<Emphasis Type="Italic">S</Emphasis> locus-linked F-box genes expressed in anthers of <Emphasis Type="Italic">Hordeum bulbosum</Emphasis>

Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S ₃ haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S ₃) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB511822–AB511825 and AB511859–AB511862.     

A new genetic locus for self-compatibility in the outcrossing grass species perennial ryegrass (Lolium perenne)

BackgroundSelf-incompatibility (SI) is a physiological mechanism that many flowering plants employ to prevent self-fertilization and maintain heterozygosity. In the grass family this is known to be controlled by a two locus (S-Z) system; however, the SI system is intrinsically leaky. Modifier genes of both the S and Z loci and a further locus, T, are known to override SI leading to self-fertilization and self-seed production. This has implications for the ecological and evolutionary success as well as the commercial breeding of grasses. Here we report a study where the genetic control of self-compatibility (SC) was determined from the results of self-pollinating an F₂ population of perennial ryegrass from two independently derived inbred lines produced by single-seed descent.Methods In vitro self-pollinations of 73 fertile plants were analysed. A genetic association analysis was made with a panel of 1863 single-nucleotide polymorphism (SNP) markers, generated through genotype-by-sequencing methodology. Markers were placed on a recombination map of seven linkage groups (LGs) created using Joinmap v.5. The seed set on self- and open-pollinated inflorescences was determined on 143 plants, including the 73 plants analysed for self-pollination response.Key ResultsSelf-pollinations revealed a bimodal distribution of percentage SC with peaks at 50 and 100 %. A single quantitative trait locus (QTL) was identified with peak association for marker 6S14665z17875_11873 that mapped to LG 6. Peak position was associated with maximum marker segregation distortion. The self-compatible plants were equally fecund after self- and open pollination.ConclusionsThis is the first report in the Poaceae family of an SC locus located on LG 6. This new SC QTL discovery, as well as indicating the complex nature of the pollen–stigma recognition process and its evolutionary significance, provides an additional source of SC for breeding perennial ryegrass.     

Implications of the Plastid Genome Sequence of Typha (Typhaceae, Poales) for Understanding Genome Evolution in Poaceae

Plastid genomes of the grasses (Poaceae) are unusual in their organization and rates of sequence evolution. There has been a recent surge in the availability of grass plastid genome sequences, but a comprehensive comparative analysis of genome evolution has not been performed that includes any related families in the Poales. We report on the plastid genome of Typha latifolia, the first non-grass Poales sequenced to date, and we present comparisons of genome organization and sequence evolution within Poales. Our results confirm that grass plastid genomes exhibit acceleration in both genomic rearrangements and nucleotide substitutions. Poaceae have multiple structural rearrangements, including three inversions, three genes losses (accD, ycf1, ycf2), intron losses in two genes (clpP, rpoC1), and expansion of the inverted repeat (IR) into both large and small single-copy regions. These rearrangements are restricted to the Poaceae, and IR expansion into the small single-copy region correlates with the phylogeny of the family. Comparisons of 73 protein-coding genes for 47 angiosperms including nine Poaceae genera confirm that the branch leading to Poaceae has significantly accelerated rates of change relative to other monocots and angiosperms. Furthermore, rates of sequence evolution within grasses are lower, indicating a deceleration during diversification of the family. Overall there is a strong correlation between accelerated rates of genomic rearrangements and nucleotide substitutions in Poaceae, a phenomenon that has been noted recently throughout angiosperms. The cause of the correlation is unknown, but faulty DNA repair has been suggested in other systems including bacterial and animal mitochondrial genomes.     

High-resolution linkage map in the vicinity of the Lp locus

Looptail (Lp) is a mutation that profoundly affects neurulation in mouse and is characterized by craniorachischisis, an open neural tube extending from the midbrain to the tail in embryos homozygous for the mutation. Lp maps to the distal portion of mouse chromosome 1, and as part of a positional cloning approach, we have generated a high-resolution linkage map of the Lp chromosomal region. For this, we have carried out extensive segregation analysis in a total of 706 backcross mice informative for Lp and derived from two crosses, (Lp/ + X SJL/J)F1 X SJL/J and (Lp/ + X SWR/J)F1 X SWR/J. In addition, 269 mice from a (Mus spretus X C57BL/6J)F1 X C57BL/6J interspecific backcross were also used to order marker loci and calculate intergene distances for this region. With these mice, a total of 28 DNA markers corresponding to either cloned genes or anonymous markers of the SSLP or SSCP-types were mapped within a 5-cM interval overlapping the Lp region, with the following locus order and interlocus distances (in cM): centromere-D1Mit110 / Atp1β1 / Cd3ζ / Cd3η / D1Mit145 — D1Hun14 / D1Mit15 — D1Mit111 / D1Mit112 — D1Mit114 — D1Mit148 / D1Mit205/ D1Mit36 / D1Mit146 / D1Mit147 / D1Mit270 / D1Hun13 — Fcgr2 — Mpp — Apoa2/Fcer1γ - Lp - D1Mit149 / Spna1/Fcer1α-Eph1-Hlx1/D1Mit62. These studies have allowed the delineation of a maximum genetic interval for Lp of 0.5 cM, a size amenable to physical mapping techniques.     

Fine genetic mapping of <Emphasis Type="Italic">cp</Emphasis>: a recessive gene for compact (dwarf) plant architecture in cucumber, <Emphasis Type="Italic">Cucumis sativus</Emphasis> L.

The compact (dwarf) plant architecture is an important trait in cucumber (Cucumis sativus L.) breeding that has the potential to be used in once-over mechanical harvest of cucumber production. Compact growth habit is controlled by a simply inherited recessive gene cp. With 150 F_2:3 families derived from two inbred cucumber lines, PI 308915 (compact vining) and PI 249561 (regular vining), we conducted genome-wide molecular mapping with microsatellite (simple sequence repeat, SSR) markers. A framework genetic map was constructed consisting of 187 SSR loci in seven linkage groups (chromosomes) covering 527.5 cM. Linkage analysis placed cp at the distal half of the long arm of cucumber Chromosome 4. Molecular markers cosegregating with the cp locus were identified through whole genome scaffold-based chromosome walking. Fine genetic mapping with 1,269 F₂ plants delimited the cp locus to a 220 kb genomic DNA region. Annotation and function prediction of genes in this region identified a homolog of the cytokinin oxidase (CKX) gene, which may be a potential candidate of compact gene. Alignment of the CKX gene homologs from both parental lines revealed a 3-bp deletion in the first exon of PI 308915, which can serve as a marker for marker-assisted selection of the compact phenotype. This work also provides a solid foundation for map-based cloning of the compact gene and understanding the molecular mechanisms of the dwarfing in cucumber.     

Identification and fine mapping of <Emphasis Type="Italic">S-d</Emphasis>, a new locus conferring the partial pollen sterility of intersubspecific F<Subscript>1</Subscript> hybrids in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The partial pollen abortion of hybrids between the indica and japonica subspecies of Asian cultivated rice is one of the major barriers in utilizing intersubspecific heterosis in hybrid rice breeding. Although a single hybrid pollen sterility locus may have little impact on spikelet fertility, the cumulative effect of several loci usually leads to a serious decrease in spikelet fertility. Isolating of the genes conferring hybrid pollen sterility is necessary to understand this phenomenon and to overcome the resulting genetic barrier. In this study, a new locus for F₁ pollen sterility, S-d, was identified on the short arm of chromosome 1 by analyzing the genetic effect of substituted segments of the near-isogenic line E11-5 derived from the japonica variety Taichung 65 (recurrent parent) and the indica variety Dee-geo-woo-gen (donor parent). The S-d locus was first mapped to a 0.8 cM interval between SSR markers PSM46 and PSM80 using a F₂ population of 125 individuals. The flanking markers were then used to identify recombinants from a population of 2,160 plants derived from heterozygotes of the primary F₂ population. Simultaneously, additional markers were developed from genomic sequence divergence in this region. Analysis of the recombinants in the region resulted in the successful mapping of the S-d locus to a 67-kb fragment, containing 17 predicted genes. Positional cloning of this gene will contribute to our understanding of the molecular basis for partial pollen sterility of intersubspecific F₁ hybrids in rice.     

Disentangling the effects of breakdown of self-incompatibility and transition to selfing in North American Arabidopsis lyrata

A breakdown of self‐incompatibility (SI) followed by a shift to selfing is commonly observed in the evolution of flowering plants. Both are expected to reduce the levels of heterozygosity and genetic diversity. However, breakdown of SI should most strongly affect the region of the SI locus (S‐locus) because of the relaxation of balancing selection that operates on a functional S‐locus, and a potential selective sweep. In contrast, a transition to selfing should affect the whole genome. We set out to disentangle the effects of breakdown of SI and transition to selfing on the level and distribution of genetic diversity in North American populations of Arabidopsis lyrata. Specifically, we compared sequence diversity of loci linked and unlinked to the S‐locus for populations ranging from complete selfing to fully outcrossing. Regardless of linkage to the S‐locus, heterozygosity and genetic diversity increased with population outcrossing rate. High heterozygosity of self‐compatible individuals in outcrossing populations suggests that SI is not the only factor preventing the evolution of self‐fertilization in those populations. There was a strong loss of diversity in selfing populations, which was more pronounced at the S‐locus. In addition, selfing populations showed an accumulation of derived mutations at the S‐locus. Our results provide evidence that beyond the genome‐wide consequences of the population bottleneck associated with the shift to selfing, the S‐locus of A. lyrata shows a specific signal either reflecting the relaxation of balancing selection or positive selection.     

Physical size of the <Emphasis Type="Italic">S</Emphasis> locus region defined by genetic recombination and genome sequencing in <Emphasis Type="Italic">Ipomoea trifida</Emphasis>, Convolvulaceae

Sporophytic self-incompatibility (SSI) in the genus Ipomoea (Convolvulaceae) is controlled by a single polymorphic S locus. We have previously analyzed genomic sequences of an approximately 300 kb region spanning the S locus of the S ₁ haplotype and characterized the genomic structure around this locus. Here, we further define the physical size of the S locus region by mapping recombination breakpoints, based on sequence analysis of PCR fragments amplified from the genomic DNA of recombinants. From the recombination analysis, the S locus of the S ₁ haplotype was delimited to a 0.23 cM region of the linkage map, which corresponds to a maximum physical size of 212 kb. To analyze differences in genomic organization between S haplotypes, fosmid contigs spanning approximately 67 kb of the S ₁₀ haplotype were sequenced. Comparison with the S ₁ genomic sequence revealed that the S haplotype-specific divergent regions (SDRs) spanned 50.7 and 34.5 kb in the S ₁ and S ₁₀ haplotypes, respectively and that their flanking regions showed a high sequence similarity. In the sequenced region of the S ₁₀ haplotype, five of the 12 predicted open reading frames (ORFs) were found to be located in the divergent region and showed co-linear organization of genes between the two S haplotypes. Based on the size of the SDRs, the physical size of the S locus was estimated to fall within the range 34–50 kb in Ipomoea.     

High-resolution mapping of the S and Z loci of Phalaris coerulescens.

Self incompatibility (SI) in Phalaris coerulescens is gametophytically determined by two unlinked multi allelic loci (S and Z). Neither the S nor Z genes have yet been cloned. As part of a map-based cloning strategy, high-resolution maps of the S and Z regions were generated from distorted segregating populations using RFLP probes from wheat, barley, oat, and Phalaris. The S locus was delimited to 0.26 cM with two boundary markers (Xwg811 and Xpsr168) and cosegregated with Xbm2 and Xbcd762. Xbcd266 was the closest marker linked to Z (0.9 cM). A high level of colinearity in the S and Z regions was found in both self-incompatible and -compatible species. The S locus was localized to the subcentromere region of chromosome 1 and the Z locus to the long arm end of chromosome 2. Several rice BAC clones orthologous to the S and Z locus regions were identified. This opens the possibility of using the rice genome sequence data to generate more closely linked markers and identify SI candidate genes. These results add further support to the conservation of gene order in the S and Z regions of the grass genomes.     

The genetic architecture of tristyly and its breakdown to self‐fertilization

The floral polymorphism tristyly involves three style morphs with a reciprocal arrangement of stigma and anther heights governed by two diallelic loci (S and M). Tristyly functions to promote cross‐pollination, but modifications to stamen position commonly cause transitions to selfing. Here, we integrate whole‐genome sequencing and genetic mapping to investigate the genetic architecture of the M locus and the genetic basis of independent transitions to selfing in tristylous Eichhornia paniculata. We crossed independently derived semi‐homostylous selfing variants of the long‐ and mid‐styled morph fixed for alternate alleles at the M locus (ssmm and ssMM, respectively), and backcrossed the F₁ to the parental ssmm genotype. We phenotyped and genotyped 462 backcross progeny using 1450 genotyping‐by‐sequencing (GBS) markers and performed composite interval mapping to identify quantitative trait loci (QTL) governing style‐length and anther‐height variation. A QTL associated with the primary style‐morph differences (style length and anther height) mapped to linkage group 5 and spanned ~13–27.5 Mbp of assembled sequence. Bulk segregant analysis identified 334 genes containing SNPs potentially linked to the M locus. The stamen modifications characterizing each selfing variant were governed by loci on different linkage groups. Our results provide an important step towards identifying the M locus and demonstrate that transitions to selfing have originated by independent sets of mating‐system modifier genes unlinked to the M locus, a pattern inconsistent with a recombinational origin of selfing variants at a putative supergene.     

Cloning and characterisation of a family of disease resistance gene analogs from wheat and barley

 The most common class of plant disease resistance (R) genes cloned so far belong to the NBS-LRR group which contain nucleotide-binding sites (NBS) and a leucine-rich repeat (LRR). Specific primer sequences derived from a previously isolated NBS-LRR sequence at the Cre3 locus, which confers resistance to cereal cyst nematode (CCN) in wheat (Triticum aestivum L.) were used in isolating a family of resistance gene analogs (RGA) through a polymerase chain reaction (PCR) cloning approach. The cloning, analysis and genetic mapping of a family of RGAs from wheat (cv ‘Chinese Spring’) and barley (Hordeum vulgare L. cvs ‘Chebec’ and ‘Harrington’) are presented. The wheat and barley RGAs contain other conserved motifs present in known R genes from other plants and share between 55–99% amino acid sequence identity to the NBS-LRR sequence at the Cre3 locus. Phylogenetic analysis of the RGAs with other cloned R genes and RGAs from various plant species indicate that they belong to a superfamily of NBS-containing genes. Two of the barley derived RGAs were mapped onto loci on chromosomes 2H (2), 5H (7) and 7H (1) using barley doubled haploid (DH) mapping populations. Some of these loci identified are associated with regions carrying resistance to CCN and corn leaf aphid. Received: 6 January 1998 / Accepted: 1 April 1998     

Development of SNP markers linked to the <Emphasis Type="Italic">L</Emphasis> locus in <Emphasis Type="Italic">Capsicum</Emphasis> spp. by a comparative genetic analysis

In pepper, the TMV resistance locus L is syntenic to the tomato I2 and the potato R3 loci on chromosome 11. In this report, we identified pepper bacterial artificial chromosome (BAC) clones corresponding to the I2 and R3 loci and developed L-linked markers using the BAC sequence information. A BAC library was screened using the tomato I2C-1 gene as a probe. The resulting clones were sorted further by PCR screening, sequencing, and genetic mapping. A linkage analysis revealed that BAC clone 082F03 could be anchored to the target region near TG36 on chromosome 11. Using the 082F03 sequence, more BAC clones were identified and a BAC contig spanning 224 kb was constructed. Gene prediction analysis showed that there were at least three I2/R3 R gene analogs (RGAs) in the BAC contig. Three DNA markers closely linked (about 1.2 cM) to the L ⁴ gene were developed by using the BAC contig sequence. The single nucleotide polymorphism marker 087H3T7 developed in this study was subjected to linkage analysis in L ⁴ - and L ³ -segregating populations together with previously developed markers. The 189D23M marker, which is known to co-segregate with L ³ , was located on the opposite side of 087H3T7, about 0.7 cM away from L ⁴ . This supports the idea that L ³ and L ⁴ may be different genes closely linked within the region instead of different alleles at the same locus. Finally, use of flanking markers in molecular breeding program for introgression of L ⁴ to elite germplasm against most aggressive tobamoviruses pathotype P_1,2,3 is discussed.     

Comparative genetic analysis of a wheat seed dormancy QTL with rice and <Emphasis Type="Italic">Brachypodium</Emphasis> identifies candidate genes for ABA perception and calcium signaling

Wheat preharvest sprouting (PHS) occurs when seed germinates on the plant before harvest resulting in reduced grain quality. In wheat, PHS susceptibility is correlated with low levels of seed dormancy. A previous mapping of quantitative trait loci (QTL) revealed a major PHS/seed dormancy QTL, QPhs.cnl-2B.1, located on wheat chromosome 2B. A comparative genetic study with the related grass species rice (Oryza sativa L.) and Brachypodium distachyon at the homologous region to the QPhs.cnl-2B.1 interval was used to identify the candidate genes for marker development and subsequent fine mapping. Expressed sequence tags and a comparative mapping were used to design 278 primer pairs, of which 22 produced polymorphic amplicons that mapped to the group 2 chromosomes. Fourteen mapped to chromosome 2B, and ten were located in the QTL interval. A comparative analysis revealed good macrocollinearity between the PHS interval and 3 million base pair (mb) region on rice chromosomes 7 and 3, and a 2.7-mb region on Brachypodium Bd1. The comparative intervals in rice were found to contain three previously identified rice seed dormancy QTL. Further analyses of the interval in rice identified genes that are known to play a role in seed dormancy, including a homologue for the putative Arabidopsis ABA receptor ABAR/GUN5. Additional candidate genes involved in calcium signaling were identified and were placed in a functional protein association network that includes additional proteins critical for ABA signaling and germination. This study provides promising candidate genes for seed dormancy in both wheat and rice as well as excellent molecular markers for further comparative and fine mapping.     

Infantile hypertrophic pyloric stenosis: evaluation of three positional candidate genes, <Emphasis Type="Italic">TRPC1</Emphasis>, <Emphasis Type="Italic">TRPC5</Emphasis> and <Emphasis Type="Italic">TRPC6</Emphasis>, by association analysis and re-sequencing

Infantile hypertrophic pyloric stenosis (IHPS) is the most common inherited form of gastrointestinal obstruction in infancy with a striking male preponderance. Infants present with vomiting due to gastric outlet obstruction caused by hypertrophy of the smooth muscle of the pylorus. Two loci specific to extended pedigrees displaying autosomal dominant inheritance have been identified. A genome scan identified loci on chromosomes 11q14–q22 and Xq23–q24 which are predicted to be responsible for a subset of smaller families with IHPS demonstrating non-Mendelian inheritance. The two linked chromosomal regions both harbour functional candidate genes which are members of the canonical transient receptor potential (TRPC) family of ion channels. Both TRPC5 (Xq23–q24) and TRPC6 (11q14–q22) have a potential role in smooth muscle control and hypertrophy. Here, we report suggestive evidence for a third locus on chromosome 3q12–q25 (Z _max = 2.7, p < 0.004), a region which harbours a third TRPC gene, TRPC1. Fine mapping of all three genes using a tagSNP approach and re-sequencing identified a SNP in the promoter region of TRPC6 and a missense variant in exon 4 of TRPC6 which may be putative causal variants.     

High-resolution mapping, cloning and molecular characterization of the Pi-k h gene of rice, which confers resistance to Magnaporthe grisea

In order to understand the molecular mechanisms involved in the gene-for-gene type of pathogen resistance, high-resolution genetic and physical mapping of resistance loci is required to facilitate map-based cloning of resistance genes. Here, we report the molecular mapping and cloning of a dominant gene (Pi-k ^h ) present in the rice line Tetep, which is associated with resistance to rice blast disease caused by Magnaporthe grisea. This gene is effective against M. grisea populations prevalent in the Northwestern Himalayan region of India. Using 178 sequence tagged microsatellite, sequence-tagged site, expressed sequence tag and simple sequence repeat (SSR) markers to genotype a population of 208 F₂ individuals, we mapped the Pi-k ^h gene between two SSR markers (TRS26 and TRS33) which are 0.7 and 0.5 cM away, respectively, and can be used in marker-assisted-selection for blast-resistant rice cultivars. We used the markers to identify the homologous region in the genomic sequence of Oryza sativa cv. Nipponbare, and a physical map consisting of two overlapping bacterial artificial chromosome and P1 artificial chromosome clones was assembled, spanning a region of 143,537 bp on the long arm of chromosome 11. Using bioinformatic analyses, we then identified a candidate blast-resistance gene in the region, and cloned the homologous sequence from Tetep. The putative Pi-k ^h gene cloned from Tetep is 1.5 kbp long with a single ORF, and belongs to the nucleotide binding site-leucine rich repeat class of disease resistance genes. Structural and expression analysis of the Pi-k ^h gene revealed that its expression is pathogen inducible.     

Construction of a BAC Contig for a 3 cM Biologically Significant Region of Mouse Chromosome 1

One QTL and genes and phenotypes have been localized in the region between 92 cM and 95cM of mouse chromosome 1. The QTL locus contributes to approximately 40% of the variation of the peak bone density between C57BL/6J (B6) and CAST/EiJ (CAST) strains. Other loci located in this chromosomal region include a neural tube defect mutant loop-tail (Lp), a lymphocyte-stimulating determinant (Lsd), and the Transgelin 2 (Tagln 2). The human chromosome region homologous to this region is 1q21-23, which also contains a QTL locus for high bone mineral density (BMD). Furthermore, it has been reported that this region may have duplicated several times in the mouse genome. Therefore, genomic sequencing of this region will provide important information for mouse genome structure, for positional cloning of mouse genes, and for the study of human homologous genes. In order to provide a suitable template for genomic sequencing by the NIH-sponsored genomic centers, we have constructed a BAC contig of this region using the RPCI-23 library. We have also identified the currently available mouse genomic sequences localized in our BAC contig. Further analysis of these sequences and BAC clones indicated a high frequency of repetitive sequences within this chromosomal area. This region also contains L1 retrotransposon sequences, providing a potential mechanism for the repetitive sequences described in the literature.     

Cloning of molecular markers for disease resistance in sunflower, Helianthus annuus L.

 A candidate-gene approach to analyse the resistance of plants to phytopathogenic fungi is presented. The resistance of sunflower (Helianthus annuus L.) to downy mildew (Plasmopara halstedii) shows a gene-for-gene interaction (monogenic resistance), whereas resistance to white rot (Sclerotinia sclerotiorum) is quantitative, with different levels of resistance for different plant parts. By homology cloning, probes were obtained homologous to some plant resistance genes (nucleotide binding site-like, NBS, genes and serine-threonine protein kinase-like, PK, genes). These clones were used as probes for linkage mapping of the corresponding genes. It was demonstrated that at least three NBS-like loci are located on linkage-group 1, in the region where downy mildew resistance loci have been described. Quantitative trait loci for S. sclerotiorum resistance to penetration or extension of the mycelium in different tissues were studied in three crosses. Major QTLs for resistance were found on linkage group 1, with up to 50% of the phenotypic variability explained by peaks at the map position of the PK locus, 25 cM from the downy mildew loci. Received: 24 September 1997 / Accepted: 21 October 1997