Flow cytometric analysis of nuclear DNA content,mitotic chromosome number and protein separation by SDS-PAGE in three accessions of African locust bean (<Emphasis Type="Italic">Parkia biglobosa</Emphasis> Benth.)

Nuclear DNA of three accessions of Parkia biglobosa collected from three locations in northern Cross River State was investigated using a Patec PA II flow cytometer equipped with an argon ion laser (488 nm), and pictures of mitotic chromosomes were taken using a digital micro-camera (Canon) placed on the eye piece of a binocular microscope at 100X oil immersion. Metaphase chromosome counts of 2n = 22 for accessions A and C and 2n = 24 for accession B, were obtained and through flow cytometry, the three accessions were confirmed to be diploids. The nuclear DNA content and genome size for the accessions were 1.5085, 1.489, and 1.5266 pg (737.7054, 728.121, and 746.5074 Mbp) for accessions A, B, and C, respectively. In another experiment, leaf samples from greenhouse-germinated seeds were analyzed for variation in the banding pattern of the protein by SDS-PAGE in the three accessions. Protein was resolved into three banding groups according to their electrophoretic mobility: slow, medium, and fast, clustering between 100–200, 40–70, and 10–25 kDa, respectively. There was 76% similarity in the banding pattern between the accessions.     

Estimating canopy cover from color digital camera image of rice field

Canopy cover (CC) is a good predictor variable for plant growth parameters such as leaf area index and aboveground biomass. A nondestructive, low-cost, and convenient method is presented for estimating CC using digital camera image analysis. CC was estimated by the ratio of plant pixels to total pixels of digital camera image of rice field. To determine the criteria for segmenting the rice plant from variable soil background, three mosaic images for rice plant, flooded/bare soil, and algae-infested background were prepared from digital camera images that were taken in various field conditions. An image analysis program was developed in Visual Basic to extract red, green, and blue (RGB) features from the mosaic images, calculate RGB-based color indices, and compute the minimum segmentation error for separating rice plant from background. When judged by the segmentation error, modified excessive green index (MEGI) showed the highest potential for segmenting rice plant from flooded/bare soil background, followed by normalized green (g) and excessive green index (EGI). At the threshold MEGI value of 0.03, the segmentation error was the lowest as 0.13%. Any single index considered was not satisfactory in segmenting rice plant from algae-infested background. However, a discriminant function of 1.2553EGI + 0.01735G − 0.01474B was successful in segmenting rice plant from flooded/bare soil and algaeinfested background with segmentation errors of 0.34 and 1.17%, respectively. CC for four rice varieties from tillering to booting stage was estimated based on the threshold value of MEGI and discriminant function and also manually using commercial software. Both estimates of CC showed good relationship of r² = 0.94, suggesting that a digital camera could be used efficiently for measuring the CC of rice field.     

Analysis of rice blast resistance gene <Emphasis Type="Italic">Pi</Emphasis>-<Emphasis Type="Italic">z</Emphasis> in rice germplasm using pathogenicity assays and DNA markers

The Pi-z gene in rice confers resistance to a wide range of races of the rice blast fungus, Magnaporthe oryzae. The objective of this study was to characterize Pi-z in 111 rice germplasm accessions using DNA markers and pathogenicity assays. The existence of Pi-z in rice germplasm was detected by using four simple sequence repeat (SSR) markers (RM527, AP4791, AP5659-1, AP5659-5) closely linked to Pi-z, and was verified using pathogenicity assays with an avirulent strain (IE1k) and two virulent races (IB33 and IB49). Among 111 germplasm accessions evaluated, 73 were found to contain the Pi-z gene using both SSR markers and pathogenicity assays. The remaining 38 germplasm accessions were found to be inconsistent in their responses to the blast races IB33, IEIk and IB49 with expected SSR marker alleles, suggesting the presence of unexpected SSR alleles and additional R gene(s). These characterized germplasm can be used for genetic studies and marker-assisted breeding for improving blast resistance in rice.     

Genetic and cytological analysis of a new spontaneous male sterility in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

A male sterile plant appeared in the radish breeding program at the Hubei Academy of Agricultural Sciences, Hubei, China. In its progeny, a two-type (half of plants male sterile, the other half male fertile) line 01GAB was established. An F₂ population of 260 plants from a cross of male-sterile 01GAB and a male fertile line 9802H segregated for male fertility in a 3:1 ratio indicating that fertility was restored by a single dominant gene, here designated RsMs. A PCR-based DNA marker specific to the male fertility Rfob gene in 9802H was absent in 01GAB. Linkage analysis placed the RsMs locus 10.7 cM away from the Rfo locus. In an F₂ population of hybrids between 01GAB and male fertile 9802B, a co-dominant DNA marker for the RSultr3.2A (a radish sulfate transporter gene) locus was linked to the RsMs locus at 1.5 cM suggesting that fertility restoration in 01GAB was located in the region with known male sterility restorers in radish. However, no maintainer for the 01GAB source of male sterility has been identified so far. Cytological observations have shown that the abnormalities in male sterile anthers first appeared in tapetum at the tetrad stage, followed by a hypertrophy of the tapetal cells at the vacuolate microspore period. These results suggest that male sterility in 01GAB is likely to be genetic in nature, or it may represent a new type of the cytoplasmic male sterility.     

Successful induction of trigenomic hexaploid <Emphasis Type="Italic">Brassica</Emphasis> from a triploid hybrid of <Emphasis Type="Italic">B.</Emphasis><Emphasis Type="Italic">napus</Emphasis> L. and <Emphasis Type="Italic">B. nigra</Emphasis> (L.) Koch

A triploid hybrid with an ABC genome constitution, produced from an interspecific cross between Brassica napus (AACC genome) and B. nigra (BB genome), was used as source material for chromosome doubling. Two approaches were undertaken for the production of hexaploids: firstly, by self-pollination and open-pollination of the triploid hybrid; and secondly, by application of colchicine to axillary meristems of triploid plants. Sixteen seeds were harvested from triploid plants and two seedlings were confirmed to be hexaploids with 54 chromosomes. Pollen viability increased from 13% in triploids to a maximum of 49% in hexaploids. Petal length increased from 1.3 cm (triploid) to 1.9 cm and 1.8 cm in the two hexaploids and longest stamen length increased from 0.9 cm (triploid) to 1.1 cm in the hexaploids. Pollen grains were longer in hexaploids (43.7 and 46.3 μm) compared to the triploid (25.4 μm). A few aneuploid offsprings were also observed, with chromosome number ranging from 34 to 48. This study shows that trigenomic hexaploids can be produced in Brassica through interspecific hybridisation of B. napus and B. nigra followed by colchicine treatment.     

Three AFLP markers tightly linked to the genic male sterility <Emphasis Type="Italic">ms</Emphasis><Subscript><Emphasis Type="Italic">3</Emphasis></Subscript> gene in chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) and conversion to a CAPS marker

Genic male sterility (GMS) has long been used as a tool for hybrid seed production in chili pepper (Capsicum annuum L.). We developed DNA markers linked to the GMS ms ₃ gene in a segregating population using bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) techniques. The segregating population was subjected to BSA-AFLP with 512 primer combinations. Three AFLP markers (Eagg/Mccc₂₇₆, Eagc/Mctt₁₇₈, and Ecag/Mtgc₂₀₄) were identified as tightly linked to the ms ₃ locus. Among them, we converted the AFLP marker Ecag/Mtgc₂₀₄ to the cleavage amplified polymorphic sequence (CAPS) marker, named GMS3-CAPS, based on sequencing analysis of internal and flanking regions for the markers between male-fertile and sterile plants. This marker will be useful for pepper breeding using the GMS system.     

Quantitative trait loci influencing fruit-related characteristics of tomato grown in high-temperature conditions

The objective of this study was to identify molecular markers linked to fruit-related traits in the tomato subjected to high temperatures. In total, 160 F₂ plants derived from a cross between a heat-tolerant breeding line, CL5915-93D4-1-0-3 (Solanum esculentum), and a heat-sensitive wild accession, L4422 (S. pimpinellifolium), were grown in a greenhouse. Six traits including fruit number, fruit weight, brix, seed number, fruit setting, and flower number were scored. The distributions of fruit number, fruit set, flower number, and seed number were skewed towards heat susceptibility which is known to be characteristic of L4422. Polymorphic bands were generated by PCR-derived methods of RAPD, ISSR and AFLP Polymorphism, the segregation ratio, and distribution over the genome of the above 3 markers were compared. Ten linkage groups, ranging 20.6–151.6 cM in size, were constructed with 62 informative markers spanning a total of 776.3 cM. Fruit-related quantitative trait loci (QTLs) were non-randomly distributed in the tomato genome. For the 6 traits investigated, 21 QTLs were dispersed on linkage groups 2–5. The genetic effects of the various QTLs were differently exhibited, in our study we have respectively found from 10.5% to 30.2% of the variation explained by the QTL for flower number (FRN4) and brix (BX2). Thirteen QTL-mapped markers were unique to 1 trait, and 4 markers were linked to more than 1 trait. Among them, QTLs linked to the I868-470 marker had effects on fruit weight and brix, and a significant positive correlation between these 2 traits was noted (r = 0.35, P < 0.05). Thus, the I868-470 marker may have the potential for simultaneous selection of high fruit weight and brix. These markers also allowed us to align genome linkage maps across distantly related species and to reveal the co-localization between these QTLs and major genes.     

Bahiagrass crop coefficients from eddy correlation measurements in central Florida

Bahiagrass (Paspalum notatum) is a warm-season grass used primarily in pastures and along highways and other low maintenance public areas in Florida. It is also used in landscapes to some extent because of its drought tolerance. Bahiagrass can survive under a range of moisture conditions from no irrigation to very wet conditions. Its well-watered consumptive use has not been reported previously. In this study, bahiagrass crop coefficients (K _c) for an irrigated pasture were determined for July 2003 through December 2006 in central Florida. The eddy correlation method was used to estimate crop evapotranspiration (ET_c) rates. The standardized reference evapotranspiration (ET_o) equation (ASCE-EWRI standardization of reference evapotranspiration task committee report, 2005) was applied to calculate ET_o values using on site weather data. Daily K _c values were estimated from the ratio of the measured ET_c and the calculated ET_o. The recommended K _c values for bahiagrass are 0.35 for January–February, 0.55 for March, 0.80 for April, 0.90 for May, 0.75 for June, 0.70 for July–August, 0.75 for September, 0.70 for October, 0.60 for November, and 0.45 for December in central Florida. The highest K _c value of 0.9 in May corresponded with maximum vapor pressure deficit conditions as well as cloud free conditions and the highest incoming solar radiation as compared to the rest of the year. During the summer (June to August), frequent precipitation events increased the cloud cover and reduced grass water use. The K _c annual trend was similar to estimated K _c values from another well-watered warm-season grass study in Florida.     

Functional genomics of microspore embryogenesis

Isolated plant microspores, when stressed and cultured in vitro, can be diverted from their normal gametophytic pathway towards sporophytic development, with the formation of haploid embryos and ultimately doubled-haploid plants. This process is called androgenesis or microspore embryogenesis, and is widely used in plant breeding programmes to generate homozygous lines for breeding purposes. Protocols for the induction of microspore embryogenesis and the subsequent regeneration of doubled haploid (DH) plants have been successfully developed for more than 200 species. These practical advances stand in stark contrast to our knowledge of the underlying molecular genetic mechanism controlling this process. The majority of information regarding the genetic and molecular control of the developmental switch from gametophytic to sporophytic development has been garnered from four intensely studied (crop) plants comprising two dicotyledonous species, rapeseed (Brassica napus) and tobacco (Nicotiana tabacum), and two monocotyledonous species, wheat (Triticum aestivum) and barley (Hordeum vulgare). In these species the efficiency of microspore embryogenesis is very high and reproducible, making them suitable models for molecular studies. In the past, molecular studies on microspore embryogenesis have focussed mainly on the identification of genes that are differentially expressed during this developmental transition and/or early in embryo development, and have identified a number of genes whose expression marks or predicts the developmental fate of stressed microspores. More recently, functional genomics approaches have been used to obtain a broad overview of the molecular processes that take place during the establishment of microspore embryogenesis. In this review we summarise accumulated molecular data obtained in rapeseed, tobacco, wheat and barley on embryogenic induction of microspores and define common aspects involved in the androgenic switch.