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 translo...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.展开更多
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...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.展开更多
Plants have developed a complicated defense mechanism during evolution to resist the harmful pathogens they encountered.The mechanism involves the interaction of the plant resistance(R)
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 ob...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.展开更多
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 ...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.展开更多
Common bean is an important but often a disease-susceptible legume crop of temperate,subtropical and tropical regions worldwide. The crop is affected by bacterial, fungal and viral pathogens. The strategy of resistanc...Common bean is an important but often a disease-susceptible legume crop of temperate,subtropical and tropical regions worldwide. The crop is affected by bacterial, fungal and viral pathogens. The strategy of resistance-gene homologue(RGH) cloning has proven to be an efficient tool for identifying markers and R(resistance) genes associated with resistances to diseases. Microsatellite or SSR markers can be identified by physical association with RGH clones on large-insert DNA clones such as bacterial artificial chromosomes(BACs). Our objectives in this work were to identify RGH-SSR in a BAC library from the Andean genotype G19833 and to test and map any polymorphic markers to identify associations with known positions of disease resistance genes. We developed a set of specific probes designed for clades of common bean RGH genes and then identified positive BAC clones and developed microsatellites from BACs having SSR loci in their end sequences. A total of 629 new RGH-SSRs were identified and named BMr(bean microsatellite RGH-associated markers). A subset of these markers was screened for detecting polymorphism in the genetic mapping population DOR364 × G19833. A genetic map was constructed with a total of 264 markers,among which were 80 RGH loci anchored to single-copy RFLP and SSR markers. Clusters of RGH-SSRs were observed on most of the linkage groups of common bean and in positions associated with R-genes and QTL. The use of these new markers to select for disease resistance is discussed.展开更多
Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia （Griseb.）, a pair of specific PCR primers FZ14P1/FZ14P2was designed to isolate candidate disease resistance gene. The pooled-PCR ...Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia （Griseb.）, a pair of specific PCR primers FZ14P1/FZ14P2was designed to isolate candidate disease resistance gene. The pooled-PCR approach was adopted using the primer pair to screen a genomic transformation-competent artificial chromosome （TAC） library derived from Z. latifolia. A positive TAC clone （ZR1） was obtained and confirmed by sequence analysis. The results indicated that ZR1 consisted of conserved motifs similar to P-loop （kinase la）, kinase 2, kinase 3a and GLPL （Gly-Leu-Pro-Leu）, suggesting that it could be a portion of NBS-LRR type of resistance gene. Using Agrobacterium-mediated transformation of Nipponbare mature embryo, a total of 48 independent transgenic To plants were obtained. Among them, 36 plants were highly resistant to the virulent bacterial blight strain PXO71. The results indicate that ZR1 contains at least one functional bacterial blight resistance gene.展开更多
Plant disease resistance gene (R gene); defense response gene encode some conserved motifs. In the present work, a PCR strategy was used to clone resistance gene analogs (RGAs); defense gene analogs (DGAs) from Sea-is...Plant disease resistance gene (R gene); defense response gene encode some conserved motifs. In the present work, a PCR strategy was used to clone resistance gene analogs (RGAs); defense gene analogs (DGAs) from Sea-island cotton variety Hai7124 using oligonucleotide primers based on the nucleotide-binding site (NBS); serine/threonine kinase (STK) in the R-gene; pathogenesis-related proteins of class 2 (PR2) of defense response gene. 79 NBS sequences, 21 STK sequences; 11 DGAs were cloned from disease-resistance cotton. Phylogenic analysis of 79 NBS-RGAs; NBS-RGAs nucleotide sequences of cotton already deposited in GenBank identified one new sub-cluster. The deduced amino acid sequences of NBS-RGAs; STK-RGAs were divided into two distinct groups respectively: Toll/Interleukin-1 receptor (TIR) group; non-TIR group, A group; B group. The expression of RGAs; DGAs having consecutive open reading frame (ORF) was also investigated; it was found that 6 NBS-RGAs; 1 STK-RGA were induced,; 1 DGA was up-regulated by infection of Verticillium dahliae strain VD8. 4 TIR-NBS-RGAs; 4 non-TIR-NBS-RGAs were arbitrarily used as probes for Southern-blotting. There existed 2–10 blotted bands. In addition, since three non-TIR-NBS-RGAs have the same hybridization pattern, we conjecture that these three RGAs form a cluster distribution in the genome.展开更多
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-si...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.展开更多
文摘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.
文摘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.
文摘Plants have developed a complicated defense mechanism during evolution to resist the harmful pathogens they encountered.The mechanism involves the interaction of the plant resistance(R)
基金supported by the funding from the National Natural Science Foundation of China(30971809)the National 973 Program of China(2004CB 117203-5)+2 种基金the National 948 Project of China[(2006-G1(A)]the National High-Tech R&D Program of China(863 Program, 2006AA100104-3)the Heilongjiang Foundation for University Key Teachers,China(1152G007)
文摘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.
基金funded by the National Natural Science Foundation of China (30771391,30700505)the Natural Science Foundation of Hebei Province,China (C2008000281)
文摘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.
文摘Common bean is an important but often a disease-susceptible legume crop of temperate,subtropical and tropical regions worldwide. The crop is affected by bacterial, fungal and viral pathogens. The strategy of resistance-gene homologue(RGH) cloning has proven to be an efficient tool for identifying markers and R(resistance) genes associated with resistances to diseases. Microsatellite or SSR markers can be identified by physical association with RGH clones on large-insert DNA clones such as bacterial artificial chromosomes(BACs). Our objectives in this work were to identify RGH-SSR in a BAC library from the Andean genotype G19833 and to test and map any polymorphic markers to identify associations with known positions of disease resistance genes. We developed a set of specific probes designed for clades of common bean RGH genes and then identified positive BAC clones and developed microsatellites from BACs having SSR loci in their end sequences. A total of 629 new RGH-SSRs were identified and named BMr(bean microsatellite RGH-associated markers). A subset of these markers was screened for detecting polymorphism in the genetic mapping population DOR364 × G19833. A genetic map was constructed with a total of 264 markers,among which were 80 RGH loci anchored to single-copy RFLP and SSR markers. Clusters of RGH-SSRs were observed on most of the linkage groups of common bean and in positions associated with R-genes and QTL. The use of these new markers to select for disease resistance is discussed.
基金supported by the National Natural Science Foundation of China (Grant No. 30760115)Transgenic Program (Grant No. 2008ZX08001-002)
文摘Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia （Griseb.）, a pair of specific PCR primers FZ14P1/FZ14P2was designed to isolate candidate disease resistance gene. The pooled-PCR approach was adopted using the primer pair to screen a genomic transformation-competent artificial chromosome （TAC） library derived from Z. latifolia. A positive TAC clone （ZR1） was obtained and confirmed by sequence analysis. The results indicated that ZR1 consisted of conserved motifs similar to P-loop （kinase la）, kinase 2, kinase 3a and GLPL （Gly-Leu-Pro-Leu）, suggesting that it could be a portion of NBS-LRR type of resistance gene. Using Agrobacterium-mediated transformation of Nipponbare mature embryo, a total of 48 independent transgenic To plants were obtained. Among them, 36 plants were highly resistant to the virulent bacterial blight strain PXO71. The results indicate that ZR1 contains at least one functional bacterial blight resistance gene.
基金supported by the National Natural Science Foundation of China(Grant Nos.30270806 and 30370899)Program for Changjiang Scholars and Innovative Research Team in University in Ministry of Education in China.
文摘Plant disease resistance gene (R gene); defense response gene encode some conserved motifs. In the present work, a PCR strategy was used to clone resistance gene analogs (RGAs); defense gene analogs (DGAs) from Sea-island cotton variety Hai7124 using oligonucleotide primers based on the nucleotide-binding site (NBS); serine/threonine kinase (STK) in the R-gene; pathogenesis-related proteins of class 2 (PR2) of defense response gene. 79 NBS sequences, 21 STK sequences; 11 DGAs were cloned from disease-resistance cotton. Phylogenic analysis of 79 NBS-RGAs; NBS-RGAs nucleotide sequences of cotton already deposited in GenBank identified one new sub-cluster. The deduced amino acid sequences of NBS-RGAs; STK-RGAs were divided into two distinct groups respectively: Toll/Interleukin-1 receptor (TIR) group; non-TIR group, A group; B group. The expression of RGAs; DGAs having consecutive open reading frame (ORF) was also investigated; it was found that 6 NBS-RGAs; 1 STK-RGA were induced,; 1 DGA was up-regulated by infection of Verticillium dahliae strain VD8. 4 TIR-NBS-RGAs; 4 non-TIR-NBS-RGAs were arbitrarily used as probes for Southern-blotting. There existed 2–10 blotted bands. In addition, since three non-TIR-NBS-RGAs have the same hybridization pattern, we conjecture that these three RGAs form a cluster distribution in the genome.
基金supported by funds provided by the Georgia Agricultural Commodity Commission for Peanuts,the National Peanut Board and the Peanut Foundation
文摘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.
