The B–A–A translocations have enabled us to simultaneously assess the possible dosage-sensitive interactions of two nonhomologous chromosome segments in affecting maize plant development. Maize B–A–A translocations contain segments of two nonhomologous essential A chromosomes in tandem arrangement attached to a segment of the long arm of a supernumerary B chromosome. By utilizing the frequent nondisjunction of the B centromere at the second pollen mitosis we produced plants containing an extra copy of the two A chromosome segments. We compared these hyperploid plants with nonhyperploid plants by measuring leaf width, plant height, ear height, internode length, stalk circumference, leaf length, and tassel-branch number in 20 paired families that involved one of the chromosome arms 1S, 1L, 4L, 5S, and 10L. One or more of the seven measured traits displayed dosage sensitivity among 17 of the 20 B–A–A translocations, which included the involvement of chromosome arms 2L, 3L, 5L, 6L, and 7L. The most obvious effect of an increased dosage of the B–A–A translocation was a significant decrease in the traits in the hyperploid plants. These effects may be either the additive effects of hyperploidy for the two chromosome segments or a result of gene interaction between them.

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MAIZE is an especially well-suited species for the study of anueploidy in plants. Maize simple B–A translocations result from reciprocal interchanges between a supernumerary B chromosome and an arm of an essential A chromosome. By utilizing the collection of simple B–A translocations the dosage of a large distal segment of 18 of the 20 maize chromosome arms, all except the long arm of chromosome 2 (2L) and the short arm of chromosome 8 (8S), can be altered (ROMAN 1947; BECKETT 1978, 1991). The dosage can be varied so that the endosperm contains two, three, or four copies and the embryo contains one, two, or three copies of the segment. The presence of an extra copy is referred to as hyperploidy while the lack of a copy of a chromosome segment (only two copies in the endosperm or only one copy in the embryo) is referred to as hypoploidy. One consequence of a change in chromosome segment copy number is a change in phenotype. Hypoploidy of any of several chromosome segments results in a reduced endosperm size, "the small kernel effect," which has been extensively analyzed (LIN 1982; BECKETT 1983; BIRCHLER and HART 1987; BIRCHLER 1993). The effects of aneuploidy on maize plant height and other morphological traits have been investigated. When the dose of several chromosome arm segments is individually increased the resulting hyperploid plants are usually altered to a modest degree (CHANG 1984; LEE et al. 1996; NEUFFER et al. 1997); when any of these 18 chromosome arm segments is individually decreased the resulting hypoploid plants are much more severely altered in their appearance and vigor (CHANG et al. 1987; BECKETT 1991; LEE et al. 1996; NEUFFER et al. 1997). It seems, therefore, that the growth and morphogenesis of maize plants is substantially buffered from the effects of aneuploidy when the dosage of a single chromosome segment is increased (hyperploidy), but often is strongly reduced when there is a decrease (hypoploidy) in dosage of that segment.

We have produced a large number of B–A–A translocations (SHERIDAN and AUGER 2006). These compound B–A translocations bear two A chromosome segments in tandem arrangement attached to a segment of the long arm of a B chromosome. Plants that are hyperploid for a B–A–A chromosome therefore contain an extra dose of both of the A chromosome segments; plants hypoploid for a B–A–A chromosome are lacking a dose of both of the A chromosome segments.

During the propagation of the newly constructed B–A–A translocations we have observed strongly modified plant phenotypes of plants hyperploid for many chromosome regions when two of these regions are simultaneously increased in dosage. Here we report on the altered phenotypes and the chromosome regions that appear to produce these changes when these regions are present in three doses.

The B–A–A translocations were constructed by bringing together one of the simple B–A translocations (BECKETT 1993) with an A–A reciprocal translocation wherein one of the chromosome arms involved in the A–A interchange is the same A chromosome arm borne on the B–A chromosome (RAKHA and ROBERTSON 1970). A crossover event in the region of shared homology of these two A chromosome segments can produce a new B–A–A chromosome. Details on the construction and detection of the B–A–A chromosomes used in this study are reported in SHERIDAN and AUGER (2006). The rare crossover products comprising the new B–A–A chromosomes were initially detected by crossing simple B–A/A–A heterozygotes pollen onto female tester stocks containing recessive kernel traits. To propagate the new B–A–A stocks the rare kernels displaying the mutant kernel phenotype were planted and the resulting progeny hyperploid plants were crossed onto the same or different kernel trait tester stocks. In most cases the cross onto the tester stock confirmed the identity of the new B–A–A translocation as evidenced by numerous nonconcordant kernels containing a recessive endosperm phenotype and a normal appearing embryo. The embryos of such kernels are hyperploid for the B–A–A translocation. These kernels were planted in families of 15 or 20 kernels in the 2007 summer nursery in Grand Forks, North Dakota, and in families of 13 kernels in the 2007–2008 winter nursery on Molokai, Hawaii.

Selection of kernels to produce hyperploid plants and nonhyperploid plants:

The selection of kernels to plant to produce the hyperploid plants was determined by the genetic kernel marker used in the female parent tester stock to propagate the B–A–A translocation stock. For example, in the case of TB-1La-5S8041, the distal segment of the B–A–A chromosome consists of the distal 90% of chromosome arm 5S bearing the dominant A2 allele while the normal chromosome 5 bore the recessive a2 allele on its short arm (5S). The tester stock was homozygous dominant for all of the aleurone color factors except that it was homozygous for the recessive a2 allele. When the tester stock was pollinated by a plant hyperploid for TB-1La-5S8041 much of the pollen contained the B–A–A chromosome. The resulting ear bore kernels with colored and colorless aleurone. Many of the kernels with colorless aleurone also had a colored embryo. The eggs of these kernels were fertilized by a hyperploid sperm containing two B–A–A chromosomes bearing the A2 allele. The resulting zygotes developed into colored embryos. The polar nuclei of these kernels fused with a hypoploid sperm that lacked any B–A–A chromosomes and therefore the resulting endosperm nuclei lacked an A2 allele, and consequently the endosperm was colorless. The same ear bore colorless kernels (colorless aleurone) and a colorless embryo. These kernels were fertilized by sperm that did not contain the A2 allele and therefore, in most cases, did not contain the B–A–A chromosome but contained the normal chromosome 5 bearing the a2 allele. Additionally on the same ear there were colored aleurone kernels with either colorless embryos or colored embryos. The former kernels result from the fertilization of the egg cell by a hypoploid sperm lacking the B–A–A chromosome and therefore an A2 allele, while the polar nuclei fused with the hyperploid sperm containing the two copies of the B–A–A chromosome bearing the A2 allele. The latter kernels could result from normal disjunction of the B–A–A chromosome during the second mitotic division in the pollen grain so that both sperm contain a B–A–A chromosome with the A2 allele. These kernels may also result from a crossover of the A2 allele from the B–A–A chromosome onto a normal A chromosome. Because chromosomes with B centromeres are regularly lost at a low rate, some of the pollen are euploid without the B translocation and can be transmitted.

The nonconcordant (colorless endosperm/colored embryo) kernels were selected for planting with a high degree of confidence that, because of their genetic marking, they contained embryos that were hyperploid for the TB-1La-5S8041 chromosome and would therefore grow into plants hyperploid for this B–A–A chromosome. In the summer nursery the kernels used for the nonhyperploid families were taken randomly from among those colored and colorless kernels that did not display the genetic markers identifying hyperploid kernels. In the cases where the tester stock was an aleurone color tester, only colored kernels were selected for planting the nonhyperploid families.

Measuring of phenotypic traits in paired families in the summer nursery:

Measurements were made in North Dakota in the summer nursery on hyperploid plants and on nonhyperploid plants grown from kernels selected from the same ear. The resulting paired families from an individual ear were planted sequentially in the nursery, with the hyperploid family planted first, followed by the nonhyperploid family. The latter family is expected to segregate for euploid plants and B–A–A hypoploid plants, as well as other types (see below). In selecting plants to measure in the hyperploid family, in as much as all the kernels planted should produce plants hyperploid for the two A chromosome arm segments borne on the B–A–A chromosome, all of the plants were considered to be suitable for obtaining measurement data. In the case of the plants in the second of the paired families, selection of the plants to be measured was required because of the segregation of different kinds of plants. For all of the plants of the 16 B–A–A stocks using aleurone color testers the source ears segregated for colored and colorless kernels, the colored kernels were selected for planting without discriminating between kernels containing colored embryos and those containing colorless embryos. These kernels generally were expected to produce a higher frequency of B–A–A hypoploid plants than produced by concordant colorless kernels, an expectation confirmed in the observations made in the winter nursery. Plants in all of the nonhyperploid families were selected on the basis of their appearance. In all cases, the plants hypoploid for the B–A–A were readily identified because of their gross abnormality. In some cases they died as seedlings or were so severely retarded in growth that they never progressed past the seedling stage. Crossing over in the hyperploid parent of the kernels used for planting could result in some plants hyperploid or hypoploid for a simple B–A translocation, which in the case of the simple B–A hypoploid plants would display reduced but less severe phenotypes than the B–A–A hypoploid plants. Because of this, among the plants that grew to the flowering stage, the taller more normal appearing plants were selected for measuring. Usually, any plant that was not obviously a B–A–A hypoploid was used for measurement.

The phenotypic traits selected for measurement were selected among those measured by LEE et al. (1996). These were (1) leaf width, (2) plant height, (3) ear height, (4) internode length, (5) stalk circumference, (6) leaf length, and (7) tassel-branch number (Table 1). The measurements were recorded to the nearest millimeter using a meter stick or a measuring tape calibrated in millimeters and centimeters. The measurements were transferred to an Excel spreadsheet. Calculations were performed to determine the total, the mean value, the standard error, and the standard error of the mean. The mean values for each measured trait of the paired families (hyperploid vs. nonhyperploid) were compared for statistically significant differences using the t-test. Photographs of mature plants at the flowering stage were taken with an Olympus 710 digital camera.

View this table: In this window In a new window   TABLE 1 Descriptions of the seven morphological traits examined for dosage sensitivity

 

Assessing plants for pollen sterility in the winter nursery:

Because of an interest in examining plants in the hyperploid and nonhyperploid families for pollen semisterility these materials were again grown for that purpose in the winter nursery. Because of adverse weather conditions it was not feasible to make measurements of traits in this planting. For the winter nursery, genetically marked hyperploid kernels were again used to plant hyperploid families, but for the nonhyperploid families some discrimination was performed in selecting kernels for planting the nonhyperploid families for 16 of the 20 families being tested. These were families where the tester stocks used to propagate the B–A–A translocations were aleurone color marker stocks. In these cases crosses of hyperploid pollen parents onto tester silks yielded ears that segregated for colored and colorless kernels. Among the colorless kernels some had colored embryos and these were used to plant the hyperploid families. The other colorless kernels had colorless embryos and these concordant kernels must have contained only the recessive aleurone color allele in both the embryo and the endosperm. None of these kernels should contain the B–A–A translocation in their embryos except for the products of rare crossover events that resulted in the B–A–A chromosome carrying the recessive allele for the tester trait.

The colored kernels all had colored aleurone but some had colored embryos and some had colorless embryos. The former could be produced by having a pollen grain containing the B–A–A chromosome (bearing the dominant aleurone color allele) undergoing a normal disjunction of the B–A–A chromosome at the second mitotic division when the generative cell divides to form two sperm cells. This failure to undergo nondisjunction would result in both sperm cells containing the dominant aleurone color allele and, consequently, both the aleurone and the embryo being colored. This full-color-type kernel could also result from transfer of the dominant color allele to a normal chromosome by its recombination with the B–A–A chromosome. The colored kernels with colorless embryos could be produced by having a pollen grain containing the B–A–A chromosome (bearing the dominant aleurone color factor) undergo nondisjunction at the second mitotic division. This failure to disjoin would result in a hyperploid sperm and a hypoploid sperm. The fertilization of the egg cell by the hypoploid sperm would result in a colorless embryo while fusion of the hyperploid sperm containing two copies of the B–A–A chromosome (bearing the dominant aleurone color allele) with the polar nuclei would result in a colored aleurone.

For all 20 B–A–A stocks grown in the winter nursery three families containing 13 kernels each were planted. For the 16 B–A–A stocks where colored and colorless kernels segregated on source ears, one family was planted with nonconcordant kernels containing colorless aleurone and colored embryos (the hyperploid family); the second family was planted with kernels containing colored aleurone, with no discrimination between kernels containing colored embryos and those containing colorless embryos; and the third family was planted with concordant colorless kernels containing colorless aleurone and colorless embryos. For the other four B–A–A stocks where other kernel marker traits were used in colorless tester stocks, one family was planted with nonconcordant kernels containing mutant endosperm and normal embryos (the hyperploid family) and the two additional families were both planted with concordant colorless kernels containing normal-appearing endosperm and embryos. These latter two families were therefore duplicate plantings.

Selection of B–A–A translocation stocks for examination:

During the propagation of most of the 64 newly created B–A–A translocation stocks (see Table 3 in SHERIDAN and AUGER 2006) in a winter nursery, we observed alterations in phenotypes of hyperploid plants of several families. Five chromosome arms, 1S, 1L, 4L, 5S, and 10L, were identified that were frequently involved in the B–A–A chromosomes associated with the altered plant phenotypes. Among the 81 B–A–A translocation stocks described in SHERIDAN and AUGER (2006), we selected the B–A–A translocation stocks that involved these five chromosome arms. These 49 B–A–A stocks and their descriptions are listed in Table 2. Plantings were made of these 49 B–A–A stocks. During the summer growing season 20 of the paired B–A–A families were identified that appeared to have differences in plant phenotypes between the hyperploid families and the nonhyperploid families. The plants in these families were subsequently examined and measurements were made on the seven selected morphological traits.

View this table: In this window In a new window   TABLE 3 Dosage-sensitive response of seven morphological traits by plants hyperploid for 20 selected B–A–A translocations

 

View this table: In this window In a new window   TABLE 2 B–A–A translocation stocks analyzed for dosage sensitivity

 

Distinguishing hyperploid and nonhyperploid kernels and plants:

The plants grown in the summer nursery had their traits measured after the completion of flowering, so it was not possible to check their pollen for semisterility. A matter of interest is the chromosome constitution of such plants. The constitution of the hyperploid plants is expected to be heterozygous for the B–A–A translocation, for an A–A translocation, and for an A–B chromosome as well as to contain normal chromosomes. These plants should exhibit pollen semisterility (BECKETT 1991) and, indeed, this has been confirmed routinely by pollen examination of hyperploid plants used for propagation. A consideration of the nonhyperploid kernels taken from an ear that provided hyperploid kernels (containing hyperploid embryos) requires a consideration of the chromosome pairing patterns and the segregation configurations of the meiotic chromosomes in the hyperploid pollen parent plants used to produce those kernels.

Four patterns of meiotic chromosome pairing and segregation:

The hyperploid pollen parent plant is crossed onto a tester plant, which is homozygous for the recessive aleurone color tester allele or heterozygous for an endosperm trait where the recessive allele for that trait is lethal. But in either case the tester stock contains only normal chromosomes. The progeny kernels and plants produced by such a cross are of several types and differ in their chromosome and genetic constitutions (Figure 1). In a plant heterozygous for a chromosome translocation the pachytene stage chromosomes of the first meiotic division can undergo either alternate disjunction or adjacent-1 or adjacent-2 disjunction (BURNHAM 1977). These three orientations are distinguished by the behavior of the centromeres: with alternate and adjacent-1 segregation homologous centromeres migrate to opposite spindle poles, while with adjacent-2 segregation, homologous centromeres migrate to the same spindle pole.

View larger version (52K): In this window In a new window Download PPT slide   FIGURE 1.— Progeny of a plant hyperploid for the B–A–A chromosome TB-1La-5S8041 crossed onto a normal female tester plant. The chromosomes are drawn to scale on the basis of the cytological map of NEUFFER et al. (1997). The five chromosomes of interest are labeled A–E. There are four common patterns of chromosome pairing and B–A–A chromosome behavior in the microsporocyte of the B–A–A hyperploid pollen parent plant. (1) The two B–A–A chromosomes pair only with each other and a B–A–A chromosome is included in the microspore product of meiosis (A). (2) The two B–A–A chromosomes pair only with each other and no B–A–A chromosome is included in the microspore (B). (3) One of the B–A–A chromosomes pairs with the normal chromosome 1 and normal chromosome 5 but there is no crossing over between these chromosomes (C). (4) One of the B–A–A chromosomes pairs with the normal chromosome 1 and normal chromosome 5 and there is a single crossover between the normal chromosome 1 and its homologous interstitial region in the B–A–A chromosome (D). There are three basic configurations of chromosome disjunction: with alternate and adjacent-1 segregation homologous centromeres migrate to opposite spindle poles, while with adjacent-2 segregation homologous centromeres migrate to the same spindle pole. Only alternate disjunction of the meiotic chromosomes will result in functional microspores and viable pollen for the first three patterns (A, B, and C), but with the fourth pattern (D) alternate segregation products containing noncrossover chromosomes will produce viable products and adjacent-1 segregation products containing crossover chromosomes will also produce viable products. See the text for additional details.

 

Progeny of crossing a hyperploid B–A–A plant onto a tester:

The progeny produced by crossing a plant hyperploid for TB-1La-5S8041 onto a tester plant homozygous for the a2 allele are shown in Figure 1. At the top of Figure 1 the five chromosomes of interest are shown and are labeled A–E. There are two basic configurations of chromosome pairing shown: (a) the B–A–A chromosomes pair only with each other (Figure 1A) and (b) one of the B–A–A chromosome pairs with the normal chromosome 1 and the normal chromosome 5 (Figure 1, C and D). In the first configuration (Figure 1A), crossing over between homologous regions of paired chromosomes does not affect the constitution of the progeny but does so with the last configuration (Figure 1D). The two recombinant chromosomes produced by crossing over are shown near the bottom of Figure 1 and are labeled F and G. Four common patterns of meiotic chromosome pairing and segregation are described below.

When the B–A–A chromosomes pair only with each other and a B–A–A chromosome is included in the microspore:

In Figure 1A the chromosome pairing configuration shows the two B–A–A chromosomes pairing together and the other four chromosomes of interest pair in a cross-shaped configuration. At anaphase I the B–A–As segregate to opposite poles and alternate segregation of the other four chromosomes results in viable pollen. It is of two types, pollen containing the 5-1 chromosome (A), the 1-B chromosome (C), and a B–A–A chromosome (E) and pollen containing the normal chromosome 1 (B), the normal chromosome 5 (D), and a B–A–A chromosome (E).

Pollen of the first type contains both the B–A–A and the 1-B chromosomes, so during the second pollen mitosis the B–A–A chromosome frequently undergoes nondisjunction to produce a hyperploid sperm (containing two B–A–A chromosomes) and a hypoploid sperm (containing no B–A–A chromosome). When this type of pollen grain functions to fertilize the egg and polar nuclei the resulting nonconcordant kernels will contain either a hyperploid embryo and a hypoploid endosperm (Figure 1, progeny I) or hypoploid embryo and hyperploid endosperm (Figure 1, progeny II).

In those cases where the B–A–A chromosome disjoins normally at the second pollen mitosis, a third type of progeny results. The pollen has a single B–A–A chromosome in each of the sperm, resulting in a kernel with a euploid embryo and euploid endosperm (Figure 1, progeny III).

A pollen grain that contains a B–A–A (E), a normal chromosome 1 (B), and a normal chromosome 5 (D) will have identical sperm, because the factors essential for nondisjunction at the second pollen mitosis are on the 1-B chromosome. Even so, these pollen grains are of no consequence since they are aneuploids and with very few exceptions do not contribute to the progeny because they are noncompetitive.

When the B–A–A chromosomes pair only with each other and no B–A–A chromosome is included in the microspore:

At a low but regular rate, B–A–A chromosomes are lost in meiosis. When alternate disjunction occurs and no B–A–A chromosome is included in the microspore, spores with only the 5-1 and the 1-B chromosomes (A and C) are deficient and will abort. Spores with the normal 1 and 5 chromosomes (B and D) will develop into euploid pollen that will compete. The endosperm and the embryo will both be euploid, and will each lack the B–A–A and 1-B chromosomes (Figure 1, progeny IV).

When one of the B–A–A chromosomes pairs with the normal chromosomes and there is no crossing over:

In the second pattern of chromosome pairing, one B–A–A chromosome is involved in the cross-shaped pairing configuration and its 1L and 5S regions pair with their homologous regions on the normal chromosomes 1L and 5S, respectively (Figure 1C). When there is no crossing over between the B–A–A chromosome (E) and normal chromosome 1 (B), then only alternate chromosome segregation, with the chromosome 5-1 (A), the 1-B chromosome (C), and the B–A–A chromosome (E) segregating from the normal chromosome 1 (B) and normal chromosome 5 (D), results in viable pollen. The pollen containing the A, C, and E chromosomes produce the same progenies as described above (Figure 1, progenies I, II, and III). Also described above, the microspore receiving the normal chromosome 1 (B), the normal chromosome 5 (D), and the other B–A–A chromosome (E) will produce noncompetitive pollen grains. If no B–A–A chromosome is included in the microspore then euploid progeny with normal pollen will result (Figure 1, progeny IV).

When one of the B–A–A chromosomes pairs with a normal chromosome and a single crossover occurs:

In the case where the B–A–A chromosome participates in the cross-shaped pachytene-stage configuration by pairing with its homologous regions on normal chromosome 1 and normal chromosome 5, a single crossover between the 1L regions of the B–A–A and the normal chromosome 1, produces two new chromatid configurations (Figure 1, chromosomes F and G).The alternate segregation products containing noncrossover chromosomes will produce viable pollen and the types of progeny will be the same as shown previously (Figure 1, progenies I, II, III, and IV). The alternate segregation products containing crossover chromosomes will all be duplicate/deficient and will abort.

Adjacent-1 segregation products containing crossover chromosomes will produce viable pollen and four new types of progeny. The microspore receiving the reciprocal adjacent-1 segregation products will contain the 1-B chromosome (C), the normal chromosome 5 (D), and the newly created simple B–A (G) crossover product (TB-1La) and will develop into viable pollen. This type of pollen will frequently undergo nondisjunction of the B–A chromosome at the second pollen mitosis to produce hyperploid and hypoploid sperm. Pollination with this type of pollen will result in nonconcordant kernels with an embryo hyperploid for the simple B–A (TB-1La) and plants with normal appearing pollen (Figure 1, progeny V), and in nonconcordant kernels containing embryos hypoploid for the 1L chromosome segment of .20-1.00 and that will produce plants with semisterile pollen (Figure 1, progeny VI). In those cases where the B–A does not undergo nondisjunction each of the sperm contains a single B–A chromosome and pollination will result in concordant kernels with an euploid embryo containing the 1-B chromosome (C), the normal chromosome 5 (D), and newly created simple B–A (G) that will produce plants with quarter-sterile pollen (Figure 1, progeny VII). The microspore receiving the 5-1 chromosome (A) and the newly created 1-5 (F) crossover product will develop into pollen that produces euploid semisterile progeny plants, provided the nonpaired B–A–A is not included in the spore (Figure 1, progeny VIII). Adjacent-1 segregation products containing noncrossover chromosomes and all adjacent-2 segregation products (where homologous centromeres segregate to the same pole) will all be duplicate/deficient and will abort.

The above-described meiotic products and progeny are not an exhaustive description of the possibilities, but are the most common products and progeny expected. Crossovers in the interstitial region between the chromosome 5 centromere and the breakpoint in chromosome arm 5S will result in meiotic products and progeny like those shown for Figure 1C when there is adjacent-1 segregation of the crossover chromosomes. See spore quartet analysis in BURNHAM (1977) for further consideration of the consequences of crossovers in the interstitial regions of interchange heterozygotes.

Dosage sensitivity:

The dosage-sensitive responses of the seven morphological traits are shown in Table 3. (See the supplemental data for the detailed data on which this table is based.) The most obvious effect of B–A–A increased dosage was a significant decrease in the trait when measured in the hyperploid plants as compared with the nonhyperploid plants.

Assessing dosage sensitivity by measurement:

Leaf width was dosage sensitive in seven cases of B–A–A hyperploidy. A decrease in leaf width was observed in the case of TB-1Sb-3L8995, TB-1Sb-4Lh, TB-6Lc-4L8764, and TB-4Lf-6L8927. In three of these cases a region of chromosome arm 4L was involved either as a long distal region (two cases) or as a long proximal region of the B–A–A chromosome. An increase in leaf width was observed in the case of plants hyperploid for TB-10L19-1La, TB-5Sc-1L070-12, and TB-6Lc-1L070-1. In all three cases a long distal region of chromosome arm 1L was the distal region of the B–A–A chromosome.

Leaf length was dosage sensitive in seven cases. A decrease in leaf length was observed in the case of hyperploidy for TB-7Lb-1L4891, TB-5Sc-1L070-12, TB-1La-5S8041, TB-4Lf-6L8927, TB-5Sc-3Lg, and TB-5La-10L006-11. Regions of chromosome arms 1L, 4L, 5S, and 10L, but not 1S, were involved in these B–A–A translocations. In the case of TB-1La-3L5267, leaf length was increased in the hyperploid plants.

Plant height was the most frequent significantly different dosage-sensitive trait measured, with 13 of the 20 B–A–A families having hyperploid plants reduced in height. These were TB-1Sb-3L8995, TB-1Sb-4Lh, TB-10L19-1La, TB-5Sc-1L070-12, TB-6Lc-1L070-1, TB-1La-5S8041, TB-6Lc-4L8764, TB-4Lf-10L6587, TB-4Lf-6L8927, TB-4Lf-3L6534, TB-5Sc-2L015-3, TB-5Sc-3Lg, and TB-5La-10L006-11.

Ear height was dosage sensitive in eight cases. In all of these cases ear height was reduced in the hyperploid plants; this reduction paralleled the reduction in plant height in the seven cases of TB-1Sb-3L8995, TB-10L19-1La, TB-5Sc-1L070-12, TB-6Lc-4L8764, TB-4Lf-10L6587, TB-4Lf-6L8927, and TB-5Sc-2L015-3, but in the case of TB-7Lb-1L4891 there was no accompanying reduction in plant height.

Internode length was dosage sensitive in nine cases, all of which resulted in a reduction of internode length in the hyperploid plants. In the seven cases of TB-10L19-1La, TB-5Sc-1L070-12, TB-6Lc-1L070-1, TB-4Lf-6L8927, TB-4Lf-3L6534, TB-5Sc-3Lg, and TB-5La-10L006-11 the reduction in internode length paralleled the reduction in height of plants hyperploid for these B–A–A translocations. In the two cases of TB-1Sb-2L4464 and TB-10L19-1d a reduction of internode length was the only significant alteration observed among the seven traits measured.

Stalk circumference was dosage sensitive in six cases. Stalk circumference paralleled the reduction in plant height in the cases of TB-1Sb-4Lh, TB-6Lc-4L8764, TB-4Lf-6L8927, and TB-5Sc-3Lg. In the cases of TB-1La-3L5267 and TB-7Lb-1L4891 stalk circumference was greater in hyperploid plants but was not accompanied by alteration in plant height.

The number of tassel branches was dosage sensitive in four cases. Reduction in tassel-branch number was accompanied by a reduction in plant height in plants hyperploid for TB-1Sb-4Lh and TB-6Lc-4L8764; while in the cases of TB-7Lb-1L4891 and TB-6Lc-1L070-1 there was an increase in the number of tassel branches.

Assessing dosage sensitivity by visual examination:

A visual examination of the plants grown in the 20 pairs of families provided a more striking contrast between the family of hyperploid plants and the family of nonhyperploid plants than provided by the measurement data. This is illustrated by Figure 2 which contains a sample of plants hyperploid for TB-1Sb-3L8995 (containing an additional copy of chromosome regions 1S.05-.49 and 3L.06-1.00) side by side with a sample of nonhyperploid plants grown from kernels from the same ear as the hyperploid plants. Whereas the measurements of morphological traits revealed significant differences in leaf width, plant height, and ear height (Table 3), an examination of the plants shown in Figure 2 makes it additionally evident that the hyperploid plants were strongly reduced in vigor and mass as compared with their nonhyperploid counterparts. This is further evident in Figure 3, which contains plants hyperploid for TB-6Lc-4L8764 (containing an additional copy of chromosome regions 6L.11-.90 and 4L.32-1.00) side by side with a sample of nonhyperploid plants. Measurements revealed significant differences in leaf width, plant height, ear height, stalk circumference, and tassel-branch number (Table 3). An examination of Figure 3 makes it evident that the hyperploid plants were less robust than their nonhyperploid counterparts. The detailed data for these two paired families are included in the supplemental data. The hyperploid plants were relatively uniform in plant height, as seen in Figure 3 and shown in the supplemental data. A relative uniformity in plant height of the hyperploid families was a regularly occurring feature among the 20 paired families that were measured.

View larger version (149K): In this window In a new window Download PPT slide   FIGURE 2.— Comparison of three plants hyperploid for TB-1Sb-3L8995 (left) with three nonhyperploid plants. All plants were grown from kernels from the same ear. Leaf width, plant height, and ear height were significantly reduced in the hyperploid plants as compared with the nonhyperploid plants.

 

View larger version (145K): In this window In a new window Download PPT slide   FIGURE 3.— Comparison of three plants hyperploid for TB-6Lc-4L8764 (left) with three nonhyperploid plants. All plants were grown from kernels from the same ear. Leaf width, plant height, ear height, stalk circumference, and tassel-branch number were significantly reduced in the hyperploid plants as compared with the nonhyperploid plants.

 

Assessing the frequency of nonhyperploid plants with normal pollen and semisterile pollen:

To obtain some understanding of the relative frequency of plants with semisterile pollen (presumed to be heterozygous for a translocation), plants with normal pollen (presumed to be homozygous for normal chromosomes), and hypoploid plants present among the nonhyperploid plants that were measured, families of plants were grown in the winter nursery using kernels from the same source ears used for the summer planting. As described in the MATERIALS AND METHODS, for the summer planting the genetically marked hyperploid kernels were the source of the hyperploid families. In the summer nursery the kernels used for the nonhyperploid families were taken randomly from among those kernels that did not display the genetic markers identifying hyperploid kernels. However, for those 16 B–A–A stocks that utilized aleurone color factors as tester traits only colored aleurone kernels were used for the nonhyperploid families.

Nearly all families segregated for semisterile plants and normal plants:

The results of examining the pollen and scoring the families for the presence of hypoploid plants in the winter nursery are shown in Table 4. The semisterile plants were distinguished from the normal plants by observing that the former contained 25–50% aborted pollen while normal plants had 95% or more normal pollen. The B–A–A hypoploid plants were judged to be hypoploid for both of the A chromosome regions borne on the B–A–A chromosome of interest, as evidenced by their severely abnormal reduction in size and their abnormal phenotypes. Among the plants examined in the 20 hyperploid families, with two exceptions, all were semisterile. These exceptional plants were likely products of miscoring kernel phenotype during seed preparation. Among the 20 paired families of nonhyperploid plants, all of them segregated for at least one semisterile plant in one or the other of the paired families except in the case of TB-4Lf-3L6534. Hypoploid plants were not expected in any of the hyperploid families and were not observed in any of them except for two hypoploid plants in the TB-5Sc-3Lg family. These two plants may have resulted from misscoring.

View this table: In this window In a new window   TABLE 4 Scoring of pollen and for hypoploidy in 2007–2008 winter nursery

 
There were 16 B–A–A translocation stocks that utilized aleurone color factors as tester traits and this provided the opportunity to plant two distinct paired nonhyperploid families. One family had colored aleurone and the other had colorless aleurone. Because the dominant color allele was borne on the B–A–A translocation chromosome and this chromosome was subject to undergoing nondisjunction and producing colored hyperploid endosperm and colorless hypoploid embryos, the colored kernel families were expected to produce more hypoploid plants than the colorless kernel families. For five of the B–A–A translocations the hypoploid plants were observed only in the nonhyperploid families grown from colored kernels; for six of the B–A–A translocations there were at least twice as many hypoploid plants observed in the families grown from colored kernels as seen in the families grown from colorless kernels; for three B–A–A translocations there was an equal distribution of hypoploid plants in the paired families; for one B–A–A translocation the hypoploid plants were observed only in a family grown from colorless kernels; and for one B–A–A translocation no hypoploid plants were observed in either of the paired nonhyperploid families.

Dosage effects on plant morphogenesis may be either additive or gene interaction effects:

In the majority of the 20 B–A–A translocations that were analyzed hyperploidy resulted in a significant difference of one or more morphological traits when compared to their nonhyperploid plant counterparts. However, there were 3 B–A–A translocations where no significant differences were found. These were TB-6Lc-1S7097, TB-1La-3L5242, and TB-10L19-9S059-10. Leaf width of the hyperploid plants was greater than their nonhyperploid counterparts for TB-10L19-1La, TB-5Sc-1L070-12, and for TB-6Lc-1L070-1. Hyperploidy for the B–A–A translocation TB-1La-3L5267 resulted in plants with leaf length and stalk circumference being greater than their nonhyperploid counterparts. These 4 and the other 13 B–A–A translocations all resulted in a reduction in development of one or more traits among the hyperploid plants. In view of the fact that 49 B–A–A translocations were initially visually screened and approximately one-third of them were found to result in a reduction in plant development, it can be concluded that many nonhomologous chromosome segments are dosage sensitive with regard to affecting development while many others appear not to be so. These data indicate that the altered plant development is not simply a result of hyperploidy but that there is a specificity of dosage sensitivity of these chromosome segments. For all seven measured traits several combinations of chromosome segments were implicated in the dosage-sensitive effects, and different combinations of segments appeared to affect different morphological traits. These differences suggest the possibility that specific gene interactions occur between the chromosome segments of different combinations. Alternatively, the dosage effects of the combinations of two chromosome segments may simply be the additive effects of hyperploidy for these two chromosome arm segments.

The B–A–A translocations have enabled us to simultaneously assess the possible dosage-sensitive interactions of two nonhomologous A chromosome segments in affecting maize plant development. The simple B–A translocations allow for manipulation of a single A chromosome segment and the analysis of trisomic plants allows for the manipulation of an entire A chromosome. The B–A–A chromosomes provide a unique opportunity for a more complex analysis of chromosome dosage sensitivity.

The validity of comparing hyperploid plants with nonhyperploid plants:

Comparing of morphological traits of the hyperploid plants with the nonhyperploid plants is appropriate for the following reasons. First, in each case the ear used as the source of the kernels planted to produce hyperploid plants was the same ear used as the source of the kernels planted to produce the nonhyperploid plants. Therefore, both sets of kernels had the same pollen parent and ear parent plants. Furthermore, the identity of the kernels selected to produce hyperploid plants was unambiguous. The B–A–A chromosome was marked with a dominant endosperm trait and the kernels selected were nonconcordant for either a color factor that was expressed in both endosperm and the embryo, or an endosperm trait that affected both endosperm phenotype and embryo or seedling viability. Consequently, the genetic markers made it possible to select nonconcordant kernels that would produce plants hyperploid for the B–A–A translocation with a high degree of confidence. This was borne out by the relatively high uniformity in phenotype within the families of hyperploid plants and the rarity of exceptional plants that appeared to be obvious contaminants.

The kernels used for producing the nonhyperploid plants were selected with a high degree of confidence that they would not produce hyperploid B–A–A plants because they did not display the endosperm marker allele expression that identifies such kernels. Although a rare crossover event in the pollen parent plant could result in exchanging the dominant marker allele with the recessive allele and result in concordant colorless kernels containing hyperploid embryos, these would be rare events to begin with, and such hyperploid plants would be expected to show up infrequently in the nonhyperloid families. This, indeed, was the case for those B–A–A translocations where the hyperploid plants displayed such a distinctly abnormal phenotype that they could obviously be distinguished.

The nonhyperploid families contained normal-appearing plants with semisterile pollen and those with normal pollen. Both kinds of plants would be expected to contain a full diploid set of genes but those producing semisterile pollen would likely also be heterozygous for a reciprocal translocation or other chromosome alteration. Some of the plants in the nonhyperploid families might contain the B–A–A translocation but not two doses of it. This type of progeny would be produced by pollination with pollen containing two doses of the segments present on the B–A–A. Such pollen would not compete well with normal pollen and therefore this type of progeny would be expected to occur with low frequency. Consequently, for purposes of comparison of the sensitivity of plant morphogenesis to the presence of three doses of two chromosome regions vs. two doses of these regions, the comparison of the plants in the hyperploid families with those in the nonhyperploid families is appropriate. Furthermore, these two groups of plants are more genetically similar than would be the case if the plants of the hyperploid families were compared in their morphological traits with the development of these traits in families of plants grown from the tester stock or an inbred line.

Pairs of chromosome regions that appear to interact:

An examination of the data in Table 3 suggest the following patterns of interaction of the five chromosome regions (1S, 1L, 4L, 5S, and 10L) that were initially identified as being frequently involved in the B–A–A chromosomes associated with altered phenotypes.

Among the four B–A–A translocations involving chromosome arm 1S, the distal region of 1S is implicated as interacting with the proximal region of 6L; the proximal region of 1S is implicated as interacting with the proximal region of 3L; and the distalmost region of 1S is implicated as interacting with the distal half of 4L.

Among the eight B–A–A translocations involving chromosome arm 1L, the proximal-middle region of 1L is implicated as interacting with the proximal-distal region of 5S; the distal region of 1L is implicated as interacting with the proximal-middle region of 6L; and most of 1L is implicated as interacting with the middle region of 7L.

Among the five B–A–A translocations involving chromosome arm 4L, the distal half of 4L is implicated as interacting with the distalmost region of 1S; most of 4L is implicated as interacting with the distal half of 3L; and the middle-distal region of 4L is implicated as interacting with most of 6L.

Among the four B–A–A translocations involving chromosome arm 5S, the middle section of 5S is implicated as interacting with the middle section of 1S, with most of 2L, and all of 3L.

Among the five B–A–A translocations involving chromosome arm 10L, the proximal region of 10L is implicated as interacting with most of 1L. The distal region of 10L is implicated as interacting with the proximal region of 4L and the proximal region of 5L.

Chromosome arms 3L and 6L were not among the five chromosome arms used as the basis of selection of the 49 B–A–A translocations selected for planting and screening for dosage-sensitive effects. However the data in Table 3 for the B–A–A translocations involving these two arms merit comment. Among the five B–A–A translocations involving chromosome arm 3L, the proximal half of 3L is implicated as interacting with 1S, the distal half of 3L is implicated as interacting with most of 4L, and most of 3L is implicated as interacting with the middle region of 5S.

Among the four B–A–A translocations involving chromosome arm 6L, most of 6L is implicated as interacting with the proximal-middle region of 4L.

Contrasting effects of hypoploidy and hyperploidy:

A comparison of the effects of hypoploidy and hyperploidy in maize reveals two interesting effects. First, hypoploidy of any of several individual chromosome segments in the endosperm results in a reduced kernel size. The chromosome arms exhibiting the most obvious effects are 1S, 1L, 4S, 5S, 7S, 7L, and 10L (BIRCHLER and HART 1987; BIRCHLER 1993). We have observed these effects when using simple B–A translocations and we have further observed that kernels with endosperm hypoploid for large regions of 2 of these arms appear to exhibit an even greater reduction in endosperm size. An example noted by ROBERTSON (1975) and observed by us is TB-1La-5S8041 with the dosage-sensitive regions being 1L.20-.80; 5S.10-1.00. However, the size of kernels containing endosperm hyperploid for the 7 arms listed above or the other 11 chromosome arms whose dosage can be manipulated with simple B–A translocations, does not appear to be altered by the additional dose of chromosome segment. There does not appear to be either a "small kernel effect" or "large kernel effect" produced by hyperploidy for either a simple B–A or a B–A–A translocation. When propagating simple B–A translocations, hypoploidy for a chromosome arm is generally associated with severe reduction in plant size and vigor (CHANG et al. 1987; BECKETT 1991; LEE et al. 1996) whereas hyperploid plants usually are altered to a modest degree and, in some cases, cannot be readily distinguished by morphological traits (CHANG 1984; LEE et al. 1996). Therefore, the altering of the dosage of a single chromosome arm segment has parallel effects on kernel and plant development in as much as in both cases hypoploidy results in a much more negative effect on endosperm and plant development than hyperploidy appears to exert on endosperm and plant development.

The second noteworthy effect relates to the effects of simultaneously altering the dosage of two segments of two nonhomologous chromosomes. When B–A–A chromosomes are employed to increase the dosage of two such segments the effects of hypoploidy on endosperm and plant development parallel those observed with the use of simple B–A translocations, but are more severe, particularly in their effects on plant phenotypes. However, in contrast to the effects of hyperploidy on plant phenotypes obtained with simple B–A translocations, the effects of hyperploidy on plant phenotypes obtained with numerous B–A–A translocations are more severe, and usually negative. An examination of the data of LEE et al. (1996) (their Table 3) shows that hyperploidy for TB-1Sb, TB-1La, TB-4Lc, and TB-10L19 was associated with tall or normal height plants, and later maturing plants. Their summary of data does show that plant height was reduced in plants hyperploid for TB-1Sb, TB-1La, and TB-4Lc, as compared to two-dose controls, while no significant difference was found for TB-10L19 (see Table 5 in LEE et al. 1996). It therefore does not appear likely that the strong effects we have observed on plant height, ear height, and other traits are simply effects of hyperploidy for a single chromosome segment. Rather, it appears likely that the more severe effects on plant morphological traits obtained when two chromosome segments are simultaneously increased in dosage are either an additive effect that is the sum of the hyperploid effects observed with hyperploidy of the simple B–A translocations involving the individual arm segments or they are a result of the interactions of genetic information borne on those two segments.

Sorting out which of the two possibilities may apply to the simultaneous hyperploidy of a particular combination of two chromosome segments will require future analysis and comparison of the effects of hyperploidy of the simple B–A translocations with the effects of hyperploidy of the B–A–A translocations. Similarly, hypoploids from B–A–A translocations could be compared with hypoploids from single B–A translocations. It does appears that, whereas the morphogenesis of the plant is fairly well buffered from negative effects of an extra copy of a chromosome segment, this modulating capacity to maintain a normal developmental program is not so operative in many cases where two chromosome segments are present in extra copies. Nevertheless, it is interesting that among the five chromosome arms (1S, 1L, 4L, 5S, and 10L) identified as arms whose dosage was frequently associated with altered plant phenotypes, all but 4L are among the arms exhibiting the strongest small kernel effects.

Further analysis of dosage-sensitive interactions:

Because this study reports data on 20 of the 49 B–A–A stocks grown for an initial survey, and these 20 stocks were selected on the basis of a visual examination conducted prior to plant maturity, a more thorough examination of the other B–A–A stocks in this group of 49 is warranted. Although these 49 stocks were selected from among the 81 B–A–A stocks available because they include all of the B–A–A stocks involving chromosome arms 1S, 1L, 4L, 5S, and 10L, it is apparent that the dosage sensitivity of segments of other chromosome arms warrants investigation.

Defining the regions of dosage-sensitive interactions:

The production of additional B–A–A translocations with different breakpoints in chromosome segments of interest should be useful in more precisely defining the regions of the segments that are responsible for the dosage-sensitive effects. An important aspect of this study is the significance of the chromosome arm breakpoints that define the chromosome segments borne on a B–A–A chromosome. These breakpoints were reported by LONGLEY (1961) on the basis of cytological examination of meiotic chromosomes of A–A chromosome translocation heterozygotes. The breakpoints are therefore cytologically determined positions along the chromosomes. It would be helpful for our research and that of other investigators if the breakpoints could be related to the maize molecular genetic map. It may be possible to identify molecular marker sites that flank the chromosome breakpoints and that have been mapped on the molecular genetic map. This would aid in the integration of the cytological map with the genetic map.

We thank J. B. Beckett, M. T. Chang, E. H. Coe, and D. S. Roberston for permission to cite their Maize News Letter articles, an anonymous reviewer for recommending inclusion of a figure containing the cross and progeny shown in Figure 1, and Elizabeth Gruman, Marc Farmer, and Joshua Wynn for technical assistance. We thank the Maize Genetics Cooperation Stock Center at University of Illinois, Urbana, Illinois, for providing translocation stocks and other stocks. This research was supported by a National Science Foundation Plant Genome Research Program grant 0321565 (to W.F.S.).

BECKETT, J. B., 1978 B-A translocations in maize I. Use in locating genes by chromosome arm. J. Hered. 69: 27–36.[Abstract/Free Full Text]

BECKETT, J. B., 1983 Kernel weight effects and transmission of a partial trisome involving the long arm of chromosome 5 in maize. Can. J. Genet. Cytol. 25: 346–353.

BECKETT, J. B., 1991 Cytogenetic, genetic and breeding applications of B-A translocations in maize, pp 491–527 in Chromosome Engineering in Plants, edited by T. TSUCHIA and P. K. GUPTA. Elsevier, Amsterdam.

BECKETT, J. B., 1993 Locating recessive genes to chromosome arm with B-A translocations, pp. 315–327 in The Maize Handbook, edited by M. FREELING and V. WALBOT. Springer-Verlag, New York.

BIRCHLER, J. A., 1993 Dosage analysis of maize endosperm development. Annu. Rev. Genet. 27: 181–204.[CrossRef][Medline]

BIRCHLER, J. A., and J. A. HART, 1987 Interaction of endosperm size factors in maize. Genetics 117: 309–317.[Abstract/Free Full Text]

BURNHAM, C. R., 1977 Discussions in Cytogenetics. Burgess Publishing Co., Minneapolis.

CHANG, M.-T., E. H. COE and J. B. BECKETT, 1987 Genetic effects of hypoploidy on kernel weight, plant height and leaf width. Maize Genet. Coop. News Lett. 61: 91–92.

CHANG, M.-T., 1984 Characterization of hyperploid B-A translocations. Maize Genet. Coop. News Lett. 58: 63–64.

LEE, E. A., L. DARRAH and E. COE, 1996 Dosage effects on morphological and quantitative traits in maize aneuploids. Genome 39: 898–908.[Medline]

LIN, B.-Y., 1982 Association of endosperm reduction with parental imprinting in maize. Genetics 100: 475–486.[Abstract/Free Full Text]

LONGLEY, A. E., 1961 Breakage points for four corn translocation series and other corn chromosome aberrations. Crop Research Bulletin No. 34–16. United States Department of Agriculture–Agricultural Research Service, Pasadena, CA.

NEUFFER, M. G., E. H. COE and S. R. WESSLER, 1997 Mutants of Maize. Cold Spring Harbor Laboratory Press, Plainview, NY.

RAKHA, F. A., and D. S. ROBERTSON, 1970 A new technique for the production of A-B translocations and their use in genetic analysis. Genetics 65: 223–240.[Free Full Text]

ROBERTSON, D. S., 1975 A new compound A-B translocation TB-5S, 1L (8041). Maize Genet. Coop. News Lett. 49: 79–81.

ROMAN, H., 1947 Mitotic nondisjunction in the case of interchanges involving the B-type chromosome in maize. Genetics 32: 391–409.[Free Full Text]

SHERIDAN, W. F., and D. L. AUGER, 2006 Construction and uses of new compound B-A-A maize chromosome translocations. Genetics 174: 1755–1765.[Abstract/Free Full Text]

Communicating editor: J. A. BIRCHLER

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