Stay-green alleles individually enhance grain yield in sorghum under drought by modifying canopy development and water uptake patterns

* Stay-green is an integrated drought adaptation trait characterized by a distinct green leaf phenotype during grain filling under terminal drought. We used sorghum (Sorghum bicolor), a repository of drought adaptation mechanisms, to elucidate the physiological and genetic mechanisms underpinning stay-green. * Near-isogenic sorghum lines (cv RTx7000) were characterized in a series of field and managed-environment trials (seven experiments and 14 environments) to determine the influence of four individual stay-green (Stg1–4) quantitative trait loci (QTLs) on canopy development, water use and grain yield under post-anthesis drought. * The Stg QTL decreased tillering and the size of upper leaves, which reduced canopy size at anthesis. This reduction in transpirational leaf area conserved soil water before anthesis for use during grain filling. Increased water uptake during grain filling of Stg near-isogenic lines (NILs) relative to RTx7000 resulted in higher post-anthesis biomass production, grain number and yield. Importantly, there was no consistent yield penalty associated with the Stg QTL in the irrigated control. * These results establish a link between the role of the Stg QTL in modifying canopy development and the subsequent impact on crop water use patterns and grain yield under terminal drought.

Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL in sorghum

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class ( Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue ( Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.

Characterization of 26 new microsatellite loci in the dugong (Dugong dugon)

Twenty-six microsatellite loci have been isolated from a dugong (Dugong dugon). The average heterozygosity was 0.52 with two to 10 alleles per locus surveyed from 50 individuals. The markers are suitable for genetic mark-recapture (PID = 5 × 10-16) in dugongs and they could also be used to quantify physical tag loss, estimate relatedness, assign paternity, elucidate population structure and identify migrants.

Relationship Between Hardness Genes and Quality in Barley (Hordeum vulgare)

Barley (Hordeum vulgare) genotypes were sequenced for polymorphism in the hardness genes, these being the three hordoindoline (hin a, hin b1 and hin b2) genes. The variation in haplotype was determined by sequencing for single nucleotide polymorphisms (SNPs). Polymorphism between each gene was then compared to grain hardness (three methods), malt quality characteristics (hot water extract and friability) and cattle feed quality. Two haplotypes were found in a set of forty barley genotypes. For hin a, two alleles were present, namely hin a1 and hin a2. However, there was no specific hin a allele that was associated with grain hardness, malt and feed quality. Barley has two hin b genes, namely hin b1 and hin b2, and the genotypes tested here had one of two alleles for each gene. However, there were no obvious effects on hardness or quality from either of these hin b alleles. Unlike wheat, where a clear relationship has been demonstrated between a number of SNPs in the wheat hardness genes and quality (soft or hard wheat), there was no such relationship for barley. Despite the wide range in hardness, malt and feed quality, there were only two haplotypes for each of the hin a, hin b1 and hin b2 genes and there was no clear relationship between grain hardness, malt or feed quality. The genotypes used in this study demonstrated that there was a low level of polymorphism in hardness genes in current commercial varieties as well as breeding lines and these polymorphisms had no impact on quality.

The genetic effective and adult census size of an Australian population of tiger prawns (Penaeus esculentus)

This study compares estimates of the census size of the spawning population with genetic estimates of effective current and long-term population size for an abundant and commercially important marine invertebrate, the brown tiger prawn (Penaeus esculentus). Our aim was to focus on the relationship between genetic effective and census size that may provide a source of information for viability analyses of naturally occurring populations. Samples were taken in 2001, 2002 and 2003 from a population on the east coast of Australia and temporal allelic variation was measured at eight polymorphic microsatellite loci. Moments-based and maximum-likelihood estimates of current genetic effective population size ranged from 797 to 1304. The mean long-term genetic effective population size was 9968. Although small for a large population, the effective population size estimates were above the threshold where genetic diversity is lost at neutral alleles through drift or inbreeding. Simulation studies correctly predicted that under these experimental conditions the genetic estimates would have non-infinite upper confidence limits and revealed they might be overestimates of the true size. We also show that estimates of mortality and variance in family size may be derived from data on average fecundity, current genetic effective and census spawning population size, assuming effective population size is equivalent to the number of breeders. This work confirms that it is feasible to obtain accurate estimates of current genetic effective population size for abundant Type III species using existing genetic marker technology.

New microsatellite loci for Carcharhinid sharks (Carcharhinus tilstoni and C. sorrah) and their cross-amplification in other shark species

Twelve microsatellite DNA markers were isolated in the spot-tail shark (Carcharhinus sorrah) and nine were isolated in Australian black-tip shark (Carcharhinus tilstoni). These loci plus 18 others developed for sharks from the genera Negaprion, Ginglymostoma, Carcharodon and Isurus were tested for amplification success on four species of Carcharhinus (including C. sorrah and C. tilstoni) and four other species representing three diverse families. Cross-amplification was most common within families. Five loci were subsequently tested for polymorphism on 50 C. sorrah and 60 C. tilstoni. The number of alleles per locus was two to 24 and the average heterozygosity was 0.54 (range 0.16-0.87) for C. sorrah and 0.64 (range 0.44-0.78) for C. tilstoni. These loci may be useful tools for genetic analyses of the Carcharhinidae.

Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea)

Background: Cultivated peanut or groundnut (Arachis hypogaea L.) is the fourth most important oilseed crop in the world, grown mainly in tropical, subtropical and warm temperate climates. Due to its origin through a single and recent polyploidization event, followed by successive selection during breeding efforts, cultivated groundnut has a limited genetic background. In such species, microsatellite or simple sequence repeat (SSR) markers are very informative and useful for breeding applications. The low level of polymorphism in cultivated germplasm, however, warrants a need of larger number of polymorphic microsatellite markers for cultivated groundnut. Results: A microsatellite- enriched library was constructed from the genotype TMV2. Sequencing of 720 putative SSR-positive clones from a total of 3,072 provided 490 SSRs. 71.2% of these SSRs were perfect type, 13.1% were imperfect and 15.7% were compound. Among these SSRs, the GT/CA repeat motifs were the most common (37.6%) followed by GA/CT repeat motifs (25.9%). The primer pairs could be designed for a total of 170 SSRs and were optimized initially on two genotypes. 104 (61.2%) primer pairs yielded scorable amplicon and 46 (44.2%) primers showed polymorphism among 32 cultivated groundnut genotypes. The polymorphic SSR markers detected 2 to 5 alleles with an average of 2.44 per locus. The polymorphic information content (PIC) value for these markers varied from 0.12 to 0.75 with an average of 0.46. Based on 112 alleles obtained by 46 markers, a phenogram was constructed to understand the relationships among the 32 genotypes. Majority of the genotypes representing subspecies hypogaea were grouped together in one cluster, while the genotypes belonging to subspecies fastigiata were grouped mainly under two clusters. Conclusion. Newly developed set of 104 markers extends the repertoire of SSR markers for cultivated groundnut. These markers showed a good level of PIC value in cultivated germplasm and therefore would be very useful for germplasm analysis, linkage mapping, diversity studies and phylogenetic relationships in cultivated groundnut as well as related Arachis species.

Polymorphic microsatellite loci for the zebra shark Stegostoma fasciatum

We report on the development and characterization of 14 polymorphic microsatellite loci in the zebra shark (Stegostoma fasciatum). Five tetranucleotide and nine dinucleotide loci were polymorphic with heterozygosities ranging from 0.400 to 0.967 and from three to 22 alleles per locus. Cross-species amplification of these zebra shark primers on four other species of orectolobid sharks was not successful.

Identification of QTL for sugar-related traits in a sweet x grain sorghum (Sorghum bicolor L. Moench) recombinant inbred population

QTL for stem sugar-related and other agronomic traits were identified in a converted sweet (R9188) × grain (R9403463-2-1) sorghum population. QTL analyses were conducted using phenotypic data for 11 traits measured in two field experiments and a genetic map comprising 228 SSR and AFLP markers grouped into 16 linkage groups, of which 11 could be assigned to the 10 sorghum chromosomes (SBI-01 to SBI-10). QTL were identified for all traits and were generally co-located to five locations (SBI-01, SBI-03, SBI-05, SBI-06 and SBI-10). QTL alleles from R9188 were detected for increased sucrose content and sugar content on SBI-01, SBI-05 and SBI-06. R9188 also contributed QTL alleles for increased Brix on SBI-05 and SBI-06, and increased sugar content on SBI-03. QTL alleles from R9403463-2-1 were found for increased sucrose content and sucrose yield on SBI-10, and increased glucose content on SBI-07. QTL alleles for increased height, later flowering and greater total dry matter yield were located on SBI-01 of R9403463-2-1, and SBI-06 of R9188. QTL alleles for increased grain yield from both R9403463-2-1 and R9188 were found on SBI-03. As an increase in stem sugars is an important objective in sweet sorghum breeding, the QTL identified in this study could be further investigated for use in marker-assisted selection of sweet sorghum.

Gene interactions constrain the course of evolution of phosphine resistance in the lesser grain borer, Rhyzopertha dominica

Phosphine, a widely used fumigant for the protection of stored grain from insect pests, kills organisms indirectly by inducing oxidative stress. High levels of heritable resistance to phosphine in the insect pest of stored grain, Rhyzopertha dominica have been detected in Asia, Australia and South America. In order to understand the evolution of phosphine resistance and to isolate the responsible genes, we have undertaken genetic linkage analysis of fully sensitive (QRD14), moderately resistant (QRD369) and highly resistant (QRD569) strains of R. dominica collected in Australia. We previously determined that two loci, rph1 and rph2, confer high-level resistance on strain QRD569, which was collected in 1997. We have now confirmed that rph1 is responsible for the moderate resistance of strain QRD369, which was collected in 1990, and is shared with a highly resistant strain from the same geographical region, QRD569. In contrast, rph2 by itself confers only very weak resistance, either as a heterozygote or as a homozygote and was not discovered in the field until weak resistance (probably due to rph1) had become ubiquitous. Thus, high-level resistance against phosphine has evolved via stepwise acquisition of resistance alleles, first at rph1 and thereafter at rph2. The semi-dominance of rph2 together with the synergistic interaction between rph1 and rph2 would have led to rapid selection for homozygosity. A lack of visible fitness cost associated with alleles at either locus suggests that the resistance phenotype will persist in the field.

Congruence in QTL for adventitious rooting in Pinus elliottii x Pinus caribaea hybrids resolves between and within-species effects

Targeting between-species effects for improvement in synthetic hybrid populations derived from outcrossing parental tree species may be one way to increase the efficacy and predictability of hybrid breeding. We present a comparative analysis of the quantitative trait loci (QTL) which resolved between from within-species effects for adventitious rooting in two populations of hybrids between Pinus elliottii and P. caribaea, an outbred F1 (n=287) and an inbred-like F2 family (n=357). Most small to moderate effect QTL (each explaining 2-5% of phenotypic variation, PV) were congruent (3 out of 4 QTL in each family) and therefore considered within-species effects as they segregated in both families. A single large effect QTL (40% PV) was detected uniquely in the F2 family and assumed to be due to a between-species effect, resulting from a genetic locus with contrasting alleles in each parental species. Oligogenic as opposed to polygenic architecture was supported in both families (60% and 20% PV explained by 4 QTL in the F 2 and F1 respectively). The importance of adventitious rooting for adaptation to survive water-logged environments was thought in part to explain oligogenic architecture of what is believed to be a complex trait controlled by many hundreds of genes.

Genetic variation within two sympatric spotted gum eucalypts exceeds between taxa variation

Population substructure and hybridization, among other factors, have the potential to cause erroneous associations in linkage disequilibrium (LD) mapping. Two closely related spotted gum eucalypts, Corymbia variegata and C. henryi (Myrtaceae) occur in sympatry in the east coast of Australia and potentially interbreed. They are morphologically similar but are distinguished as separate species based on capsule and foliage size. To determine whether they hybridize in nature and its implications for LD mapping, we investigated the level of molecular divergence between the two species at two sympatric locations separated by 300 kilometres. Very few individuals of intermediate morphology were identified, despite the two species occurring only metres apart. Analysis of genetic structure using 12 microsatellite loci showed that genetic differentiation between populations of the same species at different locations (FST = 0.07 for both species; p = 0.0001) was significantly higher than that observed between species at each location (mean FST = 0.02 and 0.04 for Cherry tree and Bunyaville respectively; p = 0.0001; all Mann-Whitney U-test p ≤ 0.01). No species-specific alleles or significant allele frequency differences were detected within a site, suggesting recurrent local gene flow between the two species. The lack of significant allele frequency differences implies no population stratification along taxonomic lines. This suggested that there is little concern for cryptic hybridization when sampling from sites of sympatry for LD mapping.

Effect of the puroindoline locus and environment on Chinese fresh noodle texture

Grain produced from doubled-haploid (DH) wheat lines, developed from a hard- and a soft-grained wheat cultivar, were bulked according to Pinb (puroindoline b) genotypes for an assessment of Chinese fresh noodle texture by a trained taste panel. Each DH line was designated as 'soft' or 'hard' grained, based on a PCR amplification of the wildtype, soft allele, or the mutant, hard allele. Theoretically, the soft and hard grain bulks represented respective Pinb alleles and an independent assortment of unlinked alleles from the parents, Sunco and Chuanyu 12. Grains from the parents and DH lines were grown at 2 locations in Queensland, Australia, and one in Sichuan, China. The grains were milled and processed for a taste panel evaluation in Chengdu, Sichuan. Results suggest the Pinb alleles had a significant effect on noodle softness and explained 30% of the variation; the 'soft' Pinb allele conferred a softer noodle texture. Location had a significant effect on noodle smoothness; wheat grain grown at Biloela, Queensland, produced a smoother noodle texture than grain grown in Sichuan. The effect of location confirms the importance of environment as a variable for this quality character. This investigation exemplifies the utility of Pinb markers for specifically altering Chinese Fresh Noodle texture.

Sorghum stay-green QTL individually reduce post-flowering drought-induced leaf senescence

Sorghum is an important source of food, feed, and biofuel, especially in the semi-arid tropics because this cereal is well adapted to harsh, drought-prone environments. Post-flowering drought adaptation in sorghum is associated with the stay-green phenotype. Alleles that contribute to this complex trait have been mapped to four major QTL, Stg1-Stg4, using a population derived from BTx642 and RTx7000. Near-isogenic RTx7000 lines containing BTx642 DNA spanning one or more of the four stay-green QTL were constructed. The size and location of BTx642 DNA regions in each RTx7000 NIL were analysed using 62 DNA markers spanning the four stay-green QTL. RTx7000 NILs were identified that contained BTx642 DNA completely or partially spanning Stg1, Stg2, Stg3, or Stg4. NILs were also identified that contained sub-portions of each QTL and various combinations of the four major stay-green QTL. Physiological analysis of four RTx7000 NILs containing only Stg1, Stg2, Stg3, or Stg4 showed that BTx642 alleles in each of these loci could contribute to the stay-green phenotype. RTx7000 NILs containing BTx642 DNA corresponding to Stg2 retained more green leaf area at maturity under terminal drought conditions than RTx7000 or the other RTx7000 NILs. Under post-anthesis water deficit, a trend for delayed onset of leaf senescence compared with RTx7000 was also exhibited by the Stg2, Stg3, and Stg4 NILs, while significantly lower rates of leaf senescence in relation to RTx7000 were displayed by all of the Stg NILs to varying degrees, but particularly by the Stg2 NIL. Greener leaves at anthesis relative to RTx7000, indicated by higher SPAD values, were exhibited by the Stg1 and Stg4 NILs. The RTx7000 NILs created in this study provide the starting point for in-depth analysis of stay-green physiology, interaction among stay-green QTL and map-based cloning of the genes that underlie this trait.

A marker-assisted backcross approach for developing submergence-tolerant rice cultivars

Submergence stress regularly affects 15 million hectares or more of rainfed lowland rice areas in South and Southeast Asia. A major QTL on chromosome 9, Sub1, has provided the opportunity to apply marker assisted backcrossing (MAB) to develop submergence tolerant versions of rice cultivars that are widely grown in the region. In the present study, molecular markers that were tightly linked with Sub1, flanking Sub1, and unlinked to Sub1 were used to apply foreground, recombinant, and background selection, respectively, in backcrosses between a submergence-tolerant donor and the widely grown recurrent parent Swarna. By the BC2F2 generation a submergence tolerant plant was identified that possessed Swarna type simple sequence repeat (SSR) alleles on all fragments analyzed except the tip segment of rice chromosome 9 that possessed the Sub1 locus. A BC3F2 double recombinant plant was identified that was homozygous for all Swarna type alleles except for an approximately 2.3-3.4 Mb region surrounding the Sub1 locus. The results showed that the mega variety Swarna could be efficiently converted to a submergence tolerant variety in three backcross generations, involving a time of two to three years. Polymorphic markers for foreground and recombinant selection were identified for four other mega varieties to develop a wider range of submergence tolerant varieties to meet the needs of farmers in the flood-prone regions. This approach demonstrates the effective use of marker assisted selection for a major QTL in a molecular breeding program.