Molecular genetic mapping of a high-lysine mutant gene (<Emphasis Type="Italic">opaque-16</Emphasis>) and the double recessive effect with <Emphasis Type="Italic">opaque-2</Emphasis> in maize

The lysin content in maize endosperm protein is considered to be one of the most important traits for determining the nutritional quality of food and feed. Improving the protein quality of the maize kernel depends principally on finding a mutant with a higher lysine content. Two high-lysine mutant lines with opaque endosperm, QCL3024 and QCL3021, were isolated from a self-cross population derived from Robertsons Mutator stocks. The gene controlling this mutation is temporarily termed opaque-16 (o16). In order to illuminate the genetic locus and effect of the o16 gene, two F_2:3 populations, one developed from a cross between QCL3024 and QCL3010 (a wild type line) and another from a cross between Qi205 (opaque-2 line) and QCL3021, were created, and F₃ seeds from the F₂ plants in the two populations were evaluated for lysine content. The distributions of lysine content and tests for their normality indicate that the lysine content in the two populations is regulated by the major gene of o16 and genes of o2 and o16, respectively. Based on two data sets of the linkage maps of the F₂ plant marker genotypes and the lysine content of F₃ seeds originating from the two F_2:3 populations, the o16 gene was located within 5 cM, at either 3 or 2.2 cM from umc1141 in the interval between umc1121 and umc1141 on the long arm of chromosome 8, depending on the recombination rate in the two populations as determined by composite interval mapping. According to the data of the F_2:3 population constructed from the o2 and o16 lines, the double recessive mutant effect was analyzed. The average lysine content of the F₃ o2o2o16o16 families identified by the umc1066 and umc1141 markers was approximately 30% higher than that of the F₃ o2o2 and o16o16 families, respectively. The lysine content of seven F₃ families among nine F₃ double recessive mutant families showed different increments, with an average increase of some 6% compared with that of the maternal o2 line. The potential application of the o16 mutant for maize high-lysine breeding may be to combine it with the o2 mutant bearing modifier genes, thus obtaining a mutant with much higher lysine content. For the purpose of pyramiding the o16 with o2 genes, the availability of closely linked markers of the o16 and o2 loci will facilitate marker-assisted selection and greatly reduce breeding time and effort.     

Characterization and genetic analysis of a low-temperature-sensitive mutant, <Emphasis Type="Italic">sy</Emphasis>-<Emphasis Type="Italic">2</Emphasis>, in <Emphasis Type="Italic">Capsicum chinense</Emphasis>

A temperature-sensitive mutant of Capsicum chinense, sy-2, shows a normal developmental phenotype when grown above 24°C. However, when grown at 20°C, sy-2 exhibits developmental defects, such as chlorophyll deficiency and shrunken leaves. To understand the underlying mechanism of this temperature-dependent response, phenotypic characterization and genetic analysis were performed. The results revealed abnormal chloroplast structures and cell collapse in leaves of the sy-2 plants grown at 20°C. Moreover, an excessive accumulation of reactive oxygen species (ROS) resulting in cell death was detected in the chlorophyll-deficient sectors of the leaves. However, the expression profile of the ROS scavenging genes did not alter in sy-2 plants grown at 20°C. A further analysis of fatty acid content in the leaves showed the impaired pathway of linoleic acid (18:2) to linolenic acid (18:3). Additionally, the Cafad7 gene was downregulated in sy-2 plants. This change may lead to dramatic physiological disorder and alteration of leaf morphology in sy-2 plants by losing low-temperature tolerance. Genetic analysis of an F₂ population from a cross between C. chinense ‘sy-2’ and wild-type C. chinense ‘No. 3341’ showed that the sy-2 phenotype is controlled by a single recessive gene. Molecular mapping revealed that the sy-2 gene is located at a genomic region of the pepper linkage group 1, corresponding to the 300 kb region of the Ch1_scaffold 00106 in tomato chromosome 1. Candidate genes in this region will reveal the identity of sy-2 and the underlying mechanism of the temperature-dependent plant response.     

Fine genetic mapping of greenbug aphid-resistance gene <Emphasis Type="Italic">Gb3</Emphasis> in <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

The greenbug, Schizaphis graminum (Rondani), is an important aphid pest of small grain crops especially wheat (Triticum aestivum L., 2n = 6x = 42, genomes AABBDD) in many parts of the world. The greenbug-resistance gene Gb3 originated from Aegilops tauschii Coss. (2n = 2x = 14, genome D^tD^t) has shown consistent and durable resistance against prevailing greenbug biotypes in wheat fields. We previously mapped Gb3 in a recombination-rich, telomeric bin of wheat chromosome arm 7DL. In this study, high-resolution genetic mapping was carried out using an F_2:3 segregating population derived from two Ae. tauschii accessions, the resistant PI 268210 (original donor of Gb3 in the hexaploid wheat germplasm line ‘Largo’) and susceptible AL8/78. Molecular markers were developed by exploring bin-mapped wheat RFLPs, SSRs, ESTs and the Ae. tauschii physical map (BAC contigs). Wheat EST and Ae. tauschii BAC end sequences located in the deletion bin 7DL3-0.82–1.00 were used to design STS (sequence tagged site) or CAPS (Cleaved Amplified Polymorphic Sequence) markers. Forty-five PCR-based markers were developed and mapped to the chromosomal region spanning the Gb3 locus. The greenbug-resistance gene Gb3 now was delimited in an interval of 1.1 cM by two molecular markers (HI067J6-R and HI009B3-R). This localized high-resolution genetic map with markers closely linked to Gb3 lays a solid foundation for map based cloning of Gb3 and marker-assisted selection of this gene in wheat breeding.     

Fine genetic mapping of <Emphasis Type="Italic">xa24</Emphasis>, a recessive gene for resistance against <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating plant bacterial disease worldwide. Different bacterial blight resistance (R) genes confer race-specific resistance to different strains of Xoo. We fine mapped a fully recessive gene, xa24, for bacterial blight resistance to a 71-kb DNA fragment in the long arm of rice chromosome 2 using polymerase chain reaction-based molecular markers. The xa24 gene confers disease resistance at the seedling and adult stages. It mediates resistance to at least the Philippine Xoo races 4, 6 and 10 and Chinese Xoo strains Zhe173, JL691 and KS-1-21. Sequence analysis of the DNA fragment harboring the dominant (susceptible) allele of xa24 suggests that this gene should encode a novel protein that is not homologous to any known R proteins. These results will greatly facilitate the isolation and characterization of xa24. The markers will be convenient tools for marker-assisted selection of xa24 in breeding programs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fine genetic mapping localizes cucumber scab resistance gene <Emphasis Type="Italic">Ccu</Emphasis> into an <Emphasis Type="Italic">R</Emphasis> gene cluster

Scab, caused by Cladosporium cucumerinum, is an important disease of cucumber, Cucumis sativus. In this study, we conducted fine genetic mapping of the single dominant scab resistance gene, Ccu, with 148 F₉ recombinant inbred lines (RILs) and 1,944 F₂ plants derived from the resistant cucumber inbred line 9110Gt and the susceptible line 9930, whose draft genome sequence is now available. A framework linkage map was first constructed with simple sequence repeat markers placing Ccu into the terminal 670 kb region of cucumber Chromosome 2. The 9110Gt genome was sequenced at 5× genome coverage with the Solexa next-generation sequencing technology. Sequence analysis of the assembled 9110Gt contigs and the Ccu region of the 9930 genome identified three insertion/deletion (Indel) markers, Indel01, Indel02, and Indel03 that were closely linked with the Ccu locus. On the high-resolution map developed with the F₂ population, the two closest flanking markers, Indel01 and Indel02, were 0.14 and 0.15 cM away from the target gene Ccu, respectively, and the physical distance between the two markers was approximately 140 kb. Detailed annotation of the 180 kb region harboring the Ccu locus identified a cluster of six resistance gene analogs (RGAs) that belong to the nucleotide binding site (NBS) type R genes. Four RGAs were in the region delimited by markers Indel01 and Indel02, and thus were possible candidates of Ccu. Comparative DNA analysis of this cucumber Ccu gene region with a melon (C. melo) bacterial artificial chromosome (BAC) clone revealed a high degree of micro-synteny and conservation of the RGA tandem repeats in this region.     

Fine mapping of a male sterility gene <Emphasis Type="Italic">ms</Emphasis>-<Emphasis Type="Italic">3</Emphasis> in a novel cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) mutant

Key message

The cucumber male sterility gene ms - 3 was fine mapped in a 76 kb region harboring an MMD1 -like gene Csa3M006660 that may be responsible for the male sterile in cucumber.

Abstract

A cucumber (Cucumis sativus L.) male sterile mutant (ms-3) in an advanced-generation inbred line was identified, and genetic analysis revealed that the male sterility trait was controlled by a recessive nuclear gene, ms-3, which was stably inherited. Histological studies suggested that the main cause of the male sterility was defective microsporogenesis, resulting in no tetrad or microspores being formed. Bulked segregant analysis (BSA) and genotyping of an F₂ population of 2553 individuals were employed used to fine map ms-3, which was delimited to a 76 Kb region. In this region, a single non-synonymous SNP was found in the Csa3M006660 gene locus, which was predicted to result in an amino acid change. Quantitative RT-PCR analysis of Csa3M006660 was consistent with the fact that it plays a role in the early development of cucumber pollen. The protein encoded by Csa3M006660 is predicted to be homeodomain (PHD) finger protein, and the high degree of sequence conservation with homologs from a range of plant species further suggested the importance of the ms-3 non-synonymous mutation. The data presented here provide support for Csa3M006660 as the most likely candidate gene for Ms-3.

     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Structural organization characteristics of the <Emphasis Type="Italic">DIP1</Emphasis> gene in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> strains mutant for the <Emphasis Type="Italic">flamenco</Emphasis> gene

Molecular cloning of the DIP1 gene located in the 20A4-5 region has been performed from the following strains with the flamenco phenotype: flam ^SS (SS) and flam ^MS (MS) characterized by a high transposition rate of retrotransposon gypsy (mdg4), flam ^{py + (P)} carrying the insertion of a construction based on the P element into the region of the flamenco gene, and flamenco ⁺. The results of restriction analysis and sequencing cloned DNA fragments has shown that strains flam ^SS , flam ^MS considerably differ from flam ^{py + (P)}, and flamenco ⁺ in the structure of DIP1. Strains flam ^SS and flam ^MS have no DraI restriction site at position 1765 in the coding region of the gene, specifically, in the domain determining the signal of the nuclear localization of the DIP1 protein. This mutation has been found to consist in a nucleotide substitution in the recognition site of DraI restriction endonuclease, which is transformed from TTTAAA into TTTAAG and, hence, is not recognized by the enzyme. This substitution changes codon AAA into AAG and is translationally insignificant, because both triplets encode the same amino acid, lysine. The DIP1 gene of strains flam ^SS and flam ^MS has been found to contain a 182-bp insertion denoted IdSS (insertion in DIP1 strain SS); it is located in the second intron of the gene. The IdSS sequence is part of the open reading frame encoding the putative transposase of the mobile genetic element HB1 belonging to the Tc1/mariner family. This insertion is presumed to disturb the conformations of DNA and the chromosome, in particular, by forming loops, which alters the expression of DIP1 and, probably, neighboring genes. In strains flamenco ⁺ and flam ^{py + (P)}, the IdSS insertion within the HB1 sequence is deleted. The deletion encompasses five C-terminal amino acid residues of the conserved domain and the entire C-terminal region of the putative HB1 transposase. The obtained data suggest that DIP1 is involved in the control of gypsy transpositions either directly or through interaction with other elements of the genome.     

High-resolution mapping and gene prediction of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> resistance gene <Emphasis Type="Italic">Xa7</Emphasis>

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F₂ mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F₂ individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes. Shen Chen and Zhanghui Huang are contributed equally to this work.     

Characterization and fine mapping of <Emphasis Type="Italic">osh15</Emphasis>(<Emphasis Type="Italic">t</Emphasis>), a novel dwarf mutant gene in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plant height is one of the most important agronomic traits of plant architecture, and also affects grain yield in rice. In this study, we obtained a novel dwarf rice mutant of japonica variety Shennong9816, designated Shennong9816d. Compared with wild-type, the Shennong9816d plant height was significantly reduced, and the tiller number significantly increased. Additionally, the mutant yield component, and the number of large and small vascular bundles were significantly decreased compared with wild-type. Genetic analysis indicated that the Shennong9816d dwarf phenotype was controlled by a recessive nuclear gene, while the plant was shown to be sensitive to gibberellic acid. Using a large F₂ population derived from a cross between Shennong9816d and the indica rice variety Habataki, the osh15(t) gene was fine mapped between RM20891 and RM20898, within a physical distance of 73.78 kb. Sequencing analysis showed that Shennong9816d carries a 1 bp mutation and a 30 bp insertion in the OSH15 region. These results suggest that osh15(t) is a novel allelic mutant originally derived from japonica variety Shennong9816, which may be useful for introducing the semi-dwarf phenotype to improve plant architecture in rice breeding practice.     

Identification and fine mapping of a stay-green gene (<Emphasis Type="Italic">Brnye1</Emphasis>) in pakchoi (<Emphasis Type="Italic">Brassica campestris</Emphasis> L. ssp. <Emphasis Type="Italic">chinensis</Emphasis>)

Key message

Using bulked segregant analysis combined with next-generation sequencing, we delimited the Brnye1 gene responsible for the stay-green trait of nye in pakchoi. Sequence analysis identified Bra019346 as the candidate gene.

Abstract

“Stay-green” refers to a plant trait whereby leaves remain green during senescence. This trait is useful in the cultivation of pakchoi (Brassica campestris L. ssp. chinensis), which is marketed as a green leaf product. This study aimed to identify the gene responsible for the stay-green trait in pakchoi. We identified a stay-green mutant in pakchoi, which we termed “nye”. Genetic analysis revealed that the stay-green trait is controlled by a single recessive gene, Brnye1. Using the BSA-seq method, a 3.0-Mb candidate region was mapped on chromosome A03, which helped us localize Brnye1 to an 81.01-kb interval between SSR markers SSRWN27 and SSRWN30 via linkage analysis in an F₂ population. We identified 12 genes in this region, 11 of which were annotated based on the Brassica rapa annotation database, and one was a functionally unknown gene. An orthologous gene of the Arabidopsis gene AtNYE1, Bra019346, was identified as the potential candidate for Brnye1. Sequence analysis revealed a 40-bp insertion in the second exon of Bra019346 in nye, which generated the TAA stop codon. A candidate gene-specific Indel marker in 1561 F₂ individuals showed perfect cosegregation with Brnye1 in the nye mutant. These results provide a foundation for uncovering the molecular mechanism of the stay-green trait in pakchoi.

     

Revealing physiological and genetic properties of a dominant maize dwarf <Emphasis Type="Italic">Dwarf11</Emphasis> (<Emphasis Type="Italic">D11</Emphasis>) by integrative analysis

Plant height is an important agronomic trait involved in lodging resistance and harvest index. The identification and characterization of mutants that are defective in plant height have implications for trait improvement in breeding programs. Two dominant maize dwarf mutants D8 and D9 have been well-characterized. Here, we report the characterization of a dominant maize dwarf mutant Dwarf11 (D11). Dwarf stature of D11 was mainly attributed to the inhibition of longitudinal cell elongation. The levels of bioactive GA₃ were significantly lower in D11. Contrarily, D8 mutant accumulates markedly higher levels of GA₃. The expression of GA biosynthetic and catabolic genes was dramatically decreased in D11. Expression variations of d8 and d9 genes were not observed in D11 mutant. Moreover, genetic suppressors of D11 were identified in inbred line Chang 7-2. Integrated omics data indicated that D11 is a novel dominant maize dwarf. The ultimate D11 gene cloning and its regulatory network elucidation may strengthen our understanding of the genetic basis of plant architecture and provide cues for breeding of crops with plant height ideotypes.     

Identification of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> in maize in northeastern China

Bremia lactucae Regel (Chromista, Peronosporaceae) is an economically destructive pathogen, which causes downy mildew disease on lettuce (Lactuca sativa L.) worldwide. The ribosomal internal transcribed spacer (ITS) of Bremia lactucae isolates was analyzed for the first time. The ITS region of lettuce downy mildew was observed to have a size of 2458 bp; thereby, having one of the longest ITS sizes recorded to date. The majority of the extremely large sized ITS2 length of 2086 was attributed to the additional presences of nine repetitive elements with lengths of 179–194 bp, which between them shared the low homology of 48–69%. Comparison of the ITS2 sequences with the B. lactucae isolates from other host plants showed that isolates present on Lactuca sativa were distinct from those on L. indica var. laciniata, as well as Hemistepta and Youngia. We suggest the high degree of sequence heterogeneity exhibited in the ITS2 region of B. lactucae may warrant the specific detection and diagnosis of this destructive pathogen or its division into several distinct species.     

Fine mapping of a male sterility gene <Emphasis Type="Italic">MS-cd1</Emphasis> in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

A dominant male sterility (DGMS) line 79-399-3, developed from a spontaneous mutation in Brassica oleracea var. capitata, has been widely used in production of hybrid cultivars in China. In this line, male sterility is controlled by a dominant gene Ms-cd1. In the present study, fine mapping of Ms-cd1 was conducted by screening a segregating population Ms79-07 with 2,028 individuals developed by four times backcrossing using a male sterile Brassica oleracea var. italica line harboring Ms-cd1 as donor and Brassica oleracea var. alboglabra as the recipient. Bulked segregation analysis (BSA) was performed for the BC₄ population Ms79-07 using 26,417 SRAP primer SRAPs and 1,300 SSRs regarding of male sterility and fertility. A high-resolution map surrounding Ms-cd1 was constructed with 14 SRAPs and one SSR. The SSR marker 8C0909 was closely linked to the MS-cd1 gene with a distance of 2.06 cM. Fourteen SRAPs closely linked to the target gene were identified; the closest ones on each side were 0.18 cM and 2.16 cM from Ms-cd1. Three of these SRAPs were successfully converted to dominant SCAR markers with a distance to the Ms-cd1 gene of 0.18, 0.39 and 4.23 cM, respectively. BLAST analysis with these SCAR marker sequences identified a collinear genomic region about 600 kb in scaffold 000010 on chromosomeA10 in B. rapa and on chromosome 5 in A. thaliana. These results provide additional information for map-based cloning of the Ms-cd1 gene and will be helpful for marker-assisted selection (MAS).     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.