Molecular Breeding

bZIP genes play important roles in the regulation of many developmental and physiological processes and adaptive responses to the environment in plants. In the present study, a novel bZIP gene, designated as BnbZIP3 (Genbank accession number: KP462877), was cloned from ramie plants based on the sequence of Unigene6582 fragment in ramie transcriptome using rapid amplification of cDNA ends (RACE) technique and real-time PCR. Results indicated that BnbZIP3 had a 999-bp open reading frame, encoding a 332 amino acid protein that containing a characteristic bZIP domain, and shared high sequence identities with bZIP factors from other plants. Translational fusion of BnbZIP3 with EGFP showed subcellular localization of the protein in nucleus. Transcripts of BnbZIP3 were found in different tissues of ramie plants. Expression of BnbZIP3 was induced by drought, high salinity and ABA treatments. The promoter of BnbZIP3 contained many cis-acting elements involved in multiple stress responses. Overexpression of BnbZIP3 in transgenic Arabidopsis plants inhibited the growth of roots under normal growth conditions, whereas it improved the growth of roots under dehydration, salinity and heavy metal Cd stress, and increased germination of seeds under drought conditions. Thus, BnbZIP3 may positively regulate stress tolerance and be used to engineer enhanced drought, salt and heavy metal tolerance of ramie varieties or other industrial crops.

The brown planthopper (BPH) is one of the most destructive insect pests of rice in Thailand. We performed a cluster analysis that revealed the existence of four groups corresponding to the variation of virulence against BPH resistance genes in 45 BPH populations collected in Thailand. Rice cultivars Rathu Heenati and PTB33, which carry Bph3, showed a broad-spectrum resistance against all BPH populations used in this study. The resistant gene Bph3 has been extensively studied and used in rice breeding programs against BPH; however, the chromosomal location of Bph3 in the rice genome has not yet been determined. In this study, a simple sequence repeat (SSR) analysis was performed to identify and localize the Bph3 gene derived from cvs. Rathu Heenati and PTB33. For mapping of the Bph3 locus, we developed two backcross populations, BC1F2 and BC3F2, from crosses of PTB33 × RD6 and Rathu Heenati × KDML105, respectively, and evaluated these for BPH resistance. Thirty-six polymorphic SSR markers on chromosomes 4, 6 and 10 were used to survey 15 resistant (R) and 15 susceptible (S) individuals from the backcross populations. One SSR marker, RM190, on chromosome 6 was associated with resistance and susceptibility in both backcross populations. Additional SSR markers surrounding the RM190 locus were also examined to define the location of Bph3. Based on the linkage analysis of 208 BC1F2 and 333 BC3F2 individuals, we were able to map the Bph3 locus between two flanking SSR markers, RM589 and RM588, on the short arm of chromosome 6 within 0.9 and 1.4 cM, respectively. This study confirms both the location of Bph3 and the allelic relationship between Bph3 and bph4 on chromosome 6 that have been previously reported. The tightly linked SSR markers will facilitate marker-assisted gene pyramiding and provide the basis for map-based cloning of the resistant gene.

Plants respond to survive under water deficit conditions via turning on or off a series of genes that further alter plant physiology and morphology, which allows a plant to tolerate, escape or avoid drought stress. These gene products are either functional (such as aquaporins and the enzymes of osmoprotectant biosynthesis) or regulatory (such as protein kinases). In order to investigate the role of maize protein kinases (PKs) in drought signal transduction pathways, genome-wide profiling and analyses of three major families of protein kinases, RLKs, CDPK-SnRKs and MAPKs, were carried out. Our selection pressure analysis found evidence for positive selection in 24 % of the ZmRLK genes, but sites under selection were predominantly located in the extracellular region. Phylogenetic analysis of CDPK-SnRKs revealed considerable conservation in protein sequences across five types—CDPKs, CRKs, PPCKs, PEPRKs and SnRKs. 19 MAPKs, 18 MAPKKs, 84 MAP3Ks and 9 MAP4Ks were identified, and the potential MAPK signaling cascades were proposed. Some PK genes based on transcriptome analysis including microarray, RNA-sequencing and real-time quantitative PCR (qRT-PCR) showed drought-induced expression. Protein structure, subcellular location and gene expression of putative ABA receptors were analyzed to provide novel molecular insights into maize drought signaling. The drought stress signal transduction pathway was first evaluated and characterized in this study. To understand microRNA (miRNA) involvement in maize drought stress response and resistance, signaling pathway-associated miRNAs and potential miRNA-target pairs were predicted. Furthermore, real-time quantification of miRNAs by stem-loop qRT-PCR demonstrated that nine miRNAs might function as positive or negative regulators in drought stress signal transduction, and these miRNAs can be used as biomarker and regulatory target in genetic analysis and improvement of drought resistance in maize. These efforts can broaden the understanding of the physiological and molecular mechanisms of resistance to drought in maize and facilitate the creation of cultivars with increased drought tolerance.

Most species of Stylosanthes (stylo) genus are important tropical pasture legumes with very versatile, widely adapted, and productive characteristics. These legumes are commercially used in various agricultural systems in many tropical and subtropical regions. However, the few molecular markers for the stylo species limit their genetic improvement. In this study, 36,558 expressed sequence tags (ESTs) have been de novo assembled using Illumina paired-end sequencing in Stylosanthes guianensis (Aubl.) Sw. to develop simple sequence repeat (SSR) markers. We searched these ESTs for SSRs and identified 4115 SSR loci from 3643 ESTs (9.96 %). Dinucleotide and trinucleotide repeat motifs were the most abundant types (30.50 and 50.33 %, respectively), whereas tetranucleotide, pentanucleotide, and hexanucleotide motifs represented <10 % of all SSRs. The motif AG/CT was the most abundant, accounting for 21.7 % of all SSRs. Moreover, 2008 SSR markers were developed using 1873 SSR-containing unigenes. A total of 115 EST-SSR markers located in the coding region were amplified using polymerase chain reaction to detect 29 S. guianensis accessions. Of these 115 markers, 96 produced reliable bands with expected sizes, and 81 markers were polymorphic, with 2–6 alleles among the 29 accessions. Analysis of the genetic diversity of all 29 accessions revealed similarity coefficients that ranged from 0.528 to 0.983. The EST-SSR markers developed in this study represent the first large-scale development of SSR markers for S. guianensis. These SSR markers will provide a valuable resource for genetic diversity studies, cultivar fingerprinting, construction of genetic maps, identification of quantitative trait loci for important traits, and molecular marker-assisted selection breeding in stylo species.

The basic leucine zippers (bZIPs) are one of the largest families of transcription factors that have been demonstrated to play diverse roles in plant growth development. These proteins are known to regulate many cellular responses in plants under stress conditions. In this study, a stress-responsive transcription factor belonging to bZIP family was cloned from finger millet (Eleusine coracana) and functionally validated in tobacco. The expression of EcbZIP60 was highly upregulated under drought, osmotic, salt and methyl viologen-induced stress in finger millet. Constitutive expression of EcbZIP60 in tobacco resulted in reduced growth of plants under normal growth conditions. However, the transgenic plants showed improved tolerance to drought stress with higher stomatal conductance and photosynthesis, resulting in improved growth. The transgenic plants also showed increased tolerance to salinity and methyl viologen-induced oxidative stress and to endoplasmic reticulum stress inducers dithiothreitol and tunicamycin. The transgenic plants showed significant upregulation of unfolded protein-responsive pathway genes such as BiP1, CRT1 and PDIL both under normal and stress conditions, although dehydrin group of genes did not show any upregulation. The results demonstrate that improved tolerance of EcbZIP60-expressing transgenic tobacco to diverse stresses could be through unfolded protein-responsive signalling pathway.

Colored wheat has piqued the interest of breeders and consumers alike. The chromosomal segment from 7E of Thinopyrum ponticum, which carries a leaf rust resistant gene, Lr19, has been rarely employed in wheat breeding operations due to its association with the Y gene, which gives a yellow tint to the flour. By prioritizing nutritional content over color preferences, consumer acceptance has undergone a paradigm change. Through marker-assisted backcross breeding, we introduced an alien segment harboring the Y (PsyE1) gene into a high yielding commercial bread wheat (HD 2967) background to generate rust resistant carotenoid biofortified bread wheat. Agro-morphological characterization was also performed on a subset of developed 70 lines having enhanced grain carotene content. In the introgression lines, carotenoid profiling using HPLC analysis demonstrated a considerable increase in β-carotene levels (up to 12 ppm). Thus, the developed germplasm caters the threat to nutritional security and can be utilized to produce carotenoid fortified wheat.

Large-scale identification of simple sequence repeat (SSR) markers has not been reported in the genus Fagopyrum due to a lack of genomic resources. High-throughput transcriptomic sequencing can provide a rich resource for development of genic-SSR markers. In our study, the immature seeds transcriptome of common buckwheat (Fagopyrum esculentum) was sequenced using the Illumina HiSeq™ 2000 platform. We obtained a total of 21 million paired-end reads after filtering. The reads were assembled into 54,947 transcripts with an average length of 1235 bp. In total, 46,126 (83.95%) transcripts showed significant hits to known proteins or domains. Among these, 43,647 (79.43%) transcripts was assigned at least one gene ontology (GO) term. A total of 12,677 (23.07%) transcripts were mapped to the 141 Kyoto Encyclopedia of Genes and Genome (KEGG) pathways. Overall, 2326 transcripts contained at least one SSR with a frequency of one SSR per 1.17 kb. Mono-nucleotide repeats were the most abundant type (45.48%) followed by tri-nucleotide repeats (35.17%). Of the 150 SSRs chosen randomly, 36 markers showed polymorphism among 24 common buckwheat accessions, and 141 genic-SSRs (94.0%) were transferable to tartary buckwheat. A low level of genetic diversity was found among 35 registered tartary buckwheat varieties in China assessed using 31 polymorphic SSR markers. Genic-SSR markers developed herein will be valuable for construction of linkage maps, genetic diversity studies, and marker-assisted selection in Fagopyrum species.

Tiller number greatly contributes to grain yield in wheat. Using ethylmethanesulfonate mutagenesis, we previously discovered the oligo-tillering mutant ot1. The tiller number was significantly lower in ot1 than in the corresponding wild type from the early tillering stage until the heading stage. Compared to the wild type, the thousand-grain weight and grain length were increased by 15.41% and 31.44%, respectively, whereas the plant height and spike length were decreased by 26.13% and 37.25%, respectively. Transcriptomic analysis was conducted at the regreening and jointing stages to identify differential expressed genes (DEGs). Functional enrichment analysis with the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases showed differential expression of genes associated with ADP binding, transmembrane transport, and transcriptional regulation during tiller development. Differences in tiller number in ot1 led to the upregulation of genes in the strigolactone (SL) and abscisic acid (ABA) pathways. Specifically, the SL biosynthesis genes DWARF (D27), D17, D10, and MORE AXILLARY GROWTH 1 (MAX1) were upregulated by 3.37- to 8.23-fold; the SL signal transduction genes D14 and D53 were upregulated by 1.81- and 1.32-fold, respectively; the ABA biosynthesis genes 9-CIS-EPOXICAROTENOID DIOXIGENASE 3 (NCED3) and NCED5 were upregulated by 1.66- and 3.4-fold, respectively; and SNF1-REGULATED PROTEIN KINASE2 (SnRK2) and PROTEIN PHOSPHATASE 2C (PP2C) genes were upregulated by 1.30- to 4.79-fold. This suggested that the tiller number reduction in ot1 was due to alterations in plant hormone pathways. Genes known to promote tillering growth were upregulated, whereas those known to inhibit tillering growth were downregulated. For example, PIN-FORMED 9 (PIN9), which promotes tiller development, was upregulated by 8.23-fold in ot1; Ideal Plant Architecture 1 (IPA1), which inhibits tiller development, was downregulated by 1.74-fold. There were no significant differences in the expression levels of TILLER NUMBER 1 (TN1) or TEOSINTE BRANCHED 1 (TB1), indicating that the tiller reduction in ot1 was not controlled by known genes. Our findings provide valuable data for subsequent research into the genetic bases and regulatory mechanisms of wheat tillering.

Rice (Oryza sativa L.) is a staple food crop globally. Brown planthopper (Nilaparvata lugens Stål, BPH) is the most destructive insect that threatens rice production annually. More than 40 BPH resistance genes have been identified so far, which provide valuable gene resources for marker-assisted breeding against BPH. However, it is still urgent to evaluate rice germplasms and to explore more new wide-spectrum BPH resistance genes to combat newly occurring virulent BPH populations. To this end, 560 germplasm accessions were collected from the International Rice Research Institute (IRRI), and their resistance to current BPH population of China was examined. A total of 105 highly resistant materials were identified. Molecular screening of BPH resistance genes in these rice germplasms was conducted by developing specific functional molecular markers of eight cloned resistance genes. Twenty-three resistant germplasms were found to contain none of the 8 cloned BPH resistance genes. These accessions also exhibited a variety of resistance mechanisms as indicated by an improved insect weight gain (WG) method, suggesting the existence of new resistance genes. One new BPH resistance gene, Bph44(t), was identified in rice accession IRGC 15344 and preliminarily mapped to a 0–2 Mb region on chromosome 4. This study systematically sorted out the corresponding relationships between BPH resistance genes and germplasm resources using a functional molecular marker system. Newly explored resistant germplasms will provide valualble donors for the identification of new resistance genes and BPH resistance breeding programs.

Durable broad-spectrum, adult-plant stem rust resistance in wheat conferred by the Sr2 gene has remained effective against Puccinia graminis f. sp tritici worldwide for more than 50 years. The Sr2 gene has been positioned on the physical map of wheat to the distal 25% portion of the short arm of chromosome 3B. Selection for this gene in wheat breeding programs within Australia has been performed so far through the use of the linked pseudo black chaff (PBC) phenotype and of the microsatellite markers Xgwm389 and Xgwm533 that flank the gene. The molecular markers flank a genetic interval of approximately 4 cM equating to a physical distance of over 10 Mbp. Recently, a 3B-specific BAC library was developed and a physical map established for this region. Analysis of the sequence of minimal tiling path-BAC clones within the region containing the Sr2 gene enabled the development of three new markers that were mapped within the Xgwm389–Xgwm533 genetic interval and tightly linked to the Sr2 gene. Screening a wide range of germplasm containing the Sr2 gene with these markers demonstrated their usefulness for marker-assisted selection in Australian wheat breeding programs.