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Advanced Techniques to Check Aneuploids

The addition of whole alien chromosomes or chromosome arms is generally detrimental to the performance of common wheat. The new aneuploids with dissected chromosomes at the sub-arm level are more useful not only for breeding purposes but also for genomic analysis. Two cytological techniques 'chromosome banding' and 'fluorescence in situ hybridization' are most useful for the identification of chromosomes and sub-arm aberrations. C-banding allows us to identify all common wheat chromosomes (Gill et al. 1991) and not only to verify the conventional wheat aneuploids in mitotic metaphase cells but also to detect deficient chromosomes. Fluorescence in situ hybridization (FISH) is useful for locating specific DNA sequences on chromosomes. The genomic in situ hybridization (GISH), which uses probes of total genomic DNA from alien species, is useful for the detection of alien chromosomes and chromosomal segments introduced into wheat. The combination of chromosome banding and FISH/GISH is an even more powerful cytological technique for identifying alien chromosomes in wheat because we can identify chromosomes and locate specific DNA sequences in the same chromosome preparation (Fig. 8.1).

Gametocidal Mechanism

Some alien chromosomes called gametocidal (Gc) chromosomes ensure their existence in common wheat in a selfish manner. When the Gc chromosome exists in the monosomic condition, two types of gametophyte are produced, those carrying the

Fig. 8.1 A mitotic metaphase cell of the barley 6H addition line of common wheat depicted by the combination of C-banding and FISH/GISH. The C-banding pattern shows that this line has a chromosomal complement of common wheat, and FISH indicates the presence of rDNA sequences in NORs, and GISH confirmed the disomic addition of barley chromosome 6H

Fig. 8.2 A schematic diagram showing the gametocidal action. Gc stands for 'gametocidal chromosome'

Gc chromosome and those without the Gc chromosome, and chromosome breakage occurs only in the latter gametophytes (Fig. 8.2). Such Gc-induced chromosomal breakage leads to either the sterility of gametes or the production of fertile gametes carrying chromosomal mutations, and the induced chromosomal mutations become stabilized in subsequent generations (Endo 1990).

Deletion Stocks of Common Wheat

Two Gc chromosomes have been used to generate deletion and dissection lines. One is chromosome 2C derived from Aegilops cylindirica Host. and the other is chromosome 3C derived from Aegilops triuncialis L. (Endo 1988). For the production of

Fig. 8.3 A C-banded mitotic metaphase cell of a progeny carrying three Gc-induced deletions of chromosomes 5A, 5B, and 7A (pointed with arrows) (Note that this plant was partial trisomic for 5B)

the deletion stocks, I started cytological selection in the progeny of the Gc (mainly 2C) monosomic addition line crossed with euploid Chinese Spring. Deletions took place in the heterozygous condition and often multiple deletions occurred in single plants as shown in Fig. 8.3. I further selected the selfed progeny of such plants to obtain single-deletion homozygotes. In some cases I ended up getting multipledeletion homozygous lines and also failed to obtain homozygotes for specific chromosome arms like the short arm of chromosome 4B. Table 8.1 summarizes the distribution of deletion breakpoints among different chromosome arms. I examined about 500 primary progeny, and in the end I identified 436 deletions and established deletion-homozygous lines for about 350 (ca. 80 %) of the deletions. This means that almost one deletion per plant occurred on average. In other words, we can expect to obtain one deletion for a specific chromosome in 4 ~ 5 % of plants we examine. The breakpoints of the deletions seem to be randomly distributed, but it is difficult to tell whether or not there are hotspots of Gc-induced chromosomal breaks. Undoubtedly the deletion stocks are useful for chromosome mapping, especially for deletion mapping of molecular markers. Werner et al. (1992) conducted deletion mapping of RFLP using the deletion stocks for the first time. Since then a series of papers on wheat chromosome deletion or bin mapping of various DNA markers using deletion stocks have been published (e.g. Qi et al. 2004). These deletion-homozygous stocks are distributed from National BioResource Project-

Wheat (shigen.nig.ac.jp/wheat/komugi/).

Table 8.1 Distribution of deletion breakpoints in different genomes, chromosome arms, and homoeologous groups in Chinese Spring wheat

Homoeologous arms

No. of deletion breakpoints

Genomes

A

B

D

Arms

S

L

S

L

S

L

Total

1

5

6

22

18

5

8

64

2

9

6

13

11

6

12

57

3

4

8

10

12

9

3

46

4

4

13

9

14

5

15

60

5

11

23

9

18

4

12

77

6

5

8

11

15

7

11

57

7

13

25

6

16

6

9

75

Subtotal

51

89

80

104

42

70

Total

140

184

112

436

Note: Data are taken from Endo and Gill (1996)

 
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