A high-density genetic map and molecular sex-typing assay for gerbils.

We constructed a high-density genetic map for Mongolian gerbils (Meriones unguiculatus). We genotyped 137 F2 individuals with a genotype-by-sequencing (GBS) approach at over 10,000 loci and built the genetic map using a two-step approach. First, we chose the highest-quality set of 485 markers to construct a robust map of 1239 cM with 22 linkage groups as expected from the published karyotype. Second, we added an additional 5449 markers onto the map based on their genotype similarity with the original markers. We used the final marker set to assemble 1140 genomic scaffolds (containing ~ 20% of annotated genes) into a chromosome-level assembly. We used both genetic linkage and relative sequencing coverage in males and females to identify X- and Y-chromosome scaffolds and from these we designed a robust and internally-controlled PCR assay to determine sex. This assay will facilitate early stage sex-typing of embryonic and young gerbils which is difficult using current visual methods. Accession ID: Meriones unguiculatus: 10047.

Accelerating public sector rice breeding with high-density KASP markers derived from whole genome sequencing of indica rice.

Few public sector rice breeders have the capacity to use NGS-derived markers in their breeding programmes despite rapidly expanding repositories of rice genome sequence data. They rely on > 18,000 mapped microsatellites (SSRs) for marker-assisted selection (MAS) using gel analysis. Lack of knowledge about target SNP and InDel variant loci has hampered the uptake by many breeders of Kompetitive allele-specific PCR (KASP), a proprietary technology of LGC genomics that can distinguish alleles at variant loci. KASP is a cost-effective single-step genotyping technology, cheaper than SSRs and more flexible than genotyping by sequencing (GBS) or array-based genotyping when used in selection programmes. Before this study, there were 2015 rice KASP marker loci in the public domain, mainly identified by array-based screening, leaving large proportions of the rice genome with no KASP coverage. Here we have addressed the urgent need for a wide choice of appropriate rice KASP assays and demonstrated that NGS can detect many more KASP to give full genome coverage. Through re-sequencing of nine indica rice breeding lines or released varieties, this study has identified 2.5 million variant sites. Stringent filtering of variants generated 1.3 million potential KASP assay designs, including 92,500 potential functional markers. This strategy delivers a 650-fold increase in potential selectable KASP markers at a density of 3.1 per 1 kb in the indica crosses analysed and 377,178 polymorphic KASP design sites on average per cross. This knowledge is available to breeders and has been utilised to improve the efficiency of public sector breeding in Nepal, enabling identification of polymorphic KASP at any region or quantitative trait loci in relevant crosses. Validation of 39 new KASP was carried out by genotyping progeny from a range of crosses to show that they detected segregating alleles. The new KASP have replaced SSRs to aid trait selection during marker-assisted backcrossing in these crosses, where target traits include rice blast and BLB resistance loci. Furthermore, we provide the software for plant breeders to generate KASP designs from their own datasets.

Inbred or Outbred? Genetic Diversity in Laboratory Rodent Colonies.

Nonmodel rodents are widely used as subjects for both basic and applied biological research, but the genetic diversity of the study individuals is rarely quantified. University-housed colonies tend to be small and subject to founder effects and genetic drift; so they may be highly inbred or show substantial genetic divergence from other colonies, even those derived from the same source. Disregard for the levels of genetic diversity in an animal colony may result in a failure to replicate results if a different colony is used to repeat an experiment, as different colonies may have fixed alternative variants. Here we use high throughput sequencing to demonstrate genetic divergence in three isolated colonies of Mongolian gerbil (Meriones unguiculatus) even though they were all established recently from the same source. We also show that genetic diversity in allegedly "outbred" colonies of nonmodel rodents (gerbils, hamsters, house mice, deer mice, and rats) varies considerably from nearly no segregating diversity to very high levels of polymorphism. We conclude that genetic divergence in isolated colonies may play an important role in the "replication crisis." In a more positive light, divergent rodent colonies represent an opportunity to leverage genetically distinct individuals in genetic crossing experiments. In sum, awareness of the genetic diversity of an animal colony is paramount as it allows researchers to properly replicate experiments and also to capitalize on other genetically distinct individuals to explore the genetic basis of a trait.

Reliability of ion accumulation and growth components for selecting salt tolerant lines in large populations of rice.

Ion accumulation and growth under salt stress was studied in two experiments in a rice mapping population derived from parents CO39 and Moroberekan with 4-fold differences in shoot Na+ accumulation. The 120 recombinant inbred lines (RILs) had differences up to 100-fold in Na+. Measurement of 'salt tolerance' (biomass production of the RILs in 100mM NaCl relative to controls) after 42 days showed a 2-fold variation in 'salt tolerance' between parents, with five RILs being more tolerant than the more tolerant parent CO39. The reliability of various traits for selecting salt tolerance in large populations was explored by measuring Na+, K+ and K+/Na+ ratios in leaf blades and sheaths after 7 or 21 days of exposure to 100mM NaCl, and their correlation with various growth components and with leaf injury. The highest correlations were found for Na+ in the leaf blade on day 21 with injury at day 42 in both experiments (r=0.7). Earlier measurements of Na+ or of injury had lower correlations. The most sensitive growth components were tiller number plant-1 and shoot water content (g water g-1 dry weight), and these were correlated significantly with Na+ and, to a lesser extent, with K+/Na+. These studies showed that exposure for at least 42 days may be needed to clearly demonstrate the beneficial effect of the trait for Na+ exclusion on growth under salinity.

Evidence from near-isogenic lines that root penetration increases with root diameter and bending stiffness in rice.

Deep rooting can be inhibited by strong layers, although there is evidence for species and cultivar (cv.) differences in their penetration ability. Here, the availability of near-isogenic lines (NILs) in rice (Oryza sativa L.) was exploited to test the hypothesis that increased root diameter is associated with greater root bending stiffness, which leads to greater root penetration of strong layers. Wax/petrolatum discs (80% strong wax) were used as the strong layer, so that strength can be manipulated independently of water status. It was found that good root penetration was consistently associated with greater root diameter and bending stiffness, whether comparisons were made between cvs or between NILs. With NILs, this effect was seen with 'research' lines bred from recombinant inbred lines of a cross between cvs Bala and Azucena and also in improved lines developed from cv. Kalinga III by introgression of parts of the genome from Azucena. Much of the bending behaviour of roots could be explained by treating them as a simple cylinder of material. In both wax disc and sand culture systems, roots that had encountered a strong layer had lower bending stiffness than roots that had not encountered a strong layer which is a novel result and not previously reported.