Analysis of genetic diversity and relationships among waxy maize inbred lines in Korea using SSR markers

Information regarding the genetic diversity and genetic relationships among elite inbred lines is necessary to improve new cultivars in maize breeding programs. In this study, genetic diversity and genetic relationships were investigated among 84 waxy maize inbred lines using 50 SSR markers. A total of 269 alleles were identified at all the loci with an average of 5.38 and a range between 2 and 13 alleles per locus. The gene diversity values varied from 0.383 to 0.923 with an average of 0.641. The cluster tree generated using the described SSR markers recognized two major groups at 32% genetic similarity. Group I included 33 inbred lines while group II included 51 inbred lines. The clustering patterns of most of the waxy maize inbred lines did not clearly agree with their source, pedigree or geographic location. The average GS among all inbred lines was 35.7 ± 10.8. Analysis of waxy maize inbred lines collected from Korea and China at 50 SSR loci revealed higher values of average number of alleles (4.9) and gene diversity (0.638) in Korean inbred lines as compared to Chinese inbred lines (3.5 and 0.563, respectively). The information obtained from the present studies would be very useful for maize breeding programs in Korea.     

Analysis of genetic distance by SSR in waxy maize

We examined the genetic diversity of 80 inbred waxy maize lines using 22 SSR molecular markers that could be used to achieve heterosis in waxy maize. Eighty inbred waxy maize lines with different phenotypes, 40 yellow, 25 white, 13 black, and two red lines were analyzed by SSR molecular marker fingerprint and cluster analysis. Using a standard genetic distance of 0.55, the 80 waxy maize inbred lines were clustered into nine groups. Among them, group II, group V, groups VII and VIII, and group IX were divided into three subgroups at a genetic distance of 0.46, into two subgroups at 0.49, into two subgroups at 0.46, and into four subgroups at 0.493, respectively. All but one of the yellow waxy maize inbred lines were clustered in groups VI, VII, VIII, and IX. Group IX (30 lines) contained 28 yellow lines; the other 11 yellow lines were distributed among groups VI, VII and VIII. Among the 25 white lines, 21 were clustered in groups III, V, VI and the third subgroup of group II. The black line N72 was in a group of its own. The black lines N75, N76 and N78 were distributed in groups VII, VIII and IX, respectively. The other nine black lines were clustered in group II. The red lines were distributed in the second subgroup of group II and there was no difference in genetic distance between them. In conclusion, there were considerable genetic differences among waxy maize inbred lines of different colors. The mean genetic distance of inbred lines of the same color was significantly less than that of lines of different colors. Therefore, we concluded that it was more accurate to determine the difference between the populations using the highly stable DNA genetic markers.     

QTL mapping of resistance to Fusarium ear rot using a RIL population in maize

Fusarium ear rot is a prevalent disease in maize, reducing grain yields and quality. Resistance breeding is an efficient way to minimize losses caused by the disease. In this study, 187 lines from a RIL population along with the resistant (87-1) and susceptible (Zong 3) parents were planted in Zhengzhou and Beijing with three replications in years 2004 and 2006. Each line was artificially inoculated using the nail-punch method. Significant genotypic variation in response to Fusarium ear rot was detected in both years. Based on a genetic map containing 246 polymorphic SSR markers with average genetic distances of 9.1 cM, the ear-rot resistance QTL were firstly analyzed by composite interval mapping (CIM). Three QTL were detected in both Zhengzhou and Beijing in 2004; and three and four QTL, respectively, were identified in 2006. The resistant parent contributed all resistance QTL. By using composite interval mapping and a mixed model (MCIM), significant epistatic effects on Fusarium ear rot as well as interactions between mapped loci and environments were observed across environments. Two QTL on chromosome 3 (3.04 bin) were consistently identified across all environments by the two methods. The major resistant QTL with the largest effect was flanked by markers umc1025 and umc1742 on chromosome 3 (3.04 bin), explaining 13–22% of the phenotypic variation. The SSR markers closely flanking the major resistance QTL will facilitate marker-assisted selection (MAS) of resistance to Fusarium ear rot in maize breeding programs.     

Population structure and genetic diversity in a commercial maize breeding program assessed with SSR and SNP markers

Information about the genetic diversity and population structure in elite breeding material is of fundamental importance for the improvement of crops. The objectives of our study were to (a) examine the population structure and the genetic diversity in elite maize germplasm based on simple sequence repeat (SSR) markers, (b) compare these results with those obtained from single nucleotide polymorphism (SNP) markers, and (c) compare the coancestry coefficient calculated from pedigree records with genetic distance estimates calculated from SSR and SNP markers. Our study was based on 1,537 elite maize inbred lines genotyped with 359 SSR and 8,244 SNP markers. The average number of alleles per locus, of group specific alleles, and the gene diversity (D) were higher for SSRs than for SNPs. Modified Roger’s distance (MRD) estimates and membership probabilities of the STRUCTURE matrices were higher for SSR than for SNP markers but the germplasm organization in four heterotic pools was consistent with STRUCTURE results based on SSRs and SNPs. MRD estimates calculated for the two marker systems were highly correlated (0.87). Our results suggested that the same conclusions regarding the structure and the diversity of heterotic pools could be drawn from both markers types. Furthermore, although our results suggested that the ratio of the number of SSRs and SNPs required to obtain MRD or D estimates with similar precision is not constant across the various precision levels, we propose that between 7 and 11 times more SNPs than SSRs should be used for analyzing population structure and genetic diversity.     

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.     

Development and mapping of SSR markers for maize

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.     

Analysis of molecular genetic diversity and population structure in Amaranthus germplasm using SSR markers

We assessed the molecular genetic diversity and population structure of Amaranthus species accessions using 11 simple sequence repeat markers. A total of 122 alleles were detected, and the number of alleles per marker (N_A) ranged from 6 to 21 with an average of 11.1 alleles. The frequency of major alleles per locus ranged from 0.148 to 0.695, with an average value of 0.496 per marker. The overall polymorphic information content values were 0.436–0.898, with an average value of 0.657. The observed heterozygosity (H_O) and expected heterozygosity (H_E) ranged from 0.056 to 0.876 and from 0.480 to 0.907, with average values of 0.287 and 0.698, respectively. The average H_O (0.240) was lower than the H_E and gene flow (Nm), and showed substantial genetic variability among all populations of amaranth accessions. The sample groupings did not strictly follow the geographic affiliations of the accessions. A similar pattern was obtained using model-based structure analysis without grouping by species type. Knowledge of the genetic diversity and population structure of amaranth can be used to select representative genotypes and manage Amaranthus germplasm breeding programs.     

Identification of functional genetic variations underlying drought tolerance in maize using SNP markers

Single nucleotide polymorphism (SNP) is a common form of genetic variation and popularly exists in maize genome. An Illumina GoldenGate assay with 1 536 SNP markers was used to genotype maize inbred lines and identified the functional genetic variations underlying drought tolerance by association analysis. Across 80 lines, 1 006 polymorphic SNPs (65.5% of the total) in the assay with good call quality were used to estimate the pattern of genetic diversity, population structure, and familial relatedness. The analysis showed the best number of fixed subgroups was six, which was consistent with their original sources and results using only simple sequence repeat markers. Pairwise linkage disequilibrium (LD) and association mapping with phenotypic traits investigated under water-stressed and well-watered regimes showed rapid LD decline within 100-500 kb along the physical distance of each chromosome, and that 29 SNPs were associated with at least two phenotypic traits in one or more environments, which were related to drought-tolerant or drought-responsive genes. These drought-tolerant SNPs could be converted into functional markers and then used for maize improvement by marker-assisted selection.     

QTL detected for grain-filling rate in maize using a RIL population

The grain-filling rate plays an important role in determining grain yield. To elucidate the genetic basis of the grain-filling rate, a set of 203 recombinant inbred lines was evaluated at two locations over 2 years. Quantitative trait loci (QTL) for grain-filling rate were detected using conditional and unconditional QTL analysis of genetic linkage maps comprising 217 SSR markers. The results showed that the grain-filling rate increased between 15 and 35 days after pollination, then decreased at the last two sampling times. Hybrids with high grain-filling rates determined the grain yield in those areas with a short growth season for maize. A total of 23 unconditional QTL for grain-filling rate were detected using the 100-kernel weight as the input data at different sampling stages. They were distributed on 10 chromosomes (except chromosome 9), and some QTL were detected at different sampling stages. In addition, nine conditional QTL were identified using the average increase in 100-kernel weight of per day between two sampling times, and six conditional QTL were detected simultaneously using the unconditional QTL mapping strategy. The QTL mapping results demonstrated that the grain-filling rate is controlled by a complex genetic mechanism, and the QTL detected at different sampling stages might be important contributors to grain yield in maize.     

Molecular genetic diversity and population structure in Lycium accessions using SSR markers

This study was conducted to assess the genetic diversity and population structure of 139 Lycium chinense accessions using 18 simple sequence repeat (SSR) markers. In total, 108 alleles were detected. The number of alleles per marker locus ranged from two to 17, with an average of six. The gene diversity and polymorphism information content value averaged 0.3792 and 0.3296, with ranges of 0.0793 to 0.8023 and 0.0775 to 0.7734, respectively. The average heterozygosity was 0.4394. The model-based structure analysis revealed the presence of three subpopulations, which was consistent with clustering based on genetic distance. An AMOVA analysis showed that the between-population component of genetic variance was less than 15.3%, in contrast to 84.7% for the within-population component. The overall F_ST value was 0.1178, indicating a moderate differentiation among groups. The results could be used for future L. chinense allele mining, association mapping, gene cloning, germplasm conservation, and designing effective breeding programs.     

Characterization of genetic diversity and population structure in wheat using array based SNP markers

Genetic diversity is crucial for successful adaptation and sustained improvement in crops. India is bestowed with diverse agro-climatic conditions which makes it rich in wheat germplasm adapted to various niches. Germplasm repository consists of local landraces, trait specific genetic stocks including introgressions from wild relatives, exotic collections, released varieties, and improved germplasm. Characterization of genetic diversity is done using morpho-physiological characters as well as by analyzing variations at DNA level. However, there are not many reports on array based high throughput SNP markers having characteristics of genome wide coverage employed in Indian spring wheat germplasm. Amongst wheat SNP arrays, 35K Axiom Wheat Breeder’s Array has the highest SNP polymorphism efficiency suitable for genetic mapping and genetic diversity characterization. Therefore, genotyping was done using 35K in 483 wheat genotypes resulting in 14,650 quality filtered SNPs, that were distributed across the B (~?50%), A (~?39%), and D (~?10%) genomes. The total genetic distance coverage was 4477.85 cM with 3.27 SNP/cM and 0.49 cM/SNP as average marker density and average inter-marker distance, respectively. The PIC ranged from 0.09 to 0.38 with an average of 0.29 across genomes. Population structure and Principal Coordinate Analysis resulted in two subpopulations (SP1 and SP2). The analysis of molecular variance revealed the genetic variation of 2% among and 98% within subpopulations indicating high gene flow between SP1 and SP2. The subpopulation SP2 showed high level of genetic diversity based on genetic diversity indices viz. Shannon’s information index (I)?=?0.648, expected heterozygosity (He)?=?0.456 and unbiased expected heterozygosity (uHe)?=?0.456. To the best of our knowledge, this study is the first to include the largest set of Indian wheat genotypes studied exclusively for genetic diversity. These findings may serve as a potential source for the identification of uncharacterized QTL/gene using genome wide association studies and marker assisted selection in wheat breeding programs.

     

Characterising a Eucalyptus cladocalyx breeding population using SNP markers

Population structure, family relatedness and inbreeding within a first-generation Eucalyptus cladocalyx breeding population were analysed with single-nucleotide polymorphism markers to underpin quantitative trait analysis and breeding program management. The breeding population, comprising families selected from wild and cultivated stands, was found to be strongly structured ( $ {{\widehat{F}}_{\mathrm{ST}}} $ ?=?18 %), with two geographically defined groups of South Australian wild subpopulations: Kangaroo Island (KI) and South Flinders Ranges (SFR). The selections from cultivated stands were shown to be derived from SFR subpopulations of SFR and had similar levels of diversity, suggesting that they were established from a broad genetic base. Relatedness and inbreeding among families was heterogeneous, ranging from completely outcrossed and predominantly half-sib (HS) to completely selfed. Families from the cultivated stands had minimal inbreeding and were close to HS on average. Among SFR subpopulations, family-average inbreeding was negatively correlated with growth, suggesting inbreeding depression (ID). Inbreeding was high, on average, in the KI subpopulations; however, evidence of ID was absent, with highly inbred families amongst the most vigorous, perhaps indicative of purging of deleterious recessive alleles in a bottleneck event. The marker-based information suggested that modification of the usual assumptions of relatedness made in undertaking quantitative analysis of the first-generation populations would be desirable.     

Analysis of genetic recombination in maize populations using molecular markers

Summary Variation in recombination rate is important to plant breeders since a major objective is to obtain favorable recombinants of linked genes. The ability to increase recombination (R) in circumstances in which favorable and unvavorable genes are linked (Corn Belt x exotic populations) and to decrease recombination when many favorable genes are linked (narrow-based, elite populations) would be of immense value. However, the concept of variation in recombination frequencies between linked genes has received limited attention despite its implications in breeding and genetic linkage studies. Molecular techniques have allowed better estimations of this variation. In this study, attempts were made to characterize: (1) the R values in the Pgm1-Adh1 and Adh1-Phi1 adjacent regions of chromosome 1 and the Idh2-Mdh2 region of chromosome 6 in F₂ families of three maize (Zea mays L.) populations; (2) the environmental effect on R values of F₂s from two populations. One population, NSO, was a Corn Belt synthetic, and the other two populations, CBMEX3 and CBCAR5, were composites from crosses between Corn Belt and exotic germ-plams.Wide ranges of estimated recombination ( ) values were observed among families in each population for all three chromsomal regions. The distribution of values for the Pgm1-Adh1 region showed that the F₂ families of each population fell into two broad categories: 0.30–0.50 and 0.02–0.20. No intermediates (0.21–0.29) were found. The distributions were almost normal for the Adh1-Phi1 and the Idh2-Mdh2 regions. It would appear that the major dispersion in the Pgm1-Adh1 region was controlled by the effects of a single gene, while the Adh1-Phi1 and Idh2-Mdh2 regions were only affected by polygenes. No correlation was found between recombination values of the two adjacent regions, indicating that the genes affecting recombination for the Pgm1-Adh1 region may be specific for that region.For the Pgm1-Adh1 region, no differences in values were found among the three populations. For the Adh1-Phi1 region, frequencies of CBMEX3 and NSO were not significantly different, but both had significantly greater values than CBCAR5. For the Idh2-Mdh2 region, CBMEX3 was significantly different from NSO. There were significant differences between some paired F₂ families within each population for each chromosome region.No significant differences in response to the two environments were detected in CBMEX3 and NSO for either region in chromosome 1.Published as Journal Paper No. 9498 of the Nebraska Agric Res Div, University of Nebraska, Lincoln, Neb. Research supported in part by USDA Competitive Grant 87-CRCR-2359     

Linkage group alignment of sorghum RFLP maps using a RIL mapping population.

Several molecular maps have been constructed in sorghum (Sorghum bicolor L. Moench) using a variety of probes from different grass species such as sorghum, maize, sugarcane, rice, oat, and barley. In order to enhance the utility of the existing mapping information by the sorghum research community, alignment and integration of all major molecular maps is necessary. To achieve this objective, a genetic map of 214 loci with a total map distance of 1200 cM was constructed using 98 F7 sorghum recombinant inbred lines (RILs) from a cross between two inbred lines, B35 and Tx7000. Few cDNA clones of sorghum and maize related to photosynthesis and drought stress were mapped on this map for the first time. Five major restriction fragment length polymorphism (RFLP) maps independently developed in this species were used for alignment purpose. The distributions of previously mapped markers were compared with their respective sorghum maps to align each of the linkage groups. In general, consistent linear order among markers was maintained in all the linkage maps. The successful alignment of these RFLP maps will now allow selection of a large number of markers for any region of the sorghum genome with many potential applications ranging from fine mapping and marker-assisted selection to map-based cloning for the improvement of sorghum and related species.     

Estimation of genetic diversity using SSR markers in sunflower

Microsatellites or simple sequence repeats (SSRs) were used for the estimation of genetic diversity among a group of 40 sunflower lines developed at the research area of Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad. Total numbers of alleles amplified by 22 polymorphic primers were 135 with an average of 6.13 alleles per locus, suggesting that SSR is a powerful technique for assessment of genetic diversity at molecular level. The expected heterozygosity (PIC) ranged from 0.17 to 0.89. The highest PIC value was observed at the locus C1779. The genetic distances ranged from 9% to 37%. The highest genetic distance was observed between the lines L50 and V3. Genetic distances were low showing lesser amount of genetic diversity among the sunflower lines.     

Gains in QTL detection using an ultra-high density SNP map based on population sequencing relative to traditional RFLP/SSR markers

Huge efforts have been invested in the last two decades to dissect the genetic bases of complex traits including yields of many crop plants, through quantitative trait locus (QTL) analyses. However, almost all the studies were based on linkage maps constructed using low-throughput molecular markers, e.g. restriction fragment length polymorphisms (RFLPs) and simple sequence repeats (SSRs), thus are mostly of low density and not able to provide precise and complete information about the numbers and locations of the genes or QTLs controlling the traits. In this study, we constructed an ultra-high density genetic map based on high quality single nucleotide polymorphisms (SNPs) from low-coverage sequences of a recombinant inbred line (RIL) population of rice, generated using new sequencing technology. The quality of the map was assessed by validating the positions of several cloned genes including GS3 and GW5/qSW5, two major QTLs for grain length and grain width respectively, and OsC1, a qualitative trait locus for pigmentation. In all the cases the loci could be precisely resolved to the bins where the genes are located, indicating high quality and accuracy of the map. The SNP map was used to perform QTL analysis for yield and three yield-component traits, number of tillers per plant, number of grains per panicle and grain weight, using data from field trials conducted over years, in comparison to QTL mapping based on RFLPs/SSRs. The SNP map detected more QTLs especially for grain weight, with precise map locations, demonstrating advantages in detecting power and resolution relative to the RFLP/SSR map. Thus this study provided an example for ultra-high density map construction using sequencing technology. Moreover, the results obtained are helpful for understanding the genetic bases of the yield traits and for fine mapping and cloning of QTLs.     

Analysis of genetic diversity and population structure of rice cultivars from Korea, China and Japan using SSR markers

A total of 29 simple sequence repeat (SSR) markers were used to analyze the genetic diversity of 150 accessions of cultivated rice (Oryza sativa L.) from Korea, China, and Japan. A total of 375 alleles were detected with an average of 12.9 per locus. The averaged values of gene diversity and polymorphism information content (PIC) for each SSR locus were 0.7001 and 0.6683, respectively. Alleles per locus in Korean rice were 8.8, whereas 8.1 and 7.2 alleles per locus were found in Chinese and Japanese rice, respectively. The mean gene diversity in Korean, Chinese, and Japanese rice was 0.6058, 0.6457, and 0.5174, respectively, whereas the mean PIC values for each SSR locus were 0.5759, 0.6138, and 0.4881, respectively. The genetic diversity of the Korean and Chinese cultivars was higher than that of the Japanese cultivars, and the genetic diversity ofjaponica was higher than that ofindica. The model-based structure analysis revealed the presence of three subpopulations, which was basically consistent with clustering based on genetic distance. An AMOVA analysis showed that the between-population component of genetic variance was less than 22% in contrast to 78% for the within-population component. The overallFST value was 0.2180, indicating a moderate differentiation among groups. The results could be used for designing effective breeding programs aimed at broadening the genetic bases of commercially grown varieties.     

Construction of a genetic linkage map in Fenneropenaeus chinensis using SNP markers

Genetic linkage maps of Fenneropenaeus chinensis were constructed using a “double pseudo-testcross” strategy with 200 single nucleotide polymorphisms (SNPs) markers. This study represents the first SNP genetic linkage map for F. chinensis. The parents and F ₁ progeny of 100 individuals were used as mapping populations. 21 genetic linkage groups in the male and female maps were identified. The male linkage map was composed of 115 loci and spanned 879.7 cM, with an average intermarker spacing of 9.4 cM, while the female map was composed of 119 loci and spanned 876.2 cM, with an average intermarker spacing of 8.9 cM. The estimated coverage of the linkage maps was 51.94% for the male and 53.77% for the female, based on two estimates of genome length. The integrated map contains 180 markers distributed in 16 linkage groups, and spans 899.3 cM with an average marker interval of 5.2 cM. This SNP genetic map lays the foundation for future shrimp genomics and genetic breeding studies, especially the discovery of gene or regions for economically important traits in Chinese shrimp.     

Genetic mapping of folate QTLs using a segregated population in maize

As essential B vitamin for humans, folates accumulation in edible parts of crops, such as maize kernels, is of great importance for human health. But its breeding is always limited by the prohibitive cost of folate profiling. The molecular breeding is a more executable and efficient way for folate fortification, but is limited by the molecular knowledge of folate regulation. Here we report the genetic mapping of folate quantitative trait loci (QTLs) using a segregated population crossed by two maize lines, one high in folate (GEMS31) and the other low in folate (DAN3130). Two folate QTLs on chromosome 5 were obtained by the combination of F₂ whole-exome sequencing and F₃ kernel-folate profiling. These two QTLs had been confirmed by bulk segregant analysis using F₆ pooled DNA and F₇ kernel-folate profiling, and were overlapped with QTLs identified by another segregated population. These two QTLs contributed 41.6% of phenotypic variation of 5-formyltetrahydrofolate, the most abundant storage form among folate derivatives in dry maize grains, in the GEMS31×DAN3130 population. Their fine mapping and functional analysis will reveal details of folate metabolism, and provide a basis for marker-assisted breeding aimed at the enrichment of folates in maize kernels.