Comparative Transcriptome Analysis Reveals the Cause for Accumulation of Reactive Oxygen Species During Pollen Abortion in Cytoplasmic Male-Sterile Kenaf Line 722HA

Cytoplasmic male sterility (CMS) is a maternally inherited trait used for hybrid production in plants, a novel kenaf CMS line 722HA was derived from the thermo-sensitive male-sterile mutant ‘HMS’ by recurrent backcrossing with 722HB. The line 722HA has great potential for hybrid breeding in kenaf. However, the underlying molecular mechanism that controls pollen abortion in 722HA remains unclear, thus limiting the full utilization of this line. To understand the possible mechanism governing pollen abortion in 722HA, cytological, transcriptomic, and biochemical analyses were carried out to compare the CMS line 722HA and its maintainer line 722HB. Cytological observations of the microspore development revealed premature degradation of the tapetum at the mononuclear stage, which resulted in pollen dysfunction. The k-means clustering analysis of differentially expressed genes (DEGs) revealed that these genes are related to processes associated with the accumulation of reactive oxygen species (ROS), including electron transport chain, F1F0-ATPase proton transport, positive regulation of superoxide dismutase (SOD), hydrogen peroxide catabolic, and oxidation-reduction. Biochemical analysis indicated that ROS-scavenging capability was lower in 722HA than in 722HB, resulting in an accumulation of excess ROS, which is consistent with the transcriptome results. Taken together, these results demonstrate that excessive ROS accumulation may affect the normal development of microspores. Our study provides new insight into the molecular mechanism of pollen abortion in 722HA and will promote further studies of kenaf hybrids.

Segregation Distortion Observed in the Progeny of Crosses Between Oryza sativa and O. meridionalis Caused by Abortion During Seed Development

Wild rice relatives having the same AA genome as domesticated rice (Oryza sativa) comprise the primary gene pool for rice genetic improvement. Among them, O. meridionalis and O. rufipogon are found in the northern part of Australia. Three Australian wild rice strains, Jpn1 (O. rufipogon), Jpn2, and W1297 (O. meridionalis), and one cultivated rice cultivar Taichung 65 (T65) were used in this study. A recurrent backcrossing strategy was adopted to produce chromosomal segment substitution lines (CSSLs) carrying chromosomal segments from wild relatives and used for trait evaluation and genetic analysis. The segregation of the DNA marker RM136 locus on chromosome 6 was found to be highly distorted, and a recessive lethal gene causing abortion at the seed developmental stage was shown to be located between two DNA markers, KGC6_10.09 and KGC6_22.19 on chromosome 6 of W1297. We name this gene as SEED DEVELOPMENT 1 (gene symbol: SDV1). O. sativa is thought to share the functional dominant allele Sdv1-s (s for sativa), and O. meridionalis is thought to share the recessive abortive allele sdv1-m (m for meridionalis). Though carrying the sdv1-m allele, the O. meridionalis accessions can self-fertilize and bear seeds. We speculate that the SDV1 gene may have been duplicated before the divergence between O. meridionalis and the other AA genome Oryza species, and that O. meridionalis has lost the function of the SDV1 gene and has kept the function of another putative gene named SDV2.

The Ficus erecta genome to identify the Ceratocystis canker resistance gene for breeding programs in common fig (F. carica)

Ficus erecta, a wild relative of common fig (F. carica), is a donor of Ceratocystis canker resistance in fig breeding programs. Interspecific hybridization followed by recurrent backcrossing is an effective method to transfer the resistance trait from wild to cultivated fig; however, this is time consuming and labor-intensive for trees, especially for gynodioecious plants such as fig. In this study, genome resources were developed for F. erecta to facilitate fig breeding programs. The genome sequence of F. erecta was determined using single-molecule real-time sequencing technology. The resultant assembly spanned 331.6 Mb with 538 contigs and an N50 length of 1.9 Mb, from which 51,806 high-confidence genes were predicted. Pseudomolecule sequences corresponding to the chromosomes of F. erecta were established with a genetic map based on single nucleotide polymorphisms from double-digest restriction-site associated DNA sequencing. Subsequent linkage analysis and whole genome resequencing identified a candidate gene for the Ceratocystis canker resistance trait. Genome-wide genotyping analysis enabled selection of female lines that possessed resistance and effective elimination of donor genome from progeny. The genome resources provided in this study will accelerate and enhance disease resistance breeding programs in fig.

The complex genetic and molecular basis of a model quantitative trait

Quantitative traits are often influenced by many loci with small effects. Identifying most of these loci and resolving them to specific genes or genetic variants is challenging. Yet, achieving such a detailed understanding of quantitative traits is important, as it can improve our knowledge of the genetic and molecular basis of heritable phenotypic variation. In this study, we use a genetic mapping strategy that involves recurrent backcrossing with phenotypic selection to obtain new insights into an ecologically, industrially, and medically relevant quantitative trait—tolerance of oxidative stress, as measured based on resistance to hydrogen peroxide. We examine the genetic basis of hydrogen peroxide resistance in three related yeast crosses and detect 64 distinct genomic loci that likely influence the trait. By precisely resolving or cloning a number of these loci, we demonstrate that a broad spectrum of cellular processes contribute to hydrogen peroxide resistance, including DNA repair, scavenging of reactive oxygen species, stress-induced MAPK signaling, translation, and water transport. Consistent with the complex genetic and molecular basis of hydrogen peroxide resistance, we show two examples where multiple distinct causal genetic variants underlie what appears to be a single locus. Our results improve understanding of the genetic and molecular basis of a highly complex, model quantitative trait.

Development and molecular characterization of wheat – Aegilops kotschyi addition and substitution lines with high grain protein, iron, and zinc

Over two billion people, depending largely on staple foods, suffer from deficiencies in protein and some micronutrients such as iron and zinc. Among various approaches to overcome protein and micronutrient deficiencies, biofortification through a combination of conventional and molecular breeding methods is the most feasible, cheapest, and sustainable approach. An interspecific cross was made between the wheat cultivar ‘Chinese Spring’ and Aegilops kotschyi Boiss. accession 396, which has a threefold higher grain iron and zinc concentrations and about 33% higher protein concentration than wheat cultivars. Recurrent backcrossing and selection for the micronutrient content was performed at each generation. Thirteen derivatives with high grain iron and zinc concentrations and contents, ash and ash micronutrients, and protein were analyzed for alien introgression. Morphological markers, high molecular weight glutenin subunit profiles, anchored wheat microsatellite markers, and GISH showed that addition and substitution of homoeologous groups 1, 2, and 7 chromosomes of Ae. kotschyi possess gene(s) for high grain micronutrients. The addition of 1U/1S had high molecular weight glutenin subunits with higher molecular weight than those of wheat, and the addition of 2S in most of the derivatives also enhanced grain protein content by over 20%. Low grain protein content in a derivative with a 2S-wheat translocation, waxy leaves, and absence of the gdm148 marker strongly suggests that the gene for higher grain protein content on chromosome 2S is orthologous to the grain protein QTL on the short arm of group 2 chromosomes.

Backcrossing Provides an Avenue for Gene Introgression from Wheat to Jointed Goatgrass (Aegilops cylindrica) in the U.S. Great Plains

Jointed goatgrass is an exotic species introduced into the western United States from Eurasia. The weed is an agricultural pest infesting winter wheat fields and causing economic loss. Common ancestry between the two species enables interspecific hybridization, thus providing a mechanism for gene flow to occur. This can facilitate the accumulation of novel genes, which could increase the wild species' competitiveness with wheat and its ability to invade novel habitats. Interest in the development of transgenic wheat cultivars has increased the concern for interspecific gene flow. Gene introgression requires recurrent backcrossing to the weedy species after the initial hybridization event. Field experiments were conducted at two locations in Colorado in 2007–2008 and 2008–2009, with jointed goatgrass acting as the sole source of viable pollen for fertilization of transplanted hybrid plants. Backcrossing rates were determined by conducting germination studies on spikes collected from a total of 206 hybrid plants. Pollination by jointed goatgrass led to the production of 463 BC1plants from seed produced on these 206 hybrid plants. Ninety-five percent confidence intervals estimate the rate of backcrossing at 0.028 to 0.306% and 0.077 to 0.604%, with medians of 0.062 and 0.152%, respectively, at the two locations. The results demonstrate that backcrossing to jointed goatgrass can occur, despite low rates of hybrid fertility. Subsequent backcrossing would make it likely that a wheat gene conferring a selective advantage will introgress into the weedy population. For the U.S. Great Plains, it is possible that transgenic wheat cultivars will be released in the future and determining proper management of these cultivars is necessary to minimize hybridization and advantageous gene introgression into weedy relatives.

Peanut resistance to Sclerotinia minor and S. sclerotiorum

The fungi Sclerotinia minor and S. sclerotiorum are the causal agents of two similar diseases of peanut (Arachis hypogaea L.). Both diseases cause significant losses in the Australian peanut industry. Development of cultivars with resistance to Sclerotinia will be an important component of integrated control. The aims of this project are to generate information that will assist in breeding for Sclerotinia resistance in peanut: to identify Sclerotinia-resistant peanut germplasm, to understand the inheritance and estimate heritability of resistance, and to test the effectiveness of identified sources of resistance against both S. minor and S. sclerotiorum. This study has clearly established that material that shows resistance to S. minor in the USA is resistant to S.�minor and likely to be resistant to S. sclerotiorum in Australia. The high level of resistance to both S. minor and S.�sclerotiorum in germplasm from Texas, particularly TxAG-4, was confirmed. VA 93B showed good resistance in the field, which is primarily due to the open bush type rather than physiological resistance. Physiological resistance to S. minor was also identified in a cultivar and a landrace from Indonesia and a rust-resistant line from Queensland. All germplasm found to have high physiological resistance to S. minor belonged to the Spanish type. Inheritance of physiological resistance to S. minor was studied using a Generation Means Analysis (GMA) of the cross TxAG-4/VA 93B and its reciprocal. The broad-sense heritability of physiological resistance on a single plant basis was estimated at 47%, much higher than earlier estimates obtained in field studies. The average gene action of Sclerotinia resistance genes from TxAG-4 was found to be additive. No dominance effects were detected in the GMA. A small but significant reciprocal effect between TxAG-4 and VA 93B indicated that VA 93B passed on some physiological resistance maternally. An experiment was conducted to confirm the value of resistance against both S. minor and S. sclerotiorum. TxAG-4 was found to have physiological resistance to both S. minor and S. sclerotiorum. This resistance was expressed against both Sclerotinia species by progeny that were selected for resistance to S. minor. On the basis of the information obtained, the comparative advantages of 3 strategies for Sclerotinia-resistant cultivar development are discussed: (1) introduction of germplasm; (2) recurrent backcrossing with screening and crossing in the BCnF1 generation; and (3) pedigree selection. At present, introduction and backcrossing are recommended as the preferred strategies.

Production of Near-Isogenic Lines and Marked Monosomic Lines in Common Wheat (Triticum aestivum) cv. Chinese Spring

Sixteen near-isogenic lines (NILs) carrying a marker gene were produced by the recurrent backcrossing method in the genetic background of common wheat (Triticum aestivum) cv. Chinese Spring (CS). Three genes from alien species showed segregation distortion. In NILs carrying a marker gene of rye (Secale cereale) or Aegilops caudata, the alien chromosome segments were detected by fluorescence in situ hybridization (FISH). The NILs were grown with replications and the effect of marker genes on plant morphology in the genetic background of CS was investigated. These NILs were further crossed with the corresponding monosomics of CS and 13 monosomic lines whose monosome carries a respective marker gene were established and named “marked monosomics.” Many of the marked monosomics were distinguishable from the disomic NILs because of the different dosage effect of the genes. The NILs are utilized for studies on gene isolation or gene regulation. Marked monosomics are useful not only for monosomic analysis but also for production of homologous chromosome substitution lines because chromosome observation is not required.

The Length of the Intact Donor Chromosome Segment Around a Target Gene in Marker-Assisted Backcrossing

Recurrent backcrossing is an established procedure to transfer target genes from a donor into the genetic background of a recipient genotype. By assessing the parental origin of alleles at markers flanking the target locus one can select individuals with a short intact donor chromosome segment around the target gene and thus reduce the linkage drag. We investigated the probability distribution of the length of the intact donor chromosome segment around the target gene in recurrent backcrossing with selection for heterozygosity at the target locus and homozygosity for the recurrent parent allele at flanking markers for a diploid species. Assuming no interference in crossover formation, we derived the cumulative density function, probability density function, expected value, and variance of the length of the intact chromosome segment for the following cases: (1) backcross generations prior to detection of a recombinant individual between the target gene and the flanking marker; (2) the backcross generation in which for the first time a recombinant individual is detected, which is selected for further backcrossing; and (3) subsequent backcross generations after selection of a recombinant. Examples are given of how these results can be applied to investigate the efficiency of marker-assisted backcrossing for reducing the length of the intact donor chromosome segment around the target gene under various situations relevant in breeding and genetic research.