Maintenance and expansion of genetic and trait variation following domestication in a clonal crop.

Clonal propagation enables favourable crop genotypes to be rapidly selected and multiplied. However, the absence of sexual propagation can lead to low genetic diversity and accumulation of deleterious mutations, which may eventually render crops less resilient to pathogens or environmental change. To better understand this trade-off, we characterize the domestication and contemporary genetic diversity of Enset (Ensete ventricosum), an indigenous African relative of bananas (Musa) and a principal starch staple for 20 million Ethiopians. Wild enset reproduction occurs strictly by sexual outcrossing, but for cultivation, it is propagated clonally and associated with diversification and specialization into hundreds of named landraces. We applied tGBS sequencing to generate genome-wide genotypes for 192 accessions from across enset's cultivated distribution, and surveyed 1340 farmers on enset agronomic traits. Overall, reduced heterozygosity in the domesticated lineage was consistent with a domestication bottleneck that retained 37% of wild diversity. However, an excess of putatively deleterious missense mutations at low frequency present as heterozygotes suggested an accumulation of mutational load in clonal domesticated lineages. Our evidence indicates that the major domesticated lineages initially arose through historic sexual recombination associated with a domestication bottleneck, followed by the amplification of favourable genotypes through an extended period of clonal propagation. Among domesticated lineages, we found a significant phylogenetic signal for multiple farmer-identified food, nutrition and disease resistance traits and little evidence of contemporary recombination. The development of future-climate adapted genotypes may require crop breeding, but outcrossing risks exposing deleterious alleles as homozygotes. This trade-off may partly explain the ubiquity and persistence of clonal propagation over recent centuries of comparative climate stability.

Genome diversity and highland-adaptative variation in Tibet barley landrace population of China.

Barley landraces accumulated variation in adapting to extreme highland environments during long-term domestication in Tibet, but little is known about their population structure and genomic selection traces. In this study, tGBS (tunable genotyping by sequencing) sequencing, molecular marker and phenotypic analyses were conducted on 1,308 highland and 58 inland barley landraces in China. The accessions were divided into six sub-populations and clearly distinguished most six-rowed, naked barley accessions (Qingke in Tibet) from inland barley. Genome-wide differentiation was observed in all five sub-populations of Qingke and inland barley accessions. High genetic differentiation in the pericentric regions of chromosomes 2H and 3H contributed to formation of five types of Qingke. Ten haplotypes of the pericentric regions of 2H, 3H, 6H and 7H were further identified as associated with ecological diversification of these sub-populations. There was genetic exchange between eastern and western Qingke but they shared the same progenitor. The identification of 20 inland barley types indicated multiple origins of Qingke in Tibet. The distribution of the five types of Qingke corresponded to specific environments. Two predominant highland-adaptative variations were identified for low temperature tolerance and grain color. Our results provide new insights into the origin, genome differentiation, population structure and highland adaptation in highland barley which will benefit both germplasm enhancement and breeding of naked barley.

The Conservation Genetics of Iris lacustris (Dwarf Lake Iris), a Great Lakes Endemic.

Iris lacustris, a northern Great Lakes endemic, is a rare species known from 165 occurrences across Lakes Michigan and Huron in the United States and Canada. Due to multiple factors, including habitat loss, lack of seed dispersal, patterns of reproduction, and forest succession, the species is threatened. Early population genetic studies using isozymes and allozymes recovered no to limited genetic variation within the species. To better explore genetic variation across the geographic range of I. lacustris and to identify units for conservation, we used tunable Genotyping-by-Sequencing (tGBS) with 171 individuals across 24 populations from Michigan and Wisconsin, and because the species is polyploid, we filtered the single nucleotide polymorphism (SNP) matrices using polyRAD to recognize diploid and tetraploid loci. Based on multiple population genetic approaches, we resolved three to four population clusters that are geographically structured across the range of the species. The species migrated from west to east across its geographic range, and minimal genetic exchange has occurred among populations. Four units for conservation are recognized, but nine adaptive units were identified, providing evidence for local adaptation across the geographic range of the species. Population genetic analyses with all, diploid, and tetraploid loci recovered similar results, which suggests that methods may be robust to variation in ploidy level.

Exome-based new allele-specific PCR markers and transferability for sodicity tolerance in bread wheat (Triticum aestivum L.).

Targeted exome-based genotype by sequencing (t-GBS), a sequencing technology that tags SNPs and haplotypes in gene-rich regions was used in previous genome-wide association studies (GWAS) for sodicity tolerance in bread wheat. Thirty-nine novel SNPs including 18 haplotypes for yield and yield-components were identified. The present study aimed at developing SNP-derived markers by precisely locating new SNPs on ~180 bp allelic sequence of t-GBS, marker validation, and SNP functional characterization based on its exonic location. We identified unknown locations of significant SNPs/haplotypes by aligning allelic sequences on to IWGSC RefSeqv1.0 on respective chromosomes. Eighteen out of the target 39 SNP locations fulfilled the criteria for producing PCR markers, among which only eight produced polymorphic signals. These eight markers associated with yield, plants m-2, heads m-2, and harvest index, including a pleiotropic marker for yield, harvest index, and grains/head were validated for its amplification efficiency and phenotypic effects in focused identification germplasm strategy (FIGS) wheat set and a doubled haploid (DH) population (Scepter/IG107116). The phenotypic variation explained by these markers are in the range of 4.1-37.6 in the FIGS population. High throughput PCR-based genotyping using new markers and association with phenotypes in FIGS wheat set and DH population validated the effect of functional SNP on closely associated genes-calcineurin B-like- and dirigent protein, basic helix-loop-helix (BHLH-), plant homeodomain (PHD-) and helix-turn-helix myeloblastosis (HTH myb) type -transcription factor. Further, genome-wide SNP annotation using SnpEff tool confirmed that these SNPs are in gene regulatory regions (upstream, 3'-UTR, and intron) modifying gene expression and protein-coding. This integrated approach of marker design for t-GBS alleles, SNP functional annotation, and high-throughput genotyping of functional SNP offers translation solutions across crops and complex traits in crop improvement programs.

Exploiting the Genomic Diversity of Rice (Oryza sativa L.): SNP-Typing in 11 Early-Backcross Introgression-Breeding Populations.

This study demonstrates genotyping-by-sequencing-based single-nucleotide polymorphism (SNP)-typing in 11 early-backcross introgression populations of rice (at BC1F5), comprising a set of 564 diverse introgression lines and 12 parents. Sequencing using 10 Ion Proton runs generated a total of ∼943.4 million raw reads, out of which ∼881.6 million reads remained after trimming for low-quality bases. After alignment, 794,297 polymorphic SNPs were identified, and filtering resulted in LMD50 SNPs (low missing data, with each SNP, genotyped in at least 50% of the samples) for each sub-population. Every data point was supported by actual sequencing data without any imputation, eliminating imputation-induced errors in SNP calling. Genotyping substantiated the impacts of novel breeding strategy revealing: (a) the donor introgression patterns in ILs were characteristic with variable introgression frequency in different genomic regions, attributed mainly to stringent selection under abiotic stress and (b) considerably lower heterozygosity was observed in ILs. Functional annotation revealed 426 non-synonymous deleterious SNPs present in 102 loci with a range of 1-4 SNPs per locus and 120 novel SNPs. SNP-typing this diversity panel will further assist in the development of markers supporting genomic applications in molecular breeding programs.

Simultaneous Improvement and Genetic Dissection of Salt Tolerance of Rice (Oryza sativa L.) by Designed QTL Pyramiding.

Breeding of multi-stress tolerant rice varieties with higher grain yields is the best option to enhance the rice productivity of abiotic stresses prone areas. It also poses the greatest challenge to plant breeders to breed rice varieties for such stress prone conditions. Here, we carried out a designed QTL pyramiding experiment to develop high yielding "Green Super Rice" varieties with significantly improved tolerance to salt stress and grain yield. Using the F4 population derived from a cross between two selected introgression lines, we were able to develop six mostly homozygous promising high yielding lines with significantly improved salt tolerance and grain yield under optimal and/or saline conditions in 3 years. Simultaneous mapping using the same breeding population and tunable genotyping-by-sequencing technology, we identified three QTL affecting salt injury score and leaf chlorophyll content. By analyzing 32M SNP data of the grandparents and graphical genotypes of the parents, we discovered 87 positional candidate genes for salt tolerant QTL. According to their functional annotation, we inferred the most likely candidate genes. We demonstrated that designed QTL pyramiding is a powerful strategy for simultaneous improvement and genetic dissection of complex traits in rice.