High-resolution crossover maps for each bivalent of Zea mays using recombination nodules

Recombination nodules (RNs) mark sites of crossing over along pachytene synaptonemal complexes (SCs). Thus, RNs provide the highest resolution cytological marker currently available for defining the frequency and distribution of crossovers along the length of chromosomes because they are observed by electron microscopy. Using the maize inbred line KYS, we have prepared an SC karyotype in which each SC was identified by relative length and arm ratio and related to the proper linkage group using inversion heterozygotes. We mapped 4272 RNs on 2080 identified SCs to produce high-resolution maps of RN frequency and distribution on each bivalent. Average RN frequency per bivalent is closely correlated with SC length. The total length of the RN map is about two-fold shorter than most linkage maps, but there is good correspondence between the relative lengths of the different maps when individual bivalents are considered. Each bivalent has a unique distribution of crossing over, but all bivalents share a high frequency of distal RNs and a severe reduction of RNs at and near kinetochores. The frequency of RNs at knobs is either similar to or higher than the average frequency of RNs along the SCs. These RN maps represent an independent measure of crossing over along maize bivalents.

Methods
SC spreads were prepared on plastic-coated slides as described by STACK and ANDERSON (2002). SC spreads were stained with 2% uranyl acetate followed by Reynold’s lead citrate (UP) or with 33% silver nitrate (SHERMAN et al. 1992). The slides were scanned using phase light microscopy, and good SC spreads on plastic were picked up onto 50 or 75 mesh grids. The grids were examined in an AEI 801 electron microscope, and SC spreads without detectable stretching and with kinetochores were photographed at a magnification of 1600X or 2500X. In total, 2080 individual SCs from 290 sets were identified and mapped with regard to RNs. Both total RN number and total SC set length (the combined length of all SCs in a cell) could be assessed for 206 of these SC sets. In the remainder of the sets, certain of the SCs could not be identified, usually due to unclear or missing kinetochores. Electron microscope negatives were scanned into a computer using a Hewlett-Packard ScanJet 4c and Adobe Photoshop (version 5.0) software. Montages of SC spreads were assembled using Photoshop. Proper tracing of each SC and the position of kinetochores and RNs were determined directly from the negatives using an 8X magnifying loupe and recorded onto prints of the montages. One lateral element from each SC was measured in micrometers using the computer program MicroMeasure (REEVES 2001). Total SC length varied from set to set, but the relative length of SCs and SC arms, i.e., arm ratios, within each set remained consistent. SCs were identified by relative lengths (percentages of total SC length for the set) and arm ratios (length of long arm divided by length of short arm). RNs were recognized using criteria of size, shape, staining intensity, and association with SC as described by STACK and ANDERSON (2002). RN positions were measured and expressed as a percentage of the arm length from the centromere. Using average lengths for each of the 10 SCs and their average arm ratios, each of the SCs was divided into 0.2 μm segments, and each observed RN was placed into one of these segments based on its original relative position from the centromere. After compiling the RN data, the genetic map length of each SC was calculated by determining the average number of RNs per SC and then multiplying by 50 (one RN = one crossover = 50 cM).