Contrasting whole-genome and reduced representation sequencing for population demographic inference: an alpine mammal example

Genomic approaches to the study of population demography rely on accurate SNP calling and by-proxy the site frequency spectrum (SFS). Two main questions for the design of such studies remain poorly investigated: do reduced genomic sequencing summary statistics reflect that of whole genome, and how do sequencing strategies and derived summary statistics impact demographic inferences? To address those questions, we applied the ddRAD sequencing approach to 254 individuals and whole genome resequencing approach to 35 mountain goat (Oreamnos americanus) individuals across the species range with a known demographic history. We identified SNPs with 5 different variant callers and used ANGSD to estimate the genotype likelihoods (GLs). We tested combinations of SNP filtering by linkage disequilibrium (LD), minor allele frequency (MAF) and the genomic region. We compared the resulting suite of summary statistics reflective of the SFS and quantified the relationship to demographic inferences by estimating the contemporary effective population size (Ne), isolation-by-distance and population structure, FST, and explicit modelling of the demographic history with δaδi. Filtering had a larger effect than sequencing strategy, with the former strongly influencing summary statistics. Estimates of contemporary Ne and isolation-by-distance patterns were largely robust to the choice of sequencing, pipeline, and filtering. Despite the high variance in summary statistics, whole genome and reduced representation approaches were overall similar in supporting a glacial induced vicariance and low Ne in mountain goats. We discuss why whole genome resequencing data is preferable, and reiterate support the use of GLs, in part because it limits user-determined filters.

Identification of Grain Size-Related QTLs in Korean japonica Rice Using Genome Resequencing and High-Throughput Image Analysis

Grain size is a key factor influencing the grain yield in rice. To identify the as-yet-unknown genes regulating grain size in Korean japonica rice, we developed a recombinant inbred line population (n = 162) from a cross between Odae (large-grain) and Joun (small-grain), and measured six traits including the thousand-grain weights of unhulled and hulled seeds, grain area, grain length, grain width and grain length-to-width ratio using high-throughput image analysis at the F8 and F9 generations. A genetic map was constructed using 248 kompetitive allele-specific PCR (KASP) markers that were polymorphic between the parental genotypes, and 29 QTLs affecting the six traits were identified, of which 15 were stable in both F8 and F9 generations. Notably, three QTL clusters affecting multiple traits were detected on chromosomes 6, 7 and 11. We analyzed whole-genome resequencing data of Odae and Joun, and selected candidate genes for the stable QTLs in the identified clusters that have high- or moderate-impact variations between Odae and Joun and encode proteins the families of which have been reported to be related to grain size regulation. These results will facilitate the identification of genes underlying the QTLs and promote molecular breeding of high-yielding Korean japonica rice varieties.

Construction and Genetic Analysis of a Worldwide Core Collection of Rapeseed

Rapeseed (Brassica napus) is an important oilseed crop, which is widely planted in the world. In a previous study, we collected 991 accessions of rapeseed from the worldwide germplasm and revealed genetic polymorphisms within these germplasm by whole-genome resequencing. However, management of such a large amount of accessions is time-consuming, laborious and costly. Therefore, we constructed a core collection of rapeseed consisting of 300 worldwide accessions based on their genetic diversity. Compared with 991 accessions, the worldwide core collection showed similar geographic distribution, the proportion of three ecotypes, nucleotide diversity and the associated SNPs of flowering time. Besides, we identified FT ortholog (BnaA02g12130D) and FLC ortholog (BnaA10g22080D) responsible for flowering time and ecotype differentiation through selective sweep analysis and genome-wide association analysis (GWAS) of flowering time using the rapeseed core collection. FT and FLC are two well-known genes regulating flowering time in Arabidopsis. These results indicate that the worldwide core collection can represent the genetic diversity of 991 worldwide accessions, which could be more efficiently used for phenotypic and genetic studies in rapeseed.

Whole-Genome Resequencing of Near-Isogenic Lines Reveals a Genomic Region Associated with High Trans-Lycopene Contents in Watermelon

Trans-lycopene is a functional phytochemical abundant in red-fleshed watermelons, and its contents vary among cultivars. In this study, the genetic basis of high trans-lycopene contents in scarlet red flesh was evaluated. Three near-isogenic lines (NILs) with high trans-lycopene contents were derived from the scarlet red-fleshed donor parent DRD and three coral red-fleshed (low trans-lycopene contents) recurrent parents. The lycopene contents of DRD (589.4 ± 71.8 µg/g) were two times higher than that of the recurrent parents, and values for NILs were intermediate between those of the parents. Coral red-fleshed lines and F1 cultivars showed low trans-lycopene contents (135.7 ± 18.0 µg/g to 213.7 ± 39.5 µg/g). Whole-genome resequencing of two NILs and their parents and an analysis of genome-wide single-nucleotide polymorphisms revealed three common introgressed regions (CIRs) on chromosomes 6, 9, and 10. Twenty-eight gene-based cleaved amplified polymorphic sequence (CAPS) markers were developed from the CIRs. The CAPS markers derived from CIR6 on chromosome 6, spanning approximately 1 Mb, were associated (R2 = 0.45–0.72) with the trans-lycopene contents, particularly CIR6-M1 and CIR6-M4. Our results imply that CIR6 is a major genomic region associated with variation in the trans-lycopene contents in red-fleshed watermelon, and CIR6-M1 and CIR6-M4 may be useful for marker-assisted selection.

Construction of a High-Density Genetic Map for Pitaya Using the Whole Genome Resequencing Approach

Pitaya (Hylocereus undatus) is one of the most economic fleshy fruit tree crops. This study aimed at producing a high-density linkage genetic map of pitaya based on the whole genome resequencing (WGrS) approach. For this purpose, a bi-parental F1 population of 198 individuals was generated and genotyped by WGrS. High-quality polymorphic 6434 single polymorphism nucleotide (SNP) markers were extracted and used to construct a high-density linkage map. A total of 11 linkage groups were resolved as expected in accordance with the chromosome number. The map length was 14,128.7 cM with an average SNP interval of 2.2 cM. Homology with the sequenced reference genome was described, and the physical and genetic maps were compared with collinearity analysis. This linkage map in addition to the available genomic resources will help for quantitative trait mapping, evolutionary studies and marker-assisted selection in the important Hylocereus species.

Whole-genome resequencing of three Coilia nasus population reveals genetic variations in genes related to immune, vision, migration, and osmoregulation

Background Coilia nasus is an important anadromous fish, widely distributed in China, Japan, and Korea. Based on morphological and ecological researches of C. nasus, two ecotypes were identified. One is the anadromous population (AP). The sexually mature fish run thousands of kilometers from marine to river for spawning. Another one is the resident population which cannot migrate. Based on their different habitats, they were classified into landlocked population (LP) and sea population (SP) which were resident in the freshwater lake and marine during the entire lifetime, respectively. However, they have never been systematically studied. Moreover, C. nasus is declining sharply due to overfishing and pollution recently. Therefore, further understandings of C. nasus populations are needed for germplasm protection. Results Whole-genome resequencing of AP, LP, and SP were performed to enrich the understanding of different populations of C. nasus. At the genome level, 3,176,204, 3,307,069, and 3,207,906 single nucleotide polymorphisms (SNPs) and 1,892,068, 2,002,912, and 1,922,168 insertion/deletion polymorphisms (InDels) were generated in AP, LP, and SP, respectively. Selective sweeping analysis showed that 1022 genes were selected in AP vs LP; 983 genes were selected in LP vs SP; 116 genes were selected in AP vs SP. Among them, selected genes related to immune, vision, migration, and osmoregulation were identified. Furthermore, their expression profiles were detected by quantitative real-time PCR. Expression levels of selected genes related to immune, and vision in LP were significantly lower than AP and SP. Selected genes related to migration in AP were expressed significantly more highly than LP. Expression levels of selected genes related to osmoregulation were also detected. The expression of NKAα and NKCC1 in LP were significantly lower than SP, while expression of NCC, SLC4A4, NHE3, and V-ATPase in LP was significantly higher than SP. Conclusions Combined to life history of C. nasus populations, our results revealed that the molecular mechanisms of their differences of immune, vision, migration, and osmoregulation. Our findings will provide a further understanding of different populations of C. nasus and will be beneficial for wild C. nasus protection.

Development and characterization of efficient xylose utilization strains of Zymomonas mobilis

Background Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level. Results Heterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment. Conclusions This study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.