Ronghua Tang

Start codon targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated peanut (Arachis hypogaea L.) genotypes.

Cultivated peanut possesses an extremely narrow genetic basis. Polymorphism is considerably difficult to identify with the use of conventional biochemical and molecular tools. For the purpose of obtaining considerable DNA polymorphisms and fingerprinting cultivated peanut genotypes in a convenient manner, start codon targeted polymorphism technique was used to study genetic diversity and relatedness among 20 accessions of four major botanical varieties of peanut. Of 36 primers screened, 18 primers could produce unambiguous and reproducible bands. All 18 primers generated a total of 157 fragments, with a mean of 8.72 ranging from 4 to 17 per primer. Of 157 bands, 60 (38.22%) were polymorphic. One to seven polymorphic bands were amplified per primer, with 3.33 polymorphic bands on average. Polymorphism per primer ranged from 14.29 to 66.67%, with an average of 36.76%. The results revealed that not all accessions of the same variety were grouped together and high genetic similarity was detected among the tested genotypes based on cluster analysis and genetic distance analysis, respectively. Further, accession-specific markers were observed in several accessions. All these results demonstrated the following: (1) start codon targeted polymorphism technique can be utilized to identify DNA polymorphisms and fingerprint cultivars in domesticated peanut, and (2) it possesses considerable potential for studying genetic diversity and relationships among peanut accessions.

Genetic Diversity in Cultivated Groundnut Based on SSR Markers

Peanut (Arachis hypogaea L.) is an important source crop for edible oil and protein. It is important to identify the genetic diversity of peanut genetic resources for cultivar development and evaluation of peanut accessions. Thirty-four SSR markers were used to assess the genetic variation of four sets of twenty-four accessions each from the four botanical varieties of the cultivated peanut. Among the tested accessions, ten to sixteen pairs of SSR primers showed polymorphisms. The maximum differentiation index, which was defined as the degree of genetic differentiation, was as high as 0.992 in the tested accessions. Each accession could be discriminated by a specific set of polymorphic SSR primers, and the intra-variety genetic distance was determined among accessions, with an average of 0.59 in var. fastigiata 0.46 in var. hypogaea 0.38 in var. vulgaris and 0.17 in var. hirsuta. Dendrogrames based on genetic distances were constructed for the four botanical varieties, which revealed the existence of different clusters. It was concluded that there was abundant intra-variety SSR polymorphism, and with more and more SSR markers being developed, the intrinsic genetic diversity would be detected and the development of genetic map and marker-assisted selection for cultivated peanut would be feasible.

Peanut Aflatoxin and Genomics Research in China: Progress and Perspectives

Peanut is an important oil and food crop in China with a unique role in agricultural development and food security. Aflatoxin contamination in peanut, normally more serious in southern parts of the country, is a crucial factor affecting sustainable development of the peanut industry. Extensive efforts have been made at several institutions in China for aflatoxin management and related genomics research. Several local peanut germplasm lines have been identified as resistant to seed infection by Aspergillus flavus or aflatoxin production. Two AFLP markers have been identified that are linked with resistance to seed invasion and one was converted into a SCAR marker for more efficient breeding application. Several new peanut cultivars with improved productivity and possessing resistance to aflatoxin contamination are extensively used in production. Integrated management approaches have been recommended to farmers based on agro-ecological characteristics in different regions. More recently, molecular techniques have been extensively used in genetic diversity assessment, investigation of genetic relationships among different germplasm groups, marker development, identification of interspecific genome introgression, gene cloning and function analysis, and genetic transformation of important traits concerning productivity, quality and food safety of peanut. Special emphasis has been placed on resistance to aflatoxin, bacterial wilt, foliar diseases and fatty acid desaturase. Perspectives of peanut genetic improvement and further research priorities are also discussed.

Phylogenetic Relationships in Genus Arachis Based on SSR and AFLP Markers

Fourteen wild species of different sections in the genus Arachis and 24 accessions of the AABB allotetraploid A. hypogaea (cultivated peanut) from several countries which belong to different botanical varieties, were analyzed by SSR and AFLP marker systems. The assay-units per system needed to distinguish among all the tested accessions were at least five for SSR or two for AFLP. The genetic distance detected by the SSR markers ranged from 0.09 to 0.95, and the mean was 0.73; and the genetic distance detected by the AFLP markers ranged from 0.01 to 0.79 with an average of 0.42. All the tested peanut SSR primer pairs were multilocus ones, and the amplified fragments per SSR marker in each peanut genome ranged from 2 to 15 with the mean of 4.77. The peanut cultivars were closely related to each other, and shared a large numbers of SSR and AFLP fragments. In contrast, the species in the genus Arachis shared few fragments. The results indicated that the cultivated peanut ( A. hypogaea L.) varieties could be partitioned into two main groups and tour subgroups at the molecular level, and that A. duranensis is one of the wild ancestors of A. hypogaea . The lowest genetic variation was detected between A. cardenasii and A. batizocoi , and the highest was detected between A. pintoi and the species in the section Arachis . The relationships among the botanical varieties in the cultivated peanut ( A. hypogaea L.) and among wild species accessions in section Arachis and those in other sections in the genus Arachis were discussed.

Molecular Profiling of Genetic Variability in Domesticated Groundnut (Arachis hypogaea L.) Based on ISJ, URP, and DAMD Markers

To detect DNA polymorphisms in the peanut, we screened 26 polymorphic primers using intron–exon splice junction (ISJ), universal rice primer (URP), and directed amplification of minisatellite region DNA (DAMD) techniques. Amplification of genomic DNA of 16 peanut accessions yielded 121 ISJ, 50 URP, and 25 DAMD fragments, of which 34, 25 and 16 were polymorphic, respectively. The range of polymorphism was 10.0–62.5%, averaging 27.7%, for ISJ; 20–80%, averaging 49.5%, for URP; and 28.6–50.0%, averaging 36.3%, for DAMD. In comparisons of multiplex ratio, average polymorphism information content, and marker index, the URP markers were relatively more efficient than ISJ and DAMD markers. Clustering results remained more or less the same with ISJ and URP markers. To the best of our knowledge, this is the first report on the study of the genetic diversity of the peanut using ISJ, URP, and DAMD markers.

Molecular Characterization of High Plant Species Using PCR with Primers Designed from Consensus Branch Point Signal Sequences

A novel method is introduced for producing molecular markers in plants using single 15- to 18-mer PCR primers designed from the short conserved consensus branch point signal sequences and standard agarose gel electrophoresis. This method was tested on cultivated peanut and verified to give good fingerprinting results in other plant species (mango, banana, and longan). These single primers, designed from relatively conserved branch point signal sequences within gene introns, should be universal across other plant species. The method is rapid, simple, and efficient, and it requires no sequence information of the plant genome of interest. It could be used in conjunction with, or as a substitute for, conventional RAPD or ISSR techniques for applications including genetic diversity analysis, phylogenetic tree construction, and quantitative trait locus mapping. This technique provides a new way to develop molecular markers for assessing genetic diversity of germplasm in diverse species based on conserved branch point signal sequences.

Rapid gene expression change in a novel synthesized allopolyploid population of cultivated peanut×Arachis doigoi cross by cDNA-SCoT and HFO-TAG technique

Abstract Allopolyploidy has played an important role in plant evolution and heterosis. Recent studies indicate that the process of wide hybridization and (or) polyploidization may induce rapid and extensive genetic and epigenetic changes in some plant species. To better understand the allopolyploidy evolutionism and the genetic mechanism of Arachis interspecific hybridization, this study was conducted to monitor the gene expression variation by cDNA start codon targeted polymorphism (cDNA-SCoT) and cDNA high-frequency oligonucleotide-targeting active gene (cDNA-HFO-TAG) techniques, from the hybrids (F1) and newly synthesized allopolyploid generations (S0–S3) between tetraploid cultivated peanut Zhongkaihua 4 with diploid wild one Arachis doigoi. Rapid and considerable gene expression variations began as early as in the F1 hybrid or immediately after chromosome doubling. Three types of gene expression changes were observed, including complete silence (gene from progenitors was not expressed in all progenies), incomplete silence (gene expressed only in some progenies) and new genes activation. Those silent genes mainly involved in RNA transcription, metabolism, disease resistance, signal transduction and unknown functions. The activated genes with known function were almost retroelements by cDNA-SCoT technique and all metabolisms by cDNA-HFO-TAG. These findings indicated that interspecific hybridization and ploidy change affected gene expression via genetic and epigenetic alterations immediately upon allopolyploid formation, and some obtained transcripts derived fragments (TDFs) probably could be used in the research of molecular mechanism of Arachis allopolyploidization which contribute to thwe genetic diploidization of newly formed allopolyploids. Our research is valuable for understanding of peanut evolution and improving the utilization of putative and beneficial genes from the wild peanut.

AhRLK1, a CLAVATA1-like leucine-rich repeat receptor-like kinase of peanut, confers increased resistance to bacterial wilt in tobacco

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease that infects hundreds of plant species. Host factors involved in disease resistance and pathogenesis remain poorly characterized. An up regulated and leucine-rich repeat receptor-like kinase characterized as CLAVATA1 and named AhRLK1 was obtained by microarray analysis in response to R. solanacearum in peanut. AhRLK1 contained presumably, a signal peptide, ten leucine-rich repeat (LRR) domains and conserved motifs of intracellular kinases. For subcellular localization, the AhRLK1 protein was visualized only in the plasma membrane. After inoculation with R. solanacearum, AhRLK1 was constantly up regulated in the susceptible variety Xinhuixiaoli but showed little changed in the resistant cultivar Yueyou92. Different hormones, including salicylic acid, abscisic acid, methyl jasmonate, and ethephon, induced expression, but expression was completely down regulated under cold and drought treatments. Transient overexpression provoked a hypersensitive response (HR) in Nicotiana benthamiana following agro-infiltration. Furthermore, in transgenic tobacco with overexpression of the gene, the resistance to R. solanacearum increased significantly. By contrast, most representative defense-responsive genes in HR, SA, JA and ET signal pathways such as NtHIN1, NtPR2, NtLOX1, and NtACS6, among others, were considerably up regulated in the AhRLK1 transgenic lines. Additionally, the EDS1 and PAD4 in the R gene signal were also up regulated in transgenic plants, but the NDR1 and NPR1 genes were down regulated. Accordingly, we suggest that AhRLK1 increases the defense response to R. solanacearum via HR and hormone defense signalling, associated with the EDS1 pathway of R gene signalling. The results provide new understanding of CLV1 function and will contribute to genetic enhancement of peanut.

A Primary Exploration of Variety Selection and Efficient Cultivation Models for Cassava/Peanut Intercropping

In this study, we determined the agronomic traits and yields of peanuts under different intercropping models by carrying out tests on variety selection, row spacing, and film mulching during sowing of intercropped peanuts. We analyzed the high-yield and comprehensive benefits in order to determine the efficient cultivation models of cassava/peanut intercropping. The results showed that among the eight tested peanut varieties, Guihua771 was the most suitable for intercropping with cassava, and its yield was 18.7% higher than Guihua17 – the local control variety – followed by Guihua22 and Guihua836. In the intercropping model selection test, the yield income of one row of cassava, intercropped with two rows of peanuts, under medium-narrow spacing (0.9-1.1m) was significantly higher than that of one row of cassava intercropped with three or four rows of peanuts with wide spacing (1.3-1.4m). The income of intercropped peanuts with film mulching was higher than that without the film, and the income from cassava and peanut intercropping 15d apart was higher than that of planting the cassava and peanuts at the same time. The best overall yield income was obtained when using Guihua771 as the peanut variety, intercropped with cassava using a row spacing arrangement of one row of cassava with two rows of peanuts, and planting the peanuts 15d ahead of the cassava, with mulching film.