Family-based association mapping in crop species

Identification of allelic variants associated with complex traits provides molecular genetic information associated with variability upon which both artificial and natural selections are based. Family-based association mapping (FBAM) takes advantage of linkage disequilibrium among segregating progeny within crosses and among parents to provide greater power than association mapping and greater resolution than linkage mapping. Herein, we discuss the potential adaption of human family-based association tests and quantitative transmission disequilibrium tests for use in crop species. The rapid technological advancement of next generation sequencing will enable sequencing of all parents in a planned crossing design, with subsequent imputation of genotypes for all segregating progeny. These technical advancements are easily adapted to mating designs routinely used by plant breeders. Thus, FBAM has the potential to be widely adopted for discovering alleles, common and rare, underlying complex traits in crop species.     

Epistatic association mapping in homozygous crop cultivars

The genetic dissection of complex traits plays a crucial role in crop breeding. However, genetic analysis and crop breeding have heretofore been performed separately. In this study, we designed a new approach that integrates epistatic association analysis in crop cultivars with breeding by design. First, we proposed an epistatic association mapping (EAM) approach in homozygous crop cultivars. The phenotypic values of complex traits, along with molecular marker information, were used to perform EAM. In our EAM, all the main-effect quantitative trait loci (QTLs), environmental effects, QTL-by-environment interactions and QTL-by-QTL interactions were included in a full model and estimated by empirical Bayes approach. A series of Monte Carlo simulations was performed to confirm the reliability of the new method. Next, the information from all detected QTLs was used to mine novel alleles for each locus and to design elite cross combination. Finally, the new approach was adopted to dissect the genetic basis of seed length in 215 soybean cultivars obtained, by stratified random sampling, from 6 geographic ecotypes in China. As a result, 19 main-effect QTLs and 3 epistatic QTLs were identified, more than 10 novel alleles were mined and 3 elite parental combinations, such as Daqingdou and Zhengzhou790034, were predicted.     

Prospects for association mapping in classical inbred mouse strains

The collection of classical inbred mouse strains displays heritable variation in a large number of complex traits. Many generations of historical recombination have contributed to the panel of classical strain genomes, raising the possibility that quantitative trait loci could be located with high resolution by correlating strain genotypes and phenotypes. Although this association mapping framework has been successful in several empirical applications, its expected performance remains unclear. We used computer simulations based on a publicly available, dense single-nucleotide polymorphism (SNP) map to measure the power and false-positive rate of association mapping on a genomic scale across 30 commonly used classical inbred strains. Expected power is (i) often low for phenotypic effect sizes that are realistic for complex traits, (ii) highly variable across the genome, and (iii) correlated with linkage disequilibrium, aspects of the allele frequency distribution, and haplotype characteristics, as predicted by theory. Simulations also demonstrate clear potential for spurious associations to be generated by unequal relatedness among the strains. These findings suggest that association mapping in the classical strains is best applied in combination with other procedures, such as QTL mapping.     

Future tools for association mapping in crop plants

Association mapping currently relies on the identification of genetic markers. Several technologies have been adopted for genetic marker analysis, with single nucleotide polymorphisms (SNPs) being the most popular where a reasonable quantity of genome sequence data are available. We describe several tools we have developed for the discovery, annotation, and visualization of molecular markers for association mapping. These include autoSNPdb for SNP discovery from assembled sequence data; TAGdb for the identification of gene specific paired read Illumina GAII data; CMap3D for the comparison of mapped genetic and physical markers; and BAC and Gene Annotator for the online annotation of genes and genomic sequences.     

Genetic architecture of maize kernel composition in the nested association mapping and inbred association panels

The maize (Zea mays) kernel plays a critical role in feeding humans and livestock around the world and in a wide array of industrial applications. An understanding of the regulation of kernel starch, protein, and oil is needed in order to manipulate composition to meet future needs. We conducted joint-linkage quantitative trait locus mapping and genome-wide association studies (GWAS) for kernel starch, protein, and oil in the maize nested association mapping population, composed of 25 recombinant inbred line families derived from diverse inbred lines. Joint-linkage mapping revealed that the genetic architecture of kernel composition traits is controlled by 21-26 quantitative trait loci. Numerous GWAS associations were detected, including several oil and starch associations in acyl-CoA:diacylglycerol acyltransferase1-2, a gene that regulates oil composition and quantity. Results from nested association mapping were verified in a 282 inbred association panel using both GWAS and candidate gene association approaches. We identified many beneficial alleles that will be useful for improving kernel starch, protein, and oil content.     

Whole-genome shotgun optical mapping of Rhodospirillum rubrum

Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a "molecular cytogenetics" approach to solving problems in genomic analysis.     

Genome-wide association mapping of three important traits using bread wheat elite breeding populations

The exponential development of molecular markers enables a more effective study of the genetic architecture of traits of economic importance, like test weight in wheat (Triticum aestivum L.), for which a high value is desired by most end-users. The association mapping (AM) method now allows more precise exploration of the entire genome. AM requires populations with substantial genetic variability of the traits of interest. The breeding lines at the end of a selection cycle, characterized for numerous traits, represent a potentially useful population for AM studies. Using three elite line populations, selected by several breeders and genotyped with about 2,500 Diversity Arrays Technology markers, several associations were identified between these markers and test weight, grain yield and heading date. To minimize spurious associations, we compared the general linear model and mixed linear model (MLM), which adjust for population structure and kinship differently. The MLM model with the kinship matrix was the most efficient. Finally, elite lines from several breeding programs had sufficient genetic variability to allow for the mapping of several chromosomal regions involved in the variation of three important traits.     

A new test for family-based association mapping with inbred lines from plant breeding programs

Association mapping holds great promise for the detection of quantitative trait loci (QTL) in plant breeding populations. The main objectives of this study were to (1) adapt the quantitative pedigree disequilibrium test to typical pedigrees of inbred lines produced in plant breeding programs, (2) compare the newly developed quantitative inbred pedigree disequilibrium test (QIPDT) with the commonly employed logistic regression ratio test (LRRT), with respect to the power and type I error rate of QTL detection, and (3) demonstrate the use of the QIPDT by applying it to flowering data of European elite maize inbreds. QIPDT and LRRT were compared based on computer simulations modeling 55 years of hybrid maize breeding in Central Europe. Furthermore, we applied QIPDT to a cross-section of 49 European elite maize inbred lines genotyped with 722 amplified fragment length polymorphism markers and phenotyped in four environments for days to anthesis. Compared to LRRT, the power to detect QTL was higher with QIPDT when using data collected routinely in plant breeding programs. Application of QIPDT to the 49 European maize inbreds resulted in a significant (P < 0.05) association located at a position for which a consensus QTL was detected in a previous study. The results of our study suggested that QIPDT is a promising QTL detection method for data collected routinely in plant breeding programs.     

Association mapping for chilling tolerance in elite flint and dent maize inbred lines evaluated in growth chamber and field experiments

Chilling sensitivity of maize is a strong limitation for its cultivation in the cooler areas of the northern and southern hemisphere because reduced growth in early stages impairs on later biomass accumulation. Efficient breeding for chilling tolerance is hampered by both the complex physiological response of maize to chilling temperatures and the difficulty to accurately measure chilling tolerance in the field under fluctuating climatic conditions. For this research, we used genome‐wide association (GWA) mapping to identify genes underlying chilling tolerance under both controlled and field conditions in a broad germplasm collection of 375 maize inbred lines genotyped with 56 110 single nucleotide polymorphism (SNP). We identified 19 highly significant association signals explaining between 5.7 and 52.5% of the phenotypic variance observed for early growth and chlorophyll fluorescence parameters. The allelic effect of several SNPs identified for early growth was associated with temperature and incident radiation. Candidate genes involved in ethylene signalling, brassinolide, and lignin biosynthesis were found in their vicinity. The frequent involvement of candidate genes into signalling or gene expression regulation underlines the complex response of photosynthetic performance and early growth to climatic conditions, and supports pleiotropism as a major cause of co‐locations of quantitative trait loci for these highly polygenic traits.     

Age-dependent transformation frequency in elite wheat varieties.

Wheat is a major world crop and as such is a primary target for improvement of agronomic characteristics via genetic engineering. Optimization of transformation is essential in order to overcome the relatively low transformation frequencies encountered with wheat. Transformation of elite wheat varieties is not always successful due to variability in regeneration and transformation frequencies between varieties. In this work, two elite wheat varieties with a relatively high embryogenic capacity were transformed by particle bombardment. A strong correlation between transformation frequency and the age of wheat donor plants was observed in both varieties. The mean transformation frequency rose from 0.7% to 5% when using immature embryos from old and young donor plants, respectively. This was observed in both varieties, the best bombardments achieving up to 7.3% frequency. Using explants at an optimal developmental stage from donor plants grown under environmentally-controlled conditions has improved the reproducibility of transformation efficiency of elite wheat varieties and leads to the production of apparently phenotypically normal, fertile, transgenic plants.     

Association mapping in an elite maize breeding population

Association mapping (AM) is a powerful approach to dissect the genetic architecture of quantitative traits. The main goal of our study was to empirically compare several statistical methods of AM using data of an elite maize breeding program with respect to QTL detection power and possibility to correct for population stratification. These models were based on the inclusion of cofactors (Model A), cofactors and population effect (Model B), and SNP effects nested within populations (Model C). A total of 930 testcross progenies of an elite maize breeding population were field-evaluated for grain yield and grain moisture in multi-location trials and fingerprinted with 425 SNP markers. For grain yield, population stratification was effectively controlled by Model A. For grain moisture with a high ratio of variance among versus within populations, Model B should be applied in order to avoid potential false positives. Model C revealed large differences among allele substitution effects for trait-associated SNPs across multiple plant breeding populations. This heterogeneous SNP allele substitution effects have a severe impact for genomic selection studies, where SNP effects are often assumed to be independent of the genetic background.     

Genome-wide association study identifies candidate genes that affect plant height in Chinese elite maize (Zea mays L.) inbred lines

Background

The harvest index for many crops can be improved through introduction of dwarf stature to increase lodging resistance, combined with early maturity. The inbred line Shen5003 has been widely used in maize breeding in China as a key donor line for the dwarf trait. Also, one major quantitative trait locus (QTL) controlling plant height has been identified in bin 5.05–5.06, across several maize bi-parental populations. With the progress of publicly available maize genome sequence, the objective of this work was to identify the candidate genes that affect plant height among Chinese maize inbred lines with genome wide association studies (GWAS).

Methods and Findings

A total of 284 maize inbred lines were genotyped using over 55,000 evenly spaced SNPs, from which a set of 41,101 SNPs were filtered with stringent quality control for further data analysis. With the population structure controlled in a mixed linear model (MLM) implemented with the software TASSEL, we carried out a genome-wide association study (GWAS) for plant height. A total of 204 SNPs (P≤0.0001) and 105 genomic loci harboring coding regions were identified. Four loci containing genes associated with gibberellin (GA), auxin, and epigenetic pathways may be involved in natural variation that led to a dwarf phenotype in elite maize inbred lines. Among them, a favorable allele for dwarfing on chromosome 5 (SNP PZE-105115518) was also identified in six Shen5003 derivatives.

Conclusions

The fact that a large number of previously identified dwarf genes are missing from our study highlights the discovery of the consistently significant association of the gene harboring the SNP PZE-105115518 with plant height (P = 8.91e-10) and its confirmation in the Shen5003 introgression lines. Results from this study suggest that, in the maize breeding schema in China, specific alleles were selected, that have played important roles in maize production.     

Recombinant inbred lines for genetic mapping in tomato

A cross between the cultivated tomato Lycopersicon esculentum and a related wild species L. cheesmanii yielded 97 recombinant inbred lines (RILs) which were used to construct a genetic map consisting of 132 molecular markers. Significant deviation from the expected 1:1 ratio between the two homozygous classes was found in 73% of the markers. In 98% of the deviating markers, L. esculentum alleles were present in greater frequency than the L. cheesmanii alleles. For most of the markers with skewed segregation, the direction of the deviation was maintained from F₂ to F₇ generations. The average heterozygosity in the population was 15%. This value is significantly greater than the 1.5% heterozygosity expected for RILs in the F₇ generation. On average, recombination between linked markers was twice as high in the RILs than in the F₂ population used to derive them. The utility of RILs for the mapping of qualitative and quantitative traits is discussed.     

Whole-genome association study on tissue tropism phenotypes in group A Streptococcus

Group A Streptococcus (GAS) has a rich evolutionary history of horizontal transfer among its core genes. Yet, despite extensive genetic mixing, GAS strains have discrete ecological phenotypes. To further our understanding of the molecular basis for ecological phenotypes, comparative genomic hybridization of a set of 97 diverse strains to a GAS pangenome microarray was undertaken, and the association of accessory genes with emm genotypes that define tissue tropisms for infection was determined. Of the 22 nonprophage accessory gene regions (AGRs) identified, only 3 account for all statistically significant linkage disequilibrium among strains having the genotypic biomarkers for throat versus skin infection specialists. Networked evolution and population structure analyses of loci representing each of the AGRs reveal that most strains with the skin specialist and generalist biomarkers form discrete clusters, whereas strains with the throat specialist biomarker are highly diverse. To identify coinherited and coselected accessory genes, the strength of genetic associations was determined for all possible pairwise combinations of accessory genes among the 97 GAS strains. Accessory genes showing very strong associations provide the basis for an evolutionary model, which reveals that a major transition between many throat and skin specialist haplotypes correlates with the gain or loss of genes encoding fibronectin-binding proteins. This study employs a novel synthesis of tools to help delineate the major genetic changes associated with key adaptive shifts in an extensively recombined bacterial species.     

影响农杆菌介导玉米优良自交系遗传转化的因素

以我国玉米骨干自交系9046,齐319,414,Mo17的幼胚为材料,在已经建立的农杆菌介导的玉米幼胚转化体系的基础上,研究了影响农杆菌介导玉米优良自交系遗传转化的因素,建立了优化的玉米优良自交系的遗传转化体系。研究结果表明,1．0—2．0mm的玉米幼胚是最适宜的转化受体;在感染液和共培养基中都加入乙酰丁香酮(200μmol／L)和抗坏血酸(50mg／L),能显著提高农杆菌对玉米的侵染能力;而感染前将幼胚高渗透压预处理未能提高转化率;延迟筛选有利于提高抗性愈伤组织的存活率。应用优化后的转化体系,获得了这4个玉米优良自交系的转基因植株,PCR阳性植株率为1．71％-4．09％。转化植株叶片总DNA的PCR和Southern杂交分析表明,T-DNA上的外源基因已经整合进了玉米基因组,并且在大多数转基因植株(71．4％)中为单位点插入。这一体系的建立,为进一步将有用基因导入玉米优良自交系奠定了基础。     

Whole-genome linkage analysis in mapping alcoholism genes using single-nucleotide polymorphisms and microsatellites

There is currently a great interest in using single-nucleotide polymorphisms (SNPs) in genetic linkage and association studies because of the abundance of SNPs as well as the availability of high-throughput genotyping technologies. In this study, we compared the performance of whole-genome scans using SNPs with microsatellites on 143 pedigrees from the Collaborative Studies on Genetics of Alcoholism provided by Genetic Analysis Workshop 14. A total of 315 microsatellites and 10,081 SNPs from Affymetrix on 22 autosomal chromosomes were used in our analyses. We found that the results from the two scans had good overall concordance. One region on chromosome 2 and two regions on chromosome 7 showed significant linkage signals (i.e., NPL >or= 2) for alcoholism from both the SNP and microsatellite scans. The different results observed between the two scans may be explained by the difference observed in information content between the SNPs and the microsatellites.     

Performance prediction of F1 hybrids between recombinant inbred lines derived from two elite maize inbred lines

Selection of recombinant inbred lines (RILs) from elite hybrids is a key method in maize breeding especially in developing countries. The RILs are normally derived by repeated self-pollination and selection. In this study, we first investigated the accuracy of different models in predicting the performance of F₁ hybrids between RILs derived from two elite maize inbred lines Zong3 and 87-1, and then compared these models through simulation using a wider range of genetic models. Results indicated that appropriate prediction models depended on genetic architecture, e.g., combined model using breeding value and genome-wide prediction (BV+GWP) has the highest prediction accuracy for high V _D/V _A ratio (>0.5) traits. Theoretical studies demonstrated that different components of genetic variance were captured by different prediction models, which in turn explained the accuracy of these models in predicting the F₁ hybrid performance. Based on genome-wide prediction model (GWP), 114 untested F₁ hybrids possibly having higher grain yield than the original F₁ hybrid Yuyu22 (the single cross between Zong3 and 87-1) have been identified and recommended for further field test.