WGNAM: whole-genome nested association mapping.

KEY MESSAGE: A powerful QTL analysis method for nested association mapping populations is presented. Based on a one-stage multi-locus model, it provides accurate predictions of founder specific QTL effects. Nested association mapping (NAM) populations have been created to enable the identification of quantitative trait loci (QTL) in different genetic backgrounds. A whole-genome nested association mapping (WGNAM) method is presented to perform QTL analysis in NAM populations. The WGNAM method is an adaptation of the multi-parent whole genome average interval mapping approach where the crossing design is incorporated through the probability of inheriting founder alleles for every marker across the genome. Based on a linear mixed model, this method provides a one-stage analysis of raw phenotypic data, molecular markers, and crossing design. It simultaneously scans the whole-genome through an iterative process leading to a model with all the identified QTL while keeping the false positive rate low. The WGNAM approach was assessed through a simulation study, confirming to be a powerful and accurate method for QTL analysis for a NAM population. This novel method can also accommodate a multi-reference NAM (MR-NAM) population where donor parents are crossed with multiple reference parents to increase genetic diversity. Therefore, a demonstration is presented using a MR-NAM population for wheat (Triticum aestivum L.) to perform a QTL analysis for plant height. The strength and size of the putative QTL were summarized enhancing the understanding of the QTL effects depending on the parental origin. Compared to other methods, the proposed methodology based on a one-stage analysis provides greater power to detect QTL and increased accuracy in the estimation of their effects. The WGNAM method establishes the basis for accurate QTL mapping studies for NAM and MR-NAM populations.

Dissecting the Genetics of Early Vigour to Design Drought-Adapted Wheat.

Due to the climate change and an increased frequency of drought, it is of enormous importance to identify and to develop traits that result in adaptation and in improvement of crop yield stability in drought-prone regions with low rainfall. Early vigour, defined as the rapid development of leaf area in early developmental stages, is reported to contribute to stronger plant vitality, which, in turn, can enhance resilience to erratic drought periods. Furthermore, early vigour improves weed competitiveness and nutrient uptake. Here, two sets of a multi-reference nested association mapping (MR-NAM) population of bread wheat (Triticum aestivum ssp. aestivum L.) were used to investigate early vigour in a rain-fed field environment for 3 years, and additionally assessed under controlled conditions in a greenhouse experiment. The normalised difference vegetation index (NDVI) calculated from red/infrared light reflectance was used to quantify early vigour in the field, revealing a correlation (p < 0.05; r = 0.39) between the spectral measurement and the length of the second leaf. Under controlled environmental conditions, the measured projected leaf area, using a green-pixel counter, was also correlated to the leaf area of the second leaf (p < 0.05; r = 0.38), as well as to the recorded biomass (p < 0.01; r = 0.71). Subsequently, genetic determination of early vigour was tested by conducting a genome-wide association study (GWAS) for the proxy traits, revealing 42 markers associated with vegetation index and two markers associated with projected leaf area. There are several quantitative trait loci that are collocated with loci for plant developmental traits including plant height on chromosome 2D (log10 (P) = 3.19; PVE = 0.035), coleoptile length on chromosome 1B (-log10 (P) = 3.24; PVE = 0.112), as well as stay-green and vernalisation on chromosome 5A (-log10 (P) = 3.14; PVE = 0.115).

Adaptive Traits to Improve Durum Wheat Yield in Drought and Crown Rot Environments.

Durum wheat (Triticum turgidum L. ssp. durum) production can experience significant yield losses due to crown rot (CR) disease. Losses are usually exacerbated when disease infection coincides with terminal drought. Durum wheat is very susceptible to CR, and resistant germplasm is not currently available in elite breeding pools. We hypothesize that deploying physiological traits for drought adaptation, such as optimal root system architecture to reduce water stress, might minimize losses due to CR infection. This study evaluated a subset of lines from a nested association mapping population for stay-green traits, CR incidence and yield in field experiments as well as root traits under controlled conditions. Weekly measurements of normalized difference vegetative index (NDVI) in the field were used to model canopy senescence and to determine stay-green traits for each genotype. Genome-wide association studies using DArTseq molecular markers identified quantitative trait loci (QTLs) on chromosome 6B (qCR-6B) associated with CR tolerance and stay-green. We explored the value of qCR-6B and a major QTL for root angle QTL qSRA-6A using yield datasets from six rainfed environments, including two environments with high CR disease pressure. In the absence of CR, the favorable allele for qSRA-6A provided an average yield advantage of 0.57 t·ha-1, whereas in the presence of CR, the combination of favorable alleles for both qSRA-6A and qCR-6B resulted in a yield advantage of 0.90 t·ha-1. Results of this study highlight the value of combining above- and belowground physiological traits to enhance yield potential. We anticipate that these insights will assist breeders to design improved durum varieties that mitigate production losses due to water deficit and CR.

Root phenotypes of young wheat plants grown in controlled environments show inconsistent correlation with mature root traits in the field.

Using a field to lab approach, mature deep-rooting traits in wheat were correlated to root phenotypes measured on young plants from controlled conditions. Mature deep-rooting root traits of 20 wheat genotypes at maturity were established via coring in three field trials across 2 years. Field traits were correlated to phenotypes expressed by the 20 genotypes after growth in four commonly used lab screens: (i) soil tubes for root emergence, elongation, length, and branching at four ages to 34 days after sowing (DAS); (ii) paper pouches 7 DAS and (iii) agar chambers for primary root (PR) number and angles at 8 DAS; and (iv) soil baskets for PR and nodal root (NR) number and angle at 42 DAS. Correlations between lab and field root traits (r2=0.45-0.73) were highly inconsistent, with many traits uncorrelated and no one lab phenotype correlating similarly across three field experiments. Phenotypes most positively associated with deep field roots were: longest PR and NR axiles from the soil tube screen at 20 DAS; and narrow PR angle and wide NR angle from soil baskets at 42 DAS. Paper and agar PR angles were positively and significantly correlated to each other, but only wide outer PRs in the paper screen correlated positively to shallower field root traits. NR phenotypes in soil baskets were not predicted by PR phenotypes in any screen, suggesting independent developmental controls and value in measuring both root types in lab screens. Strong temporal and edaphic effects on mature root traits, and a lack of understanding of root trait changes during plant development, are major challenges in creating controlled-environment root screens for mature root traits in the field.

Chemical and physical influence of sodic soils on the coleoptile length and root growth angle of wheat genotypes.

BACKGROUND AND AIMS: High exchangeable sodium percentage (ESP) and bulk density of sodic soils can reduce seedling emergence. This study examined variation in seedling coleoptile length and seminal root angle of wheat (Triticum aestivum. L) genotypes to determine whether these traits vary between genotypes that differ in their tolerance to sodic soils. METHODS: Wheat genotypes were grown in three different experiments. First, four wheat genotypes were grown using soils of three ESPs (4, 10 and 17 %) and secondly in soils of three different bulk densities (1.2, 1.4 and 1.5 g cm-3) and ESP 10 %. Thirdly, seedling coleoptile length and seminal root angle were determined for 16 genotypes grown in a soil of ESP 10 % and bulk density 1.2 g cm-2. Seminal root angle and coleoptile length measurements from the current study were compared with seedling emergence rate and force measured previously. KEY RESULTS: The seedling coleoptile length of all genotypes decreased with increasing soil ESP and bulk density, but with no significant differences between genotypes. In contrast, seminal root angles differed significantly between genotypes, but were not significantly affected by ESP or bulk density. There was an inverse relationship between the seminal root angle of the 16 genotypes and seedling emergence rate (R2 = 0.89) and also between seminal root angle and seedling emergence force (R2 = 0.61). CONCLUSIONS: Lack of significant variation in coleoptile length between genotypes suggests that this may not be a suitable characteristic to identify wheat tolerance to sodic conditions. However, a narrower seminal root angle was correlated with rate and force of seedling emergence, traits likely to improve establishment. The mechanism underlying this correlation is not yet clear. Genotypes with a narrow root angle had greater root depth. One possible mechanism might be that genotypes with narrow root angles were able to take up more soil moisture at depth, leading to a higher proportion of seedling emergence.

A Major Root Architecture QTL Responding to Water Limitation in Durum Wheat.

The optimal root system architecture (RSA) of a crop is context dependent and critical for efficient resource capture in the soil. Narrow root growth angle promoting deeper root growth is often associated with improved access to water and nutrients in deep soils during terminal drought. RSA, therefore is a drought-adaptive trait that could minimize yield losses in regions with limited rainfall. Here, GWAS for seminal root angle (SRA) identified seven marker-trait associations clustered on chromosome 6A, representing a major quantitative trait locus (qSRA-6A) which also displayed high levels of pairwise LD (r 2 = 0.67). Subsequent haplotype analysis revealed significant differences between major groups. Candidate gene analysis revealed loci related to gravitropism, polar growth and hormonal signaling. No differences were observed for root biomass between lines carrying hap1 and hap2 for qSRA-6A, highlighting the opportunity to perform marker-assisted selection for the qSRA-6A locus and directly select for wide or narrow RSA, without influencing root biomass. Our study revealed that the genetic predisposition for deep rooting was best expressed under water-limitation, yet the root system displayed plasticity producing root growth in response to water availability in upper soil layers. We discuss the potential to deploy root architectural traits in cultivars to enhance yield stability in environments that experience limited rainfall.

Greater emergence force and hypocotyl cross sectional area may improve wheat seedling emergence in sodic conditions.

Surface crusting of sodic soils is a major problem in the semi-arid tropics when rapid drying after sowing follows light showers, leading to reduced seedling emergence and grain yield. The magnitude of the force exerted by germinating seeds affects the ability of the seedlings to rupture the crust and emerge. This study aimed to determine whether the seed germination and seedling emergence force of wheat (Triticum aestivum L.) seedlings vary among different genotypes at different sodicity levels. Germination and emergence force of seedlings of four wheat genotypes was determined in assays using four solutions with sodium adsorption ratio (SAR) values ranging from 0 to 60. Seed germination and seedling emergence force varied between genotypes at different sodicity levels, with the emergence force of the coleoptile correlated to the cross sectional area of the hypocotyl. The results suggest that the selection of wheat genotypes with rapid germination, higher seedling emergence force and larger hypocotyl cross sectional area, offers a strategy to improve seedling emergence in crusted sodic soils.

Dataset on seed details of wheat genotypes, solution treatments to measure seedling emergence force and the relation between seedling force and strain.

The seed details (weight, vigor) and germination rate of 16 wheat (Triticum aestivum) genotypes in a non-limiting conditions were measured. The dataset presents seed germination rate and seed vigor of 16 wheat genotypes. The dataset also presents the concentrations of the cations to create solution treatments of various sodium adsorption ratio (SAR) and ionic strength (I). Finally, dataset presented a figure of the experimental design to measure seedling emergence force of wheat genotypes. The image of the setup and the relation between strain and force have been presented here to convert the strain of the beam into seedling emergence force. This dataset has been used in research work titled 'Greater emergence force and hypocotyl cross sectional area may improve wheat seedling emergence in sodic conditions' (Anzooman et al., 2018) [1].

A low-cost method to rapidly and accurately screen for transpiration efficiency in wheat.

BACKGROUND: Wheat (Triticum aestivum L.) productivity is commonly limited by the availability of water. Increasing transpiration efficiency (biomass produced per unit of water used, TE) can potentially lead to increased grain yield in water-limited environments ('more crop per drop'). Currently, the ability to screen large populations for TE is limited by slow, low-throughput and/or expensive screening procedures. Here, we propose a low-cost, low-technology, rapid, and scalable method to screen for TE. The method uses a Pot-in-Bucket system that allows continuous watering of the pots and frequent monitoring of water use. To investigate the robustness of the method across environments, and to determine the shortest trial duration required to get accurate and repeatable TE estimates in wheat, plants from 11 genotypes varying in phenology were sown at three dates and grown for different durations in a polyhouse with partial environmental control. RESULTS: The method revealed significant genotypic variations in TE among the 11 studied wheat genotypes. Genotype rankings for TE were consistent when plants were harvested the same day, at the flag-leaf stage or later. For these harvests, genotype rankings were consistent across experiments despite changes in environmental conditions, such as evaporative demand. CONCLUSIONS: These results indicate that (1) the Pot-In-Bucket system is suitable to screen TE for breeding purposes in populations with varying phenology, (2) multiple short trials can be carried out within a season to allow increased throughput of genotypes for TE screening, and (3) root biomass measurement is not required to screen for TE, as whole-plant TE and shoot-only TE are highly correlated, at least in wheat. The method is particularly relevant in developing countries where low-cost and relatively high labour input may be most applicable.

Selection in Early Generations to Shift Allele Frequency for Seminal Root Angle in Wheat.

A current challenge for plant breeders is the limited ability to phenotype and select for root characteristics to enhance crop productivity. The development of a high-throughput phenotyping method has recently offered new opportunities for the selection of root characteristics in breeding programs. Here, we investigated prospects for phenotypic and molecular selection for seminal root angle (SRA), a key trait associated with mature root system architecture in wheat ( L.). We first investigated genetic diversity for this trait in a panel of 22 wheat lines adapted to Australian environments. The angle between the first pair of seminal roots ranged from 72 to 106°. We then evaluated selection gain via direct phenotypic selection in early generations by comparing the resulting shift in population distribution in tail populations selected for "narrow" and "wide" root angle. Overall, two rounds of selection significantly shifted the mean root angle as much as 10°. Furthermore, comparison of allele frequencies in the tail populations revealed genomic regions under selection, for which marker-assisted selection appeared to be successful. By combining efficient phenotyping and rapid generation advance, lines enriched with alleles for either narrow or wide SRA were developed within only 18 mo. These results suggest that there is a valuable source of allelic variation for SRA that can be harnessed and rapidly introgressed into elite wheat lines.

Speed breeding for multiple quantitative traits in durum wheat.

BACKGROUND: Plant breeding requires numerous generations to be cycled and evaluated before an improved cultivar is released. This lengthy process is required to introduce and test multiple traits of interest. However, a technology for rapid generation advance named 'speed breeding' was successfully deployed in bread wheat (Triticum aestivum L.) to achieve six generations per year while imposing phenotypic selection for foliar disease resistance and grain dormancy. Here, for the first time the deployment of this methodology is presented in durum wheat (Triticum durum Desf.) by integrating selection for key traits, including above and below ground traits on the same set of plants. This involved phenotyping for seminal root angle (RA), seminal root number (RN), tolerance to crown rot (CR), resistance to leaf rust (LR) and plant height (PH). In durum wheat, these traits are desirable in environments where yield is limited by in-season rainfall with the occurrence of CR and epidemics of LR. To evaluate this multi-trait screening approach, we applied selection to a large segregating F2 population (n = 1000) derived from a bi-parental cross (Outrob4/Caparoi). A weighted selection index (SI) was developed and applied. The gain for each trait was determined by evaluating F3 progeny derived from 100 'selected' and 100 'unselected' F2 individuals. RESULTS: Transgressive segregation was observed for all assayed traits in the Outrob4/Caparoi F2 population. Application of the SI successfully shifted the population mean for four traits, as determined by a significant mean difference between 'selected' and 'unselected' F3 families for CR tolerance, LR resistance, RA and RN. No significant shift for PH was observed. CONCLUSIONS: The novel multi-trait phenotyping method presents a useful tool for rapid selection of early filial generations or for the characterization of fixed lines out-of-season. Further, it offers efficient use of resources by assaying multiple traits on the same set of plants. Results suggest that when performed in parallel with speed breeding in early generations, selection will enrich recombinant inbred lines with desirable alleles and will reduce the length and number of years required to combine these traits in elite breeding populations and therefore cultivars.

Manipulation of exercise to rest ratio within set duration on physical and technical outcomes during small-sided games in elite youth soccer players.

Training practices for elite soccer players should take into account specific technical, tactical and physical components. As a consequence of these demands small-sided games (SSGs) have become a popular conditioning tool that replicate the demands encountered during match play. The aim of this investigation was to examine how the manipulation of exercise to rest ratio, within the same overall duration, affected both physical and technical outcomes during SSGs in elite youth soccer. Twelve elite youth soccer players participated in three variations of eight minute 6v6 SSGs. The three variations included eight minutes continuous, 2×4min and 4×2min. Players perceived the continuous 8min block as the hardest (4.5±1.5AU), followed by the 2×4min (3.9±1.4AU) and the 4×2min (3.3±1.4AU), although no difference in mean HR or physical measures via GPS analysis between SSGs was evident. From the technical perspective, only goals scored reached significance, with post hoc analysis identifying the number of goals scored were significantly higher during the 4×2min and 2×4min SSGs compared to 8min continuous block. These results show that subjective ratings of exertion differed between conditions, but only minor technical manipulations were observed by adjusting work to rest ratios, with no significant effect on physical performance.

Wheats developed for high yield on stored soil moisture have deep vigorous root systems.

Many rainfed wheat production systems are reliant on stored soil water for some or all of their water inputs. Selection and breeding for root traits could result in a yield benefit; however, breeding for root traits has traditionally been avoided due to the difficulty of phenotyping mature root systems, limited understanding of root system development and function, and the strong influence of environmental conditions on the phenotype of the mature root system. This paper outlines an international field selection program for beneficial root traits at maturity using soil coring in India and Australia. In the rainfed areas of India, wheat is sown at the end of the monsoon into hot soils with a quickly receding soil water profile; in season water inputs are minimal. We hypothesised that wheat selected and bred for high yield under these conditions would have deep, vigorous root systems, allowing them to access and utilise the stored soil water at depth around anthesis and grain-filling when surface layers were dry. The Indian trials resulted in 49 lines being sent to Australia for phenotyping. These lines were ranked against 41 high yielding Australian lines. Variation was observed for deep root traits e.g. in eastern Australia in 2012, maximum depth ranged from 118.8 to 146.3cm. There was significant variation for root traits between sites and years, however, several Indian genotypes were identified that consistently ranked highly across sites and years for deep rooting traits.

Frost trends and their estimated impact on yield in the Australian wheatbelt.

Radiant spring frosts occurring during reproductive developmental stages can result in catastrophic yield loss for wheat producers. To better understand the spatial and temporal variability of frost, the occurrence and impact of frost events on rain-fed wheat production was estimated across the Australian wheatbelt for 1957-2013 using a 0.05 ° gridded weather data set. Simulated yield outcomes at 60 key locations were compared with those for virtual genotypes with different levels of frost tolerance. Over the last six decades, more frost events, later last frost day, and a significant increase in frost impact on yield were found in certain regions of the Australian wheatbelt, in particular in the South-East and West. Increasing trends in frost-related yield losses were simulated in regions where no significant trend of frost occurrence was observed, due to higher mean temperatures accelerating crop development and causing sensitive post-heading stages to occur earlier, during the frost risk period. Simulations indicated that with frost-tolerant lines the mean national yield could be improved by up to 20% through (i) reduced frost damage (~10% improvement) and (ii) the ability to use earlier sowing dates (adding a further 10% improvement). In the simulations, genotypes with an improved frost tolerance to temperatures 1 °C lower than the current 0 °C reference provided substantial benefit in most cropping regions, while greater tolerance (to 3 °C lower temperatures) brought further benefits in the East. The results indicate that breeding for improved reproductive frost tolerance should remain a priority for the Australian wheat industry, despite warming climates.

High-throughput phenotyping of seminal root traits in wheat.

BACKGROUND: Water availability is a major limiting factor for wheat (Triticum aestivum L.) production in rain-fed agricultural systems worldwide. Root system architecture has important functional implications for the timing and extent of soil water extraction, yet selection for root architectural traits in breeding programs has been limited by a lack of suitable phenotyping methods. The aim of this research was to develop low-cost high-throughput phenotyping methods to facilitate selection for desirable root architectural traits. Here, we report two methods, one using clear pots and the other using growth pouches, to assess the angle and the number of seminal roots in wheat seedlings- two proxy traits associated with the root architecture of mature wheat plants. RESULTS: Both methods revealed genetic variation for seminal root angle and number in the panel of 24 wheat cultivars. The clear pot method provided higher heritability and higher genetic correlations across experiments compared to the growth pouch method. In addition, the clear pot method was more efficient - requiring less time, space, and labour compared to the growth pouch method. Therefore the clear pot method was considered the most suitable for large-scale and high-throughput screening of seedling root characteristics in crop improvement programs. CONCLUSIONS: The clear-pot method could be easily integrated in breeding programs targeting drought tolerance to rapidly enrich breeding populations with desirable alleles. For instance, selection for narrow root angle and high number of seminal roots could lead to deeper root systems with higher branching at depth. Such root characteristics are highly desirable in wheat to cope with anticipated future climate conditions, particularly where crops rely heavily on stored soil moisture at depth, including some Australian, Indian, South American, and African cropping regions.

QTL for root angle and number in a population developed from bread wheats (Triticum aestivum) with contrasting adaptation to water-limited environments.

Root architecture traits in wheat are important in deep soil moisture acquisition and may be used to improve adaptation to water-limited environments. The genetic architecture of two root traits, seminal root angle and seminal root number, were investigated using a doubled haploid population derived from SeriM82 and Hartog. Multiple novel quantitative trait loci (QTL) were identified, each one having a modest effect. For seminal root angle, four QTL (-log10(P) >3) were identified on 2A, 3D, 6A and 6B, and two suggestive QTL (-log10(P) >2) on 5D and 6B. For root number, two QTL were identified on 4A and 6A with four suggestive QTL on 1B, 3A, 3B and 4A. QTL for root angle and root number did not co-locate. Transgressive segregation was found for both traits. Known major height and phenology loci appear to have little effect on root angle and number. Presence or absence of the T1BL.1RS translocation did not significantly influence root angle. Broad sense heritability (h (2)) was estimated as 50 % for root angle and 31 % for root number. Root angle QTL were found to be segregating between wheat cultivars adapted to the target production region indicating potential to select for root angle in breeding programs.

Cell cycle regulated phosphorylation of LIMD1 in cell lines and expression in human breast cancers.

LIMD1 is a member of the ZYXIN family of related proteins which includes AJUBA, TRIP6, LPP, WTIP, migfilin, and ZYXIN. The LIMD1 locus, 3p21.3, has been shown to undergo loss of heterozygosity in neoplastic tissues, suggesting potential tumor suppressor function. To further understand the role of LIMD1 in cancer, we have characterized endogenous expression of the LIMD1 protein and evaluated LIMD1 RNA expression in primary human breast tumors. LIMD1 levels were found to be constant throughout the cell cycle, but LIMD1 is phosphorylated during mitosis in HeLa cells. In addition, we observed colocalization of endogenous LIMD1 with vinculin at focal adhesions. In the MDA-MB435 cell line, which lacks LIMD1 expression, we detected methylation of the putative promoter region. LIMD1 mRNA expression was found to vary among primary human breast tumors; however, differences in LIMD1 expression in human breast cancers were not associated with DNA methylation of the predicted promoter region. These results suggest that some breast tumors have altered expression of LIMD1 RNA and that LIMD1 may be involved in cell anchoring via focal adhesions and in the cell cycle, particularly during mitosis.