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The 4E-ms system of producing hybrid wheat.

THERE HAVE BEEN many reports of genetic male sterilty (GMS) of wheat in the literature. Five GMS loci have been located to chromosomes so far. These loci are msl, a recessive gene located to chromosome arm 4BS (Endo et al., 1991); Ms2 and Ms3, two dominant genes located to chromosome arm 4DS and 5AS respectively (Mclntosh et al., 1998); Ms4, a dominant gene located to chromosome arm 4DS (Maan and Kianian, 2001); and ms5, a recessive gene located to chromosome arm 3AL (Klindworth et al., 2002). In 1989 we discovered a male-sterile plant in the experimental field at Lanzhou, Gansu Province of China. This plant occurred as a spontaneous mutation in the [F.sub.4] generation of our conventional cross combination No. 87(212), which had the pedigree 8186[F.sub.3]/79Jian16//852-648. This mutant, which we name Lanzhou (LZ), was subsequently shown to be inherited as a monogenic recessive (unpublished data, 1996).

Producing hybrid seeds of crops with GMS has two breeding advantages compared with cytoplasmic male sterility (Rao et al., 1990; Kaul, 1988). First, the choice of parental lines is greatly broadened: and, second the negative effects of alien cytoplasm are avoided. But the difficulty with GMS is that a pure population of male-sterile plants or seeds cannot be produced by normal crossing procedures. To overcome this problem, cytogenetic-chromosomal manipulations have been proposed to permit the production of completely male-sterile populations of plants in some crops (Rao et al., 1990). In fact, three cases of these genetic designs by chromosomal manipulation have been reported: (i) "Balanced Tertiary Trisomic" (BTT) in barley (Hordeum vulgare L.) (Ramage, 1965): (ii) "Duplicate-Deficient chromosome complements" (Dp-Df) in maize (Zea rnays L.) (Patterson, 1978); and (iii) the "XYZ system of producing hybrid wheat" (Driscoll, 1972, 1985). Each system identifies male-sterile plants from a mixture of male fertile/male-sterile plants produced by these genetic systems. But none of these systems has yet proved practical. Cytogenetically, it is easier to modify the XYZ system to add a special alien chromosome to a crop like wheat rather than to seek and modify a special chromosome that functions like BTT in barley or Dp-Df in maize.

The XYZ system of producing hybrid wheat (Driscoll, 1972) relies on the addition of chromosome 5R derived from Secale cereale L. to the male-sterile Cornerstone mutant (Driscoll, 1977). Since the dominant marker character--hairy peduncle associated with 5R--is an adult plant trait, differentiating between male-sterile plants without the marker and the male fertile plants having the marker trait will take place at a postheading plant developmental stage in the field. So it is not a practical system, as it is difficult to rogue out the male fertile plants in the short time between heading and pollen shedding. A more efficient marker character associated with an alien chromosome is needed for logically modifying the XYZ system. Seed color is an ideal choice as a marker character. The blue grain gene Ba was transferred to bread wheat from Agropyron spp. (Bolton, 1968). After chromosome 4E, derived from Agropyron elongatum ssp. ruthenicum Beldie 2n = 70, was substituted or added into the genetic background of bread wheat by wide hybridization, the 4E substitution line (4D/4E, 2n = 42) or addition line (2n = 44) expressed blue grain seed color. Blue grain is caused by a layer of blue aleurone in the seed endosperm. Li et al. (1983) studied the blue grain trait and observed that when the seeds of the 4E disomic substitution (4D/ 4E) or addition line were matured normally, the dominant blue grain gene of chromosome 4E had a "gene dosage effect," with deep-, middle-, and light-blue grains clearly conditioned by three, two, and one dosage of chromosome 4E, respectively. Cross-pollinated [F.sub.1] seeds had light-blue grains when the 4E disomic substitution (4D/4E) or addition line was used as the male parent, since the blue grain gene of chromosome 4E also produces a xenia effect (Li et al., 1983). Our objectives in this study were to transfer chromosome 4E to the LZ mutant to establish a new efficient cytogenetic system of maintaining the male sterility of the LZ mutant based on the blue grain seed color trait as a marker character associated with chromosome 4E, and then study the genetic actions of the system.


The materials used in this study were: (i) the LZ mutant 87(212) [2n = 42(msms) - 42]; (ii) a 4E disomic addition line 81529 [2n = 42(MSMS)+ 4E" = 44], developed from blue grain winter wheat line Lanmai [2n = 42(MSMS)+ 4E" = 44] and bred by Li et al. (1983) by wide hybridization; (iii) 577 common wheat lines including Chinese Spring; and (iv) 14 light-blue grain male fertile (LBGMF) lines [2n = 42(msms)+ 4E' = 43] developed during the course of this study.

Breeding of the 4E-ms system began in the field by crossing male-sterile (msms) plants of the LZ mutant as female to the blue grain line 81529 as male (Fig. 1). The light-blue [F.sub.1] seeds were space-planted in the field and each plant was harvested individually. The blue and white seeds from every self-fertilized [F.sub.1] plant were individually sorted, and [F.sub.2] seeds were planted in two adjacent rows in the field as [F.sub.2] family lines. For the blue grain rows of [F.sub.2] family lines, the self-fertile plants that segregated into white and blue grains were selected and harvested individually, and then planted as [F.sub.3] family lines using the same procedure as the [F.sub.2] generation.


The segregation rate of the three seed colors in LBGMF lines was determined by counting the percentage of white and blue seeds in all seeds from 20 self-fertilized plants sampled from the plot, then counting the percentage of self-fertile plants that were homozygous for deep blue grains in all blue grain plants of the LBGMF plot, and finally calculating the segregation rate of the three seed colors in self-fertile LBGMF lines. The transmission rate through the female of the 4E chromosome in self-fertile LBGMF lines was determined by counting the percentage of blue seeds in [F.sub.1] plants produced by artificial cross-pollination between the LBGMF line as female and a common wheat line as male. Transmission of the 4E chromosome through the male was similarly determined using the LBGMF line as male parent and the common wheat line as female. The data were tested with chi-square analysis to detect differences in seed set, segregation rate of three seed colors, and transmission rate of the 4E chromosome in 10 LBGMF lines. The seed set was determined with the formula below.</p> <pre> seed set (%) = 100{total seeds in the two outside florets of spikelets of bagged spikes/[2(total spikelets of bagged spikes)]} </pre> <p>A yield experiment was designed for examining the yield potential of LBGMF lines. The test involved four outstanding LBGMF lines, and CK Ningchun No. 4, a pure line cultivar used by local farmers in wheat production. The experimental design was a randomized block with three replications and 10-[m.sup.2] plot size. Yield data were analyzed by analysis for variance and LSD tested means.

For chromosome counts, excised root-tips were pretreated at 0 to 4[degrees]C for 24 h, then fixed in 3:1 alcohol/acetic acid for 12 to 20 h, and stored in the refrigerator until microscopic examination. Fixed root-tips were hydrolyzed in 1 M HCl for 10 rain at a constant temperature of 60[degrees]C, and then stained with standard Haema-solution. To observe metaphase I chromosome-pairing configurations, young spikes at the appropriate developmental stage were fixed in 6:3:1 absolute alcohol/chloroform/acetic acid. Anthers were squashed in 1.5% acetocarmine and pollen mother cells (PMCs) were microscopically examined to observed chromosome pairing configurations.


Breeding and Genetic Analysis

In 1992, we crossed male-sterile plants of the LZ mutant as female to the 4E disomic addition line 81529 as male. After three generations of breeding that is illustrated in Fig. 1, we tested 184 [F.sub.3] family lines and selected 13 lines where all plants from the white grain rows were male-sterile and all plants from its corresponding blue grain rows were male fertile. The progeny of most self-fertile plants in the blue grain rows segregated into white and blue grains randomly distributed in spikes; but a few self-fertile plants produced only deep-blue grains (Fig. 2). In all succeeding generations, these blue grain lines and white grain lines have performed similarly to the [F.sub.3]. These results suggested that the white grain male-sterile (WGMS) lines could be crossed as female to any cultivar of bread wheat as male (M line) for producing hybrid seed, and also that the light-blue grain male-fertile (LBGMF) lines could be used as maintainer lines for producing both WGMS and LBGMF seed by self-fertilization, and discarding the deep-blue grain male-fertile (DBGMF) seed by use of a color sorting machine. The application of the 4E-ms system to hybrid wheat seed production is illustrated in Fig. 3.


Results from cytological tests for determining chromosome number and chromosome configurations of PMCs for three color seeds from three agronomic-stable LBGMF lines 2574B, 257B, and 1376B, indicated that the WGMS, LBGMF and DBGMF lines had 2n = 42 ([21.sup.II]), 43 ([21.sup.II] + [1.sup.I]), and 44 ([22.sup.II]) respectively (Fig. 4a-4f and Table 1). It is also indicated that the 4E chromosome was inherited independently in the genetic background of bread wheat. The good male fertility observed in all LBGMF and DBGMF lines meant that there was a dominant gene(s) for male fertility in alien chromosome 4E, and this gene was expressed after chromosome 4E was added to a genetic background having the homozygous male-sterile (rosins) genotype derived from the LZ mutant. The fact that DBGMF seeds occurred in offspring of self-fertilized LBGMF lines indicated that the 4E chromosome was transmitted through both female and male gametes.


Genetic actions of the 4E-ms system

In 1996, we randomly bagged 585 spikes from 20 LBGMF lines. From the 21 839 self-fertilized grains obtained from these spikes, there were 7807 blue grains, which was 35.7% of the total. We also observed that of the 2462 total plants in the blue grain rows, there were 244 self-fertile plants that produced only deep-blue grains, which was 10.1% of the total. So DBGMF grains comprised 35.7% x 10.1% [approximately equal to] 3.6% of self-fertilized LBGMF grains on average. Therefore the average percentage of the three seed colors in the selfed progenies of the LBGMF lines was 64.3% white, 32.1% light blue, and 3.6% deep blue.

Three important traits in 10 agronomical stable LBGMF lines are shown in Table 2. Seed set of the 10 lines ranged from 56.4 to 93.2%, indicating normal self-fertility. The 10 lines had similar segregation rates of the three seed colors, with rates varying from 68.7 to 53.4% for WGMS, 29.4 to 43.3% for LBGMF, and 1.9 to 3.8% for DBGMF. The transmission rates of the 4E chromosome varied from 11.1 to 18.4% through the male to 19.0 to 25.8% through the female. Chi-square analysis indicated that most all these traits were significantly affected by the host wheat genotype ([chi square] = 387.17 > [[chi square].sub.0.01, 18] = 34.81 for the segregation rates of the three seed colors, [chi square] = 69.02 > [[chi square].sub.0.01,9] = 21.69 for the transmission rate of the 4E univalent by male, [chi square] = 129.63 > [[chi square].sub.0.01, 9] = 21.69 for seed set of 10 LBGMF lines, and [chi square] = 108.51 > [[chi square].sub.0.01, 9] = 21.69 for seed set of 10 DBGMF lines). But the transmission rate of the 4E chromosome through the female was not significantly affected by wheat genotype ([chi square] = 17.21 < [[chi square].sub.0.01, 9] = 21.69). A recent special case in our breeding program was the LBGMF line 292B, which had only 18.6% of self-fertilized seed having blue grains (Table 3), which was about half of that observed in ordinary LBGMF lines, and whose 4E chromosome transmission rate was only 5% by male and 18% by female. Because of its lower level of blue grains, line 292B is now a key germplasm line for breeding efficient LBGMF lines with fewer blue grains. We have not yet selected a LBGMF line where the transmission rate of the 4E chromosome is zero through the male, however that is a breeding goal we would like to achieve.

Since finding the LZ mutant in 1989, a total of 577 bread wheat lines were used as the male parent in crosses to the LZ mutant as female in an attempt to seek cytoplasm-specific lines that would maintain the male sterility of the LZ mutant. But, all [F.sub.1] combinations had normal fertility, with seed-set ranging from 70.9 to 96.8% and averaging 87.7% (Fig. 5). The seed-set of most combinations was higher than 80%. This compared favorably with the seed set of five self-fertilized check lines, including Ningchun No. 4, which had seed set ranging from 79.4 to 94.3% in different years and locations. So the LZ mutant was considered to be a nuclear or genetic male-sterile mutant. The same circumstances occurred in the WGMS lines of the 4E-ms system. Repeated pollinations were made to male-sterile spikes of WGMS lines with bread wheat lines as pollen donors, and the seed-set ranged from 97.5 to 100% in the cross combinations. This indicated that WGMS lines had normal female fertility.


Generally the 4E chromosome produced no significant negative effects in LBGMF lines. In our yield trial, the LBGMF line 2574B yielded 97.7% of the standard pure line cultivar Ningchun No. 4, and this difference was nonsignificant (standard error was 328.1 kg [ha.sup.-1]) (Table 3).


The establishment of the 4E-ms system of producing hybrid wheat has efficiently solved the problem of maintaining GMS in wheat, and developed a new approach and method of hybrid wheat production. Before this study Huang et al. (1988, 1991) reported a similar study to our 4E-ms system that incorporated chromosome 4E into a male-sterile wheat they found in the [F.sub.3] generation of the combination 72180/Xiaoyan No. 6. The cytogenetic mechanism is the same and the results are similar in the two independent studies, but the male-sterile mutants used are from different sources. The allelic relationships between the genes in the two recessive male-sterile mutants will be determined in the future.

In the XYZ system, not only must the alien chromosome restore fertility but also it must refrain from pairing during meiosis with any of the wheat chromosomes. Driscoll (1981) pointed out that chromosome 4H or modified 4H of barley, 2R of rye, and 4A of T. monococcure were suitable alien chromosomes for the XYZ system. We believed that there must be other alien chromosomes suitable for the XYZ system. So here we give a general name of NX-ms (where NX represents the alien chromosome added) for any system where an alien chromosome restores fertility of a nuclear male-sterility gene to produce hybrid wheat. However, there is no report on what marker gene is carried by chromosome 4H, 2R, and 4A. In this study and the similar study by Huang et al. (1988, 1991), chromosome 4E restored male fertility, was independently inherited, and proved to be an especially ideal marker gene for the NX-ms system.

Color sorting machines are available for purchase from China and Japan (Anzai, 2004). In our tests with a machine made by a Chinese company, the machines can efficiently sort blue grain seeds out from a mixture of blue and white seeds. But the machine has difficulty in efficiently sorting DBGMF seeds in mixtures with LBGMF seeds. We propose to solve this problem based on the "xenia" of the blue grain gene. A certain amount of WGMS seeds are mixed into its corresponding LBGMF line when it is replicated in the field in every year to try to keep a minimum and stable amount of DBGMF seeds in every year's self-fertilized LBGMF line. This will result in some yield loss of the WGMS seed production because of some WGMS seeds produced by natural cross-pollination in the field of the added WGMS x LBGMF.

Selecting LBGMF lines that produce fewer self-fertilized blue grains or even no deep blue grains is an important breeding objective in further improvement of the 4E-ms system. The selected LBGMF line 292B, with only 18.6% self-fertilized blue grains has been made the material basis of improving LBGMF lines with fewer blue grains. Like the modified XYZ system of producing hybrid wheat (Driscoll, 1985), the 4E chromosome would be transmitted through the male at a lower rate if chromosome 4E in the LBGMF line were replaced by a monoisosomic chromosome 4E. But it must first be proved that the gene for male fertility on 4E is located in the same arm of chromosome 4E where the gene for blue grain is also located. All of these are studies that are on going in our program.

Abbreviations: DBGMF, deep-blue grain male fertile: GMS, genetic male sterile: LBGMF, light-blue grain male fertile: LZ, Lanzhou: WGMS. white grain male sterile.


Anzai. 2004. Outline of company operations [Online]. Available at (verified 26 Aug. 2005). Anzai Manufacturing Co. Ltd., Chiba, Japan.

Bolton, E.F. 1968. inheritance of blue aleurone and purple pericarp in hexaploid wheat. Ph.D. diss. Colorado State Univ., Fort Collins, CO.

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Driscoll, C.J. 1977. Registration of Cornerstone male-sterile wheat germplasm. Crop Sci. 17:190.

Driscoll, C.J. 1981. New approaches to wheat breeding, p. 97-106. In L.T. Evens and W.J. Peacock led.) Wheat science today and tomorrow. Cambridge Univ. Press, Cambridge.

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Kuanji Zhou, * Shihong Wang, Yuqin Feng, Zhongxiang Liu, and Genxuan Wang

Kuanji Zhou and Genxuan Wang, State Key Laboratory of Arid Agroecology, Lanzhou University, Lanzhou, Gansu, China.730070. Kuanji Zhou, Shihong Wang, Zhongxiang Liu, and Yuqin Feng, Food Crops Institute, Gansu Academy of Agricultural Science. Lanzhou, Gansu, China.730070. This research was supported by the Fund of National Nature Science (30170593). Received 11 Jan. 2005. * Corresponding author (

Table 1. The chromosome configurations at metaphase I of meiosis in
pollen mother cells of three LBGMF lines segregating for three seed
color classes. ([dagger])

                               WGMS plants

LBGMF    Total plants             No. of             Seed
lines      examined            chromosomes           set


2574B         6                [21.sup.II]           0.2
257B          8                [21.sup.II]           0.0
1376B         6                [21.sup.II]           1.1

                               LBGMF plants

LBGMF    Total plants             No. of             Seed
lines      examined            chromosomes           set


2574B         10         [21.sup.II] + [1.sup.I]     86.0
257B           7         [21.sup.II] + [1.sup.I]     79.6
1376B         11         [21.sup.II] + [1.sup.I]     85.2

                               DBGMF plants

LBGMF    Total plants             No. of             Seed
lines      examined            chromosomes           set


2574B         6          [21.sup.II] + [1.sup.II]    89.1
257B          5          [21.sup.II] + [1.sup.II]    81.6
1376B         4          [21.sup.II] + [1.sup.II]    88.2

([dagger]) LBGMF, light-blue grain male fertile; WGMS, white
grain male sterile; DBGMF = deep-blue grain male fertile.

Table 2. Segregation rate of three seed colors, 4E univalent
transmission rate, and seed set in the self-fertilized progenies
of 10 LBGMF lines. ([dagger])

              Segregation rate %        4E transmission
               ([double dagger])       rate % ([section])

line      WGMS     LBGMF     DBGMF      Male     Female

389B                                    313       531
         1630       793       96         42       116
           64.7      31.5      3.8       13.4      21.8
503B                                    289       502
         1403       994       87         48       123
           56.5      40.0      3.5       16.6      24.5
515B                                    272       492
         1776       790       60         33       100
           67.6      30.1      2.3       12.1      20.3
524B                                    243       478
         1734       759       88         34        91
           67.2      29.4      3.4       14.0      19.0
576B                                    287       513
         1697       805       54         38       105
           66.4      31.5      2.1       13.2      20.5
580B                                    289       521
         1699       727       47         32       106
           68.7      29.4      1.9       11.1      20.3
585B                                    307       518
         1303       933       86         49       133
           56.1      40.2      3.7       16.0      25.7
606B                                    279       504
         1346      1047       77         47       127
           54.5      42.4      3.1       16.8      25.2
614B                                    283       488
         1306       909       80         49       121
           56.9      39.6      3.5       17.3      24.8
620B                                    272       488
         1329      1078       82         50       126
           53.4      43.3      3.3       18.4      25.8

           Seed-set % ([paragraph])

line      WGMS     LBGMF     DBGMF

389B     768       784       788
           5       505       487
           0.7      64.4      61.8
503B     782       762       774
           6       430       512
           0.8      56.4      66.2
515B     786       806       808
           8       569       553
           1.0      70.6      68.4
524B     770       750       774
           2       560       558
           0.3      74.7      72.1
576B     736       754       730
           4       612       597
          11.5      81.2      81.8
580B     714       724       716
           0       543       581
           0.0      75.0      81.2
585B     752       758       764
           0       594       636
           0.0      78.4      83.2
606B     768       738       752
           4       656       663
           0.5      88.9      88.2
614B     782       792       802
           5       738       748
           0.6      93.2      93.3
620B     750       764       760
           0       696       703
           0.0      91.1      92.5

([dagger]) LBGMF, light-blue grain male fertile; WGMS, white grain male
sterile; DBGMF, deep-blue grain male fertile.

([double dagger]) The upper value refers to the number of three colors
seeds from bagged spikes of 20 plants.

([section]) The upper and middle values refer to total
cross-pollinated seeds and total blue seeds, respectively, from 20
spikes of 20 plants.

([dagger]) The upper and middle values refer to total florets and
seeds, respectively, in the two outside spikelets of 20 bagged spikes.

Table 3. Agronomic performance and segregation rates for three seed
colors in four light-blue grain male-fertile (LBGMF) lines.

                                             Segregation rate
                                             ([double dagger])

                                       of CK
LBGMF   Plant    Seed                  ([dag-
lines   height   set       Yield       ger])     WGMS   LBGMF   DBGMF

          cm      %     [ha.sup.-1]                  %

2575B     82      91       7726       97.0       61.0   35.1     3.9
257B      76      87       7089       89.6 *     65.2   34.8     3.2
1376B     75      81       6372       80.0 **    63.9   36.1     3.6
292B      90      72       6276       78.8 **    81.4   17.1     1.5
CK        86      88       7965

* LS[D.sub.0.05] = 756.6 kg [ha.sup.-1]

** LS[D.sub.0.01] = 1100.8 kg [ha.sup.-1]

([dagger]) CK, Ningchun No. 4, a pure line cultivar.

([double dagger]) WGMS, white grain male sterile; LBGMF, light-blue
grain male fertile; DBGMF, dark-blue grain male fertile.
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Author:Zhou, Kuanji; Wang, Shihong; Feng, Yuqin; Liu, Zhongxiang; Wang, Genxuan
Publication:Crop Science
Geographic Code:1USA
Date:Jan 1, 2006
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