Development of high amylose wheat through TILLING.

BACKGROUND: Wheat (Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement. In particular, wheat starch composition is a major target for changes that could benefit human health. Starches with increased levels of amylose are of interest because of the correlation between higher amylose content and elevated levels of resistant starch, which has been shown to have beneficial effects on health for combating obesity and diabetes. TILLING (Targeting Induced Local Lesions in Genomes) is a means to identify novel genetic variation without the need for direct selection of phenotypes. RESULTS: Using TILLING to identify novel genetic variation in each of the A and B genomes in tetraploid durum wheat and the A, B and D genomes in hexaploid bread wheat, we have identified mutations in the form of single nucleotide polymorphisms (SNPs) in starch branching enzyme IIa genes (SBEIIa). Combining these new alleles of SBEIIa through breeding resulted in the development of high amylose durum and bread wheat varieties containing 47-55% amylose and having elevated resistant starch levels compared to wild-type wheat. High amylose lines also had reduced expression of SBEIIa RNA, changes in starch granule morphology and altered starch granule protein profiles as evaluated by mass spectrometry. CONCLUSIONS: We report the use of TILLING to develop new traits in crops with complex genomes without the use of transgenic modifications. Combined mutations in SBEIIa in durum and bread wheat varieties resulted in lines with significantly increased amylose and resistant starch contents.

Prospecting for Energy-Rich Renewable Raw Materials: Sorghum Stem Case Study.

Sorghum vegetative tissues are becoming increasingly important for biofuel production. The composition of sorghum stem tissues is influenced by genotype, environment and photoperiod sensitivity, and varies widely between varieties and also between different stem tissues (outer rind vs inner pith). Here, the amount of cellulose, (1,3;1,4)-ß-glucan, arabinose and xylose in the stems of twelve diverse sorghum varieties, including four photoperiod-sensitive varieties, was measured. At maturity, most photoperiod-insensitive lines had 1% w/w (1,3;1,4)-ß-glucan in stem pith tissue whilst photoperiod-sensitive varieties remained in a vegetative stage and accumulated up to 6% w/w (1,3;1,4)-ß-glucan in the same tissue. Three sorghum lines were chosen for further study: a cultivated grain variety (Sorghum bicolor BTx623), a sweet variety (S. bicolor Rio) and a photoperiod-sensitive wild line (S. bicolor ssp. verticilliflorum Arun). The Arun line accumulated 5.5% w/w (1,3;1,4)-ß-glucan and had higher SbCslF6 and SbCslH3 transcript levels in pith tissues than did photoperiod-insensitive varieties Rio and BTx623 (<1% w/w pith (1,3;1,4)-ß-glucan). To assess the digestibility of the three varieties, stem tissue was treated with either hydrolytic enzymes or dilute acid and the release of fermentable glucose was determined. Despite having the highest lignin content, Arun yielded significantly more glucose than the other varieties, and theoretical calculation of ethanol yields was 10 344 L ha-1 from this sorghum stem tissue. These data indicate that sorghum stem (1,3;1,4)-ß-glucan content may have a significant effect on digestibility and bioethanol yields. This information opens new avenues of research to generate sorghum lines optimised for biofuel production.

TILLING moves beyond functional genomics into crop improvement.

Transgenic methods have been successfully applied to trait improvement in a number of crops. However, reverse genetics studies by transgenic means are not practical in many commercially important crops, hampering investigations into gene function and the development of novel and improved cultivars. A nontransgenic method for reverse genetics called Targeting Induced Local Lesions IN Genomes (TILLING) has been developed as a method for inducing and identifying novel genetic variation, and has been demonstrated in the model plant, Arabidopsis thaliana. Recently, TILLING has been extended to the improvement of crop plants and shows great promise as a general method for both functional genomics and modulation of key traits in diverse crops.

A 50-kb plasmid rich in mobile gene sequences isolated from a marine micrococcus.

A 50,709-bp cryptic plasmid isolated from a marine Micrococcus has been sequenced and found to contain a number of putative mobile genetic elements. The coding regions for 11 putative transposases comprise approximately 17% of the total plasmid sequence. The majority of these transposases are located within a 13-kb cluster which includes a 1553-bp direct repeat consisting of a duplicated pair of transposase genes. The remaining putative ORFs showed similarity to a variety of proteins, the most notable being spider silk.

Nucleotide sequence based characterizations of two cryptic plasmids from the marine bacterium Ruegeria isolate PR1b.

Two plasmids, 76 and 148 kb in size, isolated from Ruegeria strain PR1b were entirely sequenced. These are the first plasmids to be characterized from this genus of marine bacteria. Sequence analysis revealed a biased distribution of function among the putative proteins encoded on the two plasmids. The smaller plasmid, designated pSD20, encodes a large number of putative proteins involved in polysaccharide biosynthesis and export. The larger plasmid, designated pSD25, primarily encodes putative proteins involved in the transport of small molecules and in DNA mobilization. Sequence analysis revealed uncommon potential replication systems on both plasmids. pSD25, the first repABC-type replicon isolated from the marine environment, actually contains two repABC-type replicons. pSD20 contains a complex replication region, including a replication origin and initiation protein similar to iteron-containing plasmids (such as pSW500 from the plant pathogen Erwinia stewartii) linked to putative RepA and RepB stabilization proteins of a repABC-type replicon and is highly homologous to a plasmid from the phototrophic bacterium Rhodobacter sphaeroides. Given the nature of the putative proteins encoded by both plasmids it is possible that these plasmids enhance the metabolic and physiological flexibility of the host bacterium, and thus its adaptation to the marine sediment environment.