Hidetaka Nishida

Molecular characterization of South and East Asian melon, Cucumis melo L., and the origin of Group Conomon var. makuwa and var. conomon revealed by RAPD analysis

The genetic diversity and relationship among South and East Asian melon Cucumis melo L. were studied by using RAPD analysis of 69 accessions of melon from India, Myanmar, China, Korea, and Japan. The genetic diversity was large in India, and quite small in Group Conomon var. makuwa and var. conomon from East Asia, clearly indicating a decrease in genetic variation from India toward the east. Cluster analysis based on genetic distance classified 17 groups of accessions into two major clusters: cluster I comprising 12 groups of accessions from India and Myanmar and cluster II that included five groups of accessions of Group Conomon var. makuwa and var. conomon from East Asia. Cluster I was further divided into three subclusters, of which subclusters Ib and Ic included small- and large-seed type populations, respectively. Therefore, this division was based on their seed size, not cultivation area. The large-seed type from east India was differently included in the subcluster of small-seed type (Ib). A total of 122 plants of 69 accessions were classified into three major clusters and subclusters: clusters I and II comprised melon accessions mostly from India and Myanmar, and cluster III comprised Group Conomon var. makuwa and var. conomon from East Asia. The frequency of large- and small-seed types was different between clusters I and II, also indicating genetic differentiation between large- and small-seed types. One plant of the small-seed type from east India was differently included in cluster III, and two plants from east India were classified into subcluster IV. These results clearly showed that South Asian melon is genetically differentiated by their seed size, and that small-seed type melon in east India is closely related to Group Conomon var. makuwa and var. conomon.

Phytochrome C Is A Key Factor Controlling Long-Day Flowering in Barley

A red/far-red light photoreceptor controls flowering time in barley by stimulating a flowering promoter gene in the most downstream part of the genetic pathways for flowering. The spring-type near isogenic line (NIL) of the winter-type barley (Hordeum vulgare ssp. vulgare) var. Hayakiso 2 (HK2) was developed by introducing VERNALIZATION-H1 (Vrn-H1) for spring growth habit from the spring-type var. Indo Omugi. Contrary to expectations, the spring-type NIL flowered later than winter-type HK2. This phenotypic difference was controlled by a single gene, which cosegregated only with phytochrome C (HvPhyC) among three candidates around the Vrn-H1 region (Vrn-H1, HvPhyC, and CASEIN KINASE IIα), indicating that HvPhyC was the most likely candidate gene. Compared with the late-flowering allele HvPhyC-l from the NIL, the early-flowering allele HvPhyC-e from HK2 had a single nucleotide polymorphism T1139C in exon 1, which caused a nonsynonymous amino acid substitution of phenylalanine at position 380 by serine in the functionally essential GAF (3′, 5′-cyclic-GMP phosphodiesterase, adenylate cyclase, formate hydrogen lyase activator protein) domain. Functional assay using a rice (Oryza sativa) phyA phyC double mutant line showed that both of the HvPhyC alleles are functional, but HvPhyC-e may have a hyperfunction. Expression analysis using NILs carrying HvPhyC-e and HvPhyC-l (NIL [HvPhyC-e] and NIL [HvPhyC-l], respectively) showed that HvPhyC-e up-regulated only the flowering promoter FLOWERING LOCUS T1 by bypassing the circadian clock genes and flowering integrator CONSTANS1 under a long photoperiod. Consistent with the up-regulation, NIL (HvPhyC-e) flowered earlier than NIL (HvPhyC-l) under long photoperiods. These results implied that HvPhyC is a key factor to control long-day flowering directly.

Distribution of photoperiod-insensitive alleles Ppd-B1a and Ppd-D1a and their effect on heading time in Japanese wheat cultivars

The genotypes of photoperiod response genes Ppd-B1 and Ppd-D1 in Japanese wheat cultivars were determined by a PCR-based method, and heading times were compared among genotypes. Most of the Japanese wheat cultivars, except those from the Hokkaido region, carried the photoperiod-insensitive allele Ppd-D1a, and heading was accelerated 10.3 days compared with the Ppd-D1b genotype. Early cultivars with Ppd-D1a may have been selected to avoid damage from preharvest rain. In the Hokkaido region, Ppd-D1a frequency was lower and heading date was late regardless of Ppd-D1 genotype, suggesting another genetic mechanism for late heading in Hokkaido cultivars. In this study, only 11 cultivars proved to carry Ppd-B1a, and all of them carried another photoperiod-insensitive allele, Ppd-D1a. The Ppd-B1a/Ppd-D1a genotype headed 6.7 days earlier than the Ppd-B1b/Ppd-D1a genotype, indicating a significant effect of Ppd-B1a in the genetic background with Ppd-D1a. Early-maturity breeding in Japan is believed to be accelerated by the introduction of the Ppd-B1a allele into medium-heading cultivars carrying Ppd-D1a. Pedigree analysis showed that Ppd-B1a in three extra-early commercial cultivars was inherited from ‘Shiroboro 21’ by early-heading Chugoku lines bred at the Chugoku Agriculture Experimental Station.

Molecular analysis of genetic diversity in melon landraces (Cucumis melo L.) from Myanmar and their relationship with melon germplasm from East and South Asia

Genetic diversity of Myanmar melon was evaluated by analysis of 27 RAPD markers and morphological characters using 41 accessions of melon landraces of which 36 accessions were small-seed type. The gene diversity was 0.239, higher than for group Conomon from East Asia and equivalent to Indian melon populations. Melon accessions were classified into six major clusters. The largest cluster IV comprised mainly group Conomon which was closely related to cluster V consisting of mainly group Agrestis. Most of the accessions of group Cantalupensis were grouped into clusters II or VII and were distantly related to groups Conomon and Agrestis. The genetic relationship to melon accessions from neighboring countries was analyzed. The 24 accessions of clusters IV and V were mostly clustered together with small-seed type melon of India, but the 14 accessions of clusters VI and VII were mostly clustered together with large-seed type melon of India. These results indicated that the genetic diversity of Indian melon is conserved in Myanmar. Genetic introgression among melon groups through spontaneous hybridization was also indicated and was considered important to maintain or increase the genetic diversity in Myanmar.

Diversification and genetic differentiation of cultivated melon inferred from sequence polymorphism in the chloroplast genome.

Molecular analysis encouraged discovery of genetic diversity and relationships of cultivated melon (Cucumis melo L.). We sequenced nine inter- and intra-genic regions of the chloroplast genome, about 5500 bp, using 60 melon accessions and six reference accessions of wild species of Cucumis to show intra-specific variation of the chloroplast genome. Sequence polymorphisms were detected among melon accessions and other Cucumis species, indicating intra-specific diversification of the chloroplast genome. Melon accessions were classified into three subclusters by cytoplasm type and then into 12 subgroups. Geographical origin and seed size also differed between the three subclusters. Subcluster Ia contained small-seed melon from Southern Africa and South and East Asia and subcluster Ib mainly consisted of large-seed melon from northern Africa, Europe and USA. Melon accessions of subcluster Ic were only found in West, Central and Southern Africa. Our results indicated that European melon groups and Asian melon groups diversified independently and shared the same maternal lineage with northern African large-seed melon and Southern African small-seed melon, respectively. Cultivated melon of subcluster Ic may have been domesticated independently in Africa. The presence of 11 cytoplasm types in Africa strongly supported African origin of cultivated melon and indicated the importance of germplasm from Africa.

Distribution of photoperiod-insensitive allele Ppd-A1a and its effect on heading time in Japanese wheat cultivars

The Ppd-A1 genotype of 240 Japanese wheat cultivars and 40 foreign cultivars was determined using a PCR-based method. Among Japanese cultivars, only 12 cultivars, all of which were Hokkaido winter wheat, carried the Ppd-A1a allele, while this allele was not found in Hokkaido spring wheat cultivars or Tohoku-Kyushu cultivars. Cultivars with a photoperiod-insensitive allele headed 6.9–9.8 days earlier in Kanto and 2.5 days earlier in Hokkaido than photoperiod-sensitive cultivars. The lower effect of photoperiod-insensitive alleles observed in Hokkaido could be due to the longer day-length at the spike formation stage compared with that in Kanto. Pedigree analysis showed that ‘Purple Straw’ and ‘Tohoku 118’ were donors of Ppd-A1a and Ppd-D1a in Hokkaido wheat cultivars, respectively. Wheat cultivars recently developed in Hokkaido carry photoperiod-insensitive alleles at a high frequency. For efficient utilization of Ppd-1 alleles in the Hokkaido wheat-breeding program, the effect of Ppd-1 on growth pattern and grain yield should be investigated. Ppd-A1a may be useful as a unique gene source for fine tuning the heading time in the Tohoku-Kyushu region since the effect of Ppd-A1a on photoperiod insensitivity appears to differ from the effect of Ppd-B1a and Ppd-D1a.

Loss-of-Function Mutations in Three Homoeologous PHYTOCLOCK 1 Genes in Common Wheat Are Associated with the Extra-Early Flowering Phenotype

Triticum aestivum L. cv ‘Chogokuwase’ is an extra-early flowering common wheat cultivar that is insensitive to photoperiod conferred by the photoperiod insensitive alleles at the Photoperiod-B1 (Ppd-B1) and Ppd-D1loci, and does not require vernalization for flowering. This reduced vernalization requirement is likely due to the spring habitat allele Vrn-D1 at the VERNALIZATION-D1 locus. Genotypes of the Ppd-1 loci that determine photoperiod sensitivity do not fully explain the insensitivity to photoperiod seen in ‘Chogokuwase’. We detected altered expression patterns of clock and clock-output genes including Ppd-1 in ‘Chogokuwase’ that were similar to those in an einkorn wheat mutant that lacks the clock-gene homologue, wheat PHYTOCLOCK 1 (WPCL1). Presumptive loss-of-function mutations in all WPCL1 homoeologous genes were found in ‘Chogokuwase’ and ‘Geurumil’, one of the parental cultivars. Segregation analysis of the two intervarietal F2 populations revealed that all the examined F2 plants that headed as early as ‘Chogokuwase’ had the loss-of-function wpcl1 alleles at all three homoeoloci. Some F2 plants carrying the wpcl1 alleles at three homoeoloci headed later than ‘Chogokuwase’, suggesting the presence of other loci influencing heading date. Flowering repressor Vrn-2 was up-regulated in ‘Chogokuwase’ and ‘Geurumil’ that had the triple recessive wpcl1 alleles. An elevated transcript abundance of Vrn-2 could explain the observation that ‘Geurumil’ and some F2 plants carrying the three recessive wpcl1 homeoealleles headed later than ‘Chogokuwase’. In spite of the up-regulation of Vrn-2, ‘Chogokuwase’ may have headed earlier due to unidentified earliness genes. Our observations indicated that loss-of-function mutations in the clock gene wpcl1 are necessary but are not sufficient to explain the extra-early heading of ‘Chogokuwase’.

Development of RAPD-Derived STS Markers for Genetic Diversity Assessment in Melon (Cucumis melo L.)

Random amplified polymorphic DNA (RAPD) has been used widely in diversity studies, including population structure and phylogenetics at all taxonomic levels. However, there is a problem in stability and repeatability of RAPD in some cases. Therefore, conversion of RAPD markers into new type of PCR-based marker to overcome low levels of repeatability of RAPD marker is needed. The aim of this study was to develop sequence-tagged site (STS) markers by designing specific primers based on RAPD marker sequences to provide the potential markers for analyzing genetic diversity of melon germplasm. Eight RAPD-STS markers were successfully converted from RAPD markers and have two polymorphism types: A20 and B99 showed different sizes of fragment; A22, A31, A57, B15, B71 and C00 showed presence/absence polymorphism in melon germsplasm. The applicability of new RAPD-STS markers has been demonstrated by comparing genotype analysis of 41 melon accessions using RAPD and RAPD-STS markers. Both of RAPD markers and RAPD-STS markers divided them into two major clusters. However, the RAPD-STS markers were more polymorphic than RAPD markers (polymorphic index content (PIC) values were 0.346 and 0.274, respectively). Mantels test showed significant correlation (r = 0.896, P < 0.01) between RAPD-STS dendrogram and RAPD dendrogram. Furthermore, RAPD-STS markers could give more information in population structure and identify admixture individuals by using STRUCTURE software. Eight RAPD-STS markers developed in this study are useful for genetic diversity analysis and population studies in melon.