Cloning, Expression Pattern Analysis and Subcellular Localization of Resveratrol Synthase Gene in Peanut (<i>Arachis hypogaea</i>L.)

Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico cloning and RT-PCR. Bioinformatics analysis showed that deduced amino acid sequences of the six cloned RS genes were highly conserved with a similarity from 95% to 99% when compared to the RS genes which had been deposited at the GenBank. The results of amino acid sequences analysis showed six RS proteins contained the Chal_Sti_Synt_N and ACP_Syn_III_C domains and can be classified to same family but with different evolutionary distance. Expression pattern analysis by QRT-PCR provided evidence indicating that the mRNA of six RS genes were primarily expressed in the peanut shell at different developmental stages with different expression levels, but only lower levels of them were evident in the peanut kernel. The subcellular localization of RS protein in onion epidermal cell was performed by Agrobacterium tumefaciens-mediated transformation and the green fluorescent was monitored by confocal fluorescence microscopy. The results indicated that, RS1 and RS5 were located in the nucleus and plasma membrane respectively, while the RS2, RS3, RS4 and RS6 were located in both nucleus inner membrane and plasma membrane. The data will provide basic information for elucidating the regulatory mechanisms and enzyme kinetics underlying the RS genes in the resveratrol synthase pathway.

Crop Breeding Chips and Genotyping Platforms： Progress, Challenges, and Perspectives

There is a rapidly rising trend in the development and application of molecular marker assays for gene map- ping and discovery in field crops and trees. Thus far, more than 50 SNP arrays and 15 different types of genotyping-by-sequencing （GBS） platforms have been developed in over 25 crop species and perennial trees. However, much less effort has been made on developing ultra-high-throughput and cost-effective genotyping platforms for applied breeding programs. In this review, we discuss the scientific bottlenecks in existing SNP arrays and GBS technologies and the strategies to develop targeted platforms for crop mo- lecular breeding. We propose that future practical breeding platforms should adopt automated genotyping technologies, either array or sequencing based, target functional polymorphisms underpinning economic traits, and provide desirable prediction accuracy for quantitative traits, with universal applications under wide genetic backgrounds in crops. The development of such platforms faces serious challenges at both the technological level due to cost ineffectiveness, and the knowledge level due to large genotype- phenotype gaps in crop plants. It is expected that such genotyping platforms will be achieved in the next ten years in major crops in consideration of （a） rapid development in gene discovery of important traits, （b） deepened understanding of quantitative traits through new analytical models and population designs, （c） integration of multi-layer -omics data leading to identification of genes and pathways responsible for important breeding traits, and （d） improvement in cost effectiveness of large-scale genotyping. Crop breeding chips and genotyping platforms will provide unprecedented opportunities to accelerate the development of cultivars with desired yield potential, quality, and enhanced adaptation to mitigate the effects of climate change.

Identification of ExpIdentification of Expressed Resistance Gene Analogs from Peanut (Arachis hypogaea L.) Expressed Sequence Tags

Low genetic diversity makes peanut （Arachis hypogaea L.） very vulnerable to plant pathogens, causing severe yield loss and reduced seed quality. Several hundred partial genomic DNA sequences as nucleotide-binding-site leucine-rich repeat （NBS-LRR） resistance genes （R） have been identified, but a small portion with expressed transcripts has been found. We aimed to identify resistance gene analogs （RGAs） from peanut expressed sequence tags （ESTs） and to develop polymorphic markers. The protein sequences of 54 known R genes were used to identify homologs from peanut ESTs from public databases. A total of 1,053 ESTs corresponding to six different classes of known R genes were recovered, and assembled 156 contigs and 229 singletons as peanut-expressed RGAs. There were 69 that encoded for NBS-LRR proteins, 191 that encoded for protein kinases, 82 that encoded for LRR-PK/transmembrane proteins, 28 that encoded for Toxin reductases, 11 that encoded for LRR-domain containing proteins and four that encoded for TM-domain containing proteins. Twenty-eight simple sequence repeats （SSRs） were identified from 25 peanut expressed RGAs. One SSR polymorphic marker （RGA121） was identified. Two polymerase chain reaction-based markers （Ahsw-1 and Ahsw-2） developed from RGA013 were homologous to the Tomato Spotted Wilt Virus （TSWV） resistance gene. All three markers were mapped on the same linkage group AhIV. These expressed RGAs are the source for RGA-tagged marker development and identification of peanut resistance genes.

Genome-wide SNP Genotyping Resolves Signatures of Selection and Tetrasomic Recombination in Peanut

Peanut （Arachis hypogaea; 2n = 4x = 40） is a nutritious food and a good source of vitamins, minerals, and healthy fats. Expansion of genetic and genomic resources for genetic enhancement of cultivated peanut has gained momentum from the sequenced genomes of the diploid ancestors of cultivated peanut. To facil- itate high-throughput genotyping of Arachis species, 20 genotypes were re-sequenced and genome-wide single nucleotide poiymorphisms （SNPs） were selected to develop a large-scale SNP genotyping array. For flexibility in genotyping applications, SNPs polymorphic between tetraploid and diploid species were included for use in cultivated and interspecific populations. A set of 384 accessions was used to test the array resulting in 54 564 markers that produced high-quality polymorphic clusters between diploid species, 47 116 polymorphic markers between cultivated and interspecific hybrids, and 15 897 polymorphic markers within A. hypogaea germplasm. An additional 1193 markers were identified that illuminated genomic re- gions exhibiting tetrasomic recombination. Furthermore, a set of elite cultivars that make up the pedigree of US runner germplasm were genotyped and used to identify genomic regions that have undergone pos- itive selection. These observations provide key insights on the inclusion of new genetic diversity in culti- vated peanut and will inform the development of high-resolution mapping populations. Due to its efficiency, scope, and flexibility, the newly developed SNP array will be very useful for further genetic and breeding applications in Arachis.

A Comprehensive Transcriptome Assembly of Pigeonpea （Cajanus cajan L.） using Sanger and Second-Generation Sequencing Platforms

A comprehensive transcriptome assembly for pigeonpea has been developed by analyzing 128.9 million short Illumina GA IIx single end reads, 2.19 million single end FLX/454 reads, and 18 353 Sanger expressed sequenced tags from more than 16 genotypes. The resultant transcriptome assembly, referred to as CcTA v2, comprised 21 434 transcript as- sembly contigs （TACs） with an N50 of 1510 bp, the largest one being -8 kb. Of the 21 434 TACs, 16 622 （77.5%） could be mapped on to the soybean genome build 1.0.9 under fairly stringent alignment parameters. Based on knowledge of intron junctions, 10 009 primer pairs were designed from 5033 TACs for amplifying intron spanning regions （ISRs）. By using in silico mapping of BAC-end-derived SSR loci of pigeonpea on the soybean genome as a reference, putative mapping posi- tions at the chromosome level were predicted for 6284 ISR markers, covering all 11 pigeonpea chromosomes. A subset of 128 ISR markers were analyzed on a set of eight genotypes. While 116 markers were validated, 70 markers showed one to three alleles, with an average of 0.16 polymorphism information content （PIC） value. In summary, the CcTA v2 transcript assembly and ISR markers will serve as a useful resource to accelerate genetic research and breeding applications in pigeonpea.