Isolation of Resistance Gene Analogs from Wheat Based on Conserved Domains of Resistance Genes

Two pairs of degenerate primers were designed based on nucleotide-binding site (NBS) and serine/threonine kinase domain. PCR was performed with the primers and cDNA from the Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL. Amplified products were cloned and sequenced. Nine clones with NBS and one with serine/threonine kinase domain were obtained. The NBS clones were classified to six groups according to their nucleotide sequence identities (90% or higher). These resistance gene analogs (RGAs) all have open reading frames (ORF), and their amino acid sequences show high similarity to Yr10 in wheat, Mla1 and Mla6 in barley, RPS2 in Arabidopsis and other resistance (R) genes with conserved motifs. They were preliminarily mapped on the chromosomes of homoeologous groups 1, 2 and 5 of common wheat by nulli-tetrasomic analysis. The 5'-end sequence of an RGA N5 was obtained by 5'-RACE PCR. It encodes six leucine zipper (LZ) and has high sequence similarity to RPS2.

Cloning and Characterization of a Family of Disease Resistance Gene Analogs from 6VS of Haynaldia villosa

In the present study, microdissection of 6VS and the cloning of the resistance gene analogs(RGA)from them were reported. The 6VS were microdissected with needle and 10 types of resistance gene analogs were obtained by PCR with degenerate oligonucleotide primer designed according to resistance genes. They were designated as Hvrgak1-Hvrgak10, GenBank accession numbers are AF387113-AF387121, AY040671- AY040672. Identity among RGAs was about 10-50%, and identity with cloned R gene from plants was 5-20%. Southern hybridization analysis results showed 3 RGAs, Hvrgak2, Hvrgak4, and Hvr-gak5 were linked with wheat powdery mildew resistance. These RGAs may be used as direct entrance or probes for cloning the disease resistance genes.

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.

An Integrated QTL Map of Fungal Disease Resistance in Soybean (Glycine max L. Merr):A Method of Meta-Analysis for Mining R Genes

Diseases caused by fungal pathogens account for approximately 50% of all soybean disease losses around the world. Conflicting results of fungal disease resistance QTLs from different populations often occurred. The objectives of this study were to： （i） evaluate evidence for reported fungal disease resistance QTLs associations in soybean and （ii） extract relatively reliable and useful information from the ＂real＂ QTLs and mine putative genes in soybean. An integrated map of fungal disease resistance QTLs in soybean was established with soymap 2 published in 2004 as a reference map. QTLs of fungal disease resistance developed from each of separate populations in recent 10 years were integrated into a combinative map for gene cloning and marker assisted selection in soybean. 107 QTLs from different maps were integrated and projected to the reference map with the software BioMercator 2.1. A method of meta-analysis was used to narrow down the confidence interval, and 23 ＂real＂ QTLs and their corresponding markers were obtained from 12 linkage groups （LG）, respectively. Two published R genes were found in these ＂real＂ QTLs intervals. Sequences in the ＂real＂ QTLs intervals were predicted by GENSCAN, and these predicted genes were annotated in Goblet. 228 resistance gene analogs （RGAs） in 12 different terms were mined. The results will lay the foundation for a bioinformatics platform combining abundant QTLs, and offer the basis for marker assisted selection and gene cloning in soybean.

Isolation and Characterization of NBS-LRR Class Resistance Homologous Gene from Wheat

One resistance gene analog fragment named RGA-CIN14 was isolated from TcLr19 wheat,which contains kinase-2,kinase-3a,and the GLPL motif of the NBS-spanning region,using degenerated primers according to the nucleotide binding site （NBS） conserved domain.Based on the RGA-CIN14,a full-length cDNA,CIN14,which was 2 987 bp encoding 880 amino acids,was obtained by using the method of the rapid amplification cDNA ends （RACE）.Bioinformatics analysis showed that the deduced amino acids of CIN14 protein consisted of a NB-ARC conserved domain and many leucine-rich repeats （LRR） domains.The phylogenetic tree analysis indicated a considerable identity of the protein encoded by CIN14 with that of wheat leaf rust resistance gene Lr1,but a lower similarity with Lr21.The expression profile of the CIN14 gene detected by semi-quantitative RT-PCR showed that the CIN14 gene was not induced by Puccinia triticina and it was a constitutive gene with low abundance in the wheat leaf tissue.The resistance homology sequence was successfully obtained,which provides the shortcut for cloning of the resistance gene in TcLr19 wheat.