Maurilio Alves Moreira

Characterization and genetic diversity analysis of cotton cultivars using microsatellites

Genetic diversity and the relationship between varieties are of great importance for cotton breeding. Our work was designed to estimate the informativeness of the cotton (Gossypium hirsutum L.) simple sequence repeat (SSR) microsatellite locus and to estimate the genetic distance between 53 cotton cultivars as well as to select a set of SSR primers able to differentiate between the 53 cotton cultivars studied. After extracting DNA from the 53 cultivars and characterized it using 31 pairs of SSR primers we obtained a total of 66 alleles with an average of 2.13 alleles per SSR locus and values of polymorphism information content (PIC) varying from 0.18 to 0.62, the dissimilarity coefficient varying from zero to 0.41. Statistical analysis using the unweighted pair-group method using arithmetic average (UPGMA) revealed seven subgroups which were consistent with the genealogical information available for some of the cultivars. The SSR genetic profile obtained for each of the cultivars made it possible to discriminate 52 of the 53 cultivars. This study of the genetic diversity of cotton cultivars with SSR markers support the need to introduce new alleles into the gene pool of the breeding cultivars.

Characterization of α-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. Enzymic hydrolysis of the RO is accomplished by alpha-galactosidase. While the content of RO decreases during seed germination, the activity of alpha-galactosidase increases substantially. Two alpha-galactosidases were isolated from germinating seeds by partition in an aqueous two-phase system followed by ion-exchange and affinity chromatography. One of the enzyme preparations (P1) showed a single protein with M(r) of 33 kDa, and the second (P2) had two proteins with M(r) of 31 and 33 kDa. Maximal activities against the synthetic substrate rho-nitrophenyl-alpha-D-galactopyranoside (rhoNPGal) were detected at pH 5.0-5.5 and 45-50 degrees C. Both enzymes were fairly stable at 40 degrees C, but lost most of their activities after 30 min at 50 degrees C. The K(m) values for hydrolysis of rhoNPGal by the P1 and P2 enzymes were 1.55 and 0.76 mM, respectively. The K(m) values determined for hydrolysis of raffinose and melibiose by the P2 enzyme were 5.53 and 5.34 mM, respectively and galactose was a competitive inhibitor (K(i)=0.65 mM). To different extents, both enzymes were sensitive to inhibition by galactose, melibiose, CuSO(4), and SDS. Sucrose and beta-mercaptoethanol showed discrete inhibitory effects on both enzymes.

Characterization of the anthracnose resistance gene present in Ouro Negro (Honduras 35) common bean cultivar

Ouro Negro (Honduras 35) is a highly productive Mesoamerican black seeded bean cultivar that possesses a major dominant gene conferring resistance to anthracnose (causal organism Colletotrichum lindemuthianum). In this work the anthracnose resistance gene present in Ouro Negro was characterized by studying allelic relationships to the following previously characterized anthracnose resistance genes (cultivars): Co-1 (MDRK), Co-12 (Kaboon), Co-13 (Perry Marrow), Co-2 (Cornell 49-242), Co-3 (Mexico 222), Co-4 (TO), Co-42 (SEL 1308), Co-5 (SEL1360), Co-6 (AB 136), and the resistance genes present in PI 207262 and Widusa. In addition, we determined the resistance spectrum of Ouro Negro in relation to 19 pathotypes of C. lindemuthianum. The allelism tests confirmed that the dominant anthracnose resistance gene present in Ouro Negro is positioned at a locus distinct from those with which it was compared. We propose that this new gene be named Co-10. The inoculation of Ouro Negro with the 19 pathotypes of C. lindemuthianum demonstrated that Co-10 confers resistance to pathotypes 23, 64, 67, 73, 81, 83, 87, 89, 95, 102, 117, 119, 343, 453, 1033, 1545 and 1600. The identification of Co-10 is an important contribution to bean breeding programs that are in constant need of new sources of resistance to anthracnose.

Use of molecular markers to accelerate the breeding of common bean lines resistant to rust and anthracnose

The main goal of this work was to introduce resistance genes for rust, caused by Uromyces appendiculatus, and anthracnose, caused by Colletotrichum lindemuthianum, in an adapted common bean cultivar through marker-assisted backcrossing. DNA fingerprinting was used to select plants genetically closer to the recurrent parent which were also resistant to rust and to race 89 of C. lindemuthianum. DNA samples extracted from the resistant parent (cv. Ouro Negro), the recurrent parent (cv. Rudá), and from BC1, BC2 and BC3 resistant plants were amplified by the RAPD technique. The relative genetic distances in relation to the recurrent parent varied between 9 and 59% for BC1, 7 and 33% for BC2, and 0 and 7% for BC3 resistant plants. After only three backcrosses, five lines resistant to rust and anthracnose with, approximately, 0% genetic distance in relation to the recurrent parent were obtained. These lines underwent field yield tests in two consecutive growing seasons and three of them presented a good yield performance, surpassing in that sense their parents and most of the reference cultivars tested.

Genetic diversity of Phaeoisariopsis griseola in the State of Minas Gerais, Brazil

Due to the importance of common bean angular leaf spotin the state of Minas Gerais-Brazil and to the greatvariability of the pathogen, Phaeoisariopsisgriseola, monitoring races becomes an important toolfor breeding programs aiming at genetic resistance.The pathogenic variability of 30 isolates of the P. griseola, collected from various locations in thestate of Minas Gerais, was studied using the followingcommon bean differential series (Don Timóteo,Bolón Bayo, Montcalm, G 5686, Amendoin, G 11796,BAT 332, PAN 72, Cornell 49-242, México 54, Florde Mayo and G 2858). The first trifoliate leaf wasinoculated with a 2 × 104 conidia/mL. Plants weremaintained at 20–22 °C and 95% relativehumidity for 48 hours. Symptom evaluation wasperformed 15 days after inoculation. Thirteen raceswere identified demonstrating the wide geneticvariability of the pathogen in the state of MinasGerais. Race 63.63 was the most virulent, whereas race63.23 was the most frequent (10 of 30 isolates), beingwidely distributed among the regions studied. Thevirulence phenotype indicated that the races studiedbelonged to the Mesoamerican group, which wasconfirmed when the 30 isolates were compared to Andeanand Mesoamerican standards using RAPD markers.

Inheritance of angular leaf spot resistance in common bean line BAT 332 and identification of RAPD markers linked to the resistance gene

The existence of genetic variability for angular leaf spot (ALS) resistance in the common bean germplasm allows the development of breeding lines resistant to this disease. The BAT 332 line is an important resistance source to common bean ALS. In this work we determined the inheritance pattern and identified RAPD markers linked to a resistance gene present in BAT 332. Populations F1, F2,BCs and BCr derived from crosses between BAT 332 and cultivar Rudá were used. Rudá is a commercial cultivar with carioca type grains and susceptible to ALS. The resistance of BAT 332 to race 61.41 of the pathogen was confirmed. Segregation analysis of the plants indicated that a single dominant gene confers resistance. For identification of RAPD markers linked to the resistance gene, bulk segregant analysis (BSA) was used. Two RAPD markers,OPAA07950 and OPAO12950, linked in coupling phase at 5.10 and 5.83 cM of this gene, respectively, were identified. These molecular markers are important for common bean breeders and geneticists as source of genetic information and for marker assisted selection in breeding programs.

Improved Selection with Newly Identified RAPD Markers Linked to Resistance Gene to Four Pathotypes of Colletotrichum lindemuthianum in Common Bean.

ABSTRACT Three F(2) populations derived from crosses between the resistant cultivar AB 136 and the susceptible cultivar Michelite (MiA), and one F(2) population derived from a cross between AB 136 and Mexico 222 (MeA), were used to identify markers linked to anthracnose resistance genes present in cultivar AB 136. Primer OPZ04 produced a DNA band (OPZ04(560)) linked in coupling phase to the resistance gene for pathotype 89 (8.5 +/- 0.025 cM) in one population derived from the cross MiA. In the same population, primer OPZ09 produced one band (OPZ09(950)) linked in repulsion phase (20.4 +/- 0.014 cM) to the same resistance gene. The simultaneous use of markers in coupling and in repulsion phases allowed the identification of the three genotypic classes. In the other two populations from cross MiA, OPZ04(560) was linked in coupling phase to resistance genes for pathotypes 73 (2.9 +/- 0.012 cM) and 81 (2.8 +/- 0.017 cM). In population MeA, OPZ04(560) was linked in coupling phase (7.5 +/- 0.033 cM) to resistance to pathotype 64. These data suggest that a single gene or complex locus of linked resistance genes present in cultivar AB 136 confers resistance to all four pathotypes of C. lindemuthianum.

Genetic parameters relating isoflavone and protein content in soybean seeds

Isoflavones are a class of compounds present in high amounts in soybean seeds, which can be used for prevention and treatment of several chronic diseases. Proteins present in soybean seeds are the basis for the high nutritional value and versatility of this leguminous species in animal and human feeding. The main goals of this work were to estimate heritabilities for isoflavone contents in soybean seeds and the correlation between isoflavone and protein contents. Commercial variety IAC-100 (high isoflavone and normal protein contents) and the line BARC-8 (low isoflavone and high protein contents) were crossed, and one single F1 plant derived 97 F2 seeds, which were used to obtain F3 seeds. A sample of F3 seeds from each F2 plant was used for isoflavone determination by HPLC and protein by the Kjeldahl method. Six isoflavone forms were detected: daidzin, genistin, glycitin, malonyldaidzin, malonylgenistin and malonylglycitin. Total isoflavone contents ranged from 427.92 to 965.89 μg per gram of dry seed and the protein content ranged from 45.17 to 34.95% in BARC-8 and IAC-100, respectively. Our results indicate that it is possible to select for high isoflavone content in early breeding generations because the broad sense heritabilities for the contents of the various isoflavone forms were higher than 90%. In addition, high correlation values among the contents of the individual isoflavone forms were observed (between 0.80 and 0.98). However, negative correlation values were obtained between isoflavone and protein contents, ranging from −0.51 to −0.37 for the different isoflavone forms. The correlation value of −0.47 between total isoflavone and protein contents confirmed the negative correlation between these two parameters, as reported by other authors.

Allelic relationships for genes that confer resistance to angular leaf spot in common bean

Angular leaf spot is one of the major diseases of the common bean. The extensive genetic variability of this pathogen requires the constant development of new resistant cultivars. Different sources of resistance have been identified and characterized. For the State of Minas Gerais, Brazil, four main resistance sources were found: Mexico 54, AND 277, MAR 2 and Cornell 49-242. Independent characterization of these genotypes demonstrates that resistance in all four sources is dominant and monogenic. However, there are no studies on the relationship and independence of these genes. In the present work, allelism tests were carried out to understand the relationship among the resistance genes present in these four resistance sources. The data revealed a much higher complexity in the resistance inheritance of these genes than previously reported. It was demonstrated that Cornell 49-242 possesses a dominant gene (Phg-3); Mexico 54 possesses three genes, denominated Phg-2, Phg-5 and Phg-6. In MAR 2, two genes were found, one independent designated Phg-4 and the other, an allelic form of Phg-5, denominated of Phg-52. Allelic forms were also found in AND 277, Phg-22, Phg-32 and Phg-42. These results have special importance for breeding programs aiming to pyramid resistance genes.

RAPD markers linked to a block of genes conferring rust resistance to the common bean

Abstract Rust, caused by the fungus Uromyces appendiculatus , may cause a significant loss to common bean ( Phaseolus vulgaris L.) yield.RAPD markers tightly linked to the resistance genes may be used in breeding programs to aid the development of rust-resistant b eancultivars. In this sense, the objective of the present work was to identify RAPD markers linked to a rust resistance gene block present inthe cultivar Ouro Negro. Two hundred and fourteen F 2 individuals from a cross between the resistant cultivar Ouro Negro and thesusceptible cultivar US Pinto 111 were inoculated with a mixture of eight races of U. appendiculatus . The segregation ratio obtainedsuggested that resistance is monogenic and dominant. Bulked segregant analysis was used in conjunction with the RAPD technique tosearch for markers linked to rust resistance genes. Two molecular markers flanking the rust resistance gene block were identifi ed, one at5.8 ± 1.6 cM (OX11 630 ) and the other at 7.7 ± 1.7 cM (OF10