F2 Mapping Population Research Articles

Genome-wide SNP discovery and genetic linkage map construction in sunflower (Helianthus annuus L.) using a genotyping by sequencing (GBS) approach

Recently developed plant genomics approaches (LD mapping and genome-wide selection) require many molecular markers distributed throughout the plant genome. As a result, the availability of an increasing number of markers is essential for maintaining highly efficient and accurate plant breeding programs. In this study, we identified SNP loci in sunflower using a genotyping by sequencing (GBS) approach in an intraspecific F2 mapping population. A total of 271,445,770 reads were generated by the Genome Analyzer II next-generation sequencing platform and 29.2 % of the reads were aligned to unique locations in the genome. A total of 46,278 SNP loci were identified and 7646 SNP loci were validated in an F2 population. In addition, a SNP-based linkage map was constructed. This is the first report of SNP discovery in sunflower by GBS. The SNP markers and SNP-based linkage map will be valuable molecular genetics tools for sunflower breeding.

Sylleptic branching in winter-headed apple (Malus × domestica) trees: accession-dependent responses and their relationships with other tree architectural characteristics

Well-feathered apple trees are essential for commercial orchards to optimize yields. However, most cultivars do not form these sylleptic branches readily in commercial nurseries due to high apical dominance. Several treatments exist to promote their formation in the nurseries, one of which is heading. However, not all cultivars are expected to react similarly to these treatments. We studied the branching response of 155 genotypes following heading and its relation to other architectural traits as a function of the cultivar’s genetic background. Trees were grown for two consecutive years after grafting in a nursery, and the main axes were headed in the winter following growth in year 1. After heading, a single shoot was retained when growth resumed in the second year. Plant architectural traits such as growth of the main shoot, internode length, sylleptic branching, etc. were measured before and after heading and were statistically compared using a mixed model. This model showed the effect of heading for all architectural traits studied. In addition significant genotype and genotype × treatment interactions were found. In general, genotypes that showed a more vigorous growth during the first year also reacted more vigorously to heading. Accounting for the genetic substructure of this collection, no clear distinction in tree response could be found except in a small group of individuals that belonged to an F1 mapping population. This study shows that heading is not favorable for all genotypes to promote sufficient sylleptic branching and that other methods are needed to promote branching in these accessions.

A molecular genome scan to identify DNA segments associated with live weight in Japanese quail.

Japanese quail is an animal model in biological studies and also a commercial bird for eggs and meat production. This study was conducted to map quantitative trait loci (QTL) affecting live weight in Japanese quail. An F2 mapping population was developed by crossing two diverse lines (meat type and egg layer) of Japanese quail. A total number of 34 F1 and 422 F2 progeny were produced by reciprocal crossing of eight pairs of parental birds. All the birds from three generations were genotyped for SSR markers that were spread across all the autosomal linkage groups. The studied traits were hatching weight and live weights at 1-5weeks of age. QTL analysis was conducted by the regression interval mapping. Significant QTL were detected on chromosomes 1, 2, 3 (chromosome-wide significant) and 5 (genome-wide significant, P<0.05) for body weight. Although the additive effect of the detected QTL on chromosome 5 was significant, the dominance and imprinting effects were not significant. This finding is the first report of a genome-wide significant QTL associated with live weight in Japanese quail. Our results point out to candidate DNA regions affecting live weight, a trait of great economic relevance to the Japanese quail breeding. Although these results enhance our current knowledge about the genetic control of live weight in the Japanese quail, it should be noted that the initial QTL results from the experimental designs such as backcross or F2 cannot be applied directly to the breeding programs and require further validation within the commercial lines.

Fine mapping a major obesity locus (jObes1) using a Berlin Fat Mouse × B6N advanced intercross population.

The Berlin Fat Mouse Inbred line 860 is a model for juvenile obesity. Previously, a recessive major effect locus (jObes1) on chromosome 3 between 34 and 44 Mb has been found to be responsible for 39% of the variance of total fat mass at 10 weeks in a (BFMI860 x C57BL/6NCrl) F2 population. The aim of this study was fine mapping of the jObes1 locus. An advanced intercross line (AIL) was generated from the initial F2 mapping population. Three hundred and forty-four male mice of generation 28 were excessively phenotyped and genotyped using the MegaMuga mouse chip containing 22 164 informative single-nucleotide polymorphisms. Expression of candidate genes was investigated in gonadal adipose tissue, liver and whole brain from mice of different genotype classes. Classical genetic complementation tests were performed to test candidate genes. The high mapping resolution of the AIL reduced the confidence interval for jObes1 from 10 to 0.37 Mb between 36.48 and 36.85 Mb. This region was highly significantly (logarithm (base 10) of odds (LOD) score after Benjamini and Hochberg correction (LOD(BH))>50) associated with total fat mass starting at puberty (6 weeks). Male homozygous carriers of the jObese1 BFMI allele had 3 g more fat than the other genotypes. Surprisingly, this genotype class showed lower body mass until weaning at 3 weeks (LOD(BH)=3.2). The mapped interval contains four genes. Bbs7, the most likely candidate gene that also caused obesity in the complementation test was differentially expressed in all tissues examined, whereas the neighboring cyclin A2 (Ccna2) gene showed differential expression in gonadal adipose tissue. Using an AIL, the confidence interval for jObes1 could be 27-fold reduced by finding chromosomal recombinations. Although Bbs7 is the most likely obesity gene in the jObes1 region, neighboring genes cannot be entirely excluded. Further examinations are needed to enlighten the mechanism leading to physiological consequences on body mass and fat mass in juvenile animals.

Identification of two novel powdery mildew resistance loci, Ren6 and Ren7, from the wild Chinese grape species Vitis piasezkii.

BackgroundGrapevine powdery mildew Erysiphe necator is a major fungal disease in all grape growing countries worldwide. Breeding for resistance to this disease is crucial to avoid extensive fungicide applications that are costly, labor intensive and may have detrimental effects on the environment. In the past decade, Chinese Vitis species have attracted attention from grape breeders because of their strong resistance to powdery mildew and their lack of negative fruit quality attributes that are often present in resistant North American species. In this study, we investigated powdery mildew resistance in multiple accessions of the Chinese species Vitis piasezkii that were collected during the 1980 Sino-American botanical expedition to the western Hubei province of China.ResultsA framework genetic map was developed using simple sequence repeat markers in 277 seedlings of an F1 mapping population arising from a cross of the powdery mildew susceptible Vitis vinifera selection F2-35 and a resistant accession of V. piasezkii DVIT2027. Quantitative trait locus analyses identified two major powdery mildew resistance loci on chromosome 9 (Ren6) and chromosome 19 (Ren7) explaining 74.8 % of the cumulative phenotypic variation. The quantitative trait locus analysis for each locus, in the absence of the other, explained 95.4 % phenotypic variation for Ren6, while Ren7 accounted for 71.9 % of the phenotypic variation. Screening of an additional 259 seedlings of the F1 population and 910 seedlings from four pseudo-backcross populations with SSR markers defined regions of 22 kb and 330 kb for Ren6 and Ren7 in the V. vinifera PN40024 (12X) genome sequence, respectively.Both R loci operate post-penetration through the induction of programmed cell death, but vary significantly in the speed of response and degree of resistance; Ren6 confers complete resistance whereas Ren7 confers partial resistance to the disease with reduced colony size. A comparison of the kinetics of induction of powdery mildew resistance mediated by Ren6, Ren7 and the Run1 locus from Muscadinia rotundifolia, indicated that the speed and strength of resistance conferred by Ren6 is greater than that of Run1 which, in turn, is superior to that conferred by Ren7.ConclusionsThis is the first report of mapping powdery mildew resistance in the Chinese species V. piasezkii. Two distinct powdery mildew R loci designated Ren6 and Ren7 were found in multiple accessions of this Chinese grape species. Their location on different chromosomes to previously reported powdery mildew resistance R loci offers the potential for grape breeders to combine these R genes with existing powdery mildew R loci to produce grape germplasm with more durable resistance against this rapidly evolving fungal pathogen.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0855-8) contains supplementary material, which is available to authorized users.

Identification of a novel candidate gene for rolled leaf in rice

A semi-narrow and adaxially rolled leaf mutant, rl15(t), was induced from Korean japonica rice cultivar Ilpum by chemical mutagenesis using ethyl methanesulfonate. We characterized the mutant and identified the novel gene causing the mutant phenotype. Cytological analysis of mutant leaves indicated that the adaxial leaf-rolling phenotype is due to the reduced size and number of bulliform cells in the mutant. Genetic analysis showed that the rolled leaf trait is controlled by a single recessive gene, designated rl15(t). Using an F2 mapping population generated from a cross between Milyang23 and the mutant, we mapped the candidate region to a 174 kb interval on the long arm of chromosome 1 near the centromeric region. Through whole genome sequencing in bulk and MutMap analysis, we identified the causal SNP within the candidate region. The results of RT-PCR analysis indicated that a splicing error occurred due to a base change from G to A at the beginning of the fifth intron of LOC_Os01g37837, which encodes a putative seryl-tRNA synthetase, resulting in the mutant phenotype. Further study of the rl15(t) gene will facilitate analysis of leaf architecture and morphogenesis in rice plants.

A Parthenogenesis Gene Candidate and Evidence for Segmental Allopolyploidy in Apomictic Brachiaria decumbens.

Apomixis, asexual reproduction through seed, enables breeders to identify and faithfully propagate superior heterozygous genotypes by seed without the disadvantages of vegetative propagation or the expense and complexity of hybrid seed production. The availability of new tools such as genotyping by sequencing and bioinformatics pipelines for species lacking reference genomes now makes the construction of dense maps possible in apomictic species, despite complications including polyploidy, multisomic inheritance, self-incompatibility, and high levels of heterozygosity. In this study, we developed saturated linkage maps for the maternal and paternal genomes of an interspecific Brachiaria ruziziensis (R. Germ. and C. M. Evrard) × B. decumbens Stapf. F1 mapping population in order to identify markers linked to apomixis. High-resolution molecular karyotyping and comparative genomics with Setaria italica (L.) P. Beauv provided conclusive evidence for segmental allopolyploidy in B. decumbens, with strong preferential pairing of homologs across the genome and multisomic segregation relatively more common in chromosome 8. The apospory-specific genomic region (ASGR) was mapped to a region of reduced recombination on B. decumbens chromosome 5. The Pennisetum squamulatum (L.) R.Br. PsASGR-BABY BOOM-like (psASGR–BBML)-specific primer pair p779/p780 was in perfect linkage with the ASGR in the F1 mapping population and diagnostic for reproductive mode in a diversity panel of known sexual and apomict Brachiaria (Trin.) Griseb. and P. maximum Jacq. germplasm accessions and cultivars. These findings indicate that ASGR–BBML gene sequences are highly conserved across the Paniceae and add further support for the postulation of the ASGR–BBML as candidate genes for the apomictic function of parthenogenesis.

Mapping quantitative trait loci controlling oil content, oleic acid and linoleic acid content in sunflower (Helianthus annuus L.)

Sunflower oil with high oleic acid content is in great demand due to its nutritional as well as industrial benefits. The trait is mainly controlled by dominant alleles at a major gene, Ol, with other modifiers. The objectives of this research were to map the oil content, oleic acid and linoleic acid content in sunflower seeds. An F2 mapping population from cytoplasmic male-sterile line COSF 7A (33–35 % oleic acid) and high oleic acid inbred line HO 5–13 (88–90 % oleic acid) was developed and phenotyped for oil content, oleic acid and linoleic acid content at the F2 seed level. High phenotypic and genotypic coefficients of variation were recorded for oleic acid and linoleic acid content. High heritability and high genetic advance as percent of mean was recorded for oleic acid and linoleic acid content. This indicated the presence of the additive type of gene action controlling the traits oleic acid content and linoleic acid content. The Ol gene was mapped to linkage group (LG) 14 and tightly linked to the marker HO_Fsp_b. In addition, two more quantitative trait loci (QTLs) for oleic acid content were identified in LG8 and LG9. Two QTLs for oil content and two QTLs for linoleic acid content were also identified. All these QTLs explained over 10 % of phenotypic variation. A study was conducted with 13 genotypes differing in oil quality as well as quantity over three seasons to assess the reliability of the identified QTLs over seasons. It resulted in the identification of two potential QTLs for oleic acid as well as linoleic acid content with the markers ORS 762 and HO_Fsp_b. These markers explained more than 57.6–66.6 % of phenotypic variation. Hence it can be concluded that these markers/QTLs would be useful in the marker-assisted selection breeding programme to improve oil quality. The present study also indicated the presence of at least two other genomic regions controlling oleic and linoleic acid content in sunflower.

Molecular Mapping and Candidate Gene Analysis for Numerous Spines on the Fruit of Cucumber.

Number of spines on the fruit is an important quality trait in cucumber. The inheritance and identification of molecular markers for fruit spine density gene can provide a basis for breeding and lay the foundation for gene cloning. Cucumber inbred lines NCG-122 with numerous spines and NCG-121 with few spines were used for genetic analysis and gene mapping in this study. Genetic analysis showed that the numerous spines trait in NCG-122 was qualitative, and a single recessive nuclear gene (ns) controlled this trait. The few spines trait was dominant over the numerous spines trait. In the preliminary genetic mapping of the ns gene, 8 SSR markers were found to be linked to ns, which mapped to chromosome 2 (Chr.2) of cucumber. The closest flanking markers SSR22338 and SSR11596 were linked to the ns gene, with genetic distances of 10.2 and 1.7cM, respectively. One-hundred and thirty pairs of new SSR primers and 28 pairs of Indel primers were developed based on sequence information in the preliminary mapping region of ns Fifteen SSR markers and 2 Indel markers were identified to be linked to the ns gene after analysis on the F2 mapping population using the new molecular markers. The 2 closest flanking markers, SSRns-127 and SSR04219, were 0.7 and 2.4 cM from ns, respectively. The physical distance between SSRns-127 and SSR04219 was 266.1kb, containing 27 predicted genes. Csa2G285390 was speculated as the probable candidate gene for numerous spines. The accuracy of the closest linked marker to the ns gene, SSRns-127, for MAS breeding was 95.0%.

A gene-based map of the Nod factor-independent Aeschynomene evenia sheds new light on the evolution of nodulation and legume genomes.

Aeschynomene evenia has emerged as a new model legume for the deciphering of the molecular mechanisms of an alternative symbiotic process that is independent of the Nod factors. Whereas most of the research on nitrogen-fixing symbiosis, legume genetics and genomics has so far focused on Galegoid and Phaseolid legumes, A. evenia falls in the more basal and understudied Dalbergioid clade along with peanut (Arachis hypogaea). To provide insights into the symbiotic genes content and the structure of the A. evenia genome, we established a gene-based genetic map for this species. Firstly, an RNAseq analysis was performed on the two parental lines selected to generate a F2 mapping population. The transcriptomic data were used to develop molecular markers and they allowed the identification of most symbiotic genes. The resulting map comprised 364 markers arranged in 10 linkage groups (2n = 20). A comparative analysis with the sequenced genomes of Arachis duranensis and A. ipaensis, the diploid ancestors of peanut, indicated blocks of conserved macrosynteny. Altogether, these results provided important clues regarding the evolution of symbiotic genes in a Nod factor-independent context. They provide a basis for a genome sequencing project and pave the way for forward genetic analysis of symbiosis in A. evenia.

Molecular Mapping of Stripe Rust Resistance Gene Yr76 in Winter Club Wheat Cultivar Tyee.

Tyee, one of the wheat cultivars used to differentiate races of Puccinia striiformis f. sp. tritici in the United States, was identified to have a single gene for all-stage resistance, tentatively named YrTye. To map the gene, Tyee was crossed with 'Avocet Susceptible' (AvS). Genetic analysis of the F1, F2, F2:3, and BC1 progenies confirmed a single dominant gene for resistance to race PSTv-37 that is avirulent to YrTye. A mapping population of 135 F2 plants was phenotyped with PSTv-37 and the derived F2:3 lines were tested with races PSTv-37, PSTv-40, and PSTv-79. The F2 mapping population was genotyped with simple sequence repeat (SSR) markers. A genetic map comprising 13 SSR markers located YrTye in chromosome 3AS flanked distally by SSR marker wmc11 and proximally by wmc532 at 2.6 and 3.4 cM, respectively. Amplification of Chinese Spring 3A deletion lines placed the gene in the distal bin 3AS4-0.45 to 1.00. Because YrTye is different from all formally named Yr genes in chromosomal location, we permanently name the gene Yr76. A near-isogenic line of spring common wheat was developed and selected by testing F3 lines derived from a AvS*4/Tyee cross with Tyee-avirulent and virulent races and the flanking markers. The specific SSR alleles flanking Yr76 were validated using cultivars and breeding lines with and without the gene, and showed high polymorphisms. The specificity of Yr76 is useful in differentiating P. striiformis f. sp. tritici races, and its tightly linked markers will be useful in developing resistant cultivars when combining the gene with other genes for resistance to stripe rust.

An ultra-high density genetic linkage map of perennial ryegrass (Lolium perenne) using genotyping by sequencing (GBS) based on a reference shotgun genome assembly.

High density genetic linkage maps that are extensively anchored to assembled genome sequences of the organism in question are extremely useful in gene discovery. To facilitate this process in perennial ryegrass (Lolium perenne L.), a high density single nucleotide polymorphism (SNP)- and presence/absence variant (PAV)-based genetic linkage map has been developed in an F2 mapping population that has been used as a reference population in numerous studies. To provide a reference sequence to which to align genotyping by sequencing (GBS) reads, a shotgun assembly of one of the grandparents of the population, a tenth-generation inbred line, was created using Illumina-based sequencing. The assembly was based on paired-end Illumina reads, scaffolded by mate pair and long jumping distance reads in the range of 3-40 kb, with >200-fold initial genome coverage. A total of 169 individuals from an F2 mapping population were used to construct PstI-based GBS libraries tagged with unique 4-9 nucleotide barcodes, resulting in 284 million reads, with approx. 1·6 million reads per individual. A bioinformatics pipeline was employed to identify both SNPs and PAVs. A core genetic map was generated using high confidence SNPs, to which lower confidence SNPs and PAVs were subsequently fitted in a straightforward binning approach. The assembly comprises 424 750 scaffolds, covering 1·11 Gbp of the 2·5 Gbp perennial ryegrass genome, with a scaffold N50 of 25 212 bp and a contig N50 of 3790 bp. It is available for download, and access to a genome browser has been provided. Comparison of the assembly with available transcript and gene model data sets for perennial ryegrass indicates that approx. 570 Mbp of the gene-rich portion of the genome has been captured. An ultra-high density genetic linkage map with 3092 SNPs and 7260 PAVs was developed, anchoring just over 200 Mb of the reference assembly. The combined genetic map and assembly, combined with another recently released genome assembly, represent a significant resource for the perennial ryegrass genetics community.

Fine mapping and identification of candidate genes for the sy-2 locus in a temperature-sensitive chili pepper (Capsicum chinense).

The sy - 2 temperature-sensitive gene from Capsicum chinense was fine mapped to a 138.8-kb region at the distal portion of pepper chromosome 1. Based on expression analyses, two putative F-box genes were identified as sy - 2 candidate genes. Seychelles-2 ('sy-2') is a temperature-sensitive natural mutant of Capsicum chinense, which exhibits an abnormal leaf phenotype when grown at temperatures below 24°C. We previously showed that the sy-2 phenotype is controlled by a single recessive gene, sy-2, located on pepper chromosome 1. In this study, a high-resolution genetic and physical map for the sy-2 locus was constructed using two individual F2 mapping populations derived from a cross between C. chinense mutant 'sy-2' and wild-type 'No. 3341'. The sy-2 gene was fine mapped to a 138.8-kb region between markers SNP 5-5 and SNP 3-8 at the distal portion of chromosome 1, based on comparative genomic analysis and genomic information from pepper. The sy-2 target region was predicted to contain 27 genes. Expression analysis of these predicted genes showed a differential expression pattern for ORF10 and ORF20 between mutant and wild-type plants; with both having significantly lower expression in 'sy-2' than in wild-type plants. In addition, the coding sequences of both ORF10 and ORF20 contained single nucleotide polymorphisms (SNPs) causing amino acid changes, which may have important functional consequences. ORF10 and ORF20 are predicted to encode F-box proteins, which are components of the SCF complex. Based on the differential expression pattern and the presence of nonsynonymous SNPs, we suggest that these two putative F-box genes are most likely responsible for the temperature-sensitive phenotypes in pepper. Further investigation of these genes may enable a better understanding of the molecular mechanisms of low temperature sensitivity in plants.

Targeted sequencing of a complex locus within a polyploid genome using reduced representation libraries

Apospory is a form of gametophytic apomixis in which embryos develop from unreduced embryo sacs derived from aposporous initials formed from nucellar cells of ovules to produce offspring genetically identical to the female plant. Apospory in Pennisetum squamulatum (8X) and Cenchrus ciliaris (4X) is a dominant trait controlled by a physically large, hemizygous, heterochromatic chromosomal block called the apospory-specific genomic region (ASGR). Both apomictic species are polyploid, with genome sizes estimated at 2600 to 3000 Mbp for C. ciliaris and 9400 to 10,300 Mbp for P. squamulatum. A study was conducted to determine whether duplex-specific nuclease (DSN) normalization of DNA from apomictic and sexual genotypes would reduce repetitive sequences and allow bioinformatic analysis to predict sequence contigs derived from the ASGR. DSN libraries from four genotypes were sequenced using Illumina® HiSeq 2000 technology. 39 out of 44 tested sequence characterized amplified region (SCAR) markers from in silico predicted ASGR-specific contigs were mapped to the ASGR in a Pennisetum F1 mapping population. Eighteen SCARs showed apomict-specific amplification in C. ciliaris. The successful mapping of ~90 % of the SCAR markers to the ASGR in the Pennisetum F1 mapping population shows that DSN normalization and Illumina sequencing can be used as an effective strategy for targeted mapping of a physically large locus rich in repetitive sequences, like that of the ASGR.

Genetic analysis and molecular mapping of seedling survival drought tolerance gene in lentil (Lens culinaris Medikus)

Lentil populations were developed from crosses between ‘JL-3’ (sensitive to drought stress) and ‘PDL-1’ and ‘FLIP-96-51’ (tolerant to drought stress), to study the inheritance of drought tolerance and to identify the markers associated with it. The parental types, F1, F2, F3, and backcross (BC) generations were screened for drought tolerance using seedling survivability and drought scores. The F1 hybrids responded similar to the drought-tolerant parent, indicating dominance of seedling drought tolerance over sensitivity. Segregation for seedling survival drought tolerance versus sensitivity in F2 generation was in complete agreement with monogenic 3:1 ratio. The F3 families and backcross data additionally confirmed monogenic tolerance based on seedling survival under drought. Out of 51 SSR markers screened, thirteen markers were polymorphic between the parental types. Seven markers among them were found to be associated with seedling survival drought tolerance through bulk segregant analysis. Association of these markers with seedling survival drought tolerance was further confirmed through their screening on 10 drought-tolerant and drought-sensitive genotypes. These seven markers were screened in F2 mapping population (JL-3 × PDL-1) of 101 individuals to map their position in relation to the gene for seedling survival drought tolerance. Linkage analysis mapped the seven markers within a map distance of 133.2 cM. A single major gene Sdt was identified with a LOD value of 19.9 and phenotypic variation (R 2) of 69.7 %. The Sdt locus was obtained in the marker interval of PLC_105–PBA_LC_1480 spanning 24.9 cM with the closest marker PLC_105 at a distance of 9.0 cM on the obtained linkage group. This is the first report on genetic control and linkage of SSR markers for drought tolerance in lentil. These linked markers can be used in molecular breeding programmes for introgression of seedling survival drought tolerance gene in high-yielding cultivars.

Fine Mapping and Candidate Gene Analysis of the Leaf-Color Gene ygl-1 in Maize.

A novel yellow-green leaf mutant yellow-green leaf-1 (ygl-1) was isolated in self-pollinated progenies from the cross of maize inbred lines Ye478 and Yuanwu02. The mutant spontaneously showed yellow-green character throughout the lifespan. Meanwhile, the mutant reduced contents of chlorophyll and Car, arrested chloroplast development and lowered the capacity of photosynthesis compared with the wild-type Lx7226. Genetic analysis revealed that the mutant phenotype was controlled by a recessive nuclear gene. The ygl-1 locus was initially mapped to an interval of about 0.86 Mb in bin 1.01 on the short arm of chromosome 1 using 231 yellow-green leaf individuals of an F2 segregating population from ygl-1/Lx7226. Utilizing four new polymorphic SSR markers, the ygl-1 locus was narrowed down to a region of about 48 kb using 2930 and 2247 individuals of F2 and F3 mapping populations, respectively. Among the three predicted genes annotated within this 48 kb region, GRMZM2G007441, which was predicted to encode a cpSRP43 protein, had a 1-bp nucleotide deletion in the coding region of ygl-1 resulting in a frame shift mutation. Semi-quantitative RT-PCR analysis revealed that YGL-1 was constitutively expressed in all tested tissues and its expression level was not significantly affected in the ygl-1 mutant from early to mature stages, while light intensity regulated its expression both in the ygl-1 mutant and wild type seedlings. Furthermore, the mRNA levels of some genes involved in chloroplast development were affected in the six-week old ygl-1 plants. These findings suggested that YGL-1 plays an important role in chloroplast development of maize.

Genetics and Molecular Mapping of Black Rot Resistance Locus Xca1bc on Chromosome B-7 in Ethiopian Mustard (Brassica carinata A. Braun).

Black rot caused by Xanthomonas campestris pv. campestris (Pam.) Dowson is the most destructive disease of cauliflower causing huge loss to the farmers throughout the world. Since there are limited sources of resistance to black rot in B. oleracea (C genome Brassica), exploration of A and B genomes of Brassica was planned as these were thought to be potential reservoirs of black rot resistance gene(s). In our search for new gene(s) for black rot resistance, F2 mapping population was developed in Brassica carinata (BBCC) by crossing NPC-17, a susceptible genotype with NPC-9, a resistant genotype. Out of 364 Intron length polymorphic markers and microsatellite primers used in this study, 41 distinguished the parental lines. However, resistant and susceptible bulks could be distinguished by three markers At1g70610, SSR Na14-G02 and At1g71865 which were used for genotyping of F2 mapping population. These markers were placed along the resistance gene, according to order, covering a distance of 36.30 cM. Intron length polymorphic markers At1g70610 and At1g71865 were found to be linked to black rot resistance locus (Xca1bc) at 6.2 and 12.8 cM distance, respectively. This is the first report of identification of markers linked to Xca1bc locus in Brassica carinata on B-7 linkage group. Intron length polymorphic markers provided a novel and attractive option for marker assisted selection due to high cross transferability and cost effectiveness for marker assisted alien gene introgression into cauliflower.

Mapping of Ppd-B1, a Major Candidate Gene for Late Heading on Wild Emmer Chromosome Arm 2BS and Assessment of Its Interactions with Early Heading QTLs on 3AL

Wheat heading date is an important agronomic trait determining maturation time and yield. A set of common wheat (Triticum aestivum var. Chinese Spring; CS)-wild emmer (T. turgidum L. subsp. dicoccoides (TDIC)) chromosome arm substitution lines (CASLs) was used to identify and allocate QTLs conferring late or early spike emergence by examining heading date. Genetic loci accelerating heading were found on TDIC chromosome arms 3AL and 7BS, while loci delaying heading were located on 4AL and 2BS. To map QTLs conferring late heading on 2BS, F2 populations derived from two cross combinations of CASL2BS × CS and CASL3AL × CASL2BS were developed and each planted at two times, constituting four F2 mapping populations. Heading date varied continuously among individuals of these four populations, suggesting quantitative characteristics. A genetic map of 2BS, consisting of 23 SSR and one single-stranded conformation polymorphism (SSCP) marker(s), was constructed using these F2 populations. This map spanned a genetic length of 53.2 cM with average marker density of 2.3 cM. The photoperiod-sensitivity gene Ppd-B1 was mapped to chromosome arm 2BS as a SSCP molecular marker, and was validated as tightly linked to a major QTL governing late heading of CASL2BS in all mapping populations. A significant dominance by additive effect of Ppd-B1 with the LUX gene located on 3AL was also detected. CS had more copies of Ppd-B1 than CASL2BS, implying that increased copy number could elevate the expression of Ppd-1 in CS, also increasing expression of LUX and FT genes and causing CS to have an earlier heading date than CASL2BS in long days.

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.

A genetic linkage map of coffee (Coffea arabica L.) and QTL for yield, plant height, and bean size

To breed a new variety of coffee (Coffea arabica) requires approximately 25 years due to the long generation time (5–6 years) of this perennial plant and the fact that it takes at least five generations of selection to obtain superior individuals. One way to reduce the number of generations is to use marker-assisted selection (MAS). To implement MAS, it is necessary to develop a genetic map and to identify markers associated with quantitative trait loci (QTLs) governing traits of interest. The objective of this study was to identify QTLs associated with yield, plant height, and bean size. An F2 mapping population consisting of 278 individuals was developed from a cross between Caturra × CCC1046. A total of 338 SSR were used to construct a framework linkage map. Subsequently, SNP markers were added to construct a more robust genetic map. R/qtl software was used to integrate SSR and SNP markers. The integrated linkage map consists of 22 linkage groups populated by 848 SSR and SNP markers, with a total map length of 3800 cM. F3 progenies of the mapping population were planted in five locations and evaluated for yield, plant height, and bean size. A major yield QTL was significant at two locations and a second was significant at one location. Two QTL were each detected for plant height and bean size.