Phylogenetic relationships of Aurantioideae (Rutaceae) based on RAD-Seq

The economically and nutritionally important genus Citrus belongs to the subfamily Aurantioideae in the family Rutaceae. Here, we analyzed the phylogenetic relationships of the subfamily Aurantioideae based on RAD-Seq. The RAD-Seq data produced phylogenetic trees with high support values, clear discriminations based on branch length, and elucidations of early branching events. Our genetic classification corresponded well with the classical morphological classification system and supported the subdivision of Citreae, one of two tribes of the Aurantioideae, into three subtribes—Triphasiinae, Citrinae, and Balsamocitrinae. Additionally, it was largely consistent with the subdivision of Clauseneae, the other tribe of the Aurantioideae, into three subtribes—Micromelinae, Clauseninae, and Merrillinae; the exception was Murraya paniculata. With the exception of members of primitive citrus fruit trees, namely, Severinia buxifolia and Hesperethusa crenulata, lower-level morphological groupings under subtribes based on genetic and morphological classifications corresponded well. The phylogenetic relationship between Asian “true citrus fruit trees” (genera Citrus, Poncirus, and Fortunella) and Australian/New Guinean citrus fruit trees (genera Microcitrus, Eremocitrus, and Clymenia) was inconsistent between present classification based mainly on the nuclear genome and the previous classification based on the chloroplast genome. This inconsistency may be explained by chloroplast capture. Our findings provide a valuable insight into the genetic relationships of the subfamily Aurantioideae in the family Rutaceae.     

Association analysis of the glutelin synthesis genes <Emphasis Type="Italic">GluA</Emphasis> and <Emphasis Type="Italic">GluB1</Emphasis> in a <Emphasis Type="Italic">Japonica</Emphasis> rice collection

Glutelin is the most significant seed storage protein and is regarded as an important nutrient quality trait in rice. Research on the genetic basis of the glutelin content distinction in rice will provide more choices for the diets of people with kidney disease and diabetes. The GluA and GluB1 genes play important roles in the process of glutelin synthesis. In this study, 128 Japonica rice accessions with wide geographic distributions were collected to construct the association panel. Among all the 128 accessions, both sequences of the GluA and GluB1 genes were obtained, and nucleotide polymorphisms were detected. A total of 46 SNPs and eight InDels, six SNPs and four InDels were found in the GluA and GluB1 gene sequences, respectively. Eight haplotypes and two haplotypes were classified based on the SNPs in the coding region of the GluA and GluB1 genes, respectively. Moreover, the association of the polymorphic sites in the two genes with glutelin content in the tested population was estimated. The results revealed that five SNPs in the GluA gene, one SNP and one InDel in the GluB1 gene were associated with glutelin content at a significant level (P < 0.01). Corresponding markers were also designed to check the alleles of GluA and GluB1 genes. These results suggested that polymorphisms in the GluA and GluB1 genes in rice could be utilized in molecular marker-assisted selection to improve the nutrient quality of rice breeding programmes.     

Genetics and fine mapping of a yellow-green leaf gene (<Emphasis Type="Italic">ygl</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis> L.)

Cabbage (Brassica oleracea var. capitata L.) is one of the most popular cultivated vegetables worldwide. Cabbage has rich phenotypic diversity, including plant height, head shape, head color, leaf shape and leaf color. Leaf color plays an important role in cabbage growth and development. At present, there are few reports on fine mapping of leaf color mutants in B. oleracea. In this study, a naturally occurring yellow-green leaf cabbage mutant (YL-1), derived from the self-pollinated progenies of the hybrid ‘Hosom’, was used for inheritance analysis and gene mapping. Segregation populations including F₂ and BC₁ were generated from the cross of two inbred lines, YL-1 and 01–20. Genetic analysis with the F₂ and BC₁ populations demonstrated that the yellow-green leaf color was controlled by a single recessive nuclear gene, ygl-1. Insertion–deletion (InDel) markers, designed based on the parental re-sequencing data, were used for the preliminary mapping with BSA (bulked segregant analysis) method. A genetic map constructed with 15 InDels indicated that ygl-1 was located on chromosome C01. The ygl-1 gene is flanked by InDel markers ID2 and M8, with genetic distances of 0.4 cM and 0.35 cM, respectively. The interval distance between two markers is 167 kb. Thus, it enables us to locate the ygl-1 gene for the first time in B. oleracea. This study lays the foundation for candidate gene prediction and ygl-1gene cloning.     

Larvae of the exotic predatory ladybird <Emphasis Type="Italic">Platynaspidius maculosus</Emphasis> (Coleoptera: Coccinellidae) on citrus trees: prey aphid species and behavioral interactions with aphid-attending ants in Japan

An aphidophagous ladybird, Platynaspidius maculosus (Weise) (Coleoptera: Coccinellidae), is originally distributed in China, Taiwan, and Vietnam. The ladybird has recently intruded into the southern and central parts of Japan. The present study found that the larvae of this ladybird preyed on three aphid species, Aphis spiraecola, Aphis gossypii, and Toxoptera citricidus (all Hemiptera: Aphididae), feeding on young shoots of various Citrus species in August to early October in Shizuoka Prefecture, central Japan. Laboratory rearing of the sampled larvae confirmed that the larvae completed their development (adult emergence) by consuming each of the three aphid species. The ladybird larvae were observed foraging in aphid colonies attended by one of the four ants, Lasius japonicus, Pristomyrmex punctatus, Formica japonica, and Camponotus japonicus (all Hymenoptera: Formicidae). Field observations revealed that the foraging/feeding larvae were almost completely ignored by honeydew-collecting ants even when they physically contacted each other. Thus, in Japan, the larvae of the exotic ladybird exploit colonies of the three aphid species attended by one of the four ant species on many Citrus species. On the basis of the results, I discuss the possibility of the ladybird’s reproduction on citrus trees in Japan, probable adaptations of the ladybird larvae to aphid-attending ants, and potential impacts of the ladybird on native insect enemies attacking ant-attended aphids on citrus.     

Development of a large number of SSR and InDel markers and construction of a high-density genetic map based on a RIL population of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Capsicum annuum, the most widely cultivated species of pepper, is used worldwide for its important nutritional and medicinal values. The construction of an intraspecific high-density genetic linkage map would be of practical value for pepper breeding. However, the numbers of PCR-based simple sequence repeat (SSR) and insertion/deletion (InDel) markers that are available are limited, and there is a need to develop a saturated, intraspecific linkage map. The non-redundant Capsicum species’ expressed sequence tag (EST) database from the National Center for Biotechnology Information was used in this study to develop a total of 902 usable EST-SSR markers. Additionally, 177,587 SSR loci were identified based on the pepper genomic information, including 9182 SSR loci 500 bp both upstream and downstream of coding regions. Another 4497 stable and reliable InDel loci were also developed. From 9182 SSR and 4497 InDel loci, 3356 pairs of genomic SSR primers and 1400 pairs of InDel primers that were evenly distributed in 12 chromosomes were selected. A high-density intraspecific genetic map of C. annuum was constructed using the F₁₀-generation recombinant inbred line of parents PM702 and FS871 as the mapping population, screening the selected 3356 pairs of genomic SSR primers and 1400 pairs of InDel primers and the 902 EST-SSR markers developed earlier, and 524 published SSR markers and 299 orthologous markers (including 263 COSII markers and 36 tomato-derived markers) used previously to develop an interspecific genetic map (C. annuum × C. frutescens). Eventually, a high-density complete genetic intraspecific linkage map of C. annuum containing 12 linkage groups and 708 molecular markers with a length of 1260.00 cM and an average map distance of 1.78 cM was produced. This intraspecific, high-density, complete genetic linkage map of C. annuum contains the largest number of SSR and InDel markers and the highest amount of saturation so far, and it will be of considerable significance for the breeding of improved cultivars of this important field crop in the future.     

A novel set of EST-InDel markers in <Emphasis Type="Italic">Eucalyptus</Emphasis> L’Hérit.: polymorphisms,cross-species amplification,physical positions and genetic mapping

Insertion/deletion (InDel) markers are valuable for genetic applications in plant species, and the public databases of expressed sequence tags (ESTs) have facilitated the development of genic InDel markers. In this study, we developed a novel set of 144 InDel markers in an important tree genus Eucalyptus L’Hérit. using the ESTs of GenBank. Amplicon sequencing against two parents of a mapping population (Eucalyptus urophylla S. T. Blake × E. tereticornis Smith) revealed that the InDel size ranged from 2 to 44 bases, and the dinucleotide type was the most abundant (37.3 %). The cross-species/subgenus amplification rate ranged from 62.5 % in E. tessellaris F. Muell. (subgenus Blakella) to 99.3 % in E. grandis Hill ex Maiden (subgenus Symphyomyrtus) with an average of 85.4 %. There were 121 EST-InDels (84.0 %) polymorphic among 12 individuals of E. grandis, and the mean number of alleles per polymorphic locus (N _a), observed heterozygosity (H _o), expected heterozygosity (H _e) and polymorphic information content (PIC) were 4.0, 0.278, 0.538 and 0.465, respectively. Physical positions of 143 EST-InDels were predicted on the E. grandis genome sequence. A total of 81 EST-InDels were incorporated into prior dense genetic maps of E. urophylla and E. tereticonis, and extensive synteny and colinearity were observed between E. grandis genome sequence and the mapped EST-InDel markers. These EST-InDels will provide a valuable resource of functional markers for genetic diversity evaluation, genome comparison, QTL mapping and marker-assisted breeding in Eucalyptus.     

Nucleotide polymorphisms of candidate genes of adaptive significance in the ural populations of <Emphasis Type="Italic">Larix sibirica</Emphasis> Ledeb.

The objectives of conservation and sustainable forest management require in depth study of genomes of woody plants and definition of their intraspecific genetic diversity. In recent years, an approach was developed based on the study of “candidate genes” that can potentially be involved in the formation of adaptive traits. In this study, we investigated nucleotide polymorphism of several adaptive candidate genes in the populations of Siberian larch (Larix sibirica Ledeb.) in the Urals. Representatives of this genus are among the most valuable and widely distributed forest tree species in Russia. From ten selected gene loci in the genome of L. sibirica, we isolated and investigated three loci, one of which (ABA-WDS) was sequenced in L. sibirica for the first time. The total length of the analyzed sequence in each individual amounted to 2865 bp. The length of locus alignment was from 360 bp to 1395 bp. In total, we identified 200 polymorphic positions. The most conservative is locus 4CL1-363, and the most polymorphic is locus sSPcDFD040B03103-274. The studied populations of L. sibirica are characterized by a high level of nucleotide polymorphism in comparison with other species and genuses (Picea, Pinus, Pseudotsuga, Abies) conifers plants (Hd = 0.896; π = 0.007; θ_W = 0.015). The most selectively neutral polymorphism (D _T =–0.997) was attributed to locus 4CL1-363, and polymorphism with high probability of adaptability (D _T =–1.807) was determined for the ABA-WDS locus. We identified 54 SNP markers, only five of which were nonsynonymous (9.26%) replacements. The average frequency of SNPs in the three studied loci of L. sibirica was one SNP in 53 bp. We detected unique SNP markers for eight populations, which could potentially be used to identify populations. Populations that are characterized by the highest number of unique SNP markers can be recommended for selection in order to preserve the gene pool of the species.     

Genome re-sequencing of two accessions and fine mapping the locus of <Emphasis Type="Italic">lobed leaflet margins</Emphasis> in mungbean

Mungbean (Vigna radiata L.) is a fast-growing warm-season legume crop widely distributed in Asia. A large amount of self-shading of mungbean plants could reduce its seed yield. Thus, modification of the leaflet type could affect the leaf canopy and improve yield. In this study, a classical locus, lobed leaflet margins (lma) in mungbean, was investigated, which was controlled by a semi-dominant allele. In order to map the lma, the whole genomes of AL127 carrying lma and another accession Sulu with oval leaflets were re-sequenced; 236,998 single nucleotide polymorphisms and 8896 insertion/deletions (InDels) were identified between the two accessions. Using the validated InDels and SNP markers, lma was mapped to a syntenic region about 376 kb on chromosome 3 in mungbean and on chromosome 1 in common bean, respectively. Our results provided a framework for map-based gene cloning in mungbean. Cloning the lma should shed light on the underlying molecular mechanism controlling leaf shape in legumes and further provide the molecular basis for genetic improvement on legume crops.     

Phytochemistry and bioactivity of <Emphasis Type="Italic">Citrus</Emphasis> flavonoids: a focus on antioxidant,anti-inflammatory,anticancer and cardiovascular protection activities

Epidemiological studies have suggested an inverse relationship between increased consumption of fruits and reduced risk of chronic diseases, such as cardiovascular diseases, cancer, and diabetes. Citrus fruit is one of the mostly consumed fruits worldwide, and numerous studies have revealed its remarkable health-promoting activities, such as antioxidant, anticancer, anti-inflammatory, and cardiovascular protection activities. These activities largely depend upon the diverse chemical constituents of Citrus fruits, including vitamins, minerals, terpenoids, and flavonoids. Notably, dietary flavonoids occurring in Citrus fruits have attracted growing interest due to their distinct beneficial effects on human health. In this review, we outlined the main health-related properties of Citrus flavonoids, with a focus on antioxidant, anticancer, anti-inflammation, and cardiovascular protection activities. Also the bioavailability, a critical factor that influences the biological efficacy, of Citrus flavonoids was discussed. It was believed that insights about these advances may encourage researchers to discover new phytochemical components and further study specific bioactivities from Citrus fruits.     

Map-based cloning of the dominant genic male sterile <Emphasis Type="Italic">Ms</Emphasis>-<Emphasis Type="Italic">cd1</Emphasis> gene in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

Key message

Using map-based cloning, we delimited the Ms - cd1 gene responsible for the male sterile phenotype in B. oleracea to an approximately 39-kb fragment. Expression analysis suggests that a new predicted gene, a homolog of the Arabidopsis SIED1 gene, is a potential candidate gene.

Abstract

A dominant genic male sterile (DGMS) mutant 79-399-3 in Brassica oleracea (B. oleracea) is controlled by a single gene named Ms-cd1, which was genetically mapped on chromosome C09. The derived DGMS lines of 79-399-3 have been successfully applied in hybrid cabbage breeding and commercial hybrid seed production of several B. oleracea cultivars in China. However, the Ms-cd1 gene responsible for the DGMS has not been identified, and the molecular basis of the DGMS is unclear, which then limits its widespread application in hybrid cabbage seed production. In the present study, a large BC₉ population with 12,269 individuals was developed for map-based cloning of the Ms-cd1 gene, and Ms-cd1 was mapped to a 39.4-kb DNA fragment between two InDel markers, InDel14 and InDel24. Four genes were identified in this region, including two annotated genes based on the available B. oleracea annotation database and two new predicted open reading frames (ORFs). Finally, a newly predicted ORF designated Bol357N3 was identified as the candidate of the Ms-cd1 gene. These results will be useful to reveal the molecular mechanism of the DGMS and develop more practical DGMS lines with stable male sterility for hybrid seed production in cabbage.

     

Combination of newly developed SNP and InDel markers for genotyping the <Emphasis Type="Italic">Cf-9</Emphasis> locus conferring disease resistance to leaf mold disease in the tomato

The Cf-9 gene in the tomato is known to confer resistance against leaf mold disease caused by Cladosporium fulvum, and a gene-based marker targeted to the Cf-9 allele has been widely used as a crop protection approach. However, we found this marker to be misleading in genotyping. Therefore, we developed new single-nucleotide polymorphism (SNP) and insertion and deletion (InDel) markers targeted to the Cf-9 allele in order to increase genotyping accuracy and facilitate high-throughput screening. The DNA sequences of reported Cf-9, cf-9, Cf-0, and closely related Cf-4 alleles were compared, and two functional and non-synonymous SNPs were found to distinguish the Cf-9 resistance allele from the cf-9, Cf-0, and Cf-4 alleles. An SNP marker including these two SNPs was developed and applied to the genotyping of 33 tomato cultivars by high-resolution melting analysis. Our SNP marker was able to select all three Cf-9 genotypes (resistant, heterozygous, and susceptible alleles). Interestingly, two cultivars were grouped separately from these three genotypes. To further examine this outgroup, we preformed polymerase chain reaction (PCR) on two InDel regions identified by sequence comparison of the Cf-9 and Cf-4 genes. The band patterns revealed that these two cultivars carried Cf-4 rather than Cf-9 alleles and that three cultivars classified in the Cf-9 resistance group actually carried both Cf-9 and Cf-4 genes. To determine whether these genotyping results were consistent with disease resistance phenotypes, we examined the induction of a hypersensitive response by transiently expressing the corresponding effector genes, and found that the results matched perfectly with the genotyping results. These findings indicate that the combination of our SNP and InDel markers allows resistant Cf-9 alleles to be distinguished from cf-9 and Cf-4 alleles, which will be useful for marker-assisted selection of tomato cultivars resistant to C. fulvum.     

Genetic divergence within the monotypic tree genus <Emphasis Type="Italic">Platycarya</Emphasis> (Juglandaceae) and its implications for species’ past dynamics in subtropical China

Subtropical East Asia harbours a large plant diversity that is often attributed to allopatric speciation in this topographically complex region characterized by a relative climate stability. Here, we use observations of Platycarya, a widespread subtropical Asian tree genus, to explore the consequences of past climate stability on species’ evolutionary history in subtropical China. This genus has a controversial taxonomy: while it is now prevailingly treated as monotypic, two species have been originally described, Platycarya strobilacea and P. longipes. Previous information from species distribution models, fossil pollen data and genetic data based on chloroplast DNA (cpDNA) were integrated with newly obtained genetic data from the two putative species. We used both cpDNA (psbA-trnH and trnL-F intergenic spacers, including a partial trnL gene sequence) and nuclear markers. The latter included sequences of the internal transcribed spacer region (ITS1–5.8S–ITS2) of the nuclear ribosomal DNA and random genomic single nucleotide polymorphisms. Using these nuclear genetic markers, we found interspecific genetic divergence fitting with the ‘two species’ scenario and geographically structured intraspecific variation. Using cpDNA markers, we also found geographically structured intraspecific variation. Despite deep inter- and intraspecific genetic divergence, we detected genetic admixture in southwest China. Overall, our findings of genetic divergence within Platycarya support the hypothesis of allopatric speciation. However, episodes of population interconnection were identified, at least in southwest China, suggesting that the genus has had a dynamic population history.     

Curry leaf smells better than citrus to females of <Emphasis Type="Italic">Diaphorina citri</Emphasis> (Hemiptera: Liviidae)

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama has a host range of about 20 species of the family Rutaceae, including Citrus spp. However, few studies have reported on its host preference. This study evaluated the host-choice behavior of ACP in curry leaf (Murraya koenigii L.), through free-choice test and bioassays with a type ‘Y’ olfactometer, and also characterized the volatiles involved in attracting the ACP. In the free-choice test, the number of adults per plant on curry leaf was higher than the number on citrus plants. When the ACP was tested in the olfactometer, the females showed preference for curry leaf over citrus plants. Sixteen volatile compounds were identified in citrus and curry leaves. Qualitative and quantitative differences in the compounds released by citrus and curry leaves were determined. The volatiles present in these hosts may play an important role in the attraction of D. citri. With this information, further studies should be done to develop new management strategies for the ACP.     

A novel dominant rice male sterility mutant, <Emphasis Type="Italic">OsDMS-1</Emphasis>, simultaneously controlled by independent loci on chromosomes 1, 2, and 3

We found a rice dominant genetic male-sterile mutant OsDMS-1 from tissue culture-regenerated offspring of Zhonghua 11 (japonica rice). Compared to Zhonghua 11, OsDMS-1 mutant anthers were narrow and pale and incapable of pollen release although the glume opened normally. Approximately 81.4% of this mutant pollen was small and malformed and could not be stained by iodine treatment. A paraffin section assay showed delayed degradation of the OsDMS-1 mutant tapetum without starch accumulation in the mutant pollen, ultimately leading to pollen abortion. Classical genetic analysis indicated that only one dominant gene controlled the sterility in the OsDMS-1 mutant. However, molecular mapping suggested three loci simultaneously control male sterility in this mutant: OsDMS-1A (on chromosome 1), flanked by InDel markers C1D4 and C1D5, OsDMS-1B (on chromosome 2), flanked by InDel markers C2D3 and C2D10, and OsDMS-1C (on chromosome 3), flanked by InDel markers 0315 and C3D3. Molecular mapping disagreed with classical genetic analysis regarding the number of genes controlling the OsDMS-1 mutant, indicating a novel mechanism underlying sterility in OsDMS-1. We present two hypotheses to explain this novel inheritance behavior: one is described as Parent-Originated Loci Tying Inheritance (POLTI); while the alternate hypothesis is described as Loci Recombination Lethal (LRL).     

Inheritance and gene mapping of spotted to non-spotted trait gene <Emphasis Type="Italic">CmSp-1</Emphasis> in melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L<Emphasis Type="Italic">.</Emphasis> var. <Emphasis Type="Italic">chinensis</Emphasis> Pangalo)

Melon (Cucumis melo L.) is one of the most popular and highly nutritious vegetable species within Cucurbitaceae. Because appearance is used as an important indicator of quality, the spotted to non-spotted trait associated with this product somewhat influences the buying habits of consumers. We tested a six-generation family to determine the inheritance and genetic basis of this trait. Genetic groups F₁, F₂, BC₁P₁, and BC₁P₂ were from a cross between “IM16559” (non-spotted) and “IM16553” (spotted). Our genetic analysis showed that the spotted to non-spotted trait was controlled by a single dominant gene that we named CmSp-1. Whole-genome resequencing-bulked segregant analysis (WG-BSA) demonstrated that this gene was located on the end of chromosome 2, in the intersections of 22,160,000 to 22,180,000 bp and 22,260,000 to 26,180,000 bp, an interval distance of 3.94 Mb. Insertion-deletion (InDel) markers designed based on WG-BSA data were used to map this gene. Using 13 InDel markers, we produced a genetic map indicating that CmSp-1 was tightly linked to markers I734-2 and I757, with genetic distances of 1.8 and 0.4 cM and an interval distance of 280.872 kb. The closest marker was I757. Testing of 107 different melon genotypes presented an accuracy of 84.11% in predicting the phenotype. By being able to locate CmSp-1 in melon, we can now use the findings to identify potential targets for further marker-assisted breeding and cloning projects.     

Genetic diversity revealed by physiological behavior of citrus genotypes subjected to salt stress

Different physiological behavior of a wide range of varieties and species belonging to the Citrus genus was analyzed when subjected to salt stress with the aim to seek new sources of tolerance that might be specie-specific. Our goal was to use physiological results obtained along a salt stress in order to clarify if it would be possible to associate them with the known citrus genetic diversity. For that purpose, we have selected 20 different genotypes representing the major species on the basis of the genetic diversity of Citrus genus complemented with one intergeneric hybrid Carrizo citrange (C. sinensis × P. trifoliata). A moderate salt stress of 75 mM of NaCl was applied for 12 weeks. For control plants, the main parameters contributing for more than 25 % to the diversity on the two axes of principal component analysis (PCA) were chlorophyll content, photosynthesis and Fv/Fm under light. However, the dispersal of species and varieties on the PCA did not show any particular structure. Under salt stress condition, four parameters (leaf chloride content, leaf chlorophyll content, photosynthesis and stomatal conductance) contributed more specifically to the dispersion on PCA representation with more than 15 % of contribution for each parameter. Large differences were observed within citrus genus: mandarin and pummelo presented good tolerance to salt stress while citron was very sensitive. Furthermore, all secondary genotypes that presented good tolerance to salt tolerance shared mandarin or pummelo as female parent.