Screening and identification of random amplified polymorphic DNA (RAPD) markers linked to mungbean yellow mosaic virus (MYMV) resistance in mungbean (Vigna radiata (L.) Wilczek)

Bulk segregant analysis (BSA) and random amplified polymorphic DNA (RAPD) techniques were used to analyse the F2 individuals of susceptible VBN (Gg) 2 × resistant KMG 189 to screen and identify the molecular marker linked to mungbean yellow mosaic virus (MYMV) resistant gene in mungbean. Two DNA bulks namely resistant bulks and susceptible bulks were setup by pooling equal amount of DNA from five randomly selected plants of each disease response. A total of 72 random sequence decamer oligonucleotide primers were used for RAPD analysis. Primer OPBB 05 (5′-GGGCCGAACA-3′) generated OPBB 05 260 fragment in resistant parent and their bulks but not in the susceptible parent and their bulks. Co segregation analysis was performed in resistant and susceptible F2 individuals, it confirmed that OPBB 05 260 marker was tightly linked to mungbean yellow mosaic virus resistant gene in mungbean.     

Inheritance of resistance to yellow mosaic virus in blackgram (Vigna mungo (L.) Hepper)

Summary The inheritance of resistance to mungbean yellow mosaic virus (MYMV) was studied in blackgram (Vigna mungo (L.) Hepper). The highly resistant donors Pant U-84 and UPU-2 and a highly susceptible line, UL-2, their F₁'s, F₂'s and backcrosses were grown with spreader located every 5 to 6 rows. The resistance was found to be digenic and recessive in all the crosses and free from cytoplasmic effect.     

Molecular studies on mungbean (Vigna radiata (L.) Wilczek) and ricebean (Vigna umbellata (Thunb.)) interspecific hybridisation for Mungbean yellow mosaic virus resistance and development of species-specific SCAR marker for ricebean

The aim of this study was to identify the molecular markers (SSR, RAPD and SCAR) associated with Mungbean yellow mosaic virus resistance in an interspecific cross between a mungbean variety, VRM (Gg) 1 X a ricebean variety, TNAU RED. The parental survey was carried out by using 118 markers (including 106 azuki bean primers, seven mungbean primers and five ricebean primers). This study revealed that 42 azuki bean markers (39.62%) and four mungbean markers (54.07%) showed parental polymorphism. These polymorphic markers were surveyed among the 187 F₂ plants and the results showed distorted segregation or chromosomal elimination at all the marker loci (thus, deviating from the expected 1:2:1 segregation). None of the plants harboured the homozygous ricebean allele for the markers surveyed and all of them were skewed towards mungbean, VRM (Gg) 1, allele, except a few plants which were found to be heterozygous for few markers. Among the 42 azuki bean SSR markers surveyed, only 10 markers produced heterozygotic pattern in six F₂ lines viz. 3, 121, 122, 123, 185 and 186. These markers were surveyed in the corresponding F₃ individuals, which too skewed towards the mungbean allele. In this study, one species-specific SCAR marker was developed for ricebean by designing primers from the sequenced putatively species-specific RAPD bands. A single, distinct and brightly resolved band of 400?bp was found amplified only in the resistant parent, TNAU RED, and not in any other six species or in the resistant or the susceptible bulks of the mapping population clearly indicated the identification of SCAR marker specific to the ricebean.     

Identification and confirmation of quantitative trait loci controlling resistance to mungbean yellow mosaic disease in mungbean [Vigna radiata (L.) Wilczek]

Mungbean yellow mosaic India virus (MYMIV) is a major constraint on mungbean production in South and Southeast Asia. The virus belongs to the genus Begomovirus, causing yellow mosaic disease and subsequently yield loss up to 75–100 %. The present study employed F₂ and BC₁F₁ populations derived from a cross between susceptible (BARImung 1; BM1) and resistant (BARImung 6; BM6) mungbeans to identify quantitative trait loci (QTLs) associated with resistance to MYMIV. Resistance to the virus was evaluated using F_2:3 and BC₁F_1:2 populations under field conditions in two locations in Bangladesh in 2012. A total of 1,165 simple sequence repeat markers from different legumes were used to detect the polymorphism between BM1 and BM6. Sixty-one polymorphic markers were used to construct a linkage map comprising 11 linkage groups. Composite interval mapping consistently identified two major QTLs, qMYMIV2 on linkage group 2 and qMYMIV7 on linkage group 7, conferring the resistance in both F₂ and BC₁F₁ populations. qMYMIV2 and qMYMIV7 accounted for 31.42–37.60 and 29.07–47.36 %, respectively, of the disease score variation, depending on populations and locations. At both loci, the resistant alleles were contributed by the parent BM6. qMYMIV2 appeared to be common to a major QTL for MYMIV resistance in mungbean reported previously, while qMYMIV7 is a new QTL for the resistance. The markers linked to the QTLs in this study are useful in marker-assisted breeding for development of mungbean varieties resistant to MYMIV.     

RFLP mapping of a major bruchid resistance gene in mungbean (Vigna radiata,L. Wilczek)

Summary Bruchids (genus Callosobruchus) are among the most destructive insect pests of mungbeans and other members of the genus, Vigna. Genetic resistance to bruchids was previously identified in a wild mungbean relative, TC1966. To analyze the underlying genetics, accelerate breeding, and provide a basis for map-based cloning of this gene, we have mapped the TC1966 bruchid resistance gene using restriction fragment length polymorphism (RFLP) markers. Fifty-eight F₂ progeny from a cross between TC1966 and a susceptible mungbean cultivar were analyzed with 153 RFLP markers. Resistance mapped to a single locus on linkage group VIII, approximately 3.6 centimorgans from the nearest RFLP marker. Because the genome of mungbean is relatively small (estimated to be between 470 and 560 million base pairs), this RFLP marker may be suitable as a starting point for chromosome walking. Based on RFLP analysis, an individual was also identified in the F₂ population that retained the bruchid resistance gene within a tightly linked double crossover. This individual will be valuable in developing resistant mungbean lines free of linkage drag.     

Inheritance of resistance to iron deficiency and identification of AFLP markers associated with the resistance in mungbean (Vigna radiata (L.) Wilczek)

Iron deficiency chlorosis (IDC) is a major problem reducing yield of mungbean in many countries. In this study, we crossed “KPS1”, the most popular Thai mungbean cultivar susceptible to IDC with “NM10-12”, a mungbean line from Pakistan resistant to IDC. Segregation analysis of the F₂ population revealed that the resistance is controlled by a major gene (IR) with dominant effect. Two AFLP markers, E-ACT/M-CTA and E-ACC/M-CTG were identified closely linking with the IR gene. The frequencies of these markers were assessed in 241 mungbean accessions from several countries. The accessions could be divided, in relative to total chlorophyll content of the resistant check (NM10-12) and the susceptible check (KPS1), into the resistant group with 125 accessions and the susceptible group with 116 accessions. Among 125 resistant accessions, E-ACT/M-CTA and E-ACC/M-CTG were present in 119 (95%) and 109 (87%) accessions, respectively. Both markers can identify all resistant accessions from England, Indonesia and Pakistan, but only E-ACT/M-CTA linked to all resistant accessions from Australia, India, Iraq, Taiwan and Thailand. Understanding the inheritance and identifying molecular markers linking to the IR gene can help plant breeders to improve this crop for growing in iron-deficient soils.     

Inheritance of powdery mildew (Erysiphe polygoni DC) resistance in mungbean (Vigna radiata L. Wilczek)

Detached mungbean (Vigna radiata L.Wilczek) leaves were inoculated with a conidial suspension of a local isolate (TI-1) of the powdery mildew pathogen (Erysiphe polygoni DC) under controlled environment conditions. Based on the latent period and severity of the infection, a rating scale of 0–5 was used to classify the host pathogen interactions. Reactions 0, 1 and 2 were considered resistant and referred to as R0, R1 and R2 while 3, 4 and 5 were classified as susceptible (S). RUM lines (resistant to powdery mildew) and their derivatives are crossed with several susceptible (reaction types 3–5) genotypes and the inheritance of the resistance was studied in the F₁, F₂ and F₃ generations. The results showed that powdery mildew resistance in mungbean is governed by two dominant genes designated as Pm-1 and Pm-2. When both Pm-1 and Pm-2 were present, an R0 reaction was observed after inoculation with TI-1. The resistant reaction was R1 when only Pm-1 was present and R2 in the presence of Pm-2. In the absence of both Pm-1 and Pm-2, susceptible reactions 3, 4 and 5 were observed.     

Genetic diversity of legume yellow mosaic begomoviruses in Indonesia and Vietnam

High incidences of yellow mosaic symptoms were observed in soybean and yard‐long bean crops in Indonesia in 2009 and in mungbean crops in Vietnam in 2011. All five soybean and 20 yard‐long bean samples from Java, Indonesia, and 15 mungbean samples from Vietnam with symptoms tested positive for begomovirus infection by polymerase chain reaction (PCR) with primer pair PAL1v1978B/PAR1c715H. On the basis of collection location and the nucleotide sequence comparisons of the 1.5 kb begomoviral DNA‐A components amplified, a subset of samples comprising two soybean and six yard‐long bean isolates from Indonesia and five mungbean isolates from Vietnam were taken forward for more detailed examination. Sequence comparison and phylogenetic analysis of the full‐length sequences of all Indonesian and Vietnam isolates alongside other legume‐infecting begomoviruses revealed that all the isolates from Indonesia were Mungbean yellow mosaic India virus (MYMIV) strain‐A, and all from Vietnam were Mungbean yellow mosaic virus (MYMV) strain‐B. To the best of our knowledge, this is the first identification of MYMIV and MYMV associated with yellow mosaic of legumes in Indonesia and Vietnam, respectively. The epidemiological implications and potential consequences of the emergence of legume‐infecting begomoviruses on legume production in these areas of Southeast Asia are discussed.     

Quantitative trait locus mapping reveals conservation of major and minor loci for powdery mildew resistance in four sources of resistance in mungbean [Vigna radiata (L.) Wilczek]

Powdery mildew (PM) is a common and serious disease of mungbean [Vigna radiata (L.) Wilczek]. A few quantitative trait loci (QTL) for PM resistance in mungbean have been reported. The objective of this study was to locate QTL for PM resistance in two resistant accessions V4718 and RUM5. Simple sequence repeat markers were analyzed in an F₂ population from a cross between Kamphaeng Saen 1 (KPS1; susceptible to PM) and V4718 (resistant to PM), and in F₂ and BC₁F₁ populations from a cross between Chai Nat 60 (CN60; susceptible to PM) and RUM5 (resistant to PM). Progenies of 134 F_2:3 and F_2:4 lines derived from KPS1 × V4718, and 190 F_2:3 and 74 BC₁F_1:2 lines derived from CN60 × RUM5 and CN60 × (CN60 × RUM5), respectively, were evaluated for response to PM under field conditions. Multiple interval mapping identified a major QTL on linkage group (LG) 9 and two minor QTL on LG4 for the resistance in V4718, and detected two major QTL on LG6 and LG9 and one minor QTL on LG4 for the resistance in RUM5. Comparative linkage analysis of the QTL for PM resistance in this study and in previous reports suggests that the resistance QTL on LG9 in V4718, RUM5, ATF3640 and VC6468-11-1A are the same locus or linked. One QTL on LG4 is the same in three sources (V4718, RUM5 and VC1210A). Another QTL on LG6 is the same in two sources (RUM5 and VC6468-11-1A). In addition, one QTL in V4718 on LG4 appears to be a new resistance locus. These different resistance loci will be useful for breeding durably PM-resistant mungbean cultivars.     

The allelic relationship of genes giving resistance to mungbean yellow mosaic virus in blackgram

Summary The allelic relationship of resistance genes for MYMV was studied in blackgram (V. mungo (L.) Hepper). The resistant donors to MYMV — Pant U84 and UPU 2, and their F₁, F₂ and F₃ generations — were inoculated artificially using an insect vector, whitefly (Bemisia tabaci Genn.). The two recessive genes previously reported for resistance were found to be the same in both donors.Part of Ph.D. Thesis submitted by the senior author. Research Paper No. 4271     

Development of SRAP, SRAP-RGA, RAPD and SCAR markers linked with a Fusarium wilt resistance gene in eggplant

Fusarium wilt (Fusarium oxysporum Schlecht. f. sp. melongenae) is a vascular disease of eggplant (Solanum melongena L.). The objectives of this work were (1) to confirm the monogenic inheritance of fusarium wilt resistance in eggplant, (2) to identify molecular markers linked to this resistance, and (3) to develop SCAR markers from most informative markers. We report the tagging of the gene for resistance to fusarium wilt (FOM) in eggplant using SRAP, RGA, SRAP-RGA and RAPD markers. Analysis of segregation data confirmed the monogenic inheritance of resistance. DNA from F₂ and BC₁ populations of eggplant segregating for fusarium wilt resistance was screened with 2,316 primer combinations to detect polymorphism. Three markers were linked within 2.6 cM of the gene. The codominant SRAP marker Me8/Em5 and dominant SRAP-RGA marker Em12/GLPL2 were tightly linked to each other and mapped 1.2 cM from the resistance gene, whereas RAPD marker H12 mapped 2.6 cM from the gene and on the same side as the other two markers. The SRAP marker was converted into two dominant SCAR markers that were confirmed to be linked to the resistance gene in the F_2, BC₁ and F₂ of BC₃ generations of the same cross. These markers provide a starting point for mapping the eggplant FOM resistance gene in eggplant and for exploring the synteny between solanaceous crops for fusarium wilt resistance genes. The SCAR markers will be useful for identifying fusarium wilt-resistant genotypes in marker-assisted selection breeding programs using segregating progenies of the resistant eggplant progenitor used in this study.     

Identification of quantitative trait loci associated with powdery mildew resistance in mungbean using ISSR and ISSR-RGA markers

To identify the powdery mildew (PM) resistance gene in mungbean, inter-simple sequence repeat (ISSR) markers and newly developed ISSR-anchored resistance gene analog (ISSR-RGA) markers were evaluated. When F_2:7 and F_2:8 recombinant inbred line populations derived from a cross between CN72 (susceptible cultivar in Thailand) and V4718 (resistant line from Asian Vegetable Research and Development Center) were evaluated for PM resistance under field conditions, the PM resistance gene from V4718 was found to be inherited as a single major gene. Fifteen out of 75 ISSR primers produced 27 DNA bands putatively associated with PM resistance in bulk segregant analysis (BSA). Ten ISSR primers were combined with four RGA primers homologous to the nucleotide-binding site and kinase domains of resistance (R) genes to generate 40 ISSR-RGA primer combinations. When these 40 ISSR-RGA primer combinations and 10 corresponding ISSR primers were used in BSA, 873 ISSR and 756 ISSR-RGA loci were amplified. Fifty-two of 756 ISSR-RGA loci were new, and 11 of these 23 ISSR-RGA loci were putatively associated with the PM resistance. Simple linear regression confirmed that 5 of the 27 ISSR markers and 3 of the 11 ISSR-RGA markers were significantly associated with the PM resistance gene. When these eight ISSR and ISSR-RGA markers were used for quantitative trait loci (QTL) analysis, multiple interval mapping identified a major QTL, qPMC72V18-1, explaining up to 92.4% of the phenotypic variation, flanked by I42PL229 and I85420 markers at the distance of 4 and 9 cM, respectively. These results suggest that ISSR and ISSR-RGA markers are highly efficient tools for mapping PM resistance gene in mungbean. The markers closely linked to the PM resistance gene will be useful for future marker-assisted selection to develop mungbean varieties resistant to PM.     

Quantitative trait loci mapping of Cercospora leaf spot resistance in mungbean, <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek

Cercospora leaf spot (CLS) caused by the fungus Cercospora canescens Illis & Martin is a serious disease in mungbean (Vigna radiata (L.) Wilczek), and disease can reduce seed yield by up to 50%. We report here for the first time quantitative trait loci (QTL) mapping for CLS resistance in mungbean. The QTL analysis was conducted using F₂ (KPS1 × V4718) and BC₁F₁ [(KPS1 × V4718) × KPS1] populations developed from crosses between the CLS-resistant mungbean V4718 and CLS-susceptible cultivar Kamphaeng Saen 1 (KPS1). CLS resistance in F₂ populations was evaluated under field conditions during the wet seasons of 2008 and 2009, and resistance in BC₁F₁ was evaluated under field conditions during the wet season in 2008. Seven hundred and fifty-three simple sequence repeat (SSR) markers from various legumes were used to assess polymorphism between KPS1 and V4718. Subsequently, 69 polymorphic markers were analyzed in the F₂ and BC₁F₁ populations. The results of segregation analysis indicated that resistance to CLS is controlled by a single dominant gene, while composite interval mapping consistently identified one major QTL (qCLS) for CLS resistance on linkage group 3 in both F₂ and BC₁F₁ populations. qCLS was located between markers CEDG117 and VR393, and accounted for 65.5–80.53% of the disease score variation depending on seasons and populations. An allele from V4718 increased the resistance. The SSR markers flanking qCLS will facilitate transferral of the CLS resistance allele from V4718 into elite mungbean cultivars.     

Antisense RNA approach targeting Rep gene of Mungbean yellow mosaic India virus to develop resistance in soybean

The Yellow mosaic disease is caused by Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) belonging to the genus Begomovirus of the family Geminiviridae. Yellow mosaic disease (YMD) is a major constraint to the production of soybean in South-East Asia. In India, yield losses of 10–88% had been reported due to YMD of soybean. An effort has been made to generate resistant soybean plants, by a construct targeting replication initiation protein (Rep) gene sequences of MYMIV. A construct containing the sequences of Rep gene (566?bp) in antisense orientation was used to transform cotyledonary node explants of three soybean cultivars (JS 335, JS 95-60 and NRC 37). Transformation efficiencies of 0.2, 0.21 and 0.24% were obtained with three soybean cultivars, JS 335, JS 95-60 and NRC 37, respectively. The presence of transgene in T₁ plants was confirmed by polymerase chain reaction (PCR) and sequence analysis. The level of resistance was observed by challenge inoculation with the virus in T₁ lines. The inheritance of transgene showed classical Mendelian pattern in six transgenic lines.     

Genetic localization of a bruchid resistance gene and its relationship to insecticidal cyclopeptide alkaloids, the vignatic acids, in mungbean (Vigna radiata L. Wilczek)

Bruchid resistance, controlled by a single dominant gene (Br) in a wild mungbean accession (TC1966), has been incorporated into cultivated mungbean (Vigna radiata). The resistance gene simultaneously confers inhibitory activity against the bean bug, Riptortus clavatus Thunberg (Hemiptera: Alydidae). The resultant isogenic line (BC₂₀ generation) was characterized by the presence of a group of novel cyclopeptide alkaloids, called vignatic acids. A linkage map was constructed for Br and the vignatic acid gene (Va) using restriction fragment length polymorphism (RFLP) markers and a segregating BC₂₀F₂ population. By screening resistant and susceptible parental lines with 479 primers, eight randomly amplified polymorphic DNA (RAPD) markers linked to Br were identified and cloned for use as RFLP probes. All eight RAPD-based markers, one mungbean, and four common bean genomic clones were effectively integrated around Br within a 3.7-cM interval. Br was mapped to a 0.7-cM segment between a cluster consisting of six markers and a common bean RFLP marker, Bng110. The six markers are closest to the bruchid resistance gene, approximately 0.2?cM away. The vignatic acid gene, Va, cosegregated with bruchid resistance. However, one individual was identified in the BC₂₀F₂ population that retained vignatic acids in spite of its bruchid susceptibility. Consequently, Va was mapped to a single locus at the same position as the cluster of markers and 0.2?cM away from Br. These results suggest that the vignatic acids are not the principal factors responsible for bruchid resistance in V. radiata but will facilitate the use of map-based cloning strategies to isolate the Br gene.     

Mapping a resistance gene in wheat cultivar Yangfu 9311 to yellow mosaic virus, using microsatellite markers

Wheat yellow mosaic disease, which is caused by wheat yellow mosaic bymovirus (WYMV) and transmitted by soil-borne fungus, results in severe damage on wheat (Triticum aestivum L.) production in China. For development of resistant cultivars to reduce wheat yield losses due to wheat yellow mosaic disease, resistance test and genetic analysis indicated that a single dominant gene in wheat cultivar Yangfu 9311 contributed to the resistance. Bulk segregant analysis was used to identify microsatellite markers linked to the resistance gene in an F₂ population derived from the cross Yangfu 9311 (resistant) × Yangmai 10 (susceptible). Microsatellite markers Xwmc41, Xwmc181, Xpsp3039, and Xgwm349 were co-dominantly or dominantly linked with the gene responsible for WYMV resistance at a distance of 8.1–11.6 cM. Based on the wheat microsatellite consensus map and the results from amplification of the cultivar Chinese Spring nulli-tetrasomic stocks, the resistance gene to wheat yellow mosaic disease derived from Yangfu 9311, temporarily named as YmYF, was thus mapped on the long arm of chromosome 2D (2DL).     

Infectivity analysis of two variable DNA B components of<Emphasis Type="Italic">Mungbean yellow mosaic virus-Vigna</Emphasis> in<Emphasis Type="Italic">Vigna mungo</Emphasis> and<Emphasis Type="Italic">Vigna radiata</Emphasis>

Mungbean yellow mosaic virus-Vigna (MYMV-Vig), aBegomovirus that causes yellow mosaic disease, was cloned from field-infected blackgram (Vigna mungo). One DNA A clone (KA30) and five different DNA B clones (KA21, KA22, KA27, KA28 and KA34) were obtained. The sequence identity in the 150-nt common region (CR) between DNA A and DNA B was highest (95%) for KA22 DNA B and lowest (85·6%) for KA27 DNA B. The Rep-binding domain had three complete 11 -nt (5’-TGTATCGGTGT-3′) iterons in KA22 DNA B (and KA21, KA28 and KA34), while the first iteron in KA27 DNA B (5’-ATCGGTGT-3’) had a 3-nt deletion. KA27 DNA B, which exhibited 93·9% CR sequence identity to the mungbean-infecting MYMV, also shared the 3-nt deletion in the first iteron besides having an 18-nt insertion between the third iteron and the conserved nonanucleotide. MYMV was found to be closely related to KA27 DNA B in amino acid sequence identity of BV1 (94·1%) and BC1 (97·6%) proteins and in the organization of nuclear localization signal (NLS), nuclear export signal (NES) and phosphorylation sites. Agroinoculation of blackgram (V. mungo) and mungbean (V. radiata) with partial dimers of KA27 and KA22 DNA Bs along with DNA A caused distinctly different symptoms. KA22 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in blackgram. In contrast, KA27 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in mungbean. Thus, DNA B of MYMV-Vig is an important determinant of host-range betweenV. mungo andV. radiata.     

Genetic localization of a bruchid resistance gene and its relationship to insecticidal cyclopeptide alkaloids, the vignatic acids, in mungbean ( Vigna radiata L. Wilczek)

Bruchid resistance, controlled by a single dominant gene (Br) in a wild mungbean accession (TC1966), has been incorporated into cultivated mungbean (Vigna radiata). The resistance gene simultaneously confers inhibitory activity against the bean bug, Riptortus clavatus Thunberg (Hemiptera: Alydidae). The resultant isogenic line (BC₂₀ generation) was characterized by the presence of a group of novel cyclopeptide alkaloids, called vignatic acids. A linkage map was constructed for Br and the vignatic acid gene (Va) using restriction fragment length polymorphism (RFLP) markers and a segregating BC₂₀F₂ population. By screening resistant and susceptible parental lines with 479 primers, eight randomly amplified polymorphic DNA (RAPD) markers linked to Br were identified and cloned for use as RFLP probes. All eight RAPD-based markers, one mungbean, and four common bean genomic clones were effectively integrated around Br within a 3.7-cM interval. Br was mapped to a 0.7-cM segment between a cluster consisting of six markers and a common bean RFLP marker, Bng110. The six markers are closest to the bruchid resistance gene, approximately 0.2 cM away. The vignatic acid gene, Va, cosegregated with bruchid resistance. However, one individual was identified in the BC₂₀F₂ population that retained vignatic acids in spite of its bruchid susceptibility. Consequently, Va was mapped to a single locus at the same position as the cluster of markers and 0.2 cM away from Br. These results suggest that the vignatic acids are not the principal factors responsible for bruchid resistance in V. radiata but will facilitate the use of map-based cloning strategies to isolate the Br gene. Received: 20 November 1997 / Accepted: 6 January 1998     

RAPD-based SCAR marker SCA 12 linked to recessive gene conferring resistance to anthracnose in sorghum [Sorghum bicolor (L.) Moench]

Anthracnose, caused by Colletotrichum graminicola, infects all aerial parts of sorghum, Sorghum bicolor (L.) Moench, plants and causes loss of as much as 70%. F₁ and F₂ plants inoculated with local isolates of C. graminicola indicated that resistance to anthracnose in sorghum accession G 73 segregated as a recessive trait in a cross with susceptible cultivar HC 136. To facilitate the use of marker-assisted selection in sorghum breeding programs, a PCR-based specific sequence characterized amplified region (SCAR) marker was developed. A total of 29 resistant and 20 susceptible recombinant inbred lines (RILs) derived from a HC 136 × G 73 cross was used for bulked segregant analysis to identify a RAPD marker closely linked to a gene for resistance to anthracnose. The polymorphism between the parents HC 136 and G 73 was evaluated using 84 random sequence decamer primers. Among these, only 24 primers generated polymorphism. On bulked segregant analysis, primer OPA 12 amplified a unique band of 383 bp only in the resistant parent G 73 and resistant bulk. Segregation analysis of individual RILs showed the marker OPA 12₃₈₃ was 6.03 cM from the locus governing resistance to anthracnose. The marker OPA 12₃₈₃ was cloned and sequenced. Based on the sequence of cloned RAPD product, a pair of SCAR markers SCA 12-1 and SCA 12-2 was designed using the MacVector program, which specifically amplified this RAPD fragment in resistant parent G 73, resistant bulk and respective RILs. Therefore, it was confirmed that SCAR marker SCA 12 is at the same locus as RAPD marker OPA 12₃₈₃ and hence, is linked to the gene for resistance to anthracnose.     

Molecular Confirmation of Intraspecific Tomato (<Emphasis Type="Italic">Solanum lycopersicum)</Emphasis> Hybrids and Their Evaluation Against Late Blight and Cucumber Mosaic Virus

Tomato is one of the most consumed vegetables in the world. Diseases are the number one concern in the development of high-yield and disease-resistant tomato hybrids which is the foremost priority of breeders. Present study was conducted (1) to develop DNA-based markers for genetic confirmation of tomato F₁ hybrids, (2) to utilize sequenced characterized amplified region (SCAR) marker linked to the Ph-3 gene for Phytophthora infestans resistance in tomato and (3) to evaluate male and female parental genotypes and their F₁ hybrids against late blight (LB) and cucumber mosaic virus (CMV). For molecular studies, 58 previously reported markers including RAPDs (10), SCAR (01), EST-SSR (01) and SSR (46) were applied. The SCAR marker clearly differentiated the LB3 and LB4 from Roma and T-1359 and provided evidence for Ph-3 gene. The SCAR marker was able to confirm the Ph-3 gene in the hybrids Roma × LB4, Roma × LB3, Riogrande × LB2, Riogrande × LB3 and Roma × LB7. Out of several tested primers, SSR-22 proved useful for genetic confirmation of F₁ hybrid TMS1 × Money Maker (MM). For LB, tested hybrids/genotypes were ranked as susceptible to highly susceptible with different infection percentage (IP). However, the pace of symptom development was slower in hybrid Rio × LB2, 45% IP after 10 days of inoculation compared with 85% disease in one of the parent genotypes (Riogrande). None of the tested genotypes was found resistant; however, TMS1 responded as tolerant against CMV using mechanical inoculation. Under natural field conditions, TMS1 was found resistant while hybrids TMS1 × Naqeeb and TMS1 × MM were tolerant where as others were found to be susceptible. In conclusion, all tomato hybrids were genetically confirmed using DNA-based markers. SCAR marker was useful for marker-assisted confirmation of the Ph-3 gene in parental lines and hybrids; however, this gene was unable to provide protection against the local population of P. infestans.