Genotype calling and haplotype inference from low coverage sequence data in heterozygous plant genome using HetMap

Many software packages and pipelines had been developed to handle the sequence data of the model species. However, Genotyping from complex heterozygous plant genome needs further improvement on the previous methods. Here we present a new pipeline available at https://github.com/Ncgrhg/HetMapv1) for variant calling and missing genotype imputation from low coverage sequence data for heterozygous plant genomes. To check the performance of the HetMap on the real sequence data, HetMap was applied to both the F1 hybrid rice population which consists of 1495 samples and wild rice population with 446 samples. Four high coverage sequence hybrid rice accessions and two high coverage sequence wild rice accessions, which were also included in low coverage sequence data, are used to validate the genotype inference accuracy. The validation results showed that HetMap archived significant improvement in heterozygous genotype inference accuracy (13.65% for hybrid rice, 26.05% for wild rice) and total accuracy compared with other similar software packages. The application of the new genotype with the genome wide association study also showed improvement of association power in two wild rice phenotypes. It could archive high genotype inference accuracy with low sequence coverage with a small population size with both the natural population and constructed recombination population. HetMap provided a powerful tool for the heterozygous plant genome sequence data analysis, which may help the discover of new phenotype regions for the plant species with complex heterozygous genome.

Chromosome-scale assembly of the highly heterozygous genome of red clover (Trifolium pratense L.), an allogamous forage crop species

Red clover (Trifolium pratense L.) is used as a forage crop due to a variety of favorable traits relative to other crops. Improved varieties have been developed through conventional breeding approaches, but progress could be accelerated and gene discovery facilitated using modern genomic methods. Existing short-read based genome assemblies of the ~420 Megabase (Mb) genome are fragmented into >135,000 contigs with numerous errors in order and orientation within scaffolds, likely due to the biology of the plant which displays gametophytic self-incompatibility resulting in inherent high heterozygosity. A high-quality long-read based assembly of red clover is presented that reduces the number of contigs by more than 500-fold, improves the per-base quality, and increases the contig N50 statistic by three orders of magnitude. The 413.5 Mb assembly is nearly 20% longer than the 350 Mb short read assembly, closer to the predicted genome size. Quality measures are presented and full-length isoform sequence of RNA transcripts reported for use in assessing accuracy and for future annotation of the genome. The assembly accurately represents the seven main linkage groups present in the genome of an allogamous (outcrossing), highly heterozygous plant species.

A Rare Ile-2041-Thr Mutation in the ACCase Gene Confers Resistance to ACCase-inhibiting Herbicides in Shortawn Foxtail (Alopecurus aequalis)

Understanding the mechanism of herbicide resistance is fundamental for designing sustainable weed control strategies and exploiting herbicides rationally. Shortawn foxtail is a problem grass weed infesting several important crops in China. The repeated use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has resulted in herbicide resistance in this weed. The ACCase gene of resistant individuals of a shortawn foxtail population (JSLS-1) has an Ile-2041-Thr mutation. F2 generation seeds, originated from the same heterozygous plant, were harvested, and two homozygous mutant (JSLS-1RR) and wild (JSLS-1SS) populations for the Ile-2041-Thr mutation were obtained. In whole plants, the JSLS-1RR population conferred high resistance to fenoxaprop and clodinafop, moderate resistance to haloxyfop, low resistance to pinoxaden, and no obvious resistance to clethodim and sethoxydim, compared with JSLS-1SS and a proven susceptible population (HNXY-1). A derived cleaved amplified polymorphic sequence (dCAPS) marker was developed to rapidly detect the rare Ile-2041-Thr mutation in the shortawn foxtail population. This is the first report of the cross-resistance pattern of Ile-2041-Thr mutation, and the robust dCAPS marker could quickly detect this mutation in shortawn foxtail.

(79) Direct Bulblet Regeneration from Mature Embryo: A Rapid, Efficient and Genotype-independent In Vitro Morphogenesis Pathway for Preservation of Endangered Wild Populations of Fritillaria imperialis and Fritillaria persica

Wild populations of Fritillaria sp. have dramatically decreased in Iran because of pest overflow and continual grazing. Previous studies have shown that Fritillaria cannot rapidly and efficiently propagate by traditional methods. In vitro tissue culture techniques have shown high potential for micropropagation of endangered plants. The use of bulb-scale pieces for tissue culture can result in the destruction of the endangered parent plant. Fritillaria is a heterozygous plant in which the genetic content of each embryo is different from others, even on the same plant. In this study, mature embryos of F. imperialis and F. persica were used as explant for the first time. Embryos were extracted from seeds and cultured on B5 medium supplemented with various combinations of BAP (0, 0.1, 1 mg/L), NAA (0, 0.4, 4 mg/L), and IAA (0, 0.4, 4 mg/L). Embryo explant showed low genotype dependency between different heterogenous and heterozygote populations of both F. imperialis and F. persica. The best response of bulblet regeneration in both F. imperialis and F. persica was obtained from 1 mg/L BAP + 0.4 mg/L NAA+4 mg/L IAA and direct organogenesis pathway, with 15 bulblets per explant for F. imperialia and 20 for F. persica. Because of the large number of embryos in a plant and their different genetic contents, established in vitro propagation by using embryo explant in this study can provide broad genetic resources and variations. As explained above, in vitro protocols can play a major role in rescuing F. imperialis and F. persica from extinction.

The First Genetic Linkages among Expressed Regions of the Garlic Genome

Garlic (Allium sativum L.) has been cultivated by asexual propagation since time immemorial. The discovery of male-fertile garlic accessions has opened a venue for genetic studies and improvement through sexual recombination. An S1 family of 84 plants was generated from a single male-fertile heterozygous plant from the U.S. Dept. of Agriculture Plant Introduction 540316 and used to identify the first genetic linkages in garlic based on single nucleotide polymorphisms, simple sequence repeats, and randomly amplified polymorphic DNAs. Thirty-seven markers formed nine linkage groups covering 415 centimorgans (cM) with average distance of 15 cM between loci; other 16 loci remained unlinked. A male fertility locus was placed on the map. This first genetic map of garlic is a seminal step toward the genetic improvement of garlic and eventual marker-assisted breeding.

Detection of Deleterious Genotypes in Multigenerational Studies. II. Theoretical and Experimental Dynamics with Selfing and Selection

A mathematical model was developed to help interpret genotype and allele frequency dynamics in selfing populations, with or without apomixis. Our analysis provided explicit time-dependent solutions for the frequencies at diallelic loci in diploid populations under any combination of fertility, viability, and gametic selection through meiotic drive. With no outcrossing, allelic variation is always maintained under gametic selection alone, but with any fertility or viability differences, variation will ordinarily be maintained if and only if the net fitness (fertility × viability) of heterozygotes exceeds that of both homozygotes by a substantial margin. Under pure selfing and Mendelian segregation, heterozygotes must have a twofold fitness advantage; the level of overdominance necessary to preserve genetic diversity declines with apomixis, and increases with segregation distortion if this occurs equally and independently in male and female gametes. A case study was made of the Arabidopsis act2-1 actin mutant over multiple generations initiated from a heterozygous plant. The observed genotypic frequency dynamics were consistent with those predicted by our model for a deleterious, incompletely recessive mutant in either fertility or viability. The theoretical framework developed here should be very useful in dissecting the form(s) and strength of selection on diploid genotypes in populations with negligible levels of outcrossing.