Identification of SSR markers associated with height using pool-based genome-wide association mapping in sorghum

Sorghum has been proposed as a potential energy crop. However, it has been traditionally bred for grain yield and forage quality, not traits related to bioenergy production. To develop tools for genetic improvement of bioenergy-related traits such as height, genetic markers associated with these traits have first to be identified. Association mapping has been extensively used in humans and in some crop plants for this purpose. However, genome-wide association mapping using the whole association panel is costly and time-consuming. A variation of this method called pool-based genome-wide association mapping has been extensively used in humans. In this variation, pools of individuals with contrasting phenotypes, instead of the whole panel, are screened with genetic markers and polymorphic markers are confirmed by screening the individuals in the pools. Here, we identified several new simple sequence repeats (SSR) markers associated with height using this pool-based genome-wide association mapping in sorghum. After screening the tall and short pools of sorghum accessions from the sorghum Mini Core collection developed at the International Crops Research Institute for the Semi-Arid Tropics with 703 SSR markers, we have identified four markers that are closely associated with sorghum height on chromosomes 2, 6, and 9. Comparison with published maps indicates that all four markers are clustered with markers previously mapped to height or height-related traits and with candidate genes involved in regulating plant height such as FtsZ, Ugt, and GA 2-oxidase. The mapping method can be applied to other crop plants for which a high-throughput genome-wide association mapping platform is not yet available.     

A core collection and mini core collection of Oryza sativa L. in China

The extent of and accessibility to genetic variation in a large germplasm collection are of interest to biologists and breeders. Construction of core collections (CC) is a favored approach to efficient exploration and conservation of novel variation in genetic resources. Using 4,310 Chinese accessions of Oryza sativa L. and 36 SSR markers, we investigated the genetic variation in different sized sub-populations, the factors that affect CC size and different sampling strategies in establishing CC. Our results indicated that a mathematical model could reliably simulate the relationship between genetic variation and population size and thus predict the variation in large germplasm collections using randomly sampled populations of 700?C1,500 accessions. We recommend two principles in determining the CC size: (1) compromising between genetic variation and genetic redundancy and (2) retaining the main types of alleles. Based on the most effective scheme selected from 229 sampling schemes, we finally developed a hierarchical CC system, in which different population scales and genetic diversities allow a flexible use of genetic resources. The CC, comprising 1.7% (932) of the accessions in the basic collection, retained more than 85% of both the SSR and phenotypic variations. A mini core collection, comprising 0.3% (189) of the accessions in the basic collection, retained 70.65% of the SSR variation and 76.97% of the phenotypic variation, thus providing a rational framework for intensive surveys of natural variation in complex traits in rice genetic resources and hence utilization of variation in rice breeding.     

Rapid SNP discovery and genetic mapping using sequenced RAD markers

Single nucleotide polymorphism (SNP) discovery and genotyping are essential to genetic mapping. There remains a need for a simple, inexpensive platform that allows high-density SNP discovery and genotyping in large populations. Here we describe the sequencing of restriction-site associated DNA (RAD) tags, which identified more than 13,000 SNPs, and mapped three traits in two model organisms, using less than half the capacity of one Illumina sequencing run. We demonstrated that different marker densities can be attained by choice of restriction enzyme. Furthermore, we developed a barcoding system for sample multiplexing and fine mapped the genetic basis of lateral plate armor loss in threespine stickleback by identifying recombinant breakpoints in F(2) individuals. Barcoding also facilitated mapping of a second trait, a reduction of pelvic structure, by in silico re-sorting of individuals. To further demonstrate the ease of the RAD sequencing approach we identified polymorphic markers and mapped an induced mutation in Neurospora crassa. Sequencing of RAD markers is an integrated platform for SNP discovery and genotyping. This approach should be widely applicable to genetic mapping in a variety of organisms.     

Association mapping of dynamic developmental plant height in common wheat

Drought as a major abiotic stress often occurs from stem elongation to the grain filling stage of wheat in northern China. Plant height (PH) is a suitable trait to model the dissection of drought tolerance. The purposes of the present study were to validate molecular markers for PH developmental behavior and identify elite alleles of molecular markers. After the phenotyping of 154 accessions for PH dynamic development under well-watered (WW) and drought stressed (DS) conditions, and the genotyping of 60 SSR markers from six candidate chromosome regions related to PH found in our previous linkage mapping studies, both parameters PH and drought tolerance coefficient (DTC) calculated by the conditional analysis were used for association mapping. A total of 46 significant association signals (P < 0.01) were identified in 23 markers, and phenotypic variation ranged from 7 to 50%. Among them, four markers Xgwm261-2D, Xgwm495-4B, Xbarc109-4B and Xcfd23-4D were detected under both water regimes. Furthermore, 10 markers were associated with DTC, and four with both parameters PH and DTC at the same plant development stage. The results revealed different allelic effects of associated markers; for example, the 155 bp Xgwm495-4B allele was associated with a reduced height of −11.2 cm under DS and −15.3 cm under WW, whereas the 167 bp allele exhibited increased height effects of 3.9 and 8.1 cm, respectively. This study demonstrates a strong power of joint association analysis and linkage mapping for the identification of important genes in wheat.     

Association mapping of seed quality traits using the Canadian flax (Linum usitatissimum L.) core collection

Key message

The identification of stable QTL for seed quality traits by association mapping of a diverse panel of linseed accessions establishes the foundation for assisted breeding and future fine mapping in linseed.

Abstract

Linseed oil is valued for its food and non-food applications. Modifying its oil content and fatty acid (FA) profiles to meet market needs in a timely manner requires clear understanding of their quantitative trait loci (QTL) architectures, which have received little attention to date. Association mapping is an efficient approach to identify QTL in germplasm collections. In this study, we explored the quantitative nature of seed quality traits including oil content (OIL), palmitic acid, stearic acid, oleic acid, linoleic acid (LIO) linolenic acid (LIN) and iodine value in a flax core collection of 390 accessions assayed with 460 microsatellite markers. The core collection was grown in a modified augmented design at two locations over 3 years and phenotypic data for all seven traits were obtained from all six environments. Significant phenotypic diversity and moderate to high heritability for each trait (0.73–0.99) were observed. Most of the candidate QTL were stable as revealed by multivariate analyses. Nine candidate QTL were identified, varying from one for OIL to three for LIO and LIN. Candidate QTL for LIO and LIN co-localized with QTL previously identified in bi-parental populations and some mapped nearby genes known to be involved in the FA biosynthesis pathway. Fifty-eight percent of the QTL alleles were absent (private) in the Canadian cultivars suggesting that the core collection possesses QTL alleles potentially useful to improve seed quality traits. The candidate QTL identified herein will establish the foundation for future marker-assisted breeding in linseed.     

Association mapping of agro-morphological characters among the global collection of finger millet genotypes using genomic SSR markers

Identification of alleles responsible for various agro-morphological characters is a major concern to further improve the finger millet germplasm. Forty-six genomic SSRs were used for genetic analysis and population structure analysis of a global collection of 190 finger millet genotypes and fifteen agro-morphological characters were evaluated. The overall results showed that Asian genotypes were smaller in height, smaller flag leaf length, less basal tiller number, early flowering and early maturity nature, small ear head length, and smaller in length of longest finger. The 46 SSRs yielded 90 scorable alleles and the polymorphism information content values varied from 0.292 to 0.703 at an average of 0.442. The gene diversity was in the range of 0.355 to 0.750 with an average value of 0.528. The 46 genomic SSR loci grouped the 190 finger millet genotypes into two major clusters based on their geographical origin by the both phylogenetic clustering and population structure analysis by STRUCTURE software. Association mapping of QTLs for 15 agro-morphological characters with 46 genomic SSRs resulted in identification of five markers were linked to QTLs of four traits at a significant threshold (P) level of ≤0.01 and ≤0.001. The QTL for basal tiller number was strongly associated with the locus UGEP81 at a P value of 0.001 by explaining the phenotypic variance (R ²) of 10.8 %. The QTL for days to 50 % flowering was linked by two SSR loci UGEP77 and UGEP90, explained 10 and 8.7 % of R ² respectively at a P value of 0.01. The SSR marker, FM9 found to have strong association to two agro-morphological traits, flag leaf width (P—0.001, R ²—14.1 %) and plant height (P—0.001, R ²—11.2 %). The markers linked to the QTLs for above agro-morphological characters found in the present study can be further used for cloning of the full length gene, fine mapping and their further use in the marker assisted breeding programmes for introgression of alleles into locally well adapted germplasm.     

Efficient mapping of plant height quantitative trait loci in a sorghum association population with introgressed dwarfing genes

Of the four major dwarfing genes described in sorghum, only Dw3 has been cloned. We used association mapping to characterize the phenotypic effects of the dw3 mutation and to fine map a second, epistatic dwarfing QTL on sorghum chromosome 9 (Sb-HT9.1). Our panel of 378 sorghum inbreds includes 230 sorghum conversion (SC) lines, which are exotic lines that have been introgressed with dwarfing quantitative trait loci (QTL) from a common parent. The causal mutation in dw3 associates with reduced lower internode length and an elongation of the apex, consistent with its role as an auxin efflux carrier. Lines carrying the dw3 mutation display high haplotype homozygosity over several megabases in the Dw3 region, but most markers linked to Dw3 do not associate significantly with plant height due to allele sharing between Dw3 and dw3 individuals. Using markers with a high mutation rate and the dw3 mutation as an interaction term, significant trait associations were detected across a 7-Mb region around Sb-HT9.1, largely due to higher detection power in the SC lines. Conversely, the likely QTL interval for Sb-HT9.1 was reduced to approximately 100 kb, demonstrating that the unique structure of this association panel provides both power and resolution for a genomewide scan.     

Association mapping of leaf rust resistance loci in a spring wheat core collection

Key message

We identified 15 potentially novel loci in addition to previously characterized leaf rust resistance genes from 1032 spring wheat accessions. Targeted AM subset panels were instrumental in revealing interesting loci.

Abstract

Leaf rust is a common disease of wheat, consistently reducing yields in many wheat-growing regions of the world. Although fungicides are commonly applied to wheat in the United States (US), genetic resistance can provide less expensive, yet effective control of the disease. Our objectives were to map leaf rust resistance genes in a large core collection of spring wheat accessions selected from the United States Department of Agriculture-Agricultural Research Service National Small Grains Collection (NSGC), determine whether previously characterized race-nonspecific resistance genes could be identified with our panel, and evaluate the use of targeted panels to identify seedling and adult plant resistance (APR) genes. Association mapping (AM) detected five potentially novel leaf rust resistance loci on chromosomes 2BL, 4AS, and 5DL at the seedling stage, and 2DL and 7AS that conditioned both seedling and adult plant resistance. In addition, ten potentially novel race-nonspecific resistance loci conditioned field resistance and lacked seedling resistance. Analyses of targeted subsets of the accessions identified additional loci not associated with resistance in the complete core panel. Using molecular markers, we also confirmed the presence and effectiveness of the race-nonspecific genes Lr34, Lr46, and Lr67 in our panel. Although most of the accessions in this study were susceptible to leaf rust in field and seedling tests, many resistance loci were identified with AM. Through the use of targeted subset panels, more loci were identified than in the larger core panels alone.

     

Identification of SSR markers associated with saccharification yield using pool-based genome-wide association mapping in sorghum

Saccharification describes the conversion of plant biomass by cellulase into glucose. Because plants have never been selected for high saccharification yield, cellulosic ethanol production faces a significant bottleneck. To improve saccharification yield, it is critical to identify the genes that affect this process. In this study, we used pool-based genome-wide association mapping to identify simple sequence repeat (SSR) markers associated with saccharification yield. Screening of 703 SSR markers against the low and high saccharification pools identified two markers on the sorghum chromosomes 2 (23-1062) and 4 (74-508c) associated with saccharification yield. The association was significant at 1% using either general or mixed linear models. Localization of these markers based on the whole genome sequence indicates that 23-1062 is 223 kb from a β-glucanase (Bg) gene and 74-508c is 81 kb from a steroid-binding protein (Sbp) gene. Bg is critical for cell wall assembly and degradation, but Sbp can suppress the expression of Bg as demonstrated in Arabidopsis (Yang et al. 2005). These markers are found physically close to genes encoding plant cell wall synthesis enzymes such as xyloglucan fucosyltransferase (149 kb from 74-508c) and UDP-D-glucose 4-epimerase (46 kb from 23-1062). Genetic transformation of selected candidate genes is in progress to examine their effect on saccharification yield in plants.     

Genome-wide association mapping identifies markers associated with cane yield components and sucrose traits in the Louisiana sugarcane core collection

Sugarcane is an economically important crop for both food and biofuel industries. Marker-assisted breeding in sugarcane is becoming a reality with the recent development and deployment of markers linked with disease resistance genes. Large linkage disequilibrium in sugarcane makes genome-wide association studies (GWAS) a better alternative to biparental mapping to identify markers associated with agronomic traits. GWAS was conducted on a Louisiana core collection to identify marker-trait associations (MTA) for 11 cane yield and sucrose traits using single nucleotide polymorphism (SNP) and insertion-deletion (Indel) markers. Significant (P < .05) MTAs were identified for all traits where the top ranked markers explained up to 15% of the total phenotypic variation. High correlations (0.732 to 0.999) were observed between sucrose traits and 56 markers were found consistent across multiple traits. These markers following validation in more diverse populations could be used in marker-assisted selection of clones in sugarcane breeding program in Louisiana and elsewhere.     

Genetic diversity in an African plantain core collection using AFLP and RAPD markers

Fifteen AFLP primer pairs (EcoRI+3 and MseI+3) and 60 10-mer RAPD primers were used to detect polymorphisms and assess genetic relationships in a sample of 25 plantains from diverse parts of Western and Central Africa. The discriminatory power of the AFLP technique was greater than that of the RAPD technique, since the former produced markers with greater polymorphic information content (PIC) than the latter. Hence, AFLP analysis appeared to be a more-powerful approach for identifying genetic differences among plantain accessions. In this regard, significant genetic diversity within the plantains was shown by the unweighted pair-group method of arithmetic averages (UPGMA) and the multidimensional principal coordinate (PCO) analyses. The AFLP-derived clusters indicated closer relationships between similar inflorescence types than the RAPD-derived clusters. A small group of cultivars from Cameroon were separated from the bulk of other plantains, suggesting that Cameroon may harbour accessions with useful or rare genes for widening the genetic base of breeding populations derived from the plantains. A greater effort should be directed at collecting and characterizing plantain cultivars from Cameroon.     

SNP markers linked to leaf rust and grain mold resistance in sorghum

Grain mold and rust are diseases that can significantly reduce sorghum grain yield. Breeding for resistance to these diseases is hindered by inefficient disease screening. A viable option to greatly improve breeding efficiency is to identify molecular markers or genes linked to the host resistance. In this study, we applied 14,739 single nucleotide polymorphism markers to the sorghum mini core of 242 accessions that had been evaluated for rust resistance in both greenhouse and field and for grain mold in the field for 2 years. Through association mapping we have identified two loci linked to grain mold resistance and five loci linked to rust resistance. Among the two loci linked to grain mold resistance, one contained a homolog of the maize nonhost resistance gene Rxo1. Two of rust-linked loci each contained the rust resistance gene homologous to the maize rust resistance gene Rp1-D which is the B locus (the A locus containing Pu was not linked in this study) and to the wheat rust resistance gene Lr1. The remaining loci contained genes important in other steps of the defense response, such as cyclophilins that mediate resistance response preceding hypersensitive response (HR) and Hin1 directly involved in producing HR. The results from this study will facilitate marker-assisted selection of host resistance to grain mold and rust in sorghum.     

Identification of genomic regions affecting plant height in sorghum and maize

The objective of this study was to use restriction fragment length polymorphisms (RFLPs) to determine the genetic location and effects of genomic regions controlling plant height in sorghum. F₂ plants (152) from the cross CK60 x PI229828 were used. Genomic and cDNA clones (106) identified 111 loci distributed among ten linkage groups covering 1299 cM. Interval mapping identified four regions, each in a separate linkage group. These regions may correspond to loci (dw) previously identified by alleles with qualitative effects. Also, these regions identified in sorghum may be orthologous to those previously reported for plant height in maize. Gene effects and gene action varied among genomic regions. In each region, PI229828 alleles resulted in increased plant height. Each region accounted for 9.2–28.7% of the phenotypic variation. Positive, additive effects ranged from 15 to 32cm. Tallness was dominant or overdominant and conferred by alleles from PI229828 for three quantitative trait loci (QTL). At the fourth QTL, PI229828 contributed to increased plant height, but short stature was partially dominant. One digenic interaction was significant. The presence of a PI229828 allele at one region diminished the effects of the other region. A multiple model indicated that these four regions collectively accounted for 63.4% of the total phenotypic variation. The utility of this information for germplasm conversion through backcross breeding is discussed.Journal Paper No. J. 15649 of the Iowa Agriculture and Home Economic Experiment Station, Ames, Iowa. Project No. 3134     

Association mapping of late maturity α-amylase (LMA) activity in a collection of synthetic hexaploid wheat

Late maturity α-amylase (LMA) is a genetic defect of wheat which results in the production of α-amylase, shown as substandard falling numbers, in the absence of preharvest rain and under cool temperatures during ripening. The present study is an attempt to use a whole-genome scan with DArT markers to identify chromosomal regions influencing LMA in synthetic hexaploid wheat (SHW). A high heritability estimate of 86.6% was calculated for LMA phenotype measured as optical density in a collection of 91 SHWs. Linkage disequilibrium extended up to 10 cM, and with controls for false positives, significant markers were detected at the chromosome 7B region previously linked to LMA in bread wheat, but not at the chromosome 3B region. Of potentially great interest is a region on chromosome 6B, which was identified as having a significant association with LMA phenotypes in the SHW accessions. Previous investigations suggested existence of an LMA gene on the long arm of 6B, but this is the first time it has been mapped to lie within the centromeric region of chromosome 6B, a region that harbours the Amy-1 genes and whose expression governs activity of the high pI α-amylase isoenzymes.     

Analysis of genetic mapping in a waxy/dent maize RIL population using SSR and SNP markers

A maize genetic linkage map was generated using SSR and SNP markers in a F_7:8 recombinant inbred line (RIL) population derived from a cross of waxy corn (KW7) and dent corn (Mo17). A total of 465 markers, including 459 SSR and 6 SNP markers, were assigned to 10 linkage groups which spanned 2,656.5 cM with an average genetic distance between markers of 5.7 cM, and the number of loci per linkage group ranged from 39 to 55. The SSR (85.4%) and SNP (83.3%) markers showed Mendelian segregation ratios in the RIL population at a 5% significance threshold. In linkage analysis of six SNP loci associated with kernel starch synthesis genes (ae1, bt2, sh1, sh2, su1, and wx1), all six loci were successfully mapped and are closely linked with SSR markers in chromosomes 3 (sh2), 4 (su1 and bt2), 5 (ae1), and 9 (sh1 and wx1). The SSR markers linked with genes in starch synthesis may be utilized in marker assisted breeding programs. The resulting genetic map will be useful in dissection of quantitative traits and the identification of superior QTLs from the waxy hybrid corn. Additionally, these data support further genetic analysis and development of maize breeding programs.     

Genome-wide linkage mapping of yield-related traits in three Chinese bread wheat populations using high-density SNP markers

Key message

We identified 21 new and stable QTL, and 11 QTL clusters for yield-related traits in three bread wheat populations using the wheat 90 K SNP assay.

Abstract

Identification of quantitative trait loci (QTL) for yield-related traits and closely linked molecular markers is important in order to identify gene/QTL for marker-assisted selection (MAS) in wheat breeding. The objectives of the present study were to identify QTL for yield-related traits and dissect the relationships among different traits in three wheat recombinant inbred line (RIL) populations derived from crosses Doumai?×?Shi 4185 (D?×?S), Gaocheng 8901?×?Zhoumai 16 (G?×?Z) and Linmai 2?×?Zhong 892 (L?×?Z). Using the available high-density linkage maps previously constructed with the wheat 90 K iSelect single nucleotide polymorphism (SNP) array, 65, 46 and 53 QTL for 12 traits were identified in the three RIL populations, respectively. Among them, 34, 23 and 27 were likely to be new QTL. Eighteen common QTL were detected across two or three populations. Eleven QTL clusters harboring multiple QTL were detected in different populations, and the interval 15.5–32.3 cM around the Rht-B1 locus on chromosome 4BS harboring 20 QTL is an important region determining grain yield (GY). Thousand-kernel weight (TKW) is significantly affected by kernel width and plant height (PH), whereas flag leaf width can be used to select lines with large kernel number per spike. Eleven candidate genes were identified, including eight cloned genes for kernel, heading date (HD) and PH-related traits as well as predicted genes for TKW, spike length and HD. The closest SNP markers of stable QTL or QTL clusters can be used for MAS in wheat breeding using kompetitive allele-specific PCR or semi-thermal asymmetric reverse PCR assays for improvement of GY.

     

Advantages of Amplifluor-like SNP markers over KASP in plant genotyping

Background

KASP (KBioscience Competitive Allele Specific PCR) and Amplifluor (Amplification with fluorescence) SNP markers are two prominent technologies based upon a shared identical Allele-specific PCR platform.

Methods

Amplifluor-like SNP and KASP analysis was carried out using published and own design of Universal probes (UPs) and Gene-specific primers (GSPs).

Results

Advantages of the Amplifluor-like system over KASP include the significantly lower costs and much greater flexibility in the adjustment and development of ‘self-designed’ dual fluorescently-labelled UPs and regular GSPs. The presented results include optimisation of ‘tail’ length in UPs and GSPs, protocol adjustment, and the use of various fluorophores in different qPCR instruments. The compatibility of the KASP Master-mix in both original and Amplifluor-like systems has been demonstrated in the presented results, proving their similar principles. Results of SNP scoring with rare alleles in addition to more frequently occurring alleles are shown.

Conclusions

The Amplifluor-like system produces SNP genotyping results with a level of sensitivity and accuracy comparable to KASP but at a significantly cheaper cost and with much greater flexibility for UPs with self-designed GSPs.

     

Cytonuclear epistatic quantitative trait locus mapping for plant height and ear height in maize

Plant height (PH) and ear height (EH) are important traits in maize (Zea mays L.) breeding. Previous research has indicated that these traits are influenced by quantitative trait loci (QTL). However, previous studies attempting to identify the genetic bases of PH and EH have ignored the possibility that cytoplasmic effects and cytonuclear interactions may influence these traits. The objectives of this study were to identify the cytonuclear epistatic QTL and to evaluate the contributions of cytoplasm and QTL × cytoplasm interactions to phenotypic variation of PH and EH. A reciprocal mating design was conducted to generate F2 mapping populations comprising 120 F2 plants from the direct cross (JB × Y53) and 120 F2 plants from the reciprocal cross (Y53 × JB). F2:3 mapping populations were further generated with 91 direct F2:3 families and 120 reciprocal F2:3 families (ten plants per family). The PH and EH of the above F2 and F2:3 mapping populations were evaluated in the same field at the same experimental station in 2007 and 2008. A genetic linkage map with 154 microsatellite markers was constructed, which covered 1,735.0 cM of the maize genome with an average marker spacing of 11.3 cM. A joint-analysis method incorporating the cytonuclear interaction mapping approach was proposed and performed to detect cytonuclear interacting QTL affecting PH and EH. We identified six cytonuclear epistatic QTL affecting PH and five affecting EH. The average phenotypic variance explained by the genetic components of the QTL × cytoplasm interaction for each QTL was 18 % for PH and 9 % for EH. In addition, we observed cytoplasmic effects contributing substantially to phenotypic variance, reaching 9 and 40 % of the phenotypic contributions to PH and EH, respectively.