Chromosome 1H of Betzes barley in Chinese Spring wheat causes meiotic abnormality by pre-meiotic duplication of chromosomes

Ayaka Ishihara¹, Rafiqul A. K. M. Islam², Takashi R. Endo^{1, 3, 4}, Shuhei Nasuda¹

¹ Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

² School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, SA 5064, Australia

³ Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 783 71 Olomouc, Czech Republic

⁴ Palacký University Olomouc, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 11, 783 71 Olomouc, Czech Republic

*Corresponding author: Shuhei Nasuda (E-mail: nasushu@kais.kyoto-u.ac.jp)

Abstract

Addition of chromosome 1H from barley cultivar Betzes causes sterility in Chinese Spring wheat. We observed severe distortion in meiotic metaphase I configurations in the monosomic 1H addition line. Number of chromosome increased greatly indicating several rounds of chromosome duplication in premeiotic mitosis. The meiotic cell division was arrested at the anaphase I, which was visualized by the stretched chromosomes connected at the telomeric ends. Immunostaining of the meiotic cells using an α-tubulin antibody indicated that premature cytokinesis took place before separation of homologous chromosomes and that the bipolar spindles are formed in the irregularly formed dyads. 

Key words: wheat-barley addition, chromosome 1H, sterility, meiotic arrest

Barley (Hordeum vulgare L.; 2n = 2x = 14, genome formula HH) chromosome addition lines of common wheat (Triticum aestivum L., 2n = 6x = 42, genome formula AABBDD) have been established to broaden the spectrum of genetic diversity for improvement of wheat. Beyond their practical importance in agriculture, barley chromosome addition lines are good research resources in Triticeae genetics and genomics. In a wheat-barley addition line, each barely chromosome is sorted in wheat background, thus the addition lines allow allocation of barley genes and molecular markers to each barley chromosome (for review, see Molnár-Láng et al. 2014). Recently published draft genome sequence of barely, aimed to reveal all gene-associated sequences in barley, also relies on the wheat-barley addition lines in generation of chromosome-specific survey sequences (the International Barley Genome Sequencing Consortium, the IBGSC, Mayer et al. 2012).

Islam and his colleagues did pioneering studies of wheat-barley addition. They produced six wheat-barley disomic addition lines of common wheat for barley chromosomes 2H to 7H by crossing Chinese Spring (CS) wheat and Betzes barley (Islam et al. 1981; Islam 1983). Islam and Shepherd (2000) produced the seventh addition line of the CS wheat-Betzes barley combination carrying one intact and the short arm of chromosome 1H together with a pair of 6H chromosomes. Interestingly, only this chromosomal constitution allowed the fertile addition of chromosome 1H to common wheat. Islam and Shepherd (2000) demonstrated that single addition of 1H caused sterility, which could be rescued by the presence of 6H. The sterility of 1H addition is suspected to be due to meiotic abnormality.

In the season 2012-2013 we screened 35 plants of the progeny of the cross double monosomic addition of Betzes barley chromosomes 1H and 6H in CS (21”W + 1’1H + 1’6H, female) and CS (male).  Chromosome constitutions in root tip cells were tested by the FISH/GISH protocol as described elsewhere (Ishihara et al. in press). We found one plant that had chromosome 1H monosomically (Figure 1). Other progeny possessed 6H (12 plants), 1H and 6H (2 plants), 1H and the long arm of 6H (1 plant), and neither of 1H and 6H (19 plants). In the season 2013-2014, we screened 59 plants of the progeny and found that seven had chromosome 1H, 12 had only chromosome 6H, seven had both 1H and 6H, and 33 had neither 1H nor 6H. These monosomic 1H addition plants grew normally to reproductive stages.

Selfed- and crossed-seed fertilities of the 1H monosomic addition were very low. When it was selfed, ninety-seven florets bore no seed. When manually pollinated with CS pollen, it bore two seed out of 289 florets (0.7%), contrasting to the crossed-seed fertility of the double monosomic addition line 1H and 6H (30.3%, 47 seed out of 155 florets). The sterility of the monosomic 1H addition was on both female and male sides. Cytological observation of the meiotic metaphase I (MI) of the pollen mother cells (PMCs) in the monosomic 1H addition line revealed that numbers of chromosomes were increased (Figure 2). Large numbers of chromosomes prevented us to correctly determine the meiotic chromosome configurations in MI, however, it was clear that chromosome numbers were more than the expected 21” +1’ configuration and that the chromosome numbers varied greatly. Interestingly, most of chromosomes formed bivalents, indicating that random increase of individual chromosomes, which results in the increase of univalents, was not the case. Since the MI plates were normally formed, we speculate that increase of chromosome happened in premeiotic mitosis.

Increase in chromosome number and/or chromosome doubling is not necessarily causes sterility but could produce fertile gamete with aneuploidy. In the case of 1H monosomic addition line, the progress of meiosis was blocked at anaphase I (AI) where the stretched bivalents were often observed (Figure 3). The bivalents seemed to be under tention between two polls. We examined organization of spindles by immunostaining of the spindle by the α-tubulin antibody (methodology of the immunostaining can be found in Matsuoka et al. 2013). We could not find clear bipolar spindle in MI (Figure 4). Cytokinesis happened precociously before the completion of anaphase I. The resulting irregular dyads contained nonseparated homologues where the bipolar spindles formed. These observations indicated that increase in chromosome number before entering to MI and abnormal separation of chromosomes at AI might be the cause of sterility.

One of the fertile progeny of the 1H monosomic addition crossed with CS had septaploid chromosome number, indicating that the 1H monosomic addition line formed unreduced hexaploid gamete (data not shown). We also observed the expected 21” +1’ MI configuration very rarely in the PMC, which could also give rise of fertile gamete (data not shown). The very scarce crossed seed fertility in the 1H monosomic addition could be the results of hybridization of those very rare female gametes.

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