Segregant Analysis Method Research Articles

Bulk Segregant Analysis for the Identification of Shoot Fly Resistance Linked Molecular Marker in Sorghum [Sorghum bicolor (L.) Moench

Sorghum (Sorghum bicolor (L.) Moench) is one of the most important crops in the semi-arid regions of the world. One of the important biotic constraints to sorghum production in India is the shoot fly which attacks sorghum at the seedling stage. The study was undertaken during 2021–22 at the Centre for Millets Research, Sardarkrushinagar Dantiwada Agricultural University, Deesa, Gujarat, India to assess linked molecular markers for sorghum shoot fly resistance using the bulk segregant analysis (BSA) method with simple sequence repeat (SSR) marker from developed F2 mapping population with two genetically diverse parental lines, SWARNA (susceptible to shoot fly) and IS 18551 (resistant to shoot fly). Sixty-five SSRs primers pair were used for the parental polymorphism survey using two contrasting parents to detect the primers exhibiting polymorphism. Eight out of sixty-five primers showed polymorphism (12.30%) between two contrasting parental lines in sorghum. Two out of eight polymorphic SSRs primer pairs i.e., Xtxp 67 and Xgap 88 were found polymorphic between resistance and susceptibility in parents and bulks and thus reported to be putatively linked with shoot fly. Bulk segregant analysis (BSA) was extended to identify the traits controlled by minor genes with additive effects, which increased the power and efficiency of this molecular technique to construct genetic map in the sorghum crop improvement program. The identified SSRs markers i.e., Xtxp 67 and Xgap 88 might be useful to screen resistance for shoot fly infestation in future sorghum improvement program.

The WD40 domain-containing protein Ehd5 positively regulates flowering in rice (Oryza sativa).

Heading date (flowering time), which greatly influences regional and seasonal adaptability in rice (Oryza sativa), is regulated by many genes in different photoperiod pathways. Here, we characterized a heading date gene, Early heading date 5 (Ehd5), using a modified bulked segregant analysis method. The ehd5 mutant showed late flowering under both short-day and long-day conditions, as well as reduced yield, compared to the wild type. Ehd5, which encodes a WD40 domain-containing protein, is induced by light and follows a circadian rhythm expression pattern. Transcriptome analysis revealed that Ehd5 acts upstream of the flowering genes Early heading date 1 (Ehd1), RICE FLOWERING LOCUS T 1 (RFT1), and Heading date 3a (Hd3a). Functional analysis showed that Ehd5 directly interacts with Rice outermost cell-specific gene 4 (Roc4) and Grain number, plant height, and heading date 8 (Ghd8), which might affect the formation of Ghd7-Ghd8 complexes, resulting in increased expression of Ehd1, Hd3a and RFT1. Taken together, these results demonstrate that Ehd5 functions as a positive regulator of rice flowering and provide insight into the molecular mechanisms underlying heading date.

Epistatic interaction between CsCEN and CsSHBY in regulating indeterminate/determinate growth of lateral branch in cucumber

Two genetic loci, det-ma (CsCEN) and det-lb, showed epistatic interaction on indeterminate/determinate growth of LB in cucumber. CsSHBY was identified as the candidate gene for det-lb locus. Plant architecture depends on the spatial regulation of meristems from both main axis (MA) and lateral branches (LBs). Fate (indeterminate or determinate) of these meristems is a crucial source of architectural diversity determining crop productivity and management. CENTRORADIALIS/TERMINAL FLOWER 1/SELF-PRUNING (CETS) gene family have been well known as pivotal regulators for indeterminate/determinate growth of MA. Nevertheless, genes that regulate LB indeterminacy/determinacy remained unclear. Cucumber (Cucumis sativus L.) has typical monopodial growth and multiple lateral branches. Both MA and LBs had indeterminate or determinate growth, and indeterminate/determinate growth of LB was controlled by two distinct loci, det-ma (CsCEN) and det-lb. In our study, based on bulked segregant analysis (BSA) method, the det-lb locus was mapped on a 60.6kb region on chromosome 1 harboring only one gene CsaV3_1G044330, which encoded a putative vacuolar-sorting protein (designated as CsSHBY). Multipoint mutations in CsSHBY were identified in D082 and D226, compared with CCMC, including nonsynonymous SNP mutations and a 6-bp deletion in exons. Further, qPCR showed that CsSHBY was highly expressed in lateral bud of CCMC, suggesting that CsSHBY might play an active role in regulating indeterminate/determinate growth of LB. Genetic analyses showed that det-ma (CsCEN) had an epistatic effect on det-lb (CsSHBY), and CsCEN could activate CsSHBY promoter by Dual luciferase and GUS activity assays. Meanwhile, Cscen or Csshby was found to influence auxin contents and CsYUCs and CsPINs expression levels. These findings provided new insights into precisely optimizing plant architecture for yield improvements.

Mapping and characterization of a novel adult-plant leaf rust resistance gene LrYang16G216 via bulked segregant analysis and conventional linkage method.

A novel adult-plant leaf rust resistance gene LrYang16G216 on wheat chromosome 6BL was identified and mapped to a 0.59cM genetic interval by BSA and conventional linkage method. Leaf rust (Puccinia triticina) is one of the most devastating fungal diseases of wheat (Triticum aestivum L.). Discovery and identification of new resistance genes is essential to develop disease-resistant cultivars. An advanced breeding line Yang16G216 was previously identified to confer adult-plant resistance (APR) to leaf rust. In this research, a recombinant inbred line (RIL) population was constructed from the cross between Yang16G216 and a highly susceptible line Yang16M6393, and genotyped with exome capture sequencing and 55K SNP array. Through bulked segregant analysis (BSA) and genetic linkage mapping, a stable APR gene, designated as LrYang16G216, was detected and mapped to the distal region of chromosome arm 6BL with a genetic interval of 2.8cM. For further verification, another RIL population derived from the cross between Yang16G216 and a susceptible wheat variety Yangmai 29 was analyzed using the enriched markers in the target interval, and LrYang16G216 was further narrowed to a 0.59cM genetic interval flanked by the KASP markers Ax109403980 and Ax95083494, corresponding to the physical position 712.34-713.94Mb in the Chinese Spring reference genome, in which twenty-six disease resistance-related genes were annotated. Based on leaf rust resistance spectrum, mapping data and physical location, LrYang16G216 was identified to be a novel and effective APR gene. The LrYang16G216 with linked markers will be useful for marker-assisted selection in wheat resistance breeding.

Inheritance of spike color in einkorn wheat (Triticum monococcum L.)

Aim: specify the spike color inheritance in einkorn wheat (Triticum monococcum L.) hybrids. Methods: reciprocal hybrids between the black-spikeed UA0300282 and white-spikeed UA0300311 cultivated einkorn accessions were created with the use of the “single cross” method. Four generations were analyzed using the segregation analysis method: P1, P2, F1, and F2 at autumn and spring sowing. Results: it was found that for the combination UA0300311 × UA0300282 at autumn sowing, the most suitable inheritance model is MX2-EA-AD, which implies the presence of two main genes with an equal additive effect plus polygene systems with an additive-dominant effect. In the plants of spring sowing, spike color is described by the MX2-CD-AD model, which suggests the presence of two major genes with full dominant effect plus polygenes with additive-dominant effect. In the reciprocal combination UA0300282 × UA0300311, the optimal model that describes best the spike color dispersion in plants of autumn sowing is MX2-ADI-AD, which suggests the presence of two main genes with an additive-dominant-epistatic effect plus polygenes with the additive-dominant effect. Distribution of the spring-sowing plants in terms of the spike color is well described by the MX2-ADI-ADI model – two main genes with an additive-dominant-epistatic effect plus a system of polygenes also with an additive-dominant-epistatic effect. The genes manifest themselves differently in the trait control depending on the weather conditions determined by the sowing time. In the group of direct combination plants (UA0300311 × UA0300282) of autumn sowing, heritability determined by the main gene is 97%, while that determined by polygenes is 2.7%; at spring sowing, these values are 67% and 32% respectively. In the reciprocal combination (UA0300282 × UA0300311) of autumn sowing, the main genes heritability effect is 99%, and the polygenic system accounts for 1%; in plants of spring sowing, respectively, 72%, and 28%. Conclusions: on the basis of the spike color expressiveness in the crossing combination of the einkorn kinds of wheat UA0300311 × UA0300282, the parental forms differ in two main genes and polygenes. The ratio of spike color heritability components depends on the growing conditions: at autumn sowing, 97–99 % of heritability is determined by the main genes, the polygenes account for 1–3 % of phenotypic variability; at spring sowing, the heritability component increases to 28–33 % due to the polygenic complex.

Fine mapping of a major QTL qPA7-1 for low hydrocyanic acid content in sorghum-sudangrass hybrid.

The purpose of this study was to study the genetic mechanism of low hydrocyanic acid (HCN) content. The segregation of HCN content trait in fresh stems and leaves was determined in the sorghum (Sorghum bicolor (L.) Moench)-sudangrass (Sorghum sudanense (Piper) Stapf) hybrid F2 population (N=1200), also used to detect a quantitative trait locus (QTL) for HCN content. Our hypothesis was that the additive effect of QTL was negative, showing that QTL was associated with low HCN. In the present research, a total of 11 simple sequence repeats (SSR) polymorphic primers were screened, and four SSR markers associated with low HCN content were developed based on the bulked segregant analysis method. A high-resolution genetic linkage group of the previously known qPA7-1 locus of the low HCN trait was constructed by analyzing different populations, families, and recombinants. Then, the QTL qPA7-1 of sorghum-sudangrass hybrid was fine-mapped to a 203.6kb region between markers SORBI4G4-120 and SORBI4G4-680, and seven candidate genes for low HCN were predicted in this region based on sequence comparison with the sorghum reference genome. According to gene annotation, the candidate genes related to low HCN content may be different from those involved in the known regulation mode of sorghum dhurrin biosynthesis and metabolism.

The Formation of Hollow Trait in Cucumber (Cucumis sativus L.) Fruit Is Controlled by CsALMT2.

The hollow trait is crucial for commercial quality of cucumber (Cucumis sativus L.) fruit, and its molecular regulatory mechanism is poorly understood due to its environmental sensitivity. In the previous research, we obtained the hollow and the non-hollow materials of ecotype cucumbers of South China, which were not easily affected by the external environment through a systematic breeding method. In this study, first, we proposed to use the percentage of the hollow area as the criterion to compare the hollow characteristics between two materials, and to analyze the formation mechanism of early hollow trait from the perspective of cytology. The results showed that the hollow trait occurred in the early stage of fruit development, and formed with the opening of carpel ventral zipped bi-cell layer, which formed rapidly from 2 to 4 days, and then slowed to a constant rate from 14 to 16 days. Meanwhile, the different genetic populations were constructed using these materials, and fine mapping was performed by bulked segregant analysis (BSA) and kompetitive allele specific PCR (KASP) method. The Csa1G630860 (CsALMT2), encoding protein ALMT2, was determined as a candidate gene for regulating the hollow trait in fruit. Furthermore, the expression profile of CsALMT2 was analyzed by qRT-PCR and fluorescence in situ hybridization. The expression of CsALMT2 had obvious tissue specificity, and it was abundantly expressed in the ovule development zone inside the fruit. In the hollow material of cucumber fruit, the expression of CsALMT2 was significantly downregulated. The subcellular localization in tobacco leaves indicated that CsALMT2 was distributed on the plasma membrane. In conclusion, in this study, for the first time, we found the regulatory gene of hollow trait in cucumber fruit, which laid the foundation for subsequent research on the molecular mechanism of hollow trait formation in cucumber fruit, and made it possible to apply this gene in cucumber breeding.

Fine mapping of qDB.A03, a QTL for rapeseed branching, and identification of the candidate gene.

Increasing the yield of rapeseed is required to meet the rapidly expanding demand for both edible vegetable oil and biofuel. Branching, an important determinant of yield potential in rapeseed, is controlled by a series of quantitative trait loci (QTLs). To explore the genetic mechanism regulating the natural variation of branching, a BC1F1 population derived from a cross between dense branching 2 (dense branching line) and L72 (normal branching line) was used to map QTL conferring branching in rapeseed. A major QTL, qDB.A03, for branching-related traits was identified by the BeadChip Array assisted bulked segregation analysis method, which was subsequently validated by the classical QTL mapping approach, and fine mapped to the 256kb region. This interval contains 56 annotated or predicted genes, 8 of which are candidates for controlling the branching trait. Comparative and expression analysis revealed four promising candidate genes for qDB.A03. Fine mapping and identification of the candidate genes for qDB.A03 represents the first step toward unraveling the genetical and molecular mechanisms controlling branching in rapeseed.

Genomic-Assisted Marker Development Suitable for CsCvy-1 Selection in Cucumber Breeding.

Cucumber is a widely grown vegetable crop plant and a host to many different plant pathogens. Cucumber vein yellowing virus (CVYV) causes economic losses on cucumber crops in Mediterranean countries and in some part of India such as West Bengal and in African countries such as Sudan. CVYV is an RNA potyvirus transmitted mechanically and by whitefly (Bemisia tabaci) in a semipersistent manner. Control of this virus is heavily dependent on the management of the insect vector and breeding virus-resistant lines. DNA markers have been used widely in conventional plant breeding programs via marker-assisted selection (MAS). However, very few resistance sources against CVYV in cucumber exist, and also the lack of tightly linked molecular markers to these sources restricts the rapid generation of resistant lines. In this work, we used genomics coupled with the bulked segregant analysis method and generated the MAS-friendly Kompetitive allele specific PCR (KASP) markers suitable for CsCvy-1 selection in cucumber breeding using a segregating F2 mapping population and commercial plant lines. Variant analysis was performed to generate single-nucleotide polymorphism (SNP)-based markers for mapping the population and genotyping the commercial lines. We fine-mapped the region by generating new markers down to 101 kb with eight genes. We provided SNP data for this interval, which could be useful for breeding programs and cloning the candidate genes.

Inheritance of marsh spot disease resistance in cranberry common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris) is an annual legume crop that is grown worldwide for its edible dry seeds and tender pods. Marsh spot (MS) of the seeds is a physio-genic stress disease affecting seed quality in beans. Studies have suggested that this disease involves a nutritional disorder caused by manganese deficiency, but the inheritance of resistance to this disease has not been reported. A biparental genetic population composed of 138 recombinant inbred lines (RILs) was developed from a cross between an MS resistant cultivar ‘Cran09’ and an MS susceptible cultivar ‘Messina’. The 138 RILs and their two parents were evaluated for MS resistance during five consecutive years from 2015 to 2019 in sandy and heavy clay soils in Morden, Manitoba, Canada. The MS incidence (MSI) and the MS resistance index (MSRI) representing disease severity were shown to be both highly correlated heritable traits that had high broad-sense heritability values (H2) of 86.5% and 83.2%, respectively. No significant differences for MSI and MSRI were observed between the two soil types in all five- (MSI) or four-year (MSRI) data collection, but significant correlations among years were observed despite MS resistance was moderately affected by year. The MSIs and MSRIs displayed a right-skewed distribution, indicating a mixed genetic model involving a few major genes and polygenes. Using the joint segregation analysis method, the same four major genes with additive-epistasis effects showed the best fit for both traits, explaining 84.4% and 85.3% of the phenotypic variance for MSI and MSRI, respectively. For both traits, the M1, M2, M3 and m4 acted as the favorable (resistant) alleles for the four genes where M and m represent two alleles of each gene. However, due to epistatic effects, only the individuals of the M1M2M3M4 haplotype appeared to be highly resistant, whereas those of the m1m2m3M4 haplotype were the most susceptible. The m4 allele significantly suppressed the additive effects of M1M2M3 on resistance, but decreased susceptibility due to the additive effects of m1m2m3. Further quantitative trait locus (QTL) mapping is warranted to identify and validate individual genes and develop molecular markers for marker-assisted selection of resistant cultivars.

MULTI-FLORET SPIKELET 4 (MFS4) Regulates Spikelet Development and Grain Size in Rice

In rice, the spikelet is the basic unit of inflorescence, and its development is important for determining the grain yield and quality. We reported a rice spikelet mutant multi-floret spikelet 4 (mfs4) which resulted in the production of extra floral organs or a whole extra floret, and elongated sterile lemmas. The results suggested that the mutation of the MFS4 gene interfered with spikelet meristem determinacy and floral organ identity. In addition, the plant height and the grain length and width in the mfs4 mutant were all less than those in the wild type. Using the bulked segregant analysis method, the MFS4 gene was localized in a 557-kb region on the long arm of chromosome 1. Sequence analysis showed that there was a C-base deletion at the open reading frame of LOC_Os01g67430. Further tests indicated that a wild type copy of LOC_Os01g67430 was able to reverse the mfs4 defects, which indicated that LOC_Os01g67430 was the MFS4 gene. The MFS4 gene encodes a lipase located in the mitochondria and is expressed strongly in the young inflorescence. qRT-PCR results showed that the expression of some genes that were known to regulate spikelet meristem determinacy and grain size were decreased in the mfs4 mutant, which indicated that the MFS4 gene regulates spikelet meristem determinacy and grain size by modulating the expression of these genes.

Genetic analysis and molecular mapping of Rp, a mutant gene encoding red pericarp in rice (Oryza sativa L.)

Coloured rice has pigments deposited in the grain pericarp; red rice is the most common type of coloured rice. Red rice is rich in essential nutrients and has been grown and consumed in China for a long time. In this study, we report the genetic characterisation and preliminary molecular mapping of a mutant gene encoding red pericarp in rice (Oryza sativa L.). To analyse the genetic basis of the red pericarp mutant, a reciprocal cross between GER-3 (red pericarp, indica cv.) and 898 (white pericarp, indica cv.) was made. The genetic analysis results confirmed that there was only one dominant gene, temporarily designated Rp (Red pericarp) controlling the segregation of the red pericarp in the F<sub>2</sub> population. For the molecular mapping of Rp, an F<sub>2</sub> population derived from an inter-subspecific cross between Gene Engineering Rice-3 (GER-3) and C418 (japonica cv., white pericarp) was constructed. The genotype of the pericarp colour of the F<sub>2</sub> individuals in the mapping population was validated by progeny testing of the F<sub>2:3</sub> families. Simple sequence repeat (SSR) markers and the bulked segregation analysis (BSA) method were used; Rp was mapped to the short arm of chromosome 7 between the SSR markers RM21182 and RM21268, with a genetic distance of 3.5 and 12.0 cM, respectively. In this paper, the potential origin of the red pericarp mutant gene Rp was also discussed.

Fine Mapping of a Locus Underlying the Ectopic Blade-Like Outgrowths on Leaf and Screening Its Candidate Genes in Rapeseed (Brassica napus L.).

Leaf is an important organ for higher plants, and the shape of it is one of the crucial traits of crops. In this study, we investigated a unique aberrant leaf morphology trait in a mutational rapeseed material, which displayed ectopic blade-like outgrowths on the adaxial side of leaf. The abnormal line 132000B-3 was crossed with the normal line 827-3. Based on the F2:3 family, we constructed two DNA pools (normal pool and abnormal pool) by the bulked segregant analysis (BSA) method and performed whole genome re-sequencing (WGR), obtaining the single-nucleotide polymorphism (SNP) and insertion/deletion (InDel) data. The SNP-index method was used to calculate the Δ(SNP/InDel-index), and then an association region was identified on chromosome A10 with a length of 5.5 Mbp, harboring 1048 genes totally. Subsequently, the fine mapping was conducted by using the penta-primer amplification refractory mutation system (PARMS), and the associated region was narrowed down to a 35.1-kbp segment, containing only seven genes. These seven genes were then analyzed according to their annotations and finally, BnA10g0422620 and BnA10g0422610, orthologs of LATE MERISTEM IDENTITY1 (LMI1) gene from Arabidopsis and REDUCED COMPLEXITY (RCO) gene from its relative Cardamine hirsuta, respectively, were identified as the candidate genes responding to this blade-like outgrowth trait in rapeseed. This study provides a novel perspective into the leaf formation in Brassica plants.

Identification of QTL for resistance to leaf blast in foxtail millet by genome re-sequencing analysis.

Three QTL for resistance to leaf blast were identified on chromosomes 1, 2, and 8 of the foxtail millet cultivar Yugu 5. Leaf blast disease of foxtail millet (Setaria italica) is caused by Pyricularia spp., can infect all the aboveground parts of plants, and is the most frequently observed blast disease in China. Lack of information on genetic control of disease resistance impedes developing leaf blast-resistant cultivars. An F6 recombinant inbred line (RIL) population from the cross Yugu 5 × Jigu 31 was phenotyped for its reactions to leaf blast in six field trials in the naturally diseased nurseries. An ultra-density genetic linkage map was constructed using 35,065 single nucleotide polymorphism (SNP) markers generated by sequencing of the RIL population. Three QTL, QLB-czas1, QLB-czas2, and QLB-cazas8, were detected in the genomic intervals of 276.6kb, 1.62Mb, and 1.75Mb on chromosomes 1, 2, and 8 of Yugu 5, which explained 14-17% (2 environments), 9% (5 environments), and 12-20% (6 environments) of the phenotypic variations. Bulked segregant analysis (BSA) and RNA sequencing (BSR-Seq) method identified common SNPs that fell in the genomic region of QLB-czas8, providing additional evidence of localization of this QTL. Three and 19 predicted genes were annotated to be associated with disease resistance in the genomic intervals for QLB-czas2 and QLB-czas8. Due to their unique positions, these QTL appear to be new loci conferring resistance to leaf blast. The identification of these new resistance QTL will be useful in cultivar development and the study of the genetic control of blast resistance in foxtail millet.

Sentiment analysis from travellers’ reviews using enhanced conjunction rule based approach for feature-specific evaluation of hotels

The evolution of the internet has steered an enormous amount of travel reviews of hotels on the web. People referring to these reviews are often overloaded and confused by the sheer amount of information available. Sentiment analysis techniques have been successful in aggregating the reviews, extracting their sentiments and thereby minimizing the information overload. But lacking in specific feature-based sentiment analysis has restricted customers in getting the actual scenario of hotels entirely. This paper presents a prospective design on lexicon-based approach for feature-based sentiment analysis of travel reviews on hotels or resorts. In particular, an enhanced form of conjuncture-based approach is proposed to segregate sentences into relevant clauses, identifying the feature and the sentiment value associated with it. Overall sentiment score for features like food, service, and location of a hotel is being calculated. The experiment results show significantly better accuracy and precision than the conventional text segregation and sentiment analysis methods, namely trigram and conjunction rule based approach.

QOil-3, a major QTL identification for oil content in cottonseed across genomes and its candidate gene analysis

Cotton (Gossypium spp.) is an important resource of fiber and oil. Oil content in cottonseed is getting more and more attention. In present study, a genetic map with 388 molecular markers was constructed to identify QTL for oil content in cottonseed. An immortal double backcross population including 376 individuals was generated from the crosses between HS46, MAR-188 and their 188 RILs. A total of 8 QTL associated with oil content were identified. One of them, qOil-3 for oil content explaining a collective phenotypic variance of 5.24 % was detected in four different environments of two years. It has only significant additive main effect from embryo genome and maternal additive main effect, implying high value in genetic breeding. The position for qOil-3 with the internal of approximately 2 cM on chromosome A03 overlapped the position of QTL for oil content by bulked segregant analysis and whole-genome re-sequencing method. Based on the sequence and gene expression analysis between two parents including HS46 and MAR-188 as well as DY-pool and GY-pool, two candidate genes Gh_A03G0701 and Gh_A03G0699 at the overlaps of qOil-3 were screened. Gh_A03G0701 was predicted to encode the 3-ketoacyl-CoA enzyme that is related to synthesis of oil. Two SNPs occurred in the exons of this gene between the extreme oil content pool, as well as between the parents HS46 and MAR-188. Gh_A03G0699 has four mutation loci in downstream sequence between HS46 and MAR-188 as well as GY-pool and DY-pool. Both candidate genes might serve as the foundation for further studies of the molecular mechanisms in natural variation of oil content and the genetic breeding for higher oil content in cottonseed.

Rapid Identification of a Genomic Region Conferring Dwarfism in Rapeseed (Brassica napus L.) YA2016-12

Plant height is a vital agronomic trait for crops, including oilseed crops such as rapeseed (Brassica napus L.). It affects the crop yield, oil content, and lodging resistance in rapeseed. In this study, we investigated a dwarf trait controlled by a semi-dominant allele in rapeseed. A dwarf line, YA2016-12, was crossed with a tall line, G184-189, and an F2 population was established. Forty of the tallest plants and 40 of the shortest plants from the F2 population were selected and two DNA pools (tall and dwarf) were constructed by the bulked segregant analysis (BSA) method. The two DNA pools and two parental DNAs were then re-sequenced. A sliding window analysis was used to calculate the Δ(SNP-index) and discover an association region on chromosome A03 with a length of 12.4 Mb. Within this region, we found 1225 genes, including 811 genes with non-synonymous or frameshift mutations between YA2016-12 and G184-189. Alignment to known plant height-related orthologs in Arabidopsis thaliana, as well as KEGG pathway and gene ontology annotations, was used to identify nine candidate genes (BnaA03g31770D, BnaA03g37960D, BnaA03g24740D, BnaA03g40550D, BnaA03g26120D, BnaA03g35130D, BnaA03g42350D, BnaA03g25610D, and BnaA03g39850D) involved in gibberellin or cytokinin signaling. Identification of the causal gene for this trait, and of genetic markers linked to favorable alleles, has potential utility for marker-assisted selection to breed rapeseed varieties with improved height.

Genetic variation and development of a SCAR marker of anemone-type flower in chrysanthemum

An anemone shape featuring elongated and pigmented disk florets is an important target in breeding for flower shape in chrysanthemum. A good understanding of the inheritance of anemone-related traits and specific molecular markers are vital for accelerating the breeding progress. In this study, two segregating intercultivar F1 populations of the anemone-type ‘Nannong Xuefeng’ × the nonanemone-type ‘QX096’ (80 lines) and the same anemone-type ‘Nannong Xuefeng’ × ‘Monalisa’ (144 lines) were employed to investigate the genetic variation of anemone traits, and the anemone-specific markers were developed via the bulked segregant analysis (BSA) method by constructing two extreme bulks from each 10 anemone-type and nonanemone-type F1 lines, respectively. A moderate high level of variation coefficient (~ 24 to 57%) was obtained for most tubular floret traits relevant to the anemone type in both segregating populations. The anemone morphology of the hybrids was intermediate between that of their parents, and some transgressive individuals were observed, indicating great potential for the selection of lines with the desirable anemone type. In the BSA, four sequence-related amplified polymorphism (SRAP) primer combinations were identified as informative between the bulks of the anemone and nonanemone F1 individuals. After cloning and sequencing, only one SRAP marker, i.e., M11E1_272, was successfully converted to the sequence-characterized amplified region (SCAR) marker SCAR168. The SCAR marker was validation in the two F1 populations demonstrated that the marker SCAR168 could truly discriminate anemone and nonanemone types with a high coincidence ratio 87.86~92.5%. In addition, a general linear model (GLM)-based association analysis (P < 0.01) revealed that the SCAR marker exhibited a positive effect on the anemone traits and explained 20.83~33.66% phenotypic variation. Results of the current study show the inheritance pattern of anemone traits, and the developed anemone-specific SCAR marker paves the way for the marker-assisted selection of flower shape in chrysanthemum.

Identification and Confirmation of New Molecular Markers Linked to The Fertility Restorer Genes in Sugar Beet

It is of special importance to track the fertility restoring alleles in plants such as sugar beet in which cytoplasmic male sterility (CMS) is used to produce hybrid seed, so that the possibility of detection of presence and absence of specific restoring alleles facilitates to a great extent the development of new O-type lines. For tagging of fertility restorer genes in sugar beet, an F2 population was used. At first, fertility scoring of F2 single plants in the field was done and the population was divided to four groups including fertile, half fertile, yellow sterile, and white sterile. Then, leaf sampling from the F2 single plants was accomplished for DNA extraction. Polymorphism of random primers of RAPD and STS primers was investigated in fertile and sterile bulks by bulk segregant analysis method. Several RAPD and STS primers produced polymorphism in fertile and sterile bulks. After testing the selected primers on the DNA of single plants of the bulk, F2 population and other sugar beet genotypes, one STS repulsion marker and one coupling marker were identified to distances of 17 and 8% recombination frequencies, respectively. In this research, the molecular basis of the genomic regions of fertility restoring loci of sugar beet was studied for the first time in Iran. These markers could be used for preliminary selection of O-type plants from pollinator populations and hence the time and costs related to pair crosses for identification of O-type character could be saved.

Abstract 618: Identification of Novel Disease Genes for Metabolic Syndrome

Factors that underlie the association of diverse traits of the metabolic syndrome have remained largely unknown. The strong heritable nature of this condition provides an exceptional avenue for discovery of the disease mechanisms by using modern techniques of human molecular genetics. Strikingly, efforts by genome-wide association studies to identify common variants that show association with two or more traits have largely failed. Growing evidence indicates excess of rare genetic variants through recent exponential population growth, raising the possibility for their causal role in common diseases. With the advent of high throughput sequencing the power for identification of functional rare variants has dramatically increased. By biasing for extreme phenotypes and utilizing traditional method of segregation analysis we have discovered a number of disease genes for metabolic syndrome and coronary artery disease. A major benefit of this strategy is that true causal relationships has been established between rare variations in a gene, its cognate pathways and the disease of interest. These, in turn, have provided fundamental new insight into pathogenesis of poorly understood diseases such as plaque erosion, and logical starting points for identifying new targets and pathways for therapeutic intervention. By dissecting cognate pathways, we have identified novel targets, including Dyrk1B kinase, a novel incretin and components of Wnt signaling pathway to treat CAD, diabetes and hyperlipidemia and have implemented those succesfuly in animal models.