Brazilian Society of Plant Breeding. Printed in Brazil
Interchanges in popcorn (
Zea mays
L.) involving the
nucleolus organizer chromosome
Maria Suely Pagliarini1*, Gléia Laverde Ricci1, Neide da Silva1, and Carlos Alberto Scapim2
ABSTRACT - The analysis of microsporogenesis in endogamous plants of popcorn (S5 to S7) showed several and distinct interchanges which involve the nucleolus organizer (chromosome 6). The detection of cells with interchanges was facilitated by the presence of two nucleoli of different sizes in contrast to normal ones with a single big nucleolus. Interchange points do not always seem to be at the same place. Whereas in several situations the interchange point clearly involved more than two chromosome pairs, a simple terminal translocation seemed to occur in others. During diplotene, a cross-shaped configuration connected with the nucleoli was observed in some meiocytes. Some heteromorphic bivalents were found during diakinesis, after which meiosis progressed normally to the end and gave rise to apparently normal tetrads with one normal nucleolus in each microspore. Tests of pollen viability in fixed pollen grains showed 100% stainability in normal and in affected plants. This is the first report on chromosome interchanges in popcorn.
Key words:popcorn, interchange, chromosome 6, nucleolus organizer, meiosis.
1 Departmento de Genética e Biologia Celular, Universidade Estadual de Maringá (UEM), 87020-900, Maringá, PR, Brasil. *E-mail: mspagliarini@uem.br 2 Departmento de Agronomia, UEM
INTRODUCTION
Karyotypes may undergo structural rearrangements during the cell cycle. Among the rearrangements reported in animals and plants, translocations have been extensively described and are the most easily recognized during meiosis (Sybenga 1975). Translocation is the transfer of a chromosome segment from its original position to another one in the genome. The most common type is the reciprocal translocation or interchange in which two chromosomes are broken and subsequently exchange blocks of chromatin (Swanson et al. 1981). In heterozygotes, when all homologous segments have paired at pachytene, a typical cross-shaped configuration is formed. Depending on the formation or non-formation of
chiasmata in these segments, different configurations will appear at diakinesis or metaphase I after the pairing cross opens up (Sybenga 1975). Translocations in maize have been widely reported since 1930, and most of them involve chromosome 6 which carries the nucleolus organizer (Burnham 1962). Burnham (1950) analyzed 27 heterozygote translocation stocks involving chromosome 6.
Reciprocal chromosomal translocations have been successfully used to establish linkage relationships between translocation breakpoints and the genes that control important traits (Singh 1993). However, the use of stocks with translocations in linkage studies is labor-intensive and slow as it requires minutious work under the microscope. After 1970, B-A interchanges in maize were largely reported (Singh 1993). The interchanges Received 14 June 2006
were obtained by translocations between a member of the standard “A chromosome” set and a type of supernumerary “B chromosome” found in certain maize lines. B-A interchanges are also very useful for genetic analysis for locating specific genes (Beckett 1978, Carlson 1986).
Cytogenetics studies are indispensable during breeding programs in allogamous plants to obtain homozygosis. The meiotic stability of maize inbred lines is an important consideration in view of their extensive use in genetics and plant breeding research. Microsporogenesis is a genetic controlled process (Baker et al. 1976, Golubovskaya 1979, 1989) that culminates in the formation of four haploid cells. When an allogamous plant is submitted to self-pollination, many genes, including those involved in the control of meiotic processes, enter homozygosis causing inbred depression. Although many factors may affect the fertility of inbred lines, some data indicate that at least part of the fertility depression may be attributed to meiotic irregularities (Smith and Murphy 1986, Defani-Scoarize et al. 1995a, b, Pagliarini et al. 2002).
An important objective of a maize breeding program is to develop new inbred lines that combine well to achieve higher grains yields and superior agronomic performance in hybrid combinations. In such breeding programs, the choice of parents is crucial, because this decision will determine the probability of establishing a new superior line (Hallauer and Miranda Filho 1988). In a breeding program to develop hybrids adapted to the edaphoclimatic conditions of the state Paraná (Brazil), one popcorn variety (CMS-43) was chosen to obtain inbred lines. In the current paper we are reporting the occurrence of interchanges involving chromosome 6 (nucleolus organizer chromosome) in endogamous plants derived from CMS-43.
MATERIALS AND METHODS
Plants of a popcorn population (S0) from the
National Research Center of Maize and Sorghum/ Embrapa (Sete Lagoas, state of Minas Gerais), named CMS-43, were self-pollinated over seven generations (S1 to S7) to obtain homozygous inbred lines for hybridization. The experimental trials were conducted on an Experimental Farm of the Universidade Estadual de Maringá (Maringá, state of Paraná).
Inflorescences for meiotic study were collected and fixed in a mixture of ethanol 95% and acetic acid (3:1) for 24 hours, transferred to 70% alcohol and stored under refrigeration until use. Microsporocytes were prepared by squashing and staining with 0.5% propionic carmine. Pollen viability in normal plants and in those with the interchange was investigated by using different staining types (propionic carmine, potassium iodine, tetrazolium chloride (TTC), Alexander’s procedure, and methylene blue) in fixed pollen grains. Results obtained for interchanges were analyzed by ANOVA. Photomicrographs were taken under a Wild Leitz microscope using Kodak Imagelink – HQ, ISO 25 black and white film.
RESULTS
During cytological analysis of microsporogenesis under light microscopy, the nine plants of S5 to S7
generations presented a complex system of interchanges involving chromosome 6. The number of cells affected by the interchange was low and variable among plants (Table 1). Analysis of the results showed significant differences among meiotic phases, but not among generations (Table 2).
The detection of cells with interchange was facilitated by two nucleoli of different sizes in contrast to the normal ones with a single big nucleolus. When chromosome 6 organizes the nucleolus in normal cells, its terminal chromomeres are usually not seen (Figure 1 a). However, in interchange plants three terminal chromomeres were easily detected by their localization above the big and the small nucleoli (Figure 1 b, c).
The interchange point could also be seen on the paired chromosomes (Figure 1 b to g), albeit not always in the same place. In some cases, there was a short segment of chromosomes from the NOR to the interchange point (Figure 1b, c, g), whereas in others the distance between them was long (Figure 1 d, f). In some meiocytes, the terminal segment with chromomeres above the NOR was longer than usual (Figure 1 e) and in several situations, the interchange point clearly involved more than two chromosome pairs (Fig. 1 g), including chromosome 6. In others, on the other hand, a simple terminal translocation occurred (Figure 1 f).
Table 1. Percentage of plants with interchange involving chromosome 6 in endogamous popcorn plants
Ta b l e 2. Va r i a n c e a n a l y s i s i n v o l v i n g g e n e r a t i o n s w i t h interchanges
df MS F
Generations 2 367.6717 2.486 Phases 2 295.2716 20.007 * Generations x phases 4 83.0494 0.561 Residue 72 147.9105
* Significant at 5% probability by test F
Source of variation
such crosses. In diakinesis, there was a predominance of bivalents. In a few cases, a quadrivalent configuration was associated with the nucleolus (Figure 1j), while some heteromorphic bivalents were found during diakinesis (Figure 1 l). In metaphase I, a few meiocytes showed a quadrivalent configuration (Figure l). After diakinesis, the meiosis progressed normally up to the end giving rise to apparently normal tetrads with one normal nucleolus in each microspore. Tests of pollen viability in fixed pollen grains showed 100% stainability in normal and in affected plants with all dyes.
DISCUSSION
At least 15 naturally occurring cases of chromosomal interchange in genetic stocks and in breeding material had been established in corn until 1942 (Burnham 1962). Accumulated data in literature suggest that the nucleolus organizer chromosome (chromosome 6 in maize) is the most susceptible to interchanges (Zeller 1973, Singh 1993). Burnham (1950) reported the result of an extensive study involving 27 translocations only in chromosome 6, with breakage
occurring at different loci: (i) in the short arm between the centromere and the nucleolus organizer; (ii) in the long arm or in the satellite; (iii) in the nucleolus organizer. Although we could not identify the chromosomes involved in the interchange with chromosome 6, the cases illustrated in Figure 1 clearly show that the interchanges occurred at different sites.
The most important consequences of meiotic behavior in heterozygote interchanges result from the metaphase I orientation and the subsequent anaphase I segregation of the configuration. The relative frequencies of the different orientation types (adjacent-1, adjacent-2, and alternate segregation) depend on a number of factors (Sybenga 1975, Rickards 1983, Sybenga and Rickards 1987). If the three types of orientation occurred at random, it would be expected that a translocation would lead to inviability in approximately two-thirds of the gametes. In maize, where the phenomenon has been extensively studied, the frequency of inviable seeds or defective pollen is close to 50%, suggesting that alternate orientation at metaphase occurs with a frequency close to 50% (Burnham 1962, Swanson et al. 1981, Singh 1993). However, a low spore abortion had been reported in a number of plant species with heterozygote interchanges (Yamashita 1947, Burnham 1953,1962, Barton 1954). In the present inbred lines, tests of pollen viability in fixed pollen grains showed total pollen stainability suggesting 100% fertility. No difference between normal and interchange-carrying plants was found in tests with different stains. We believe that the use of fixed pollen grains is not adequate for testing pollen viability, but vital pollen was not available.
% of cells with interchange plant-1
1 2 3 4 5 6 7 8 9 S5 Pachytene 22 40 55 33 23 34 38 40 6 Diplotene 11 5 6 20 11 25 25 20 1 Diakinesis 0 6 4 15 3 15 12 8 0
S6 Pachytene 5 10 4 6 32 16 29 28 38 Diplotene 5 5 2 5 11 5 16 26 18 Diakinesis 0 5 0 0 3 0 1 1 71
S7 Pachytene 4 0 17 8 32 60 55 32 16 Diplotene 30 6 4 24 50 13 0 16 0 Diakinesis 2 3 2 20 14 4 0 5 0
Figure 1. Aspects of interchanges involving chromosome 6 in popcorn plants. a. Normal pachytene with a single big nucleolus. b to e. Meiocytes in pachytene showing interchange involving chromosome 6. There are two nucleoli of different sizes, the chromomeres above them and the interchange point (arrow); note also differences in interchange point on the chromosomes. f. A terminal translocation (arrow). g. A translocation set involving more than two chromosome pairs (arrow). h, i. Diplotene with translocation involving chromosome 6. Observe the cross-shaped configuration in I. j. A quadrivalent in diakinesis. k. A heteromorphic bivalent in diakinesis (arrow).
After analyzing several translocations in chromosome 6 in maize, Burnham (1950) concluded that the number of spore abortion depended on the frequency of crossing-over in interstitial segments. When non-disjunction occurs at chromosome 6, the spore receiving the extra chromosome has two nucleolus organizers and potentially two nucleoli, often fused into one, while the spore receiving no organizer shows scattered small bodies of nucleolar material, referred to as a diffuse nucleolus. No spore with diffuse nucleolus was found in any of the spores analyzed in normal and affected plants. One normal nucleolus was detected in all of them. Similar to the plants under study, quadrivalents are not always found in heterozygote interchanges in maize (Sybenga 1992). Small interchanged segments may fail to have chiasmata in some meiocytes and two bivalents are formed when chiasmata are present in the non-interchanged arms. Bivalents may be heteromorphic, depending on the relative sizes of the different chromosomes and chromosome segments. According to the same author, depending on the formation or non-formation of chiasmata in the segments under interchange, different configurations will appear at diakinesis or metaphase I after the opening of the pairing cross. The chiasma keeps the four chromosomes together, but the original cross shape is not maintained when the homologues are separated from each other at the end of pachytene. Remnants of the cross may be visible at diplotene, but not at diakinesis, which explains why the difference in the number of abnormal cells is so great between these phases. Chiasmata also follow a genotype-specific system of frequency and localization, which is affected by pairing problems encountered around the pairing cross.
Simple and multiple translocations were found in the inbred lines under analysis. According to Sybenga (1992), it is not uncommon that a chromosome be involved in more than one interchange. The meiotic behavior of simple terminal translocations differs from that of interchanges only by a three-armed configuration instead of a four-armed pairing cross. There are only few reports of meiosis of simple terminal translocation, and one case was reported in rye (Sybenga and Verhaar 1980). In maize, simple translocation has been registered by Burnham (1930).
Interchanges in nature have played a major role in the evolution and speciation of several crop species. They can occur in somatic or meiotic cells spontaneously or be induced by ionizing radiation or other mutagens. Interchanges in maize have been detected in the progeny of homozygous plants for the sticky gene (Beadle 1937), and in plants carrying the Activator and Dissociation mutation-controlling elements (McClintock 1951). The causes of trans-locations were not known in the plants under analysis. Maize plants from the National Research Center of Maize and Sorghum/Embrapa, generally show more meiotic instability than plants of other breeding companies (Defani-Scoarize et al. 1995a, b, 1996, Pagliarini et al. 2002). One explanation for this instability may be the recent origin and status of such populations, which, as a rule, are still in initial stages of breeding and not yet subjected to selection for seed production.
ACKNOWLEDGEMENTS
This study was supported by PRONEX/ FUNDAÇÃO ARAUCÁRIA – No. 1227 -33/04
RESUMO-Análise da microsporogênese em plantas endogâmicas de milho pipoca (S5 a S7) revelaram distintas translocações envolvendo o cromossomo organizador do nucléolo (cromossomo 6). A detecção de células portadoras de translocação foi facilitada pela presença de dois nucléolos de diferentes tamanhos em contraste com células normais que apresentavam um único nucléolo. Os pontos de translocação não ocorreram sempre no mesmo local. Translocações terminais simples e translocações envolvendo mais que dois pares de cromossomos também foram observados. Em alguns meiócitos, durante o diplóteno, uma configuração em forma de cruz foi observada conectando os dois nucléolos. Bivalentes heteromórficos foram observados durante a diacinese. A partir da metáfase I a meiose progrediu normalmente originando tétrades aparentemente normais com apenas um nucléolo em cada micrósporo. Testes de viabilidade em grãos de pólen fixados mostraram a mesma
Translocações em milho pipoca (
Zea mays
L.)
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coloração em plantas normais e em plantas portadoras de translocação. Esta é a primeira descrição de translocação em milho pipoca.
