Bulk Segregant Analysis Approach Research Articles

A k-mer-based bulked segregant analysis approach to map seed traits in unphased heterozygous potato genomes.

High-throughput sequencing-based methods for bulked segregant analysis (BSA) allow for the rapid identification of genetic markers associated with traits of interest. BSA studies have successfully identified qualitative (binary) and quantitative trait loci (QTLs) using QTL mapping. However, most require population structures that fit the models available and a reference genome. Instead, high-throughput short-read sequencing can be combined with BSA of k-mers (BSA-k-mer) to map traits that appear refractory to standard approaches. This method can be applied to any organism and is particularly useful for species with genomes diverged from the closest sequenced genome. It is also instrumental when dealing with highly heterozygous and potentially polyploid genomes without phased haplotype assemblies and for which a single haplotype can control a trait. Finally, it is flexible in terms of population structure. Here, we apply the BSA-k-mer method for the rapid identification of candidate regions related to seed spot and seed size in diploid potato. Using a mixture of F1 and F2 individuals from a cross between 2 highly heterozygous parents, candidate sequences were identified for each trait using the BSA-k-mer approach. Using parental reads, we were able to determine the parental origin of the loci. Finally, we mapped the identified k-mers to a closely related potato genome to validate the method and determine the genomic loci underlying these sequences. The location identified for the seed spot matches with previously identified loci associated with pigmentation in potato. The loci associated with seed size are novel. Both loci are relevant in future breeding toward true seeds in potato.

QTL Mapping of Soybean (Glycine max) Vine Growth Habit Trait.

The vine growth habit (VGH) is a notable property of wild soybean plants that also holds a high degree of importance in domestication as it can preclude using these wild cultivars for breeding and improving domesticated soybeans. Here, a bulked segregant analysis (BSA) approach was employed to study the genetic etiology of the VGH in soybean plants by integrating linkage mapping and population sequencing approaches. To develop a recombinant inbred line (RIL) population, the cultivated Zhongdou41 (ZD41) soybean cultivar was bred with ZYD02787, a wild soybean accession. The VGH status of each line in the resultant population was assessed, ultimately leading to the identification of six and nine QTLs from the BSA sequencing of the F4 population and F6-F8 population sequence mapping, respectively. One QTL shared across these analyzed generations was detected on chromosome 19. Three other QTLs detected by BSA-seq were validated and localized to the 90.93 kb, 2.9 Mb, and 602.08 kb regions of chromosomes 6 and 13, harboring 14, 53, and 4 genes, respectively. Three consistent VGH-related QTLs located on chromosomes 2 and 19 were detected in a minimum of three environments, while an additional six loci on chromosomes 2, 10, 13, and 18 were detected in at least two environments via ICIM mapping. Of all the detected loci, five had been reported previously whereas seven represent novel QTLs. Together, these data offer new insights into the genetic basis of the VGH in soybean plants, providing a rational basis to inform the use of wild accessions in future breeding efforts.

Genetic mapping and molecular characterization of the delayed green gene dg in watermelon (Citrullus lanatus).

Leaf color mutants are common in higher plants that can be used as markers in crop breeding and are important tools in understanding regulatory mechanisms of chlorophyll biosynthesis and chloroplast development. Genetic analysis was performed by evaluating F1, F2 and BC1 populations derived from two parental lines (Charleston gray with green leaf color and Houlv with delayed green leaf color), suggesting that a single recessive gene controls the delayed green leaf color. In this study, the delayed green mutant showed a conditional pale green leaf color at the early leaf development but turned to green as the leaf development progressed. Delayed green leaf plants showed reduced pigment content, photosynthetic, chlorophyll fluorescence parameters, and impaired chloroplast development compared with green leaf plants. The delayed green (dg) locus was mapped to 7.48 Mb on chromosome 3 through bulk segregant analysis approach, and the gene controlling delayed green leaf color was narrowed to 53.54 kb between SNP130 and SNP135 markers containing three candidate genes. Sequence alignment of the three genes indicated that there was a single SNP mutation (G/A) in the coding region of ClCG03G010030 in the Houlv parent, which causes an amino acid change from Arginine to Lysine. The ClCG03G010030 gene encoded FtsH extracellular protease protein family is involved in early delayed green leaf development. The expression level of ClCG03G010030 was significantly reduced in delayed green leaf plants than in green leaf plants. These results indicated that the ClCG03G010030 might control watermelon green leaf color and the single SNP variation in ClCG03G010030 may result in early delayed green leaf color development during evolutionary process.

Fine Mapping of Clubroot Resistance Loci CRA8.1 and Candidate Gene Analysis in Chinese Cabbage (Brassica rapa L.).

Clubroot is caused by Plasmodiophora brassicae, which threatens Brassicaceae crop production worldwide. In recent years, there has been an outbreak and rapid spread of clubroot in many major cruciferous crop-producing areas of China. In this study, we identified a cabbage material DingWen (DW) with different resistant capabilities from Huashuang5R (H5R) and Huayouza62R of Brassica napus, which are currently used as the main resistant cultivars for clubroot management in China. We used a next-generation sequencing-based bulked segregant analysis approach, combined with genetic mapping to identify clubroot-resistant (CR) genes from F1 population generated from a cross between the DW (CR) and HZSX (clubroot susceptible). The CR locus of DW (named CRA8.1) was mapped to a region between markers A08-4346 and A08-4853, which contains two different loci CRA8.1a and CRA8.1b after fine mapping. The CRA8.1b loci contain a fragment of 395 kb between markers A08-4624 and A08-4853 on A08 chromosome, and it is responsible for the resistance to PbZj and PbXm isolates. However, together with CRA8.1a, corresponding to a 765-kb region between markers A08-4346 and A08-4624, then it can confer resistance to PbXm+. Finally, through expression analysis between resistant and susceptible materials, two genes encoding TIR-NBS-LRR proteins (BraA08g039211E and BraA08g039212E) and one gene encoding an RLP protein (BraA08g039193E) were identified to be the most likely CR candidates for the peculiar resistance in DW.

High-resolution genetic mapping reveals cis-regulatory and copy number variation in loci associated with cytochrome P450-mediated detoxification in a generalist arthropod pest.

Chemical control strategies are driving the evolution of pesticide resistance in pest populations. Understanding the genetic mechanisms of these evolutionary processes is of crucial importance to develop sustainable resistance management strategies. The acaricide pyflubumide is one of the most recently developed mitochondrial complex II inhibitors with a new mode of action that specifically targets spider mite pests. In this study, we characterize the molecular basis of pyflubumide resistance in a highly resistant population of the spider mite Tetranychus urticae. Classical genetic crosses indicated that pyflubumide resistance was incompletely recessive and controlled by more than one gene. To identify resistance loci, we crossed the resistant population to a highly susceptible T. urticae inbred strain and propagated resulting populations with and without pyflubumide exposure for multiple generations in an experimental evolution set-up. High-resolution genetic mapping by a bulked segregant analysis approach led to the identification of three quantitative trait loci (QTL) linked to pyflubumide resistance. Two QTLs were found on the first chromosome and centered on the cytochrome P450 CYP392A16 and a cluster of CYP392E6-8 genes. Comparative transcriptomics revealed a consistent overexpression of CYP392A16 and CYP392E8 in the experimental populations that were selected for pyflubumide resistance. We further corroborated the involvement of CYP392A16 in resistance by in vitro functional expression and metabolism studies. Collectively, these experiments uncovered that CYP392A16 N-demethylates the toxic carboxamide form of pyflubumide to a non-toxic compound. A third QTL coincided with cytochrome P450 reductase (CPR), a vital component of cytochrome P450 metabolism. We show here that the resistant population harbors three gene copies of CPR and that this copy number variation is associated with higher mRNA abundance. Together, we provide evidence for detoxification of pyflubumide by cytochrome P450s that is likely synergized by gene amplification of CPR.

Characterization of genetic determinants of the resistance to phylloxera, Daktulosphaira vitifoliae, and the dagger nematode Xiphinema index from muscadine background

BackgroundMuscadine (Muscadinia rotundifolia) is known as a resistance source to many pests and diseases in grapevine. The genetics of its resistance to two major grapevine pests, the phylloxera D. vitifoliae and the dagger nematode X. index, vector of the Grapevine fanleaf virus (GFLV), was investigated in a backcross progeny between the F1 resistant hybrid material VRH8771 (Vitis-Muscadinia) derived from the muscadine R source ‘NC184–4’ and V. vinifera cv. ‘Cabernet-Sauvignon’ (CS).ResultsIn this pseudo-testcross, parental maps were constructed using simple-sequence repeats markers and single nucleotide polymorphism markers from a GBS approach. For the VRH8771 map, 2271 SNP and 135 SSR markers were assembled, resulting in 19 linkage groups (LG) and an average distance between markers of 0.98 cM. Phylloxera resistance was assessed by monitoring root nodosity number in an in planta experiment and larval development in a root in vitro assay. Nematode resistance was studied using 10–12 month long tests for the selection of durable resistance and rating criteria based on nematode reproduction factor and gall index. A major QTL for phylloxera larval development, explaining more than 70% of the total variance and co-localizing with a QTL for nodosity number, was identified on LG 7 and designated RDV6. Additional QTLs were detected on LG 3 (RDV7) and LG 10 (RDV8), depending on the in planta or in vitro experiments, suggesting that various loci may influence or modulate nodosity formation and larval development. Using a Bulked Segregant Analysis approach and a proportion test, markers clustered in three regions on LG 9, LG 10 and LG 18 were shown to be associated to the nematode resistant phenotype. QTL analysis confirmed the results and QTLs were thus designated respectively XiR2, XiR3 and XiR4, although a LOD-score below the significant threshold value was obtained for the QTL on LG 18.ConclusionsBased on a high-resolution linkage map and a segregating grapevine backcross progeny, the first QTLs for resistance to D. vitifoliae and to X. index were identified from a muscadine source. All together these results open the way to the development of marker-assisted selection in grapevine rootstock breeding programs based on muscadine derived resistance to phylloxera and to X. index in order to delay GFLV transmission.

Molecular Mapping and Candidate Gene Analysis for GA3 Responsive Short Internode in Watermelon (Citrullus lanatus).

Plants with shorter internodes are suitable for high-density planting, lodging resistance and the preservation of land resources by improving yield per unit area. In this study, we identified a locus controlling the short internode trait in watermelon using Zhengzhouzigua (long internode) and Duan125 (short internode) as mapping parents. Genetic analysis indicated that F1 plants were consistent with long internode plants, which indicates that the long internode was dominant over the short internode. The observed F2 and BC1 individuals fitted the expected phenotypic segregation ratios of 3:1 and 1:1, respectively. The locus was mapped on chromosome 9 using a bulked segregant analysis approach. The region was narrowed down to 8.525 kb having only one putative gene, Cla015407, flanking by CAPS90 and CAPS91 markers, which encodes gibberellin 3β-hydroxylase (GA 3β-hydroxylase). The sequence alignment of the candidate gene between both parents revealed a 13 bp deletion in the short internode parent, which resulted in a truncated protein. Before GA3 application, significantly lower GA3 content and shorter cell length were obtained in the short internode plants. However, the highest GA3 content and significant increase in cell length were observed in the short internode plants after exogenous GA3 application. In the short internode plants, the expression level of the Cla015407 was threefold lower than the long internode plants in the stem tissue. In general, our results suggested that Cla015407 might be the candidate gene responsible for the short internode phenotype in watermelon and the phenotype is responsive to exogenous GA3 application.

Bulked segregant analysis RNA-seq (BSR-Seq) validated a stem resistance locus in Aegilops umbellulata, a wild relative of wheat.

Many disease resistance genes that have been transferred from wild relatives to cultivated wheat have played a significant role in wheat production worldwide. Ae. umbellulata is one of the species within the genus Aegilops that have been successfully used as sources of resistance genes to leaf rust, stem rust and powdery mildew. The objectives of the current work was to validate the map position of a major QTL that confers resistance to the stem rust pathogen races Ug99 (TTKSK) and TTTTF with an independent bi-parental mapping population and to refine the QTL region with a bulk segregant analysis approach. Two F2 bi-parental mapping populations were developed from stem rust resistant Ae. umbellulata accessions (PI 298905 and PI 5422375) and stem rust susceptible accessions (PI 542369 and PI 554395). Firstly, one of the two populations was used to map the chromosome location of the resistance gene. Later on, the 2nd population was used to validate the chromosome location in combination with a bulk segregant analysis approach. For the bulk segregant analysis, RNA was extracted from a bulk of leaf tissues of 12 homozygous resistant F3 families, and a separate bulk of 11 susceptible homozygous F3 families derived from the PI 5422375 and PI 554395 cross. The RNA samples of the two bulks and the two parents were sequenced for SNPs identification. Stem rust resistance QTL was validated on chromosome 2U of Ae. umbellulata in the same region in both populations. With bulk segregant analysis, the QTL position was delimited within 3.2 Mbp. Although there were a large number of genes in the orthologous region of the detected QTL on chromosome 2D of Ae. tauschii, we detected only two Ae. umbellulata NLR genes which can be considered as a potential candidate genes.

Identification and Mapping of the Clubroot Resistance Gene CRd in Chinese Cabbage (Brassica rapa ssp. pekinensis).

The rapid spread of clubroot disease, which is caused by Plasmodiophora brassicae, threatens Brassicaceae crop production worldwide. Breeding plants that have broad-spectrum disease resistance is one of the best ways to prevent clubroot. In the present study, eight Chinese cabbage germplasms were screened using published clubroot-resistant (CR) loci-/gene-linked markers. A CR gene Crr3 potential carrier “85-74” was detected which linked to marker BRSTS61; however, “85-74” shows different responses to local pathogens “LAB-19,” “LNND-2,” and “LAB-10” from “CR-73” which harbors Crr3. We used a next-generation sequencing-based bulked segregant analysis approach combined with genetic mapping to detect CR genes in an F2 segregant population generated from a cross between the Chinese cabbage inbred lines “85-74” (CR) and “BJN3-1” (clubroot susceptible). The “85-74” line showed resistance to a local pathogen “LAB-19” which was identified as race 4; a genetic analysis revealed that the resistance was conferred by a single dominant gene. The CR gene which we named CRd was mapped to a 60 kb (1 cM) region between markers yau389 and yau376 on chromosome A03. CRd is located upstream of Crr3 which was confirmed based on the physical positions of Crr3 linked markers. The identification of CRd linked markers can be applied to marker-assisted selection in the breeding of new CR cultivars of Chinese cabbage and other Brassica crops.

A QTL with Major Effect on Reducing Stripe Rust Severity Detected From a Chinese Wheat Landrace.

Stripe rust, a devastating disease of wheat worldwide, can be controlled by use of diverse wheat resistance resources. To find new quantitative trait loci (QTL) for resistance to stripe rust, Qing Shumai (a Chinese winter wheat landrace possessing slow rusting resistance) was crossed with the susceptible line Mingxian 169. The parents and 276 recombinant inbred lines (RILs) from the cross were evaluated in five environments involving two locations (Gansu and Shandong provinces, China) and four autumn-sown wheat seasons (2008 to 2012). Disease severities on Qing Shumai were lower than 25%, contrasting with approximately 90% on Mingxian 169. The RILs varied in rust intensity in a continuous and monomodal distribution. A bulked segregant analysis approach using 2,344 simple sequence repeat (SSR) markers mapped a major QTL to the long arm of chromosome 6D (hereby designated as QYr.cau-6DL). An SSR marker (gpw5179, https://wheat.pw.usda.gov/GG2/index.shtml ) was identified as being tightly linked with QYr.cau-6DL. Combination between QYr.cau-6DL and the stripe rust-resistance gene Yr18 was examined using 160 F2:3 families of Qing Shumai × RL6058 (a near-isogenic line for Yr18 in the genetic background of the spring wheat Thatcher). The combination elevated the resistance consistently across both winter and spring wheat backgrounds, acting synergistically without undesired epistasis.

Use of Bulk Segregant Analysis for Varietal Identification and Genetic Diversity Estimation in Pakistani Basmati and Non-Basmati Rice Cultivars through Molecular Markers

Identification of alleles for a particular variety through Bulk Segregant Analysis (BSA) is a valuable technique that probes differences specifically in distinguishing traits. Our study was an attempt to develop variety specific marker which differentiate aromatic rice cultivars from non-aromatic. Bulk Segregant Analysis (BSA) approach with RAPD and ISSR assay was undertaken to trace the variety specific alleles among 2 group of popular Pakistani Basmati and non-Basmati rice genotypes to check adulteration by rice exporters. Out of 160 RAPDs and 30 ISSR used in BSA, 29 RAPD and 18 ISSR revealed consistent polymorphisms and were used to assort the advanced disparities amongst 10 varieties. Overall 262 of 359 random amplified alleles and 116 of 151 inter simple sequence alleles were found polymorphic. The number of alleles generated from RAPD and ISSR marker ranged from 5 to 19 and 3-18 respectively. Pair-wise genetic similarity among the varieties with jaccard coefficients was 0.85 for RAPDs and 0.79 for ISSRs. UPGMA dendrogram based on cluster analysis of individual and combine genetic similarity co-efficient resolved 10 rice cultivars into 2 major groups, Basmati and Non-basmati. Moreover, a total of 3 variety specific alleles of 650, 800, 1075 bp were amplified by random primers OPN-05 and OPE-09. While ISSR generated five (UBC-814, UBC-811, UBC-808, UBC-808a and UBC-82) rare alleles for specific genotypes with band sizes of 1500, 1400, 250, 950 and 720 bp respectively. Direct sequencing of rare allelic amplii¬ed bands in Sequence Characterized Amplii¬ed Regions (SCARs) and sequence tagged sites (STS) might behave useful for recreating phylogenic trees with aim of preserving the integrity of Basmati rice.

Identification of Gene Related to Hard Bunch Phenotype in Oil Palm (Elaeis guineensis Jacq.)

<p>ABSTRACT</p><p>Molecular genetic analysis of hard bunch phenomenon in oil palm was done in order to elucidate the role of genetic factor underlying hard bunch in oil palm plantation. The aim of this study was to identify the AFLP primer combination that co-segregates with hard bunch phenotype related gene in oil palm. Molecular analysis was done by bulk segregant analysis approach. DNA was isolated from leaves of the normal and hard bunch palm. DNA from ten individual palms from each category were pooled and used as a template. A total of 56 AFLP primer combinations were selected for selection of polymorphic primer, and as a result it was found that 22 AFLP primer combinations (39.28%) were polymorphic. A total of 48 individual of palm DNA containing 24 individual for each group were further genotyped by those 22 polymorphic markers. Of these, one AFLP primer combination (E-ACC/M-CTG) was obtained as a co-segregated marker that distinguished the hard bunch DNA from the normal one. Based on the analysis of the target sequence aligned to the oil palm DNA sequences available in database, we found that our sequence has similarity with Ty-1 copia retrotransposon. This sequence distribute in all 16 linkage group of oil palm genome.</p><p>Keywords: abnormal fruits, AFLP, oil palm, Ty-1 copia retrotransposon</p>

Identification of QTLs and possible candidate genes conferring sheath blight resistance in rice (Oryza sativa L.).

Sheath blight, caused by the pathogenic fungus Rhizoctonia solani Kühn, is one of the most devastating diseases in rice. Breeders have always faced challenges in acquiring reliable and absolute resistance to this disease in existing rice germplasm. In this context, 40 rice germplasm including eight wild, four landraces, twenty- six cultivated and two advanced breeding lines were screened utilizing the colonized bits of typha. Except Tetep and ARC10531 which expressed moderate level of resistance to the disease, none could be found to be authentically resistant. In order to map the quantitative trait loci (QTLs) governing the sheath blight resistance, two mapping populations (F2 and BC1F2) were developed from the cross BPT-5204/ARC10531. Utilizing composite interval mapping analysis, 9 QTLs mapped to five different chromosomes were identified with phenotypic variance ranging from 8.40 to 21.76%. Two SSR markers namely RM336 and RM205 were found to be closely associated with the major QTLs qshb7.3 and qshb9.2 respectively and were attested as well in BC1F2 population by bulk segregant analysis approach. A hypothetical β 1–3 glucanase with other 31 candidate genes were identified in silico utilizing rice database RAP-DB within the identified QTL region qshb9.2. A detailed insight into these candidate genes will facilitate at molecular level the intricate nature of sheath blight, a step forward towards functional genomics.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-0954-2) contains supplementary material, which is available to authorized users.

Yr60, a Gene Conferring Moderate Resistance to Stripe Rust in Wheat.

Stripe rust, caused by Puccinia striiformis f. sp. tritici W., is a devastating disease of wheat worldwide. A new stripe rust resistance gene with moderate seedling and adult plant resistance was mapped using an F5 recombinant inbred line (RIL) population developed from the cross of the resistant parent 'Almop' with the susceptible parent 'Avocet'. The parents and RILs were phenotyped for seedling stripe rust response variation in a greenhouse and in field trials at Toluca, Mexico for 2 years. Almop showed moderate levels of resistance at both seedling and adult plant stages compared with the highly susceptible response of Avocet. The distribution of homozygous resistant, homozygous susceptible, and segregating RILs conformed to segregation at a single locus. Seedlings and adult plant responses were correlated, indicating that the same gene conferred resistance at both stages. A bulk segregant analysis approach with widely distributed simple sequence repeat (SSR) markers mapped the resistance gene to the distal region of the long arm of chromosome 4A. The SSR marker wmc776 cosegregated with this gene, whereas markers wmc219 and wmc313 were tightly linked and both located at 0.6 centimorgans. The resistance locus was designated Yr60.

Identification of candidate genes for rice grain aroma by combining QTL mapping and transcriptome profiling approaches

The present report is in continuation to our earlier reports on the identification and fine mapping of three aroma QTLs in basmati rice using a bi-parental mapping population derived from a cross between Pusa 1121, a basmati rice variety, and Pusa 1342, a non-aromatic rice variety. We used a combination of genetic mapping and transcriptome profiling to narrow down the number of differentially expressed genes in rice to identify potential candidate genes for rice grain aroma. Highly aromatic and non-aromatic recombinant inbred lines (RILs) were identified through sensory analysis of mature milled grains. RILs with similar phenotypes were bulked together using bulk segregant analysis approach which drastically reduced the number of differentially expressed genes from 4016 to 1344. The transcriptome profiles generated were analyzed through Affymetrix rice genome array containing probe sets designed from all the predicted rice gene sequences. Microarray-based transcriptome profiling revealed one down-regulated gene co-located in QTL region aro3.1 on chromosome 3, eight genes co-located in the aro4.1 region on chromosome 4 and the badh2 gene on chromosome 8 to be differentially expressed in the aromatic parent and aromatic bulk. These genes are the most suitable candidates for future validation and development of new molecular functional markers to facilitate marker assisted breeding.

Mapping of two suppressors of OVATE (sov) loci in tomato

Tomato fruit shape varies significantly in the cultivated germplasm. To a large extent, this variation can be explained by four genes including OVATE. While most varieties with the OVATE mutation bear elongated fruits, some accessions carry round fruit, suggesting the existence of suppressors of OVATE in the germplasm. We developed three intraspecific F2 populations with parents that carried the OVATE mutation but differed in fruit shape. We used a bulk segregant analysis approach and genotyped the extreme classes using a high-throughput genotyping platform, the SolCAP Infinium Assay. The analyses revealed segregation at two quantitative trait loci (QTLs), sov1 and sov2. These loci were confirmed by genotyping and QTL analyses of the entire population. More precise location of those loci using progeny testing confirmed that sov1 on chromosome 10 controlled obovoid and elongated shape, whereas sov2 on chromosome 11 controlled mainly elongated fruit shape. Both loci were located in intervals of <2.4 Mb on their respective chromosomes.

QDTY12.1: a locus with a consistent effect on grain yield under drought in rice

BackgroundSelection for grain yield under drought is an efficient criterion for improving the drought tolerance of rice. Recently, some drought-tolerant rice varieties have been developed using this selection criterion and successfully released for cultivation in drought-prone target environments. The process can be made more efficient and rapid through marker-assisted breeding, a well-known fast-track approach in crop improvement. QTLs have been identified for grain yield under drought with large effects against drought-susceptible varieties. Most of the identified QTLs show large QTL × environment or QTL × genetic background interactions. The development of mapping populations in the background of popular high-yielding varieties, screening across environments, including the target environments, and the identification of QTLs with a consistent effect across environments can be a suitable alternative marker-assisted breeding strategy. An IR74371-46-1-1 × Sabitri backcross inbred line population was screened for reproductive-stage drought stress at the International Rice Research Institute, Philippines, and Regional Agricultural Research Station, Nepalgunj, Nepal, in the dry and wet seasons of 2011, respectively. A bulk segregant analysis approach was used to identify markers associated with high grain yield under drought.ResultsA QTL, qDTY12.1, significantly associated with grain yield under reproductive-stage drought stress was identified on chromosome 12 with a consistent effect in two environments: IRRI, Philippines, and RARS, Nepalgunj, Nepal. This QTL explained phenotypic variance of 23.8% and contributed an additive effect of 45.3% for grain yield under drought. The positive QTL allele for qDTY12.1 was contributed by tolerant parent IR74371-46-1-1.ConclusionsIn this study, qDTY12.1 showed a consistent effect across environments for high grain yield under lowland reproductive-stage drought stress in the background of popular high-yielding but drought-susceptible recipient variety Sabitri. qDTY12.1 was also reported previously [Crop Sci 47:507–516, 2007] to increase grain yield under upland reproductive-stage drought stress situations. qDTY12.1 is the only QTL reported so far in rice to have shown a large effect against multiple recipient genetic backgrounds as well as under highly diverse upland and lowland rice ecosystems. qDTY12.1 can be successfully introgressed to improve grain yield under drought of popular high-yielding but drought-susceptible lowland as well as upland adapted varieties following marker-assisted breeding.

Molecular Characterization of Non-flowering Perennial Sorghum spp. Hybrids

Aims: The goal of this study was to characterize recently identified, non-flowering, putative tetraploid Sorghum spp. hybrids utilizing bulked segregant analysis with SSRs and compare them to S. bicolor, S. halepense, and triploid putative Sorghum spp. hybrids. Confirmed species hybrids between S. bicolor and S. halepense would provide resources for investigating risks of invasiveness and transgene escape alongside potential for identifying novel perennial Sorghum feedstocks of value. Study design: Bulked segregant analysis of Sorghum species and S. species hybrids. Place and Duration of Study: Department of Soil & Crop Sciences; Texas AM College Station, TX, USA; 2009 2011 Methodology: A bulked segregant analysis approach was conducted using SSRs mined from the S. bicolor genome sequence. Bulked samples of S. bicolor, typical flowering S. halepense, non-flowering tetraploid putative Sorghum spp. hybrids, and triploid putative Sorghum spp. hybrids were surveyed to identify both unique markers specific to each bulk and markers indicative of S. bicolor genetic material introgressed into the Sorghum spp. hybrids. Results: Thirty-nine and 23 markers were found to be unique to the S. bicolor and typical flowering S. halepense bulks, respectively. These unique markers could be utilized in breeding programs to identify interspecific hybrids. A subset of 23 unique SSRs were found in the non-flowering tetraploid putative Sorghum spp. hybrid, and they may be useful in the characterization of the non-flowering phenotype. Conclusion: Markers identified in this study provide: 1) species-specific tools for confirmation of interspecific Sorghum spp. hybrids and quantification of gene flow between Sorghum spp., 2) candidate gene and genomic region resources for dissection of the nonResearch Article American Journal of Experimental Agriculture, 2(1): 9-20, 2012 10 flowering phenotype, and 3) Sorghum spp. hybrid-specific markers suitable towards development of perennial biofuel and forage sorghum feedstocks.

Detection and mapping of quantitative trait loci for the contents of the terpenoid indole alkaloids, vindoline and catharanthine, in the leaves of Catharanthus roseus using bulk segregant analysis

SummaryA mapping population consisting of 191 F2:7 recombinant inbred lines (RILs) of Catharanthus roseus was used for segregation analysis of DNA markers, leading to the construction of a genetic linkage map of 174 DNA markers placed on eight linkage groups (LG). These markers were present at an average inter-marker distance of 10.2 cM. Leaves harvested from cultivated C. roseus plants are the main source of vindoline (V) and catharanthine (C), which are chemically condensed during commercial synthesis of the widely-used anti-cancer drugs, vincristine and vinblastine. To detect and map the major genes/quantitative trait loci (QTL) that control the accumulation of V and C in C. roseus leaves, a bulk segregant analysis approach was used. The above mapping population of RILs was phenotyped for their leaf alkaloid profiles and the distributions of V and C contents were used to assemble two pairs of contrasting bulks of RILs, one each for V and C accumulation in extremely low or extremely high amounts. Primers for the 174 already- mapped markers were PCR amplified on the bulks. Among the markers that discriminated between the high-C and low-C bulks, and the high-V and low-V bulks, those that amplified all members of their specific bulk were identified. This led to the detection of two QTLs (V1 and V2) and their co-segregating/linked DNA markers for high-V content, and one QTL (C1) and its linked DNA markers for high-C content. These three QTLs were then mapped. V1 and V2 were observed to lie on LG1, and C1 was on LG6. The QTLs V1 and V2 had large additive effects. These, and C1, appear to be suitable for marker assisted selection.

Aanlysis of short photo-periodic sensitive genic male sterility and molecular mapping of rpms3(t) gene in rice (Oryza sativa L.) using SSR markers

A research was conducted on the pollen fertility of rice sterile lines D52S and D38S responsive to photoperiod during the sensitive stage under natural and controlled conditions. Bulk segregant analysis (BSA) and recessive class approach were applied to identify DNA markers that co-segregate with gene conferring male-sterility in D52S mutant rice. The results showed that in day-light higher or equal to 14.00 h, D52S and D38S rice pollen were fertile; however, they were sterile when day-length was less than 14.00 h. They were therefore considered to be short photo-periodic sensitive genic male sterile lines(Short PGMS lines). Under short day-light conditions, the pollen fertility segregation of F2 populations from crosses between D52S/Shuhui527 and D52S/Gui99showed 3:1 ratio of fertile to sterile plants suggestingthat male sterility in D52S was controlled by one recessive gene. Two markers RM244 and RM216 located on chromosome number 10 co-segregated completely with the rpms locus. The locus was mapped to the interval between SSR markers RM2571 (6.6 cM) and RM244 (4.6 cM).