Gene prediction for leaf margin phenotype and fruit flesh color in pineapple (Ananas comosus) using haplotype-resolved genome sequencing.

Pineapple (Ananas comosus (L.) Merr.) is one of the most economically important tropical fruit species. The major aim of the breeding programs in several countries, including Japan, is quality improvement, mainly for the fresh market. 'Yugafu', a Japanese cultivar with distinctive pipe-type leaf margin phenotype and white flesh color, is popular for fresh consumption. Therefore, genome sequencing of 'Yugafu' is expected to assist pineapple breeding. Here, we developed a haplotype-resolved assembly for the heterozygous genome of 'Yugafu' using long-read sequencing technology and obtained a pair of 25 pseudomolecule sequences inherited from the parental accessions 'Cream pineapple' and 'HI101'. The causative genes for leaf margin and fruit flesh color were identified. Fine mapping revealed a 162-kb region on CLG23 for the leaf margin phenotype. In this region, 20 kb of inverted repeat was specifically observed in the 'Cream pineapple' derived allele, and the WUSCHEL-related homeobox 3 (AcWOX3) gene was predicted as the key gene for leaf margin morphogenesis. Dominantly repressed AcWOX3 via RNAi was suggested to be the cause of the pipe-type leaf margin phenotype. Quantitative trait locus (QTL) analysis revealed that the terminal region of CLG08 contributed to white flesh and low carotenoid content. Carotenoid cleaved dioxygenase 4 (AcCCD4), a key gene for carotenoid degradation underlying this QTL, was predicted as the key gene for white flesh color through expression analysis. These findings could assist in modern pineapple breeding and facilitate marker-assisted selection for important traits.

Genomic characterization of a rare Carica papaya X chromosome mutant reveals a candidate monodehydroascorbate reductase 4 gene involved in all-hermaphrodite phenomenon.

Sex form is one of the most important characteristics in papaya cultivation in which hermaphrodite is the preferable form. Self-pollination of H*-TSS No.7, an inbred line derived from a rare X chromosome mutant SR*, produced all-hermaphrodite progeny. The recessive lethal allele controlling the all-hermaphrodite phenomenon was proposed to be the recessive Germination suppressor (gs) locus. This study employed next-generation sequencing technology and genome comparison to identify the candidate Gs gene. One specific gene, monodehydroascorbate reductase 4 (MDAR4) harboring a unique polymorphic 3 bp deletion in H*-TSS No.7 was identified. The function of MDAR4 is known to be involved in the hydrogen peroxide (H2O2) scavenging pathway and is associated with seed germination. Furthermore, MDAR4 showed higher expression in the imbibed seeds than that in the dry seeds indicating its potential role in the seed germination. Perhaps this is the very first report providing the evidences that MDAR4 is the candidate of Gs locus in H*-TSS No.7. In addition, Gs allele-specific markers were developed which would be facilitated for breeding all-hermaphrodite lines.

SSR markers developed using next-generation sequencing technology in pineapple, Ananas comosus (L.) Merr.

Simple sequence repeat (SSR) markers provide a reliable tool for the identification of accessions and the construction of genetic linkage maps because of their co-dominant inheritance. In the present study, we developed new SSR markers with next-generation sequencing using the Roche 454 GS FLX+ platform. Five hundred SSR primer sets were tested for the genetic identification of pineapple, including 100 each for the di-, tri-, tetra-, penta-, and hexa-nucleotide motif SSRs. In total, 160 SSR markers successfully amplified fragments and exhibited polymorphism among accessions. The SSR markers revealed the number of alleles per locus (ranging from 2 to 13), the expected heterozygosity (ranging from 0.041 to 0.823), and the observed heterozygosity (ranging from 0 to 0.875). A total of 117 SSR markers with tri- or greater nucleotide motifs were shown to be effective at facilitating accurate genotyping. Using the SSR markers, 25 accessions were distinguished genetically, with the exception of accessions 'MD-2' and 'Yonekura'. The developed SSR markers could facilitate the establishment of efficient and accurate genetic identification systems and the construction of genetic linkage maps in the future.

Long-read bitter gourd (Momordica charantia) genome and the genomic architecture of nonclassic domestication.

The genetic architecture of quantitative traits is determined by both Mendelian and polygenic factors, yet classic examples of plant domestication focused on selective sweep of newly mutated Mendelian genes. Here we report the chromosome-level genome assembly and the genomic investigation of a nonclassic domestication example, bitter gourd (Momordica charantia), an important Asian vegetable and medicinal plant of the family Cucurbitaceae. Population resequencing revealed the divergence between wild and South Asian cultivars about 6,000 y ago, followed by the separation of the Southeast Asian cultivars about 800 y ago, with the latter exhibiting more extreme trait divergence from wild progenitors and stronger signs of selection on fruit traits. Unlike some crops where the largest phenotypic changes and traces of selection happened between wild and cultivar groups, in bitter gourd large differences exist between two regional cultivar groups, likely reflecting the distinct consumer preferences in different countries. Despite breeding efforts toward increasing female flower proportion, a gynoecy locus exhibits complex patterns of balanced polymorphism among haplogroups, with potential signs of selective sweep within haplogroups likely reflecting artificial selection and introgression from cultivars back to wild accessions. Our study highlights the importance to investigate such nonclassic example of domestication showing signs of balancing selection and polygenic trait architecture in addition to classic selective sweep in Mendelian factors.

Genetic diversity and relatedness of mango cultivars assessed by SSR markers.

Assessment of genetic diversity and relatedness is an essential component of germplasm characterization and use. We analyzed 120 mango (Mangifera indica L.) genetic resources in Japan for their parentage, cultivar identification, genetic relatedness, and genetic diversity, using 46 polymorphic simple sequence repeat (SSR) markers. Ten sets of three SSR markers could successfully distinguish 83 genotypes with the exception of synonymous and identical accessions. We successfully assessed parentage, newly identifying or reconfirming both parents of 11 accessions, and revealing over 30 cultivars as offspring of 'Haden'. Genetic relatedness and diversity analyses revealed three distinct clusters. Two clusters correspond to the groups of USA and India, which are closely related. The other includes accessions from Southeast and East Asia. The results agree with the previous identification of genetically distinct Indian and Southeast Asian types, and suggest that the Florida accessions, which originated from hybrids between those two types, are more closely related to the Indian type.

Genome sequencing of the staple food crop white Guinea yam enables the development of a molecular marker for sex determination.

BACKGROUND: Root and tuber crops are a major food source in tropical Africa. Among these crops are several species in the monocotyledonous genus Dioscorea collectively known as yam, a staple tuber crop that contributes enormously to the subsistence and socio-cultural lives of millions of people, principally in West and Central Africa. Yam cultivation is constrained by several factors, and yam can be considered a neglected "orphan" crop that would benefit from crop improvement efforts. However, the lack of genetic and genomic tools has impeded the improvement of this staple crop. RESULTS: To accelerate marker-assisted breeding of yam, we performed genome analysis of white Guinea yam (Dioscorea rotundata) and assembled a 594-Mb genome, 76.4% of which was distributed among 21 linkage groups. In total, we predicted 26,198 genes. Phylogenetic analyses with 2381 conserved genes revealed that Dioscorea is a unique lineage of monocotyledons distinct from the Poales (rice), Arecales (palm), and Zingiberales (banana). The entire Dioscorea genus is characterized by the occurrence of separate male and female plants (dioecy), a feature that has limited efficient yam breeding. To infer the genetics of sex determination, we performed whole-genome resequencing of bulked segregants (quantitative trait locus sequencing [QTL-seq]) in F1 progeny segregating for male and female plants and identified a genomic region associated with female heterogametic (male = ZZ, female = ZW) sex determination. We further delineated the W locus and used it to develop a molecular marker for sex identification of Guinea yam plants at the seedling stage. CONCLUSIONS: Guinea yam belongs to a unique and highly differentiated clade of monocotyledons. The genome analyses and sex-linked marker development performed in this study should greatly accelerate marker-assisted breeding of Guinea yam. In addition, our QTL-seq approach can be utilized in genetic studies of other outcrossing crops and organisms with highly heterozygous genomes. Genomic analysis of orphan crops such as yam promotes efforts to improve food security and the sustainability of tropical agriculture.

Draft genome sequence of bitter gourd (Momordica charantia), a vegetable and medicinal plant in tropical and subtropical regions.

Bitter gourd (Momordica charantia) is an important vegetable and medicinal plant in tropical and subtropical regions globally. In this study, the draft genome sequence of a monoecious bitter gourd inbred line, OHB3-1, was analyzed. Through Illumina sequencing and de novo assembly, scaffolds of 285.5 Mb in length were generated, corresponding to ∼84% of the estimated genome size of bitter gourd (339 Mb). In this draft genome sequence, 45,859 protein-coding gene loci were identified, and transposable elements accounted for 15.3% of the whole genome. According to synteny mapping and phylogenetic analysis of conserved genes, bitter gourd was more related to watermelon (Citrullus lanatus) than to cucumber (Cucumis sativus) or melon (C. melo). Using RAD-seq analysis, 1507 marker loci were genotyped in an F2 progeny of two bitter gourd lines, resulting in an improved linkage map, comprising 11 linkage groups. By anchoring RAD tag markers, 255 scaffolds were assigned to the linkage map. Comparative analysis of genome sequences and predicted genes determined that putative trypsin-inhibitor and ribosome-inactivating genes were distinctive in the bitter gourd genome. These genes could characterize the bitter gourd as a medicinal plant.

Current status of tropical fruit breeding and genetics for three tropical fruit species cultivated in Japan: pineapple, mango, and papaya.

Tropical fruit crops are predominantly produced in tropical and subtropical developing countries, but some are now grown in southern Japan. Pineapple (Ananas comosus), mango (Mangifera indica) and papaya (Carica papaya) are major tropical fruits cultivated in Japan. Modern, well-organized breeding systems have not yet been developed for most tropical fruit species. Most parts of Japan are in the temperate climate zone, but some southern areas such as the Ryukyu Islands, which stretch from Kyushu to Taiwan, are at the northern limits for tropical fruit production without artificial heating. In this review, we describe the current status of tropical fruit breeding, genetics, genomics, and biotechnology of three main tropical fruits (pineapple, mango, and papaya) that are cultivated and consumed in Japan. More than ten new elite cultivars of pineapple have been released with improved fruit quality and suitability for consumption as fresh fruit. New challenges and perspectives for obtaining high fruit quality are discussed in the context of breeding programs for pineapple.

Development of plants resistant to Papaya leaf distortion mosaic virus by intergeneric hybridization between Carica papaya and Vasconcellea cundinamarcensis.

In this study, we confirmed that Vasconcellea cundinamarcensis resists Papaya leaf distortion mosaic virus (PLDMV), and used it to produce intergeneric hybrids with Carica papaya. From the cross between C. papaya and V. cundinamarcensis, we obtained 147 seeds with embryos. Though C. papaya is a monoembryonic plant, multiple embryos were observed in all 147 seeds. We produced 218 plants from 28 seeds by means of embryo-rescue culture. All plants had pubescence on their petioles and stems characteristic of V. cundinamarcensis. Flow cytometry and PCR of 28 plants confirmed they were intergeneric hybrids. To evaluate virus resistance, mechanical inoculation of PLDMV was carried out. The test showed that 41 of 134 intergeneric hybrid plants showed no symptoms and were resistant. The remaining 93 hybrids showed necrotic lesions on the younger leaves than the inoculated leaves. In most of the 93 hybrids, the necrotic lesions enclosed the virus and prevented further spread. These results suggest that the intergeneric hybrids will be valuable material for PLDMV-resistant papaya breeding.

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.

A cytochrome P450, OsDSS1, is involved in growth and drought stress responses in rice (Oryza sativa L.).

Cytochrome P450s are among the largest protein coding gene families in plant genomes. However, majority of the genes remain uncharacterized. Here, we report the characterization of dss1, a rice mutant showing dwarfism and reduced grain size. The dss1 phenotype is caused by a non-synonymous point mutation we identified in DSS1, which is member of a P450 gene cluster located on rice chromosome 3 and corresponds to the previously reported CYP96B4/SD37 gene. Phenotypes of several dwarf mutants characterized in rice are associated with defects in the biosynthesis or perception of the phytohormones gibberellins (GAs) and brassinosteroids (BRs). However, both GA and BR failed to rescue the dss1 phenotype. Hormone profiling revealed the accumulation of abscisic acid (ABA) and ABA metabolites, as well as significant reductions in GA19 and GA53 levels, precursors of the bioactive GA1, in the mutant. The dss1 contents of cytokinin and auxins were not significantly different from wild-type plants. Consistent with the accumulation of ABA and metabolites, germination and early growth was delayed in dss1, which also exhibited an enhanced tolerance to drought. Additionally, expressions of members of the DSS1/CYP96B gene cluster were regulated by drought stress and exogenous ABA. RNA-seq-based transcriptome profiling revealed, among others, that cell wall-related genes and genes involved in lipid metabolism were up- and down-regulated in dss1, respectively. Taken together, these findings suggest that DSS1 mediates growth and stress responses in rice by fine-tuning GA-to-ABA balance, and might as well play a role in lipid metabolism.

Genome sequence comparison reveals a candidate gene involved in male-hermaphrodite differentiation in papaya (Carica papaya) trees.

The sex type of papaya (Carica papaya) is determined by the pair of sex chromosomes (XX, female; XY, male; and XY(h), hermaphrodite), in which there is a non-recombining genomic region in the Y and Y(h) chromosomes. This region is presumed to be involved in determination of males and hermaphrodites; it is designated as the male-specific region in the Y chromosome (MSY) and the hermaphrodite-specific region in the Y(h) chromosome (HSY). Here, we identified the genes determining male and hermaphrodite sex types by comparing MSY and HSY genomic sequences. In the MSY and HSY genomic regions, we identified 14,528 nucleotide substitutions and 965 short indels with a large gap and two highly diverged regions. In the predicted genes expressed in flower buds, we found no nucleotide differences leading to amino acid changes between the MSY and HSY. However, we found an HSY-specific transposon insertion in a gene (SVP like) showing a similarity to the Short Vegetative Phase (SVP) gene. Study of SVP-like transcripts revealed that the MSY allele encoded an intact protein, while the HSY allele encoded a truncated protein. Our findings demonstrated that the SVP-like gene is a candidate gene for male-hermaphrodite determination in papaya.

Mapping of the gynoecy in bitter gourd (Momordica charantia) using RAD-seq analysis.

Momordica charantia is a monoecious plant of the Cucurbitaceae family that has both male and female unisexual flowers. Its unique gynoecious line, OHB61-5, is essential as a maternal parent in the production of F1 cultivars. To identify the DNA markers for this gynoecy, a RAD-seq (restriction-associated DNA tag sequencing) analysis was employed to reveal genome-wide DNA polymorphisms and to genotype the F2 progeny from a cross between OHB61-5 and a monoecious line. Based on a RAD-seq analysis of F2 individuals, a linkage map was constructed using 552 co-dominant markers. In addition, after analyzing the pooled genomic DNA from monoecious or gynoecious F2 plants, several SNP loci that are genetically linked to gynoecy were identified. GTFL-1, the closest SNP locus to the putative gynoecious locus, was converted to a conventional DNA marker using invader assay technology, which is applicable to the marker-assisted selection of gynoecy in M. charantia breeding.

Digital transcriptome analysis of putative sex-determination genes in papaya (Carica papaya).

Papaya (Carica papaya) is a trioecious plant species that has male, female and hermaphrodite flowers on different plants. The primitive sex chromosomes genetically determine the sex of the papaya. Although draft sequences of the papaya genome are already available, the genes for sex determination have not been identified, likely due to the complicated structure of its sex-chromosome sequences. To identify the candidate genes for sex determination, we conducted a transcriptome analysis of flower samples from male, female and hermaphrodite plants using high-throughput SuperSAGE for digital gene expression analysis. Among the short sequence tags obtained from the transcripts, 312 unique tags were specifically mapped to the primitive sex chromosome (X or Y(h)) sequences. An annotation analysis revealed that retroelements are the most abundant sequences observed in the genes corresponding to these tags. The majority of tags on the sex chromosomes were located on the X chromosome, and only 30 tags were commonly mapped to both the X and Y(h) chromosome, implying a loss of many genes on the Y(h) chromosome. Nevertheless, candidate Y(h) chromosome-specific female determination genes, including a MADS-box gene, were identified. Information on these sex chromosome-specific expressed genes will help elucidating sex determination in the papaya.

SuperSAGE: powerful serial analysis of gene expression.

SuperSAGE is a variant of the Serial Analysis of Gene Expression (SAGE) technology, based on counting transcripts by sequencing analysis of short sequence tags. In SuperSAGE, 26 bp tags are extracted from cDNA using the Type III restriction endonuclease EcoP15I. The use of a longer tag size in SuperSAGE allows a secure tag-to-gene annotation in any eukaryotic organism. We have succeeded in combining SuperSAGE and high-throughput sequencing technology (Now- or Next-Generation Sequencing, NGS) in an approach we call High-throughput SuperSAGE (HT-SuperSAGE). This approach allows deep transcriptome analysis and multiplexing, while reducing time, cost, and effort for the analysis. In this chapter, we present the detailed HT-SuperSAGE protocol for both the Illumina Genome Analyzer and also the AppliedBiosystems SOLiD sequencer.

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.