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Joseph J. Rothleutner, Mara W. Friddle, and Ryan N. Contreras

determined by analysis of nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI) using Pisum sativum ‘Ctirad’ (2C = 8.76 pg) as an internal standard, the difference between genome size estimates between fluorochromes and bp

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Brian M. Schwartz, Ryan N. Contreras, Karen R. Harris-Shultz, Douglas L. Heckart, Jason B. Peake, and Paul L. Raymer

subsequently reanalyzed for 2C nuclear DNA content, nuclei were stained with a 2.0 mL solution containing 0.3% ribonuclease (RNase) and 0.6% propidium iodide (PI). Samples were incubated at 4 °C for a minimum of 1 h and analyzed by flow cytometry at Oregon

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Ryan N. Contreras and Kimberly Shearer

– AT%. Table 4. Base pair composition of 48 Acer taxa determined by comparing holoploid (2C) genome size determined using flow cytometry analysis of nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) using Pisum

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Rengong Meng and Chad Finn

Nuclear DNA flow cytometry was used to differentiate ploidy level and determine nuclear DNA content in Rubus. Nuclei suspensions were prepared from leaf discs of young leaves following published protocols with modifications. DNA was stained with propidium iodide. Measurement of fluorescence of 40 genotypes, whose published ploidy ranged from diploid to dodecaploid, indicated that fluorescence increased with an increase in chromosome number. Ploidy level accounted for 99% of the variation in fluorescence intensity (r 2 = 0.99) and variation among ploidy levels was much higher than within ploidy levels. This protocol was used successfully for genotypes representing eight different Rubus subgenera. Rubus ursinus Cham. and Schldl., a native blackberry species in the Pacific Northwest, which has been reported to have 6x, 8x, 9x, 10x, 11x, and 12x forms, was extensively tested. Genotypes of R. ursinus were predominantly 12x, but 6x, 7x, 8x, 9x, 11x, and 13x forms were found as well. Attempts to confirm the 13x estimates with manual counts were unsuccessful. Ploidy level of 103 genotypes in the USDA-ARS breeding program was determined by flow cytometry. Flow cytometry confirmed that genotypes from crosses among 7x and 4x parents had chromosome numbers that must be the result of nonreduced gametes. This technique was effective in differentiating chromosome numbers differing by 1x, but was not able to differentiate aneuploids. Nuclear DNA contents of 21 diploid Rubus species from five subgenera were determined by flow cytometry. Idaeobatus, Chamaebatus, and Anaplobatus were significantly lower in DNA content than those of Rubus and Cylactis. In the Rubus subgenus, R. hispidus and R. canadensis had the lowest DNA content and R. sanctus had the highest DNA content, 0.59 and 0.75 pg, respectively. Idaeobatus had greater variation in DNA content among diploid species than the Rubus subgenus, with the highest being from R. ellipticus (0.69 pg) and lowest from R. illecebrosus (0.47 pg).

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J.R. Bohac

Sweetpotato, Ipomoea batatas is in the morning glory family, Convolvulaceae, genus Ipomoea, group Batatas. It has many wild Ipomoea relatives that serve as a reservoir of many needed pest and stress-resistance genes. A major barrier to introgression of useful genes is the ploidy gap—sweetpotato is a hexaploid and wild Ipomoeas are diploids and tetraploids. The wild species can be successfully crossed using 2n pollen or by first increasing ploidy by colchicine treatment. The ploidy of such hybrid offpsring can be determined by DNA flow cytometry. My objective was to develop a technique to determine DNA content in Ipomoea and values for DNA content for the major Ipomoea species using the EPIC flow cytometer with a UV detector. Nuclei were extracted and pretreated with cellulase and pectolyase before staining with propidium iodide (PI). A highly linear relationship was found between the DNA content determined by DNA flow cytometry and the ploidy of the closest sweetpotato relatives as determined by chromosome counts. These species were diploid I. trifida, tetraploid I. batatas, and hexaploid I. batatas. DNA content was most similar among other diploid Ipomoea species in the group Batatas and was significantly different in other Ipomoeas not in group Batatas.

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Guangxin Liu, Yue Lan, Haoyang Xin, Fengrong Hu, Zhuhua Wu, Jisen Shi, and Mengli Xi

Lily (Lilium L.) species produce among the most important cut flowers worldwide. China has ≈55 species of Lilium. Although many plants from this genus have been used in hybridization efforts, their cytology has remained unclear. The goal of the current study was to characterize the chromosomes of Lilium rosthornii Diels. Root tips were used to characterize Giemsa C-banding, propidium iodide (PI) banding, and 45S rDNA locations. The karyotype of L. rosthornii belongs to type 3B. C-banding revealed polymorphic banding patterns with the following formula: 2n = 24 = CI = 4C + 14CI+ + 2I+ +2I+ 2. Two of the four 45S rDNA hybridization sites were located at pericentromeric positions on the two short arms of the homologues of chromosome 1, and the other two were located on the long arms of one chromosome 6 homolog and one chromosome 11 homolog. Six of the eight PI bands were detected in the centromeres of the homologues of chromosomes 1, 5, and 8, and the other two PI bands were detected on the long arms of one chromosome 6 and one chromosome 11. Lilium rosthornii showed enriched banding in both Giemsa C-banding and PI painting. Interestingly, not all 45S rDNA was located in homologous chromosomal locations. These results may provide reference data for L. rosthornii for use in further Lilium breeding.

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Hirotoshi Tsuda, Hisato Kunitake, Yo Aoki, Akiko Oyama, Takuya Tetsumura, Haruki Komatsu, and Katsunori Yoshioka

We tested efficient in vitro methods for screening the genotypes with higher pH tolerance using multiple shoots of intersectional hybrids between Vaccinium corymbosum ‘Spartan’ and V. bracteatum. The response of the four hybrid clones tested to different pH levels was clone-dependent in vitro. An apparent difference was found in the rooting rate among the hybrid clones even at higher pH levels; the rooting rates of JM4 (91%) at pH 8.0 indicated a significantly high value compared with other clones (JM1: 24%, JM2: 9%, JM3: 8%, ‘Spartan’: 0%). Furthermore, JM4 showed constantly high rooting rates (91% to 100%) at all pH levels with no significant differences. Similar differences in the root characters of the hybrids were also confirmed by checking the viability of roots using fluorescein diacetate (FDA)/propidium iodide (PI) staining after dipping the roots of in vitro-produced shoots in liquid medium at different pH levels for 6 hours. These results suggest that an in vitro screening method using the rooting rate of multiple shoots and the viability test of roots by FDA/PI staining as a marker could become a very useful tool for the selection of germplasm with tolerance to higher pH within a short time using small planting spaces. In addition, JM4, which showed a high rooting rate at pH 8.0, could be useful in breeding new cultivars with higher pH tolerance.

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Josue Ortega-Ortega, Francisco Arturo Ramírez-Ortega, Roberto Ruiz-Medrano, and Beatriz Xoconostle-Cázares

Coffee is an important crop worldwide, grown on about 10 million hectares in tropical regions including Latin America, Africa, and Asia. The genus Coffea includes more than 100 species; most are diploid, except for C. arabica, which is allotetraploid and autogamous. The genetic diversity of commercial coffee is low, likely due to it being self-pollinating, in addition, the widespread propagation of few selected cultivars, such as Caturra, Bourbon, and Typica. One approach is the analysis of genome size in these cultivars as a proxy to study its genetic variability. In the present work, genome size of 16 cultivars was assessed through high-resolution flow cytometry (FCM). Nuclear DNA was analyzed using a modified procedure that uses propidium iodide (PI) and 4′,6′-diamino-2-phenylindole dihydrochloride hydrate (DAPI) staining. The C. arabica cultivars investigated possessed a nuclear DNA content ranging from 2.56 ± 0.016 pg for Typica, to 3.16 ± 0.033 pg for ICATU, which had the largest genome size. All cultivars measured using both fluorochromes had greater estimates with DAPI than PI. The proportion of the genome composed of guanosine and cytosine (GC%) among the cultivars evaluated in this study ranged from 37.03% to 39.22%. There are few studies of genome size by FCM of distinct important C. arabica cultivars, e.g., hybrids and artificial crosses. Thus, this work could be valuable for coffee breeding programs. The data presented here are intended to expand the genomic understanding of C. arabica and could link nuclear DNA content with evolutionary relationships such as diversification, hybridization and polyploidy.

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Hamidou F. Sakhanokho and Nurul Islam-Faridi

, Germany) with a mercury lamp (HBO-100; Partec). The flow cytometer had been modified and upgraded with a filter combination (03-340-1000; Partec) adapted to recognize both propidium iodide (PI) and ultraviolet stains depending on its configuration. 4

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Ying Wang, Cale A. Bigelow, and Yiwei Jiang

analyzed by the Cytomics Expo32 software. The amount of propidium iodide (PI) intercalating in the double-stranded DNA is proportional to DNA content for the sample. The fluorescence intensity of PI measured by the flow cytometer is also proportional to DNA