Acid soil is a worldwide problem to plant production. Acid toxicity is mainly caused by a lack of essential nutrients in the soil and excessive toxic metals in the plant root zone. Of the toxic metals, aluminum (Al) is the most prevalent and most toxic. Plant species have evolved to variable levels of tolerance to aluminum enabling breeding of high Al-tolerant cultivars. Physiological and molecular approaches have revealed some mechanisms of Al toxicity in higher plants. Mechanisms of plant tolerance to Al stress include: 1) exclusion of Al from the root tips, and 2) absorbance, but tolerance of Al in root cells. Organic acid exudation to chelate Al is a feature shared by many higher plants. The future challenge for Al tolerance studies is the identification of novel tolerance mechanisms and the combination of different mechanisms to achieve higher tolerance. Molecular approaches have led to significant progress in explaining mechanisms and detection of genes responsible for Al tolerance. Gene-specific molecular markers offer better options for marker-assisted selection in breeding programs than linked marker strategies. This paper mainly focuses on recent progress in the use of molecular approaches in Al tolerance research.

Maize-specific pyruvate orthophosphate dikinase (PPDK) was overexpressed in rice independently or in combination with the maize C₄-specific phosphoenolpyruvate carboxylase (PCK). The wild-type (WT) cultivar Kitaake and transgenic plants were evaluated in independent field and tank experiments. Three soil moisture treatments, well-watered (WW), moderate drought (MD) and severe drought (SD), were imposed from 9 d post-anthesis till maturity. Leaf physiological and biochemical traits, root activities, biomass, grain yield, and yield components in the untransformed WT and two transgenic rice lines (PPDK and PCK) were systematically studied. Compared with the WT, both transgenic rice lines showed increased leaf photosynthetic rate: by 20%-40% under WW, by 45%-60% under MD, and by 80%-120% under SD. The transgenic plants produced 16.1%, 20.2% and 20.0% higher grain yields than WT under the WW, MD and SD treatments, respectively. Under the same soil moisture treatments, activities of phosphoenolpyruvate carboxylase (PEPC) and carbonic anhydrase (CA) in transgenic plants were 3-5-fold higher than those in WT plants. Compared with ribulose-1,5-bisphosphate carboxylase, activities of PEPC and CA were less reduced under both MD and SD treatments. The transgenic plants also showed higher leaf water content, stomatal conductance, transpiration efficiency, and root oxidation activity and a stronger active oxygen scavenging system than the WT under all soil moisture treatments, especially MD and SD. The results suggest that drought tolerance is greatly enhanced in transgenic rice plants overexpressing C₄ photosynthesis enzymes. This study was performed under natural conditions and normal planting density to evaluate yield advantages on a field basis. It may open a new avenue to drought-tolerance breeding via overexpression of C₄ enzymes in rice.

Given the important roles of miRNAs in post-transcriptional gene regulation, identification of differentially expressed miRNAs will facilitate the elucidation of molecular mechanisms underlying kernel development. In this study, we constructed a small RNA library to comprehensively represent the full complement of individual small RNAs and to characterize miRNA expression profiles in pooled ears of maize (Zea mays L.) at 10, 15, 20, 22, 25 and 30 days after pollination (DAP). At least 21 miRNAs were differentially expressed. The differential expression of three of these miRNAs, i.e., miR528a, miR167a and miR160b, at each stage was verified by qRT-PCR. The results indicated that these miRNAs might be involved in kernel development. In addition, the predicted functions of target genes indicated that most of the target genes are involved in signal transduction and cell communication pathways, particularly the auxin signaling pathway. The expression of candidate germination-associated miRNAs was analyzed by hybridization to a maize genome microarray, and revealed differential expression of genes involved in plant hormone signaling pathways. This finding suggests that phytohormones play a critical role in the development of maize kernels. We found that in combination with other miRNAs, miR528a regulated a putative laccase, a Ring-H2 zinc finger protein and a MADS box-like protein, whereas miR167a and miR160b regulated multiple target genes, including ARF (auxin response factor), a member of the B3 transcription factor family. All three miRNAs are important for ear germination, development and physiology. The small RNA transcriptomes and mRNA obtained in this study will help us gain a better understanding of the expression and function of small RNAs in the development of maize kernel.

Genomic DNA and cDNA sequences of an isoamylase gene were isolated and characterized from the rye genome. The full-lengths of the rye isoamylase gene are 7351 bp for genomic DNA and 2364 bp for cDNA. There are 18 exons and 17 introns in the genomic sequence, which shares a similar organization with homologous genes from Aegilops tauschii, maize, rice and Arabidopsis. Exon regions of rye and other plant isoamylase genes are more conserved than the introns. High sequence similarity (> 95%) was observed in mature proteins of isoamylase genes originating from rye, Ae. tauschii, wheat and barley. The transcript profile revealed that rye isoamylase is mainly expressed in the seed endosperm with a maximum level at the middle developmental stage (15 DPA). A phylogenetic tree based on the deduced aa sequences of mature proteins from rye and other plant isoamylases indicated that rye isoamylase is more closely related to Ae. tauschii wDBE1 and wheat iso1. This is the first report on identification and characterization of the isoamylase gene from rye, making it possible to explore the roles of this enzyme for amylopectin development in rye and triticale.

The backcross (BC) breeding strategy has been increasingly used for developing high yielding varieties with improved abiotic stress tolerances in rice. In this study, 189 Huang-Hua-Zhan (HHZ) introgression lines (ILs) developed from three different selection schemes were evaluated for yield related traits under drought stress and non-stress conditions in the target and off-season winter nursery environments to assess the selection efficiency of BC breeding for improving different complex traits, and led us to five important results. The first result indicated that the primary target traits should be selected first in the target environments (TEs) in order to achieve the maximum genetic gain. Secondly, BC breeding for drought tolerance (DT) in rice was almost equally effective by strong phenotypic selection in the main target environments and in the winter-season of Hainan. Thirdly, exploiting genetic diversity in the subspecific gene pools is of great importance for future genetic improvement of complex traits in rice. Fourthly, considerable genetic gain can be effectively achieved by selection for secondary target traits among the ILs with the primary traits. Finally, the developed ILs provide useful materials for future genetic/genomic dissection and molecular breeding of complex traits.

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most destructive diseases of rice (Oryza sativa L.) worldwide. The type III secretion system (T3SS) of Xoo, encoded by the hrp (hypersensitive response and pathogenicity) genes, plays critical roles in conferring pathogenicity in host rice and triggering a hypersensitive response (HR) in non-host plants. To investigate the major genes conferring the pathogenicity and avirulence of Xoo, we previously constructed a random Tn5-insertion mutant library of Xoo strain PXO99^A. We report here the isolation and characterization of a Tn5-insertion mutant PXM69. Tn5-insertion mutants were screened on indica rice JG30, which is highly susceptible to PXO99^A, by leaf-cutting inoculation. Four mutants with reduced virulence were obtained after two rounds of screening. Among them, the mutant PXM69 had completely lost virulence to the rice host and ability to elicit HR in non-host tobacco. Southern blotting analysis showed a single copy of a Tn5-insertion in the genome of PXM69. PCR walking and sequencing analysis revealed that the Tn5 transposon was inserted at nucleotide position 70,192-70,201 in the genome of PXO99^A, disrupting the type III hrc (hrp-conserved) gene hrcQ, the first gene in the D operon of the hrp cluster in Xoo. To confirm the relationship between the Tn5-insertion and the avirulence phenotype of PXM69, we used the marker exchange mutagenesis to create a PXO99^A mutant, ΔhrcQ::KAN, in which the hrcQ was disrupted by a kanamycin-encoding gene cassette at the same site as that of the Tn5-insertion. ΔhrcQ::KAN showed the same phenotype as mutant PXM69. Reintroduction of the wild-type hrcQ gene partially complemented the pathogenic function of PXM69. RT-PCR and cellulase secretion assays showed that the Tn5-disruption of hrcQ did not affect transcription of downstream genes in the D operon and function of the type II secretion system. Our results provide new insights into the pathogenic functions of clustered hrp genes in Xoo.

Using newly developed methods and software, association mapping was conducted for chromium content and total sugar in tobacco leaf, based on four -omics datasets. Our objective was to collect data on genotype and phenotype for 60 leaf samples at four developmental stages, from three plant architectural positions and for three cultivars that were grown in two locations. Association mapping was conducted to detect genetic variants at quantitative trait SNP (QTS) loci, quantitative trait transcript (QTT) differences, quantitative trait protein (QTP) variability, and quantitative trait metabolite (QTM) changes, which can be summarized as QTX locus variation. The total heritabilities of the four -omics loci for both traits tested were 23.60% for epistasis and 15.26% for treatment interaction. Epistasis and environment × treatment interaction had important impacts on complex traits at all -omics levels. For decreasing chromium content and increasing total sugar in tobacco leaf, six methylated loci can be directly used for marker-assisted selection, and expression of ten QTTs, seven QTPs and six QTMs can be modified by selection or cultivation.

Grain shape as a major determinant of rice yield and quality is widely believed to be controlled by quantitative trait loci (QTL). We have identified a novel gene “GS2” to largely regulate grain length and width in rice. The GS2 allele in the big-grain rice line ‘CDL’ functioned in a dominant manner. In the present study, we employed a chromosome walking strategy in the residual heterozygous lines from recombinant inbred population between cultivar “R1126” and CDL, and located the GS2 gene in an interval of ~ 33.2 kb flanked by marker GL2-35-1 and GL2-12 in the long arm of rice chromosome 2. According to genome annotations, three putative gene loci, LOC_Os02g47280, LOC_Os02g47290 and LOC_Os02g47300, exist in this candidate region. In addition, allelic analysis with previously reported genes demonstrated that GS2 was novel for regulating rice grain shape. These results will help promote the cloning and functional characterization of the GS2 gene and further develop linked markers to be used in marker-assisted breeding.

Starch is a crucial component determining the processing quality of wheat (Triticum aestivum L.)-based products. Wheat starch generally contains A-type and B-type starch granules, having different effects on starch properties and end-use qualities. In the present study, 240 recombinant inbred lines (RILs) derived from a PH82-2/Neixiang 188 cross were grown in Anyang, Henan, China, during three cropping seasons. A-type and B-type granule contents were determined using a laser diffraction particle size analyzer, defined as the percentage of total starch volume. A total of 195 SSR and STS markers were used to construct a genetic map. QTL analysis was performed by composite interval mapping. Three QTL for A-type starch granule content were mapped on chromosomes 1DL, 7BL and 4AL, explaining 5.6%, 5.2% and 3.8% of the phenotypic variation, respectively. These results provide useful information for improving starch quality in common wheat.