Tiller and spikelet numbers are important agronomic traits affecting wheat grain yield, but the molecular mechanisms controlling these traits are largely unknown. We have identified a gene (Wheat Tiller-1, WT-1) that regulates numbers of these two very important agronomic traits. While trying to understand the early events of tiller development in wheat, cross section analysis of the crown region showed that differentiation of the tiller buds and apical meristem into spikelets occurs during early seedling stages. The gene was identified by VIGS silencing using sequence around the VHIID motif of the LS gene of tomato that controls branching. VIGS gene silencing, first using the tomato sequence and then from the gene identified from wheat resulted in uniculm and reduce tiller number phenotype. Overall, the WT-1 showed only 37.6% predicted protein similarity to the LS gene although the VHIID motif was conserved. The gene has three structural copies one each on the three wheat homoeologous group 7 chromosomes. Although share >98% sequence similarity, the three gene copies showed different expression pattern in various tissues and growth stages. Silencing of the gene via stable RNAi showed reduction in both tiller and spikelet number. SEM analysis of the RNAi plants showed that silencing of WT-1 reduced the tiller bud initiation. Among the progeny of independent RNAi events, variation in both spikelet and tiller numbers correlated with the level of reduction in the gene expression, showing role of the gene in controlling tiller number and spikelet number per spike.

Modern maize hybrids often contain biotech and native traits. To-date all biotech traits have been randomly inserted in the genome. Consequently, developing hybrids with multiple traits is expensive, time-consuming, and complex. Here we report using CRISPR-Cas9 to generate a complex trait locus (CTL) to facilitate trait stacking. A CTL consists of multiple preselected sites positioned within a small well-characterized chromosomal region where trait genes are inserted. We generated individual lines, each carrying a site-specific insertion landing pad (SSILP) that was targeted to a preselected site and capable of efficiently receiving a transgene via recombinase-mediated cassette exchange. The selected sites supported consistent transgene expression and the SSILP insertion had no effect on grain yield. We demonstrated that two traits residing at different sites within a CTL can be combined via genetic recombination. CTL technology is a major step forward in the development of multi-trait maize hybrids.

Summary An efficient Agrobacterium ‐mediated site‐specific integration ( SSI ) technology using the flipase/flipase recognition target ( FLP / FRT ) system in elite maize inbred lines is described. The system allows precise integration of a single copy of a donor DNA flanked by heterologous FRT sites into a predefined recombinant target line ( RTL ) containing the corresponding heterologous FRT sites. A promoter‐trap system consisting of a pre‐integrated promoter followed by an FRT site enables efficient selection of events. The efficiency of this system is dependent on several factors including Agrobacterium tumefaciens strain, expression of morphogenic genes Babyboom ( Bbm ) and Wuschel2 ( Wus2 ) and choice of heterologous FRT pairs. Of the Agrobacterium strains tested, strain AGL 1 resulted in higher transformation frequency than strain LBA 4404 THY ‐ (0.27% vs. 0.05%; per cent of infected embryos producing events). The addition of morphogenic genes increased transformation frequency (2.65% in AGL 1; 0.65% in LBA 4404 THY ‐). Following further optimization, including the choice of FRT pairs, a method was developed that achieved 19%–22.5% transformation frequency. Importantly, >50% of T0 transformants contain the desired full‐length site‐specific insertion. The frequencies reported here establish a new benchmark for generating targeted quality events compatible with commercial product development.

Development of transgenic cell lines or organisms for industrial, agricultural, or medicinal applications involves inserting DNA into the target genome in a way that achieves efficacious transgene expression without a deleterious impact on fitness. The genomic insertion site is widely recognized as an important determinant of success. However, the effect of chromosomal location on transgene expression and fitness has not been systematically investigated in plants. Here we evaluate the importance of transgene insertion site in maize and soybean using both random and site-specific transgene integration. We have compared the relative contribution of genomic location on transgene expression levels with other factors, including cis-regulatory elements, neighboring transgenes, genetic background, and zygosity. As expected, cis-regulatory elements and the presence/absence of nearby transgene neighbors can impact transgene expression. Surprisingly, we determined not only that genomic location had the least impact on transgene expression compared to the other factors that were investigated but that the majority of insertion sites recovered supported transgene expression levels that were statistically not distinguishable. All 68 genomic sites evaluated were capable of supporting high-level transgene expression, which was also consistent across generations. Furthermore, multilocation field evaluation detected no to little decrease in agronomic performance as a result of transgene insertion at the vast majority of sites we evaluated with a single construct in five maize hybrid backgrounds.

Polyploidy is a major evolutionary process in eukaryotes, yet the expression balance of homeologs in natural polyploids is largely unknown. To study this expression balance, the expression patterns of 2180 structurally well-characterized genes of wheat were studied, of which 813 had the expected three copies and 375 had less than three. Copy numbers of the remaining 992 ranged from 4 to 14, including homeologs, orthologs, and paralogs. Of the genes with three structural copies corresponding to homeologs, 55% expressed from all three, 38% from two, and the remaining 7% expressed from only one of the three copies. Homeologs of 76–87% of the genes showed differential expression patterns in different tissues, thus have evolved different gene expression controls, possibly resulting in novel functions. Homeologs of 55% of the genes showed tissue-specific expression, with the largest percentage (14%) in the anthers and the smallest (7%) in the pistils. The highest number (1.72/3) of homeologs/gene expression was in the roots and the lowest (1.03/3) in the anthers. As the expression of homeologs changed with changes in structural copy number, about 30% of the genes showed dosage dependence. Chromosomal location also impacted expression pattern as a significantly higher proportion of genes in the proximal regions showed expression from all three copies compared to that present in the distal regions.

Barley stripe mosaic virus (BSMV)-based virus-induced gene silencing (VIGS) is an effective strategy for rapid functional analysis of genes in wheat leaves, but its utility to transiently express genes, and silencing in other tissues including root, flower, and developing grains, has not been demonstrated in monocots. We monitored green fluorescent protein (GFP) expression to demonstrate the utility of BSMV as a transient expression vector and silenced genes in various wheat tissues to expand VIGS utility to characterize tissue-specific genes. An antisense construct designed for coronatine insensitive1 (COI1) showed an 85% decrease in COI1 transcript level in roots accompanied by a 26% reduction in root length. Similarly, silencing of seed-specific granule-bound starch synthase by antisense and hairpin constructs resulted in up to 82% reduction in amylose content of the developing grains. VIGS of meiosis-specific genes demonstrated by silencing wheat homologue of disrupted meiosis cDNA1 (DMC1) by an antisense construct resulted in a 75–80% reduction in DMC1 transcript level accompanied by an average of 37.2 univalents at metaphase I. The virus-based transient GFP expression was observed in the leaf, phloem, and root cortex at 10–17 days post-inoculation. A novel observation was made that 8–11% of the first selfed generation progeny showed VIGS inheritance and that this proportion increased to 53–72% in the second and to 90–100% in the third generations. No viral symptoms were observed in the progeny, making it possible to study agronomic traits by VIGS. VIGS inheritance is particularly useful to study genes expressing during seed germination or other stages of early plant growth.

Seven winter wheat (Triticum aestivum L.) germplasm lines carrying the Wsm1 gene conferring resistance to Wheat streak mosaic virus (WSMV)—Alliance‐Wsm1 (Reg. No. GP‐858, PI 653710), Arrowsmith‐Wsm1 (Reg. No. GP‐859, PI 653711), Goodstreak‐Wsm1 (Reg. No. GP‐860, PI 653712), Harry‐Wsm1 (Reg. No. GP‐861, PI 653713), Millennium‐Wsm1 (Reg. No. GP‐862, PI 653714), Wahoo‐Wsm1 (Reg. No. GP‐863, PI 653715), and Wesley‐Wsm1 (Reg. No. GP‐864, PI 653716)—were codeveloped by Washington State University, Pullman, WA; the University of Nebraska, Lincoln, NE; and the USDA‐ARS. These seven different winter wheat cultivars were selected to provide more sources of effective resistance to WSMV in winter wheat cultivars of Nebraska and adjoining states. Resistance to WSMV is conferred by the Wsm1 gene, which was translocated from Thinopyrum intermedium (Host) Barkworth & D.R. Dewey [Agropyron intermedium (Horst.) Beauv.] into wheat. The STSJ15 marker was used to select for the gene in the backcross progeny until the BC4F1 generation. In BC4F2 generation, screening for disease resistance was done using the Sidney 81 isolate of WSMV, along with the recurrent parents. Lines showing high levels of resistance to WSMV were further selected for seed increase and field evaluation. These lines may serve as a winter wheat source of WSMV resistance and may be used for gene pyramiding and for studying the effect of the Wsm1 gene in different backgrounds.

SUMMARY Regardless of genetic makeup, a female honey bee becomes a queen or worker depending on the food she receives as a larva. For decades, it has been known that nutrition and juvenile hormone (JH) signaling determine the caste fate of the individual bee. However, it is still largely unclear how these factors are connected. To address this question, we suppressed nutrient sensing by RNA interference (RNAi)-mediated gene knockdown of IRS (insulin receptor substrate) and TOR (target of rapamycin) in larvae reared on queen diet. The treatments affected several layers of organismal organization that could play a role in the response to differential nutrition between castes. These include transcript profiles, proteomic patterns, lipid levels, DNA methylation response and morphological features. Most importantly, gene knockdown abolished a JH peak that signals queen development and resulted in a worker phenotype. Application of JH rescued the queen phenotype in either knockdown, which demonstrates that the larval response to JH remains intact and can drive normal developmental plasticity even when IRS or TOR transcript levels are reduced. We discuss our results in the context of other recent findings on honey bee caste and development and propose that IRS is an alternative substrate for the Egfr (epidermal growth factor receptor) in honey bees. Overall, our study describes how the interplay of nutritional and hormonal signals affects many levels of organismal organization to build different phenotypes from identical genotypes.

Centromeric regions of higher eukaryotes are comprised mainly of tandem and non-tandem repeat sequences with variable copy number, spacing, order and orientation; are heterochromatic in nature, and are believed to be devoid of actively transcribing genes. Here, we report an actively transcribing wheat homolog of HSP70 gene that maps in the functional wheat centromere, and copy number of which seems to change in response to centromeric breaks. The HSP70 gene physically maps on the short arm of chromosomes 1A and 1D of Chinese Spring (CS) and 1R of rye. Whereas, on chromosome 1B in both ‘CS’ and Pavon background, the gene maps in the functional centromere as evident from its presence in both cytologically confirmed true ditelosomic lines Dt1BS and Dt1BL. Sequence comparison of 11 ESTs showed three sequence patterns suggesting that all three homoeologous copies of the gene are expressing. The cDNA-single stranded conformation polymorphism analysis confirmed expression of the ‘CS’ 1B copy of the gene. Observed in two independently developed Dt1BL lines, the 1B copy number of the gene showed three to fivefold increase in response to chromosomal breaks around the centromere. Putative gene duplications seem to involve large chromosomal segments as only one of the ten restriction enzymes used for DNA gel-blot analysis showed unique extra fragment band in the Dt1BL line. Further investigations are warranted to uncover the nature and mechanism of these duplications.

A marker-assisted background selection (MABS)-based gene introgression approach in wheat (Triticum aestivum L.) was optimized, where 97% or more of a recurrent parent genome (RPG) can be recovered in just two backcross (BC) generations. A four-step MABS method was developed based on 'Plabsim' computer simulations and wheat genome structure information. During empirical optimization of this method, double recombinants around the target gene were selected in a step-wise fashion during the two BC cycles followed by selection for recurrent parent genotype on non-carrier chromosomes. The average spacing between carrier chromosome markers was <4 cM. For non-carrier chromosome markers that flanked each of the 48 wheat gene-rich regions, this distance was approximately 12 cM. Employed to introgress seedling stripe rust (Puccinia striiformis f. sp. tritici) resistance gene Yr15 into the spring wheat cultivar 'Zak', marker analysis of 2,187 backcross-derived progeny resulted in the recovery of a BC(2)F(2ratio3) plant with 97% of the recurrent parent genome. In contrast, only 82% of the recurrent parent genome was recovered in phenotypically selected BC(4)F(7) plants developed without MABS. Field evaluation results from 17 locations indicated that the MABS-derived line was either equal or superior to the recurrent parent for the tested agronomic characteristics. Based on these results, MABS is recommended as a strategy for rapidly introgressing a targeted gene into a wheat genotype in just two backcross generations while recovering 97% or more of the recurrent parent genotype.