated generation technique.

Resistance breeding for rice blast is an economic strategy for protecting rice crops against this disease. Genes with nucleotide-binding site leucine-rich repeat (NBS-LRR) structures are known to contribute to disease resistance. Here, we identified a candidate resistance gene, named Pior4(t), associated with leaf and panicle blasts in an introgression line carrying the chromosome 4 segment of wild rice (Oryza rufipogon Griff.) backcrossed with the cultivar ‘Nipponbare’ (Oryza sativa L.). Mapping analysis based on leaf blast severity confirmed that Pior4(t) was localized in the 177-kb NBS-LRR cluster region. To identify the Pior4(t) sequence, mutant lines were generated by knocking out a candidate NBS-LRR gene in a homozygous line carrying Pior4(t), M18, using CRISPR/Cas9-mediated genome editing. Leaf blast resistance was lost in the mutant lines lacking the corresponding Os04g0620950 N-terminal sequence of the M18 line. The result suggested that the counterpart NBS-LRR gene in the M18 line is involved in resistance to leaf blast. Pior4(t) showed homology to Pi63 in the resistant cultivar ‘Kahei’, and an NBS-LRR gene in the resistant cultivar ‘Mine-haruka’ carrying Pi39(t). These results suggest that the NBS-LRR gene is a candidate gene of Pior4(t) and is present on the long arm of chromosome 4.

“Tanpo”, a Japanese rice landrace widely cultivated approximately 120 years ago in Akita Prefecture, exhibits a shorter, wider, thicker, and heavier grain compared to Akitakomachi. Microscopic analysis has revealed that the epidermal cells of Tanpo spikelet hulls are narrower and shorter, with an increased number of cells in the grain width direction, thus resulting in a distinctive grain shape. In a genetic analysis of an F₂ population derived from a cross between Tanpo and Akitakomachi, the Tanpo GW5 allele was found to determine the grain shape in a recessive manner. The GW5 allele in Tanpo is a loss-of-function allele because it generates a stop codon immediately after the start codon with a 100-bp deletion within the first exon. Because the GW5 protein suppresses glycogen synthase kinase 2 (GSK2), a negative regulator of brassinosteroid (BR) signaling, GW5 deficiency in Tanpo results in reduced BR signaling. As a result, the expansion of epidermal cells was suppressed, while the radial cell division was promoted, which led to thicker and shorter spikelet hulls and, ultimately, the characteristic grain shape of Tanpo. The identification of this unique allele in the Tanpo landrace provides a valuable resource for breeding new rice varieties with unique grain characteristics.